Infection Biology of Chlamydia pneumoniae
Leslie Bailey
Department of Molecular BiologyLaboratory for Molecular Infection Medicine Sweden (MIMS)Department of Public Health and Clinical Medicine, Cardiology
Umeå UniversityUmeå 2008
Department of Molecular BiologyDepartment of Public Health and Clinical Medicine, Cardiology
Department of Community Medicine and Rehabilitation, GeriatricsUmeå University
SE-901 87, Umeå, Sweden
Copyright © 2008 by Leslie BaileyISBN: 978-91-7264-532-5
Printed by Print & Media, Umeå University, Umeå, Sweden, 2008, 2004192
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To Lisa and Elliot
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ABSTRACT
There are two main human pathogens in the family of
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TABLE OF CONTENTS
LIST OF PUBLICATIONS .............................................................................................. v Additional papers not included in thesis ................................................................. v
INTRODUCTION ............................................................................................................ 1 Chlamydiaceae ........................................................................................................ 1 Historical view of Chlamydia ................................................................................. 1 TWAR – an orienteer-related disease? ................................................................... 2 Taxonomy ............................................................................................................... 2
Structure of Chlamydia in general and C. pneumoniae in particular ........................... 4 Morphology ............................................................................................................ 4 LPS ......................................................................................................................... 4 Peptidoglycan .......................................................................................................... 4 Polymorphic membrane proteins ............................................................................ 5 Major outer membrane protein ............................................................................... 5 Heat shock proteins ................................................................................................. 6
The developmental cycle ............................................................................................. 7 The key players of Chlamydia ................................................................................ 7 Attachment .............................................................................................................. 8 Internalization ......................................................................................................... 8 Internalization: Endocytosis - mediated via activation of the actin cytoskeleton ... 8 The inclusion - a chlamydial protected site ............................................................. 9 Inclusion membrane proteins ................................................................................ 10 Primary differentiation and proliferation .............................................................. 10 Re-differentiation .................................................................................................. 11 Exit from the host cell ........................................................................................... 11 Bacteria and iron ................................................................................................... 12 Chlamydia and iron ............................................................................................... 12 Chlamydial persistence ......................................................................................... 12
Clinical manifestations .............................................................................................. 14 Acute infection ...................................................................................................... 14 Chronic infection .................................................................................................. 14
C. pneumoniae and atherosclerosis ........................................................................... 15 Atherosclerosis ...................................................................................................... 15 Atherosclerosis and animal models ....................................................................... 15 Lipoprotein (a) as a mouse model for atherosclerosis ........................................... 16 C. pneumoniae in atherosclerosis .......................................................................... 17 Antimicrobial trials have questioned the C. pneumoniae infection theory ........... 17 C. pneumoniae and inflammation of the vascular system ..................................... 18
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Pro-inflammatory cytokines .................................................................................. 18 C. pneumoniae and pro-inflammatory cytokines .................................................. 19 Bacterial infection of bone demonstrates similar cytokine induction as in
atherosclerosis ....................................................................................................... 19 The type three secretion system (T3SS) .................................................................... 21
Historical observations of a possible T3SS ........................................................... 21 First evidence of a chlamydial T3SS .................................................................... 22 Composition of the chlamydial T3S injectisome .................................................. 23 Basal apparatus ..................................................................................................... 23 The Chlamydia translocon .................................................................................... 24 Chaperones of the T3SS ........................................................................................ 25 Transcriptional regulation of T3SS chaperones .................................................... 26 The needle complex .............................................................................................. 26 Gating of T3S machinery ...................................................................................... 27 Effector proteins.................................................................................................... 28 Other secreted proteins ......................................................................................... 29 Extracellular T3SS ................................................................................................ 30 Intracellular T3SS ................................................................................................. 30 Temporal transcription of T3SS genes .................................................................. 31 Stress of the T3SS ................................................................................................. 32
Antibiotics ................................................................................................................. 33 Antimicrobial resistance is a global problem ........................................................ 33 Antibiotic treatment of C. pneumoniae infection .................................................. 33
Small molecules ........................................................................................................ 34 Chemical genetics and small chemical molecules ................................................ 34 Small molecules as inhibitors of virulence ........................................................... 34 Small Molecules and inhibition of T3SS .............................................................. 34
OBJECTIVES OF THIS THESIS ............................................................................. 36 RESULTS AND DISCUSSION ..................................................................................... 37
Paper I ....................................................................................................................... 37 Screening of T3SS inhibitors using Y. pseudotuberculosis T3SS ......................... 37 INP0010 inhibits C. pneumoniae intraceullar propagation ................................... 38 INP0010 shows diverse effect in C. trachomatis .................................................. 38 INP0400 is active against both C. pneumoniae and C. trachomatis. .................... 38 Treatment with INP0010 inhibits secretion of putative T3SS effector proteins ... 39
Paper II ...................................................................................................................... 40 INP0400 inhibits proliferation but not primary differentiation ............................. 40 INP400 inhibits secretion of putative effectors ..................................................... 40 INP0400 treatment during the late cycle promotes bacterial dissociation from
the inclusion membrane ........................................................................................ 41
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Paper III ..................................................................................................................... 42 DNA is preferable to RNA for normalization of gene expression ........................ 42 INP0010 decreases initiation of transcription ....................................................... 43 Expression of T3SS in the presence of INP0010 .................................................. 43
Paper IV .................................................................................................................... 44 INP0010 inhibits early- and mid-developmental cycle ......................................... 44 Iron initiates proliferation but not terminal re-differentiation in the presence of
INP0010 ................................................................................................................ 44 INP0010 blocks secretion of IncB into the host cell cytoplasm ............................ 45
Paper V ...................................................................................................................... 47 C. pneumoniae infection decreases bone mineral density in mice ........................ 47 C. pneumoniae growth in hFOBs increases production of IL-6 and expression
of RANKL ............................................................................................................ 47 CONCLUSIONS ............................................................................................................ 49 ACKNOWLEDGEMENTS ............................................................................................ 50 REFERENCES ............................................................................................................... 52
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LIST OF PUBLICATIONS This thesis is based on the following articles and manuscripts which will be referred
to by their roman numerals I-V.
I. Bailey L, Gylfe Å, Sundin C, Muschiol S, Elofsson M, Nordström P,
Henriques-Normark B, Lugert R, Waldenström A, Wolf-Watz H, and
Bergström S. Small molecule inhibitors of type III secretion in Yersinia block the
Chlamydia pneumoniae infection cycle. FEBS Lett. 2007 Feb 20;581(4):587-95.
II. Muschiol S, Bailey L, Gylfe Å, Sundin C, Hultenby K, Bergström S,
Elofsson M, Wolf-Watz H, Normark S and Henriques-Normark B. A small-
molecule inhibitor of type III secretion inhibits different stages of the infectious
cycle of Chlamydia trachomatis. Proc Natl Acad Sci U S A. 2006 Sep
26;103(39):14566-71.
III. Bailey L, Engström P, Önskog T, Bergström S and Johansson J. The T3SS-
inhibitor INP0010 decreases transcription initiation and modulates mRNA stability
during early development in Chlamydia pneumoniae. Submitted.
IV. Bailey L, Muschiol S, Engström P, Nordström P Henriques-Normark B,
Waldenström A, Gylfe Å, Elofsson M, Wolf-Watz H and Sven Bergström. The
Type three secretion: a possible mechanism for Chlamydia pneumoniae to utilize
intracellular iron acquisition - Demonstrated by using the T3SS-inhibitor INP0010.
Manuscript.
V. Bailey L, Engström P, Nordström A, Waldenström A, Bergström S and
Nordström P. Chlamydia pneumoniae Infection Results in Generalized Bone Loss
in Mice. Submitted.
Additional papers not included in thesis
(VI). Steptoe A, Shamaei-Tousi A, Gylfe A, Bailey L, Bergström S, Coates AR,
Henderson B. Protective effect of human heat shock protein 60 suggested by its
association with decreased seropositivity to pathogens.Clin Vaccine Immunol. 2007
Feb;14(2):204-7.
(VII). Ovchinnikova O, Gylfe Å, Bailey L, Nordström A, Bergström S,
Waldenström A, Hansson GK and Nordström N. Osteoprotegerin Promotes
Fibrous Cap Formation in Atherosclerotic Lesions of apoE deficient Mice.
Submitted.
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INTRODUCTION
Chlamydiaceae There are two main species (spp.) of the family Chlamydiaceae that are common
pathogens in humans. Two of the spp, Chlamydia trachomatis and C. pneumoniae, are
common pathogens in humans, whereas the other species occur mainly in animals
(summarized in Table 1). The sexually transmitted C. trachomatis has been isolated
only from humans and is comprised of two human biovars (trachoma and
lymphogranuloma venereum, LGV), including a total of 18 serovars, whereas C.
pneumoniae consists of one human biovar (TWAR) and two animal biovars, one
infecting horses (biovar equine) and the other infecting frogs1 and koalas (biovar
koala)2.
Historical view of Chlamydia Initially, these bacteria were first believed to be viruses, due to their obligate
intracellular requirements. In 1964, Schechter et al., revealed the truth about Chlamydia
when he found the presence of both RNA and DNA as well as cell wall structures,
demonstrated by electron microscopy3. However, Chlamydia was grouped with
Rickettsia until the genus Chlamydia was established and two different strains,
Chlamydia trachomatis and Chlamydia psittaci were isolated4.
Since then, several new chlamydial strains have been isolated. The first report of a
Chlamydia pneumoniae-like bacterium was described in 1965, believed to be an
atypical strain of C. psittaci. The bacterium was isolated from the eye of a child during a
trachoma vaccine trial in Taiwan, and thus given the name TW-1835. The first report of
C. pneumoniae causing clinical manifestations came in 1983, when the bacterium was
isolated in the United States from a throat swab of a university student suffering from
pharyngitis. This isolate was termed AR-39, due to its isolation as an Acute Respiratory
pathogen6. This group of organisms was eventually called TWAR, which was an
acronym for the first two isolates, i.e. TW-183 and AR-39. At that time, TWAR was
considered a human C. psittaci strain spreading from human to human without an avian
or mammalian host 6. Interestingly, TWAR was found to be a serologically unique
group among the known C. psittaci isolates. These showed differential and milder
pathogenic properties when grown in cell culture or inoculated into mice or chicken
embryos. In 1989, the strain was identified as a separate species within the genus
Chlamydia and named C. pneumoniae7. To fulfill the criteria to become a member of
the genus Chlamydia, the strain had to meet the following standards: obligate
intracellular parasitism, unique developmental cycle, share genus-specific
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lipopolysaccharide (LPS), and complement fixation antigen and comparable guanine-to-
cytosine content7.
TWAR – an orienteer-related disease? In the 90’s, TWAR was believed to be the common cause of sudden unexpected cardiac
death (SUD) among orienteers in Sweden8-10
. During the years 1979-92, 16 young
Swedish orienteers, of whom 14 were of the top elite level, suffered SUD, most of them
while exercising. Serological examination in all 5 cases where serum was available for
screening with a wide range of microbial antigens revealed positive serology for C.
pneumoniae10
. Accordingly, in 2001, it was concluded that Swedish elite orienteers do
not have a higher exposure rate to C. pneumoniae than blood donors11
. Therefore, C.
pneumoniae is no longer considered an orienteer-related disease, mostly due to its high
sero-prevalence in humans. Moreover, Wesslen et al. suggested that sub-acute infection
or re-activation of Bartonella played an important pathogenetic role in the Swedish
orienteers who suffered SUD12
. On the contrary, there is no association between elite
orienteers and prevalence of Bartonella antibody positivity in Denmark13
. Thus, these
results neither support nor contradict the possibility that microorganisms were involved
in some of the SUDs among young Swedish orienteers in the 90’s.
Taxonomy In 1999, a revised taxonomy of the family Chlamydiaceae was suggested based on 16S
and 23S rRNA sequence similarity clusters14
. Everett et al. recommended that the genus
Chlamydia be divided into two genera, Chlamydia and Chlamydophila. Altogether, the
revised genus family contains nine species (Table 1). However, the new reclassification
has been considered unnecessary by the Chlamydia research community15
. The authors
disagreed on the need for a new genus called Chlamydophila. Their objection to
division of the species into two separate genuses was based on the small differences
among genomes of C. trachomatis, C. psittaci and C. pneumoniae, and the report from
Tanner et al, which presents a taxonomically and phylogenetically coherent grouping
into one genus16
. Another aspect that was considered important was that the well-
recognized name Chlamydia had been accepted by the public and any change of the
name would cause confusion. In agreement with the arguments against the genus
division, the old classification has been used in the present work.
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TABLE 1. The family Chlamydiaceae as proposed by Everett et al. 199914
Species Typical host (s) Route of entry
Chlamydia
C. trachomatis Human Pharyngeal, ocular, genital, rectal
C. suis Swine Pharyngeal
C. muridarium Mouse and hamster Pharyngeal, genital
Chlamydophila*
C. psittaci Birds Pharyngeal, ocular, genital
C. pneumoniae Human, koala, horse, frog Pharyngeal, ocular
C. pecorum Domastic animals, koalas Oral
C. felis House cat Pharyngeal, ocular, genital
C. caviae Guinea pig Pharyngeal, ocular, genital, urethral
C.abortus Mammals Oral, genital
Modified from2. The Chlamydophila classification is not refered to in this thesis*
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Structure of Chlamydia in general and C. pneumoniae in particular
Morphology C. pneumoniae does not only differ in genomic content when compared to C.
trachomatis17,18
. Morphologically, C. pneumoniae can be distinguished from other
chlamydial species by the pear-shaped structure of its elementary bodies (EBs) as well
as a loose outer membrane19
. However, the pear-shaped appearance is not a
characteristic feature of all C. pneumoniae isolates20-23
. Thus, Wolf et al. demonstrated
by scanning electron microscopy the presence of a large periplasmic space between
the
cytoplasmic and the outer membranes of C. pneumoniae elementary bodies (EBs)24
.
LPS C. pneumoniae, like other Gram-negative bacteria, has LPS in its cell membrane.
Chlamydial LPS is a genus-specific group of antigens, which is shown to be surface-
exposed and present in both EBs and RBs25
. Therefore, it is suitable as a marker for
chlamydial infections. However, the chlamydial version of LPS seems to have lower
endotoxin activity than enterobacterial LPS and therefore is less potent as an inducer of
inflammation26,27
. Structurally and physiologically, chlamydial LPS is similar to the
rough form of LPS in enterobacteria, but is not identical among chlamydial spp.28,29
.
Peptidoglycan During all developmental stages, Chlamydia appears to be surrounded by two layers
constituting the cell membrane, a characteristic feature of Gram-negative bacteria.
However, unlike other Gram-negative bacteria, Chlamydia does not have a
peptidoglycan layer in the periplasmic space30-32
. In contrast, its cell envelope contains
penicillin-binding proteins, and the presence of peptide cross-links analogous to those
between peptidoglycan backbones has been suggested30
. It has been known for a long
time that the growth of Chlamydia is sensitive to penicillin, despite the absence of
peptidoglycan33
. This paradox is known as the chlamydial anomaly32
. Furthermore,
genomic sequence comparison of C. pneumoniae and C. trachomatis revealed the
presence of genes for peptidoglycan synthesis, membrane assembly and recycling,
respectively17,18
. Peptidoglycan has been suggested to be necessary for RB cell division
and to be produced during growth within host cells34,35
. However, many questions still
remain unanswered regarding how, when and why peptidoglycan is synthesized in
Chlamydia.
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Polymorphic membrane proteins
One of the main components of the chlamydial outer membrane is the polymorphic
membrane proteins (Pmps) localized on the surface of Chlamydia36
. This family of
proteins comprises a significant portion of the genome, approximately 4% in C.
pneumoniae18
. Overall there are 21 Pmp genes in C. pneumoniae18
. All of the 21 genes
of C. pneumoniae have been shown to be transcribed and encode large proteins that are
all phylogenetically related to one of six basic subtypes: Pmp A, B, D, E, G and H37
.
The exact function of the Pmps is not fully known, although several studies indicate that
some Pmps are potentially surface-exposed and immunogenic38,36,39
. Accordingly,
Pmp20 and Pmp21 increase the production of the pro-inflammatory cytokines
interleukin-6 (IL-6), IL-8 and monocyte chemoattractant protein-1 (MCP-1) in human
endothelial cells40
.
Major outer membrane protein The major outer membrane protein (MOMP, gene product of ompA) is the most
abundant protein in the outer membrane of Chlamydia. It was first described and
characterized in 1981 by Caldwell et al. in C. trachomatis41
. MOMP is thought to
maintain the structural integrity of EBs via disulfide bond cross-linking within the EB
outer membrane. MOMP is a multifunctional protein also considered to form pore-like
structures since it shares several biochemical properties with classical porins42
and is
believed to have a role in the infectious process43
. Initially, it was believed that C.
pneumoniae MOMP was different from the protein in C. trachomatis, in the sense of a
diverse antigenic immune determinant in C. pneumoniae MOMP44
. However, further
studies have shown that the MOMP of C. pneumoniae is indeed surface-exposed and an
immunogenic protein45
. The reason why earlier reports demonstrated opposing results
could probably be explained by conformational changes of epitopes that are easily
destroyed by detergents when the antigens are processed for immunoblotting or other
analyses45
. The antigenic properties of MOMP have been extensively studied since the
discovery that purified MOMP (the first chlamydial immunogenic molecule) was
capable of raising antibodies which could neutralize the infectivity of C. trachomatis in
vitro46
. MOMP is now one of the major candidates for development of a Chlamydia
vaccine 47
.
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Heat shock proteins Chlamydia contains heat shock proteins (Hsps) in the outer membrane complexes of
both EBs and RBs48,49
. The genes encoding Hsp10, Hsp60 (homologues to E. coli
GroEL) and Hsp70 (homologues to E. coli DnaK) are implicated in chlamydial
pathogenesis50-53
. These genes are constitutively expressed throughout the
developmental cycle54,55
. The Hsps are highly conserved within chlamydial species,
including C. pneumoniae48,56
. GroEL from C. pneumoniae has been detected in
atherosclerotic plaques57
and serum antibodies to GroEL correlate with the ability of C.
pneumoniae to be detected in plaques58
. Furthermore, cross-reactivity of GroEL
antibodies with human Hsp 60 has been demonstrated59,60
. Thus, this supports a role for
C. pneumoniae infection in the pathogenesis of atherosclerotic heart disease (discussed
further in section C. penumoniae and atherosclerosis).
7
The developmental cycle
The key players of Chlamydia Chlamydiae are intracellular bacteria that have a unique biphasic developmental cycle
with two distinct morphological and functional forms (Fig. 1). The extracellular,
infectious form (0.3 μm) is called the elementary body (EB). The EB is metabolically
inert and the nucleoid is highly compacted in EBs due to the condensation of nuclear
material by the bacterial histone-like proteins Hc1 and Hc2, gene products of hctA and
hctB, respectively61-63
. The intracellular, replicating form (1.0 μm) is called the
reticulate body (RB) and is the metabolically active form of Chlamydia. RBs consist of
homogenous internal material and the cytoplasm appears granular with diffuse, fibrillar
nucleic acids64
. Moreover, there is an intermediate state during transition from RB to
EB, called the intermediate body (IB). The length of the complete cycle is dependent on
the infecting strain, the host cell, and the environmental conditions, varying from 48 to
96 hours in cell culture models. However, the focus here will be describing the
developmental cycle of C. pneumoniae.
FIGURE 1. Chlamydial forms. Electron micrograph of the C. pneumoniae isolate T45 in HEp-2 cell cultures
at 48 hours post infection. Elementary body (EB), reticular body (RB) and intermediate body (IB).
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Attachment The receptors and chlamydial ligands involved in the attachment of EBs to host cell
membranes are undefined. The initial interactions of EBs of many, but not all,
chlamydial strains and species with the host cell appear to be through reversible,
electrostatic interactions with heparan sulphate-like glycosaminoglycans (reviewed in
reference 72). Accordingly, N-glycanase treatment decreased the attachment and
infectivity of C. pneumoniae in human epithelial and endothelial cells, suggesting the
importance of chlamydial glycans containing a high-mannose oligosaccharide for entry,
in vitro and in vivo65,66
. Moreover, Puolakkainen and co-workers proposed that the
mannose 6-phosphate/insulin-like growth factor 2 receptor could serve as a receptor
specifically for C. pneumoniae attachment67,68
. In addition, infectivity was inhibited
when the mannose 6- phosphate receptor was blocked by retinoic acid68
. Chlamydial
surface-exposed proteins are also anticipated to be involved in attachment, for example
of MOMP43
, Omp2, Hsp7069
and PmpD/Pmp270
.
Internalization After attachment, the EB is rapidly internalized into the host cell cytoplasm. There are
several models proposing how EBs enters the host. For example, in 1978, Byrne and
Moulder described the entry of C. psittaci and C. trachomatis into non-professional
phagocytic cells as a process termed “parasite-mediated endocytosis”71
. Lipid-raft
mediated uptake has been implicated as another alternative entry mechanism. However,
because of conflicting results further work is needed to clarify the relevance of lipid-raft
mediated uptake72
. Thus, the internalization step is a crucial step for chlamydial survival
and development of pathogenesis. The mechanism however is still unknown.
Internalization: Endocytosis - mediated via activation of the
actin cytoskeleton Chlamydia is highly dependent on reorganizing the actin cytoskeleton to facilitate their
entry into host cells in vitro73
. C. pneumoniae induces transient microvillar hypertrophy
upon binding to epithelial cells74
. Chlamydia is known to induce tyrosine
phosphorylation of several proteins regulating eukaryotic signaling pathways75-77
.
Several different signaling pathways regulating actin dynamics and organization, such
as PI3-kinase (PI3K) and MEK-ERK kinases, as well as the Rho family of GTPases,
have been shown to be targeted by Chlamydia74,77
. Although C. pneumoniae and C.
trachomatis induce similar morphological changes in microvillar rearrangements, they
target distinct signaling pathways mediating cell alterations. C. pneumoniae uptake has
been shown to be PI3K-dependent involving MAPK kinase ERK1/2 activity74
. In
contrast, C. trachomatis entry involves TARP phosphorylation78
(described in T3SS
section) and subsequently host signal transduction pathways recruiting Rac and
activation of WAVE2/Arp2/379
.
9
The inclusion - a chlamydial protected site When the EB have been endocytosed and localized inside the host cell, EBs are
protected within a modified vacuole, called the inclusion, throughout the developmental
cycle (Fig. 2). In this compartment the bacteria can avoid fusion with vesicular
trafficking pathways of the host cell and internalization of lyzosomes and endosomes
(reviewed in reference 77). Trafficking of the EB to the peri-nuclear region and the
Golgi area is dependent on early chlamydial gene expression80
. Morphologically,
C. pneumoniae inclusions are smaller in size when compared to inclusions of
C. trachomatis. Moreover, multiple inclusions are seen during C. pneumoniae infection,
whereas C. trachomatis inclusions normally fuse.
FIGURE 2. Developmental cycle of C. pneumoniae. See text for abbreviations.
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Inclusion membrane proteins In the inclusion membrane, there exists a group of Chlamydia-specific proteins called
inclusion membrane proteins (Incs). The first report of an Inc protein was demonstrated
in C. psittaci and named IncA81
. Since then, six other Incs, from IncB to IncG, have
been characterized82,83
. Inc proteins are not well conserved between Chlamydia spp.18
.
However, Incs display similarities in their hydropathy profiles indicating a structural
homology, comprised of a large bi-lobed hydrophobic region of approximately 50–80
amino acids84
. It is hypothesized that the major hydrophobic domain in each Inc protein
is embedded in the inclusion membrane. Toh et al. have identified 90 putative Inc
proteins in C. pneumoniae and 36 in C. trachomatis based on the general structure of
the predicted proteins85
. The potential to export such a large number of Incs to the
inclusion membrane suggests that the inclusion membrane may have several functions
in vesicle trafficking, inclusion development, avoidance of lysosomal fusion, nutrient
acquisition and signaling associated with EB-RB-EB reorganization77,86
. Since Incs in
general lack a signal sequence, the T3SS has been postulated, and to some extent
demonstrated, to translocate Incs (more in the T3SS section). IncA has been shown to
have a function in the homotypic fusion of inclusions during C. trachomatis infection87
.
IncA and G of C. trachomatis have been shown to be phosporylated by undefined host
kinases88,89
. IncG has further been shown to interact with the eukaryotic protein 14-3-
3β, implicated in a number of signal transduction pathways by taking advantage of their
ability to alter the intracellular distribution of bound ligands89
. However, the function of
the interaction remains unknown.
Primary differentiation and proliferation Around 8-12 hours post-infection (p.i), C. pneumoniae EBs start to differentiate into
RBs. This occurs before intracellular replication/proliferation/growth begins by binary
fission24
(own observations). It has been suggested that de novo protein expression is
required to initiate intracellular growth, as differentiation can be blocked by the addition
of antibiotic inhibitors of transcription or translation, e.g. antibiotics belonging to the
class of macrolides 90
. But the underlying signal(s) are yet undefined. Between 12 to 48
hours p.i., RBs multiply and are highly dependent on host cells supplying iron, amino
acids and energy80,91
. However, there are differences in the specific needs for nutrients
and energy between species and strains. For example, C. psittaci and C. trachomatis
have a complete set of genes required for tryptophan biosynthesis, whereas these genes
are absent in the C. pneumoniae genome17,18
. Moreover, naturally occurring amino acids
differentially influence the development of C. pneumoniae and C. trachomatis92
. There
are morphological differences in the localization of RBs within the inclusion between C.
pneumoniae and C. trachomatis during equivalent stages of development. RBs of C.
pneumoniae are located throughout the entire lumen of the inclusion, whereas
C. trachomatis RBs are typically tightly juxtaposed to the inclusion membrane24
.
11
Certain circumstances, for example nutrient deficiency, may result in morphological
alterations of RBs and the emergence of enlarged, atypical chlamydial forms termed
“persistent forms.”
Re-differentiation After multiple rounds of division, C. pneumoniae RBs start to re-differentiate back to
infectious EBs around 36-48 hours p.i24
(own observations). During re-differentiation of
RBs to EBs, IBs can be detected. Expression of a number of late-cycle genes occurs
during the re-differentiation process and is described in the review by Abdelrahman and
Belland 64
. However, the signal(s) regulating this event is unknown and one speculative
mechanism has been proposed involving the T3SS (discussed in the T3SS section).
Exit from the host cell When the intracellular growth of C. pneumoniae is completed, infectious EBs are
released into the cytoplasm to initiate new cycles in new host cells by exocytosis or host
cell lysis (72-92 hours p.i, Fig. 3). Chlamydial exit has not been extensively studied,
until recently. Hybiske et al., describes lysis of the host cell membrane as a sequential
process in which the inclusion ruptures before the cell membrane93
. The same authors
described a second mechanism for host cell entry of C. trachomatis and C. caviae as a
packaged-release mechanism, called extrusion93
. The extrusion of Chlamydia involves
the large Chlamydia-containing vacuole pinching off and extruding out of the cell. This
leaves the host cell intact, with a residual bacteria-containing inclusion. The
implications of this exit strategy have not been fully explored or demonstrated for C.
pneumoniae.
FIGURE 3. Growth of C. pneumoniae isolate T45 in HEp-2 cells.
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Bacteria and iron Iron is an important growth factor for pathogenic bacteria, with the exception of the
Lyme disease agent, Borrelia burgdorferi94
. Intracellular bacteria, such as Listeria and
Mycobacteria, depend on intracellular iron sources that are host-derived95-97
. Highly
sophisticated iron-uptake systems have been identified in most bacterial species with
iron-binding siderophores as the major mechanism for bacteria to respond to iron
limitation. For instance, Mycobacteria express at least three known iron uptake systems
and Listeria can access cytoplasmic iron from ferritin98
. However, no iron uptake
mechanism has so far been identified in Chlamydia.
Chlamydia and iron One of the best understood pathways of iron delivery into the eukaryotic host cell is via
the iron-saturated form of transferrin, holo-transferrin, and its high affinity binding to
the transmembrane transferrin receptor99
. Intracellular iron is transferred to the
endosomal pathway (early endosomes) by acidification and an NADH-dependent
reductase activity which converts ferric iron to ferrous iron, which is next exported into
the cytoplasm 99
. Chlamydia escapes this pathway when neither the nascent nor the
mature inclusion fuses with the early endosome90,96,97,100-102
. However, the early
endosome, containing transferrin, is recruited early during the infection and stays
closely associated with the chlamydial inclusion through the entire developmental cycle.
This might suggest a possible chlamydial source of iron, since Chlamydia has been
shown to utilize holo-transferrin as an iron-source95,97
. Moreover, YtgA, a predicted
periplasmic metal-binding protein, has recently been proposed to be involved in iron
acquisition in Chlamydia103
. Furthermore, the divalent cation-dependent regulator A
(DcrA) homologue of ferric uptake regulator (Fur) in E. coli has also been suggested to
be involved in yet unexplained molecular regulatory mechanisms104,105
.
Chlamydial persistence Physicians and the medical community have long thought that long-term chronic or
repeated chlamydial infection might make Chlamydia-related diseases worse.
Accordingly, it has been proposed that C. pneumoniae and C. trachomatis are capable
of causing long-term infections continuing for months or even years in the absence of
treatment. Moreover, when Chlamydia enters a persistent state associated with an
incomplete developmental cycle, the infection is difficult to eradicate with antibiotics or
could, even worse, enter persistence due to antibiotic treatment106
. It has been suggested
that persistence is induced as a result of Chlamydia lacking some essential amino acids,
such as tryptophan, which is essential for complete chlamydial development. How well
these in vitro models really support the in vivo situation is unclear.
13
Exposing chlamydial infections in vitro, with interferon-gamma (IFN-) has become a
conventional procedure to promote persistence (reviewed in reference 91). IFN-
induces tryptophan depletion through activation of the host tryptophan-degrading
enzyme indoleamine 2, 3-dioxygenase (IDO) resulting in the inability of RBs to
differentiate into EBs. Interestingly, C. trachomatis uses a chlamydial tryptophan
synthase to counteract the absence of tryptophan when the amino acid is removed from
the cell culture medium, suggesting the ability to synthesize tryptophan in vivo107
.
However, C. pneumoniae lacks the trpBA operon encoding the tryptophan synthase and
uses another mechanism for tryptophan synthesis108
. Iron depletion, using the iron
chelator deferoxamine-mesylate (DAM), has also been shown to induce persistence of
Chlamydia, in vitro95,97,109-111
. The key clinical question is still open: Does long-term
chronic or repeated infection lead to the intermittent appearance of disease? And how
relevant are the in vitro models described above? Can the models truly mimic the
presence of a rigorous host immune response battling the pathogen to persist in vivo?
••
14
Clinical manifestations
Acute infection C. pneumoniae is a respiratory pathogen that causes upper and lower respiratory tract
diseases and invades the epithelial cells of the respiratory tract and lung tissue. In the
acute infection, C. pneumoniae is transmitted from person to person via respiratory
secretions and droplets112
. Transmission is relatively inefficient, and the incubation
period may be several weeks, which slows down the spread of outbreaks113
. The
majority of C. pneumoniae infections are asymptomatic or mild upper respiratory tract
infections, most of which are mild and self-restricted and therefore often go
undiagnosed114
. For example, in a collaborative study we analyzed 500 asymptomatic
subjects for prevalence of C. pneumoniae infection. In total, 63% were IgG-positive,
indicative of a previous C. pneumoniae infection115
. Accordingly, other studies have
demonstrated a sero-prevalence around 40% to 70% in the adult population globally116
(reviewed in reference 116). Thus, the majority of humans are infected with C.
pneumoniae at some stage of their lives. However, the incubation period of infection
due to C. pneumoniae can be several weeks, which is longer than that of many other
respiratory pathogens117
. Involvement of C. pneumoniae infection has been described in
pneumonia, acute bronchitis, common cold, persistent cough, pharyngitis, sinusitis and
otitis media118(reviewed in reference 118).
Chronic infection In addition to acute respiratory infections, several chronic respiratory tract inflammatory
diseases have been associated with C. pneumoniae infection. These include chronic
bronchitis and chronic obstructive pulmonary disease (COPD) as well as sarcoidosis
(reviewed in reference 118). Several studies also address an association between C.
pneumoniae and asthma119(reviewed in reference 119). Furthermore, C. pneumoniae
may play a role in neurological disorders such as multiple sclerosis120
. C. pneumoniae
has also been associated with reactive arthritis, where C. pneumoniae DNA and mRNA
were found in synovial material from patients suffering from inflammatory joint
disease121,122
. In addition to respiratory tract infections, C. pneumoniae has been
associated with cardiovascular diseases, in particular in the development of
atherosclerosis.
15
C. pneumoniae and atherosclerosis
Atherosclerosis Atherosclerosis develops as a reaction to damage of the arterial wall
123. It is now
considered an inflammatory response to retain and modify lipids in the vessel wall. Both
innate and adaptive immune defense mechanisms are believed to be important in
pathogenesis124,125
. Classical risk factors for developing atherosclerosis are
hypercholesterolemia, smoking, diabetes mellitus and hypertension as well as
environmental and genetic factors123
. However, exactly how the different established
risk factors contribute to the initiation and/or progression of the atherosclerotic lesions
is unclear. This has led to studies of other possible factors that could be involved in the
etiology and pathogenesis of atherosclerosis and its complications. An alternative
explanation that has raised a lot of controversy is the atherosclerotic infection theory.
This hypothesis was formulated at the beginning of the last century when Frothingham
suggested that, “The sclerosis of old age may simply be a summation of lesions arising
from infectious or metabolic toxins”126
. However, this idea did not gain much interest
until the end of the seventies when Fabricant and co-workers127
showed that chickens
experimentally infected with a herpes virus developed vascular lesions that resembled
atherosclerosis in man. Later studies have especially pointed out C. pneumoniae, but
also Helicobacter pylori, Herpes simplex virus and cytomegalovirus as possible primary
etiological factors or co-factors in the pathogenesis of atherosclerotic diseases like
ischemic heart disease and cerebrovascular disease116
.
Atherosclerosis and animal models Mice are the most frequently used animals in experimental atherosclerosis studies.
These rodents are easy to breed, and importantly, it is possible to knock out and replace
endogenous genes in mice as well as create transgenic models128
. Wild-type mice are
normocholesterolemic, and most lipids in the blood are carried by High Density
Lipoprotein (HDL), which is known to be protective against atherosclerosis in
humans123
. Atherosclerotic lesions do not develop spontaneously in mice, and
hypercholesterolemia has to be induced either by genetic manipulation or special diets
containing high levels of fat and cholesterol. Lesion formation in mice is restricted to
the aortic root or aortic sinus area, and the lesions are mainly early stage fatty lesions
with invading smooth muscle cells (SMC) as a characteristic feature128
. Apolipoprotein
E (ApoE) deficient mice are the most commonly used genetically modified mouse
model since they develop atherosclerosis spontaneously when fed rodent chow129
.
Moreover, these mice have total cholesterol levels about five times higher than wild-
type mice and at 10 weeks of age develop atherosclerotic lesions in the aorta and
coronary and pulmonary arteries128,129
. In addition, ApoE-deficient mouse models of C.
••
16
pneumoniae infection have produced contradictory results. Studies demonstrate either
increased atherosclerosis or no effect on progression130-133
.
Lipoprotein (a) as a mouse model for atherosclerosis Dahlén and co-workers have shown an exceptionally strong statistical association
between plasma levels of lipoprotein (a) (Lp(a)) and atherosclerosis134
. Lp(a) has so far
only been found in humans, some monkeys and in hedgehogs135
. Lp(a) is very similar to
Low Density Lipoprotein (LDL) in its constitution. Lp(a) contains, just like LDL, apo-B
100, but Lp (a) differs from LDL in that it contains an extra large protein,
apolipoprotein (a) (apo(a)) that is linked to apoB by a disulphide bridge. Accordingly,
mice cannot produce Lp(a). Therefore, a double transgenic mouse expressing human
apo(a)/apoBH produces Lp(a) and develops atherosclerosis136
. Thus, these mice can be
used in an atherosclerotic model to investigate whether C. pneumoniae infection in
combination with Lp(a) accelerates the development of atherosclerosis.
In addition, 20-week-old Lp(a) mice sacrificed at 10 weeks p.i. with C. pneumoniae
following three repeated inoculations, did not show any macroscopic plaque formations
in the aortic cusps of the aortic root, after staining with oil red. However, infiltration of
macrophages and possibly foam cells was observed (Fig. 4A-B). Moreover, when
compared to ApoE-deficient mice at same age, the Lp(a) mice display less pronounced
lesions of atherosclerotic plaques (Fig. 4C). Accordingly, Berg et al. reported that Lp(a)
mice develop atherosclerotic lesions after 46 weeks of age when fed a normal diet137
.
Thus, these mice were considered to be time-consuming and difficult to work with as a
model for spontaneous development of atherosclerosis.
FIGURE 4. Photomicrographs of cross-sections stained for lipids in the aortic root of 20-week-old mice.
(A) Accumulation of lipoid or foam cells expanding the subendothelial space in aortic cusp of a Lp(a) double
transgenic mouse. The wall of the aorta is thickened by this accumulation of material (magnification 100).
(B) A closer view of the area marked in (A) (magnification 400). (C) Oil red staining shows accumulation of
lipids within atherosclerotic plaques in the three aortic cusps.
17
C. pneumoniae in atherosclerosis Among the chronic inflammatory conditions associated with C. pneumoniae,
atherosclerosis is the most widely studied. The first report of an association of C.
pneumoniae with atherosclerosis was published in 1988. Saikku and co-workers
demonstrated elevated levels of C. pneumoniae antibodies in patients suffering from
myocardial infarctions and coronary heart disease compared to controls138
. Since then,
more than 50 seroepidemiological reports have strengthened the early findings of
Saikku. However, large scale serological prospective studies investigating the
association with C. pneumoniae and the risk of coronary events did not find any
connection (reviewed in reference 116). Moreover, other strong associations exist
between C. pneumoniae infection and atherosclerosis as demonstrated by detection of
the organism within atherosclerotic lesions, but not in adjacent normal tissue by
immunohistochemistry, polymerase chain reaction and electron microscopy139-141
.
Viable C. pneumoniae have also been cultured from atheromatous plaques, suggesting a
more causal relationship142-145
. Animal models demonstrate that C. pneumoniae can
either initiate lesion development or cause exacerbation of lesions in rabbit and mouse
animal models, respectively146 (reviewed in reference 146).
Antimicrobial trials have questioned the C. pneumoniae
infection theory To better understand the pathogenic significance of C. pneumoniae in the development
of atherosclerosis, several pharmacological intervention trails with antibiotics have been
performed. Initially, clinical antibiotic interventions were promising, with some
protective effects reported147 (reviewed in reference 147). However, most of these studies
were considered to suffer from major weaknesses such as small patient groups and
treatment courses too short to be effective against chronic C. pneumoniae infection.
Therefore, two larger prospective clinical trials were performed148,149
. Despite long-term
treatment with a bactericidal antibiotic effective against C. pneumoniae, no reduction in
the rate of cardiovascular events was observed148
. Accordingly, a one-year course of
weekly azithromycin did not alter the risk of cardiac events among patients with stable
coronary artery disease149
. However, these studies have been questioned as antibiotic
treatment is not effective against chronic and persistent C. pneumoniae infections106,150-
152. Another consideration is whether the atherosclerotic process already has been
initiated prior to antibiotic treatment. Thus, taking antibiotics too late in the
inflammatory process is unlikely to have an effect and factors unaffected by antibiotics
are likely to obscure any beneficial anti-chlamydial effect153
. Therefore, C. pneumoniae
infections remain a reasonable risk factor for development of atherosclerosis.
••
18
C. pneumoniae and inflammation of the vascular system The ability of C. pneumoniae to infect several cell types and to disseminate into various
tissues after respiratory infection is fundamental for the various pathogenic
consequences. Endothelial cells become activated upon infection with C. pneumoniae
and increase production of inflammatory mediators and express adhesion molecules
such as intercellular adhesion molecule (ICAM-1) and vascular cell adhesion molecule-
1 (VCAM-1) which subsequently leads to the transendothelial migration of leukocytes
to the inflammatory site154
. Additionally, C. pneumoniae-infected endothelial cells
increase secretion of monocyte chemoattractant protein-1 (MCP-1) and IL-8 which
stimulate the transendothelial migration of monocytes and neutrophils as well as
monocyte differentiation into macrophages (reviewed in reference 154). Moreover C.
pneumoniae has been found in foam cells and is believed to mediate oxidation of
LDL155
. In addition, Rödel and collaborators have published several reports on the
ability of C. pneumoniae to infect and proliferate in SMC156-158
. Taken together, the
collective activation of several cell types in the vascular system would create a
sustained inflammatory environment. Contributing to the initiated inflammatory
response following the infection is the production of a cascade of pro-inflammatory
cytokines e.g. IL-1β, IL-6, IL-8 and tumor necrosis factor (TNF-α) that helps sustain the
state of the inflammatory site by acting as cell signaling molecules.
Pro-inflammatory cytokines The first cytokine to be discovered was IL-1 in the early 1950’s
159. In general, cytokines
are regulatory proteins presented by leukocytes and other cell types such as fibroblasts,
epithelial cells, osteoblasts, e.g. used for cell-cell communication. Cytokines do not only
act in a paracrine manner but can also signal in an autocrine fashion and activate the cell
that is producing them. Cytokines produced during a bacterial infection are e.g. the pro-
inflammatory cytokines IL-1β, IL-6, IL-8. These cytokines exhibit pleiotropic actions
that include the regulation of host innate and adaptive immune responses and the
modulation of inflammatory responses160
. Moreover, these pro-inflammatory cytokines
reveal biological activity including osteoclastgenesis stimulating bone resorption161
(reviewed in 161). On the other hand, cytokine responses by epithelial or phagocytic
cells can be disrupted by some pathogenic bacteria, such as Yersinia enterocolitica and
Pseudomonas aeruginosa, eventually enabling their escape from the host's immune
system160,162
. A number of studies have shown that cytokines can affect the growth of a
variety of bacteria inside host cells. For example, TNF- stimulates the growth of
Mycobacterium tuberculosis in human monocytes163
. IL-6 has been shown to increase
the growth of Mycobacterium in macrophages, thereby inhibiting T cell activation164
.
However, not much is described regarding whether or not a bacterium benefits from the
production of pro-inflammatory cytokines. It is speculative, but it could be a good
strategy for recruitment of immune cells to the site of infection to subsequently infect
19
these cells. In this manner, the infection could enter the circulation and spread to distant
tissues.
C. pneumoniae and pro-inflammatory cytokines As mentioned earlier, C. pneumoniae is believed to be associated with manifestations of
inflammatory chronic diseases such as atherosclerosis, asthma, neurological disorders
and arthritis. C. pneumoniae has been shown to infect and proliferate in vascular cells
e.g. endothelial cells, smooth muscle cells, macrophages and in atherosclerotic
lesions140,165
. Several in vitro experiments reveal that the infection induces expression
and release of pro-inflammatory cytokines such as TNF- , IL-1 and IL-6 in vascular
cells166-170
. Moreover, C. pneumoniae infection of respiratory cells is shown to induce
IL-8 (CXCL8) production171,172
. Recently, it was reported that C. pneumoniae infection
of microglial cells and astrocyte cells increased production of MCP-1, IL-1, IL-6 and
TNF-173
.
Bacterial infection of bone demonstrates similar cytokine
induction as in atherosclerosis Clinical studies have revealed that bacterial infection of bone tissue is a possible
explanation for imbalanced bone remodeling, but the mechanisms responsible for these
observations are not defined161
. Interestingly, the inflammatory response of bone
destruction resembles the pathogenesis of atherosclerosis. Several studies have
demonstrated that Staphylococcus aureus and Salmonella can invade and persist within
cultured murine and human osteoblasts and the infection promotes the ability to produce
IL-1, IL-6 and TNF-, known to initiate the inflammation that results in progressive
bone destruction174-179
.
The bone resorption and remodelling system is regulated through increased expression
of the receptor activator of NF-B ligand (RANKL180
), its receptor (RANK) and the
synchronized production of a decoy receptor named osteoprotegerin (OPG181
). Under
normal circumstances, the maintenance of bone tissue depends on the dynamic interplay
between RANKL/OPG regulated by osteoblasts and the activation of bone resorption
mediated via RANK expressed on pre-osteoclasts. In bone disorders, bone resorption
exceeds bone formation and the result will be bone destruction. Interestingly,
microorganisms like S. aureus, Salmonella and the peroidontitis-associated bacterium
Actinobacillus actinomycetemcomitans have been previously shown to induce the
expression of RANKL in osteoblast-like cells and synovial fibroblasts mediating
osteoclast activation, therefore they are suggested to be mediators of
osteoclastgenesis177,182,183
.
••
20
Accordingly, a case study reported two patients suffering from unexplained chronic
anaemia with detected C. pneumoniae in their bone marrow184
. This could be of interest,
as osteoblast progenitor cells are generated from multi-potent cells that differentiate e.g.
into mature osteoblasts and are thereafter recruited to the bone-forming sites.
Moreover, accumulating evidence indicates a pathopyhsiological link between
atherosclerosis and osteoporosis185-192
. We have performed a study demonstrating that
OPG promotes fibrous cap formation in atherosclerotic lesions of ApoE-deficient mice
(manuscript VII in this thesis). Could infections, in particular C. pneumoniae infection,
be implicated in the pathogenesis of both diseases?
21
The type three secretion system (T3SS)
Bacterial infection is dependent on pathogenic traits that benefit survival and
proliferation of the bacterium in its host. Bacterial proteins involved in pathogenesis
need to be anchored to the bacterial cell wall, or delivered across the cell wall. Such
actions demand special bacterial protein secretion systems. One such example is the
type three secretion system (T3SS). This system allows Gram-negative bacteria in a
close contact with a host cell, to transport proteins from the bacterium into the
cytoplasm of the eukaryotic host cell. This transport system was first studied and
characterized in pathogenic Yersina spp.193
. However, T3SS was first visualized in
Salmonella194
followed by enteropathogenic E. coli195
and Shigella196
. Thereafter,
T3SSs have been characterized in many additional bacteria 197
. All human pathogenic
bacteria possessing a T3SS use it to translocate effector proteins into host cells. These
host cell-localized effectors subsequently modulate host cellular functions198
. The T3SS
is a complex apparatus, consisting of more than 25 proteins spanning the entire bacterial
envelope. The exterior component of all T3SSs is the syringe-like structure protruding
out from the bacterium, known as the needle complex or injectisome. The needle
complex projects from the bacterium and mediates delivery of the bacterial effector
proteins through its channel. Secretion by this mechanism is contact-dependent199
.
I will focus further on the composition and regulation of the T3SS in the section
describing chlamydial T3SS. However, many assumptions of the chlamydial T3SS are
based on information on assembly and regulation of the T3SS in Yersinia.
Historical observations of a possible T3SS In 1973, 15 years before the first visualization of the T3SS in Salmonella
194, Matsumoto
published electron micrographs showing rosette-like structures and projections at the
surface of C. psittaci200
. Each of these rosettes had a diameter of about 20 nm and
appeared to be arranged in a radial formation of nine subunits. However, the research
community was doubtful of these observations and speculated that the projections were
artifacts formed during purification of EBs rather than natural structures. From the years
1975 to 1981, Matsumoto continued studying and analyzing his observations 201,202
.
Hence, in 1982, he published a new freeze-deep-etching technique that rendered
examination in more detail. In his new electron micrographs he noticed that each
projection on the RB surface emerged from the center of a flower-like structure, about
30 nm in diameter. These flower-like structures appeared to be a radial arrangement of
nine leaves with a projection reaching out from the bacterial surface (Fig. 5). Further,
the morphology of the RB projections was identical to the projections on EBs. Each
projection was anchored in to the cytoplasmic membrane of the Chlamydia, from where
it protruded out through the inclusion membrane of the cell. All RBs had these
••
22
projections and the number of projections peaked 10 h after infection after which they
gradually decreased to approximately the same number of projections as on EBs203,204
.
Comparable structures have thereafter been observed on the surface of all other human-
pathogenic Chlamydia (C. trachomatis205
and C. pneumoniae23
). Today, many other
researchers apply these early observations from Matsumoto to support their own
hypotheses and results on T3SS in Chlamydia.
FIGURE 5. Early observations of Matsumoto – the chlamydial projections. A: T3S projections (arrows)
are viewed from the cytosolic side of the infected cell extending across the inclusion membrane from
underlying RBs. B: Projections on the RB surface emerged from the center of a flower-like structure, in an
arrangement of nine leaves (insert). C. caviae were examined by scanning and transmission electron
microscopy (TEM). Micrographs are modified and reprinted with permission from Trends in Microbiology
(Picture A: Copyrigh 2007) and ASM (Picture B: Copyright 1982), respectively.
First evidence of a chlamydial T3SS The first report of a chlamydial T3SS came in 1997
206. By sequencing a fragment of the
C. psittaci genome the authors detected four open reading frames that were predicted to
encode proteins with similarity to T3S apparatus components CdsU and CdsV (contact-
dependent secretion), CopN (Chlamydia outer protein homologue of YopN) and Scc1
(specific Chlamydia chaperone). Analysis of the C. trachomatis serovar D genome later
confirmed the results from Hsia et al., that Chlamydia possesses a complete set of genes
that could encode a T3S apparatus17
. Later, the same authors analyzed the genome
sequence of C. pneumoniae CWL029, revealing same repertoire of T3S-related genes as
seen in C. trachomatis18
. Interestingly, the T3S-associated genes were scattered
throughout the genome. This contrasted to other Gram-negative bacteria whose T3S-
23
genes are typically located at one distinct locus of the chromosome, termed the
pathogenicity island, or on a virulence plasmid17,207
. A common feature of T3SS genes
is their low G/C content relative to the rest of the genome. This is not the case for
Chlamydia, whose entire genome is low in G/C206,208,209
. Taken together, these early
reports have been the starting-point for intensive research on the chlamydial T3SS.
Composition of the chlamydial T3S injectisome Several C. pneumoniae genes are homologous to genes coding for units resembling the
T3S apparatus of Yersinia and other known T3SS (summarized in Fig 6). Function has
been predicted based upon studies of their homologues in Yersinia, for which the T3SS
is the best characterized. As mentioned earlier, the T3S apparatus has a complex
architecture with the majority of its key players believed to be associated with the
bacterial membrane. Many of the apparatus components, in Yersinia referred to as the
Yersinia secretion-Yersinia outer proteins system (Ysc-Yops), are well conserved
among bacteria207
. Accordingly, T3S-associated genes of Chlamydia were originally
labeled with references to the Ysc-Yop nomenclature. However, this nomenclature has
been revised by Fields et al.210
based on the convention of contact-dependent secretion
(Cds) in Chlamydia, proposed by Hsia et al206
. Therefore, the apparatus components
will be denoted as Cds in this work.
Basal apparatus In Chlamydia, the predicted basal body of the T3SS is anchored to the inner membrane
of the bacterium. The integral membrane protein CdsV (also known as LcrD and
previously termed Cds2) is predicted to form a central protein-conducting channel
across the cytoplasmic membrane based upon work done in Yersinia pestis207,211
. Other
proteins suggested to be involved in the formation of such a channel include CdsD,
CdsR, CdsS, CdsT and CdsU (previously termed Cds1). Recently, CdsD was reported
to be phosphorylated by PknD, an integral membrane protein with a kinase activity
domain located in the cytoplasm212
. The CdsU homologue, YscU, was recently
described as an essential component of the export apparatus of the Yersinia injectisome.
YscU cleavage is needed to acquire a conformation allowing recognition of a subset of
secreted substrates, termed the translocators that form a translocon213
. However, cdsU
transcripts have not been detected at any time-point throughout the developmental cycle
in C. pneumoniae54
, whereas they have been detected in C. trachomatis 210
. These
findings suggest variable importance of CdsU in Chlamydia. CdsL and CdsN are
predicted to cooperate in regulating translocation according to their function in Yersinia
and other T3SS 214
. CdsN mediates ATP hydrolysis providing the energy for secretion
and CdsL might function as its regulator, as in Yersinia215
. CdsJ is an outer membrane
lipoprotein spanning the periplasmic space and therefore possibly bridges the inner and
outer membranes. The final component of the basal apparatus is the distal outer
••
24
membrane protein CdsC. CdsC, which is the homologue of YscC in Yersinia, belongs to
the secretin family of proteins and forms a stable ring-like structure in the outer
membrane216
. In other systems, these outer-ring structures are thought to function as
transport channels for macromolecules. Interestingly, this structure shares similarities to
the rosette-like structures observed by Matsumoto217
.
FIGURE 6. Schematic illustration of the chlamydial T3S-injectisome. The putative structure of the
chlamydial injectisome is derived by comparison with homologus proteins of the Yersinia T3S apparatus. The
figure is modified from (www.genome.jp – KEGG). See text for abbreviations.
The Chlamydia translocon To be able to transport effector proteins through the eukaryotic cell membrane,
microorganisms with a T3SS apparatus have to be in close contact with the host cell.
Upon cell contact the bacteria are believed to insert a channel-complex into the cell
plasma membrane that allows translocation of proteins into the cell218 (reviewed in
reference 218). This complex is commonly refered to as the translocon. In Yersinia,
YopB and YopD interact at the interface between bacteria and host cell, and have been
suggested to form a translocon pore. This pore complex allows the direct Yop effector
translocation into the target cell219
. A third translocator in Yersinia, LcrV (low calcium
response protein V) is localized at the distal tip of the needle where it forms a bridge
that connects the needle to the translocon220
. So far, no LcrV homologue has been
reported in Chlamydia.
25
YopB and YopD in Yersinia have homologues in C. pneumoniae and C. trachomatis,
termed Chlamydia outer proteins CopB and CopD208
. This assertion is not based upon
sequence homology between these proteins but rather similar size and hydrophobicity
profiles. Moreover, both CopB and CopD lack signal sequences and lie in close
proximity to the postulated T3SS chaperones, Scc2 (also termed LcrH-1) and Scc3 (also
termed LcrH-2, both discussed in more detail in the next section). Thus, their predicted
function is to act as a gating mechanism facilitating translocation of secreted effectors
across the inclusion membrane. CopB has been detected in the C. trachomatis inclusion
membrane, consistent with its presumed function as a T3S translocator221
. Additionally,
CopD has been demonstrated to be translocated in the heterologous system of the SPI-2
T3SS of S. enterica (serovar Typhimurium)222
. Interestingly, a paralogue of CopB has
been described, named CopB2223
. CopB2 can be detected in the host cell cytosol,
probably demonstrating distinct functions when compared to other known translocators.
However further studies are needed to evaluate its function. Taken together, this
suggests that Chlamydia most certainly acquires a translocon in the inclusion membrane
to complete delivery of effectors.
Chaperones of the T3SS The T3S chaperones can be grouped into different classes according to their binding
affinities to their corresponding cognate substrates (reviewed in reference 220). As the
name implies, effector-class chaperones bind to one or at most a few translocated
effectors. In contrast, translocator-class T3S chaperones bind to the proteins that form
the translocon. Common to these two classes is their ability to facilitate secretion by
guiding proteins to the T3S-apparatus and keeping them in an unfolded state prior to
secretion. They also prevent premature interactions between different T3S components.
Some of the best-studied T3S-associated chaperones are those grouped in the
translocator class of chaperones224
. In Y. pseudotuberculosis, LcrH (low calcium
response protein H) belonging to the chaperones termed Sycs, binds and stabilizes
YopB and YopD in the bacterial cytoplasm prior to secretion225
. In Chlamydia,
orthologs to syc-gene products have been found and are believed to be specific
Chlamydia chaperones according to their structural similarities to known chaperones in
other T3SSs206,210,221
. In the first report of a possible T3SS of Chlamydia, Scc1
(homologous to SycE in Yersinia) was discovered206,210,221
. SycE is thought to act as a
stabilizing chaperone for YopE, a anti-phagocytic factor produced by Yersinia226
.
Recently, it has been demonstrated in C. trachomatis serovar L2 that the two
chaperones Scc2 (LcrH-1) and Scc3 (LcrH-2), homologous to LcrH, were associated
with the same translocator protein, YopD, in a heterologous Yersinia T3SS model
223.
Moreover, LcrH in Yersinia interacts with YopB and YopD at a unique binding site
established by the folding of its three tandem tetratricopeptide repeats (TPRs)
••
26
facilitating protein-protein interactions227
. Both Scc2 and Scc3 of Chlamydia possess a
TRP domain223
. Additionally, it was shown that Scc2 interacts with CopN, and is
suggested to serve as a chaperone for this protein228
. Recently, two new chaperones
were discovered and shown to be involved in regulation of CdsF in C. trachomatis.
These were denoted as CdsE and CdsG according to their similarities to the
Pseudomonas spp. needle chaperones PscE and PscG, respectively229
. However, these
chaperones only share 66% and 67% similarity to the predicted protein sequences in C.
pneumoniae, respectively, as determined by the multiple sequence alignment program
ClustalW2 (author contribution).
Transcriptional regulation of T3SS chaperones In 2000, Subtil et al. speculated that scc2 and scc3 could be expressed at different time-
points during the infection, for example in EBs at the entry phase and during
proliferation in RBs, and enable the sequential translocation of structurally related
proteins208
. Five years later, Byrne et al. proposed that protein products of the scc2
operon are likely to function during attachment since these genes are
expressed
following EB arrangement and the protein products of the scc3 operon likely function
during intracellular survival230
. Temporal expression studies with C. pneumoniae
revealed that Scc3 was one of the few T3S genes expressed as early as 1.5 h following
infection and expressed continuously during the developmental cycle. In contrast, Scc2
was apparently not to be expressed until late in the developmental cycle, suggesting a
different function for each of the gene products228
. Expression of copB, and copB2 are
both mid- to late-expressed T3S genes. SicA, the chaperone of the translocators SipB
and SipC of Salmonella, activates the expression of late SPI-1 T3S genes encoding only
late effector proteins. This occurs by SicA forming a regulatory complex with a
transcriptional activator InvF231
. Interestingly, the Scc homolog LcrH in Yersinia also
acts as a negative regulator of Yop expression, in disagreement with its chaperone
function as a stabilizator of the translocon232
. Further, LcrH has been shown to interact
with YscY in Yersinia233
. Thus, this implicates the multi-functional activity of T3S
chaperones as stabilizers, activator pilots and as regulator(s) of the T3SS. It is tempting
to speculate that Sccs can function as negative regulators of the Chlamydia translocon
or even possess more distinct purposes.
The needle complex Bacteria possessing a T3S apparatus can simply be described as bacteria using a needle
to become virulent. Early on in the field of Chlamydia research, the most firmly
established hypothesis concerning this needle structure was that the projections function
as channels that facilitate the uptake of nutrients from the host cytosol by Chlamydia, an
idea designated as the “soup-through-a-straw” hypothesis234
. The needle-like structure,
referred to as the needle complex, has been shown by scanning transmission electron
27
microscopy to consist of approximately 150 YscF subunits and the tip complex is
formed by three to five LcrV monomers in Yersinia enterocolitica235
. Moreover,
purified needles from Pseudomonas aeruginosa detached from the bacterial surface
were 60-80 nm in length and 7 nm in width, resembling purified YscF needles from
Yersinia236
. Recently, using a bioinformatics approach, gene CT666 in C. trachomatis
was revealed to be homologous to yscF in Yersinia, termed cdsF229
. The open reading
frame for cdsF is located within an operon encoding other putative components of the
apparatus (Fig. 7). It was further shown by immunoblotting and electron microscopy
that CdsF was concentrated in the outer membrane of EBs and surface-exposed as a
component of an extracellular needle-like projection. During infection, CdsF was
detectible in the inclusion membrane with a punctate distribution adjacent to membrane-
associated RBs229
. CdsF of C. trachomatis shares 89% similarity to the predicted
protein sequence in C. pneumoniae, suggesting the existence of CdsF in this species as
well (author contribution).
Gating of T3S machinery CopN is the protein corresponding to YopN of Yersinia. It is proposed to be the lid of
the T3SS apparatus in the inclusion membrane regulating the gating function of
T3SS221
. CopN of C. trachomatis was the first protein shown to be translocated in a
T3S-dependent manner in a heterologus system using Y. enterocolitica221
and later S.
typhimurium SPI-1222
. CopN has been associated with the inclusion membrane during
intracellular growth, supporting the idea of a gating mechanism. In Yersinia, YopN can
form a complex with TyeA and its chaperones SycN and YscB237
. Subsequently, this
complex, under non-permissive conditions, is presumed to block secretion, as YopN
cannot be secreted while interacting with TyeA. When dissociated from TyeA, YopN is
released opening the gate for translocation of Yops. However, the underlying signal(s)
regulating the release of YopN from TyeA is undefined237
. This plug-like mechanism is
extremely interesting from a chlamydial point of view. Messenger RNA of copN is
known to be expressed late in the developmental cycle of both C. pneumoniae and C.
trachomatis54,217,238
. J. Peters et al., suggests a theory based on unpublished data, where
CopN was not expressed in EBs, only late in normal RBs. Therefore it, is hypothesized
that CopN can provoke RB detachment (concerning C. trachomatis, author’s
annotation) and shutting off the T3SS as a consequence54,217
. This is in agreement with
their mathematical theory, later described in the intracellular T3SS section. Their
assumption is that there is a possible function for the regulation of CopN involved as a
T3SS on/off switch important for the re-differentiation of infectious EBs. Whether or
not detachment from the inclusion membrane is sufficient enough to regulate the switch
is speculative. However, more functional studies on CopN and a possible complex
interaction with a TyeA-like protein are needed. Scc3 is a possible chaperone
regulating such a complex since it has been demonstrated to interact with CopN
228.
••
28
Effector proteins Often, effector proteins of Chlamydia are referred to as secreted substrates in the
literature. However, proteins secreted by the T3SS in other species are divided into two
classes of substrates/proteins according to their function and time of action. First, there
are those proteins secreted when the apparatus assembled and ready to secrete the
translocators, which generates the pore formed in the cell membrane. After this, the
effectors are then delivered through the T3S-apparatus, either to the inclusion
membrane or into the cell cytoplasm. Therefore, in this section, I have separated the
predicted Chlamydia effectors into a single class of secreted proteins (summarized in
Table 2).
There is not a common function for all known T3S effector proteins. For instance, Yops
secreted by Yersinia, have one mission, targeting intracellular host cell signaling
pathways to benefit their survival outside the cell and escape from the innate immune
system. Additionally, Salmonella Sips secreted by the SP-1 T3SS induce cell invasion,
while Sses secreted by the SP-2 T3SS support their proliferation in macrophages
(reviewed in reference 207). The diversity among T3SS provides, in the context of
Chlamydia, both pros and cons. It would have been much simpler if effectors were
homologues between different bacteria or if chlamydial T3SS genes were located at a
single location, as in other pathogens constituting a T3SS. However, surrogate T3SS
have been successful in revealing secretion of potential chlamydial effectors (Table 2).
Using heterologous T3SS the inclusion membrane proteins IncA, IncB and IncC were
predicted to be T3S-mediated effector proteins of C. pneumoniae. This was possible by
constructing hybrid genes by fusing the 5' part of the chlamydial inc-gene of interest in
frame with the calmodulin-dependent adenylate cyclase (Cya) gene of Bordetella
pertussis and subsequently measuring the production and secretion of hybrid proteins239
.
Interestingly, the transcription of incA, incB and incC has been shown to occur earlier
than transcription of genes encoding components of the T3SS itself 240
. Additionally,
IncC of C. trachomatis, has been shown to be secreted through heterologous T3SS210
. In
a randomized search of proteins with unknown function conserved among C.
pneumoniae, C. trachomatis and C. caviae, 24 new candidate proteins were found.
None of the predicted proteins belonged to the Inc family of proteins. Out of these 24
candidates, four were shown to be secreted as full-length proteins by a T3S mechanism
in S. flexneri. One of the protein homologs of Cpn705 was found to be secreted in the
cytoplasm of infected cells241
. Pkn5 was originally identified and postulated to be T3SS-
related when the genome of C. trachomatis was sequenced17
. In one of the known T3S
sub-clusters (Fig. 7) the pkn5 gene is located upstream of cdsC and encodes a putative
serine/threonine kinase208
. Pkn5 has been shown to be translocated via the SPI-2 T3SS
of S. enterica222
. Recently a new member was included in the family of Chlamydia
effectors242
. The protein CT847 was revealed to be secreted in a T3SS-mediated model
29
of Y. pseudotuberculosis. Moreover, the protein interacted in the cell cytoplasm with a
eukaryotic protein termed GPIC (Grap2 cyclin D-interacting protein). CT847 shares
62% sequence similarity to C. pneumoniae gene product Cpn1004 (author contribution).
TABLE 2. Chlamydial effectors
C. pneumoniae C. trachomatis
Effector Location Secreted Gene-
number Secreted
Gene-
number Possible function
IncAa Inclusion Yes
Cpn0186
Cpn0595 Yes CT119
Membrane protein
IncBa Inclusion Yes Cpn0291 No CT232
Membrane protein/host cell
interactions*
IncCa/b
Inclusion Yes Cpn0292 Yes CT233 Membrane protein
Pkn5c S/T kinase Yes Cpn0703 No CT673 S/T kinase activity
TARPd Surface of EBs No Cpn0572 Yes CT456 Invasion
Hypothetical proteind Unknown No Cpn1004 Yes CT847
Interaction with
eukaryotic GPIC
Hypothetical proteina
Secreted to
cytoplasm Yes Cpn0705 No CT671 Host cell interaction(s)
Hypothetical proteina Unknown Yes Cpn0725 No CT652.1 Unknown
Hypothetical proteina Unknown Yes Cpn0859 No CT718 Unknown
Hypothetical proteina Unknown Yes Cpn1005 No CT848 Unknown
Effector proteins secreted in heterologues system: S. flexneria, Y. enterocoliticab, S. typhimuriumc, Y.
pseudotuberculosisd. For references; see text.
Other secreted proteins There are other proteins proposed to be secreted into the cytoplasm of host cells. For
instance, Cap1 (homologous to Cpn0648) is a protein of unknown function recognized
by cytotoxic T-cells243
and CADD (homologous to Cpn0761) has been shown to be
involved in modulation of host cell apoptosis and to be located in close contact with the
inclusion244
. Another protein identified as secreted is the chlamydial protease-like
activity factor (CPAF). CPAF has the ability to cleave a eukaryotic transcription
factor245
. Translocation of CPAF does not seem to occur via the T3SS (referred to246
and
unpublished data by A. Subtil). There are two genes coding for products with unknown
function in C. pneumoniae, Cpn0796 and Cpn0797 and have no homologues in C.
trachomatis. Both are found to be secreted in the host cell cytoplasm247,248
. However,
••
30
whether or not any of these proteins are T3SS-related is unknown. With ongoing efforts
to develop novel tools to better study and characterize virulence mechanisms in
Chlamydia, it is likely that the predicted function of many effector proteins will be
updated in the future.
Extracellular T3SS Most T3SSs characterized in other spp. are functional upon host cell contact e.g. when
internalized or phagocytosed. This has also been postulated as a function for the
chlamydial T3SS. The first report of a T3SS-translocated protein that was tyrosine-
phosphorylated upon cell contact was a protein named Tir (translocated intimin
receptor) described in enteropathogenic E. coli (EPEC249
). A tyrosine-phosphorylated
effector protein has also been found in C. trachomatis, named TARP (translocated
actin-recruiting phosphoprotein78
). TARP is translocated through T3S by Y.
pseudotuberculosis and is demonstrated to be involved in the recruitment of actin,
leading to internalization of C. trachomatis. Moreover, it has been reported that C.
muridarum, C. caviae, and C. pneumoniae TARP fails to become phosphorylated at
tyrosine residues at the site of entry initiating internalization, therefore indicating the
action of TARP is species-specific for C. trachomatis250
. However, all chlamydial
species examined to date show recruitment of actin to the site of entry. Thus, Chlamydia
likely shares at least some common mechanism(s) for entry.
Intracellular T3SS Chlamydia are strictly intracellular bacteria that rely on mechanisms facilitating their
required host environmental needs. Therefore, it is not remarkable that many predictions
and assumptions suggest employment of a T3SS to maintain intracellular survival. It
may be essential to understand the involvement of T3S in the development of
Chlamydia. In addition, the function of T3SS in other pathogens is most often crucial
during invasion of cells and escaping the innate immune system, and usually not for
intracellular proliferation207
.
The genome has been completely sequenced for the Parachlamydia-related
Acanthamoeba symbiont UWE25251
. UWE25 is thought to have diverged from the
family of chlamydiaceae about 700 million years ago and also encodes a complete
T3SS251
. Thus, it is possible that the common ancestor of the T3SS evolved from
Parachlamydia. The T3SS is described as a contact-dependent secretion system. It
follows that Bavoil and colleagues have formed an hypothesis using a mathematical
model that proposes that RBs grow strictly in contact with the inclusion membrane and
that this contact is mediated by the T3S apparatus217,252
. As the inclusion compartment
grows, the T3S activity decreases for every RB until the RB detaches from the inclusion
membrane, and this detachment from the chlamydial inclusion membrane constitutes
31
the signal for late RB-to-EB differentiation. Moreover, this could explain why
chlamydial development, initially almost synchronous, becomes asynchronous in the
mature inclusion as EBs differentiate from RBs253
.
Temporal transcription of T3SS genes T3SS genes in Chlamydia exhibit temporal expression throughout the developmental
cycle54,210,221,240
. These genes have been clustered into three major classes of genes,
termed early-, mid- and late-cycle genes54,254
. The early-cycle genes expressed in C.
pneumoniae include cdsC, cdsS, cdsL, cdsJ and scc3. The genes expressed during mid-
developmental cycle are cdsD, cdsN and cdsR, and finally the late-cycle genes include
copN, scc1, scc2, and cdsT54
(Fig. 7). Recently, a fourth classes of genes temporally
expressed in the developmental cycle was described and defined as tardy genes110
. The
tardy gene class was established and defined based on a transcriptional profile that
differed from the late genes110
. In the study by Mayer and co-workers, there is a
difference in the classification of some genes, as for example scc3 was moved to the
mid-cycle class of genes and scc2 was placed in the tardy class. Lugert et al., also
demonstrated a different temporal expression profile, where transcription of cdsN, copN
and scc1 was detected 8 h p.i in comparison to Slepenkin et al. who were able to detect
cdsN at the time of infection, whereas copN and scc1 were not transcribed before 12 h
p.i.54,240
. These differences in transcriptional kinetics can probably be explained by the
sensitive methodology/technique of RT-PCR and different inoculation doses. The fact
that scc2 is expressed in the tardy class of genes suggests a regulatory function in the
early phase of the following infectious cycle, coding for early EB proteins. This
speculation is supported by the study where Mayer et al. compared their expression data
with data from the proteomic study of EBs of Vandahl et al.255
. Their study
demonstrate that genes coding for EB proteins were primarily connected with products
in late gene clusters, whereas transcripts coding for EB mRNAs were mainly connected
with tardy clusters110,255
. Proteomic analyses of C. pneumoniae revealed that all
components of T3S-apparatus were present in EBs255
, whereas only cdsC was found in
the outer membrane complex proteome and proposed to be surface-exposed256
.
••
32
FIGURE 7. Organization of the T3SS gene cluster in the C. pneumoniae TW183 genome. Gene size is
not displayed to scale and location shown on the genomic illustration is not exact (modified from54,257).
Stress of the T3SS When Chlamydia are stressed by the cell-mediated immune system, T3SS-modulated
processes may become of importance. For instance, IFN--treated C. pneumoniae-
infected cells decrease transcription of scc1, scc2 and cdsV54
. In contrast, scc3
expressed throughout the normal developmental cycle, did not appear to be affected by
IFN- treatment54,55
. In C. psittaci, transcriptional levels of incA were analyzed during
early-mid developmental cycle (12 h p.i.) and incA was demonstrated to be up-regulated
when the infection was treated with IFN- or when iron was depleted using desferal258
.
In contrast, we have demonstrated that INP0010 treatment reduced the expression of
incA and incB transcripts in C. pneumoniae 12 h p.i. (paper III in this thesis). Moreover,
an extensive study combining the microarray-technique with qRT-PCR demonstrated a
down-regulation of the T3SS-associated genes scc1, scc2 and cdsC in an iron-induced
persistence model110
. In addition, Ouellette et al. reported that transcription of scc2 and
additional genes involved in bacterial growth were up-regulated during IFN-γ
persistence259
in contrast to previous works demonstrating an inhibition of transcription
following IFN-γ stimulation238,258,260
. Thus, transcriptional profiles of T3SS-associated
genes change under different stress-induced conditions and remain to be further
investigated.
33
Antibiotics
Antimicrobial resistance is a global problem
Traditional antibiotics are used for their capacity to kill bacteria or inhibit their growth.
However, antimicrobial resistance has become a global problem and is probably one of
the most essential assignments for microbiologists and physicians in combating the
bacterial infections. Therefore, increasing efforts are present challenging new
antimicrobial strategies compensating the use of traditional antibiotics and the inability
of microorganisms to circumvent its action. Consequently, in 2002, the European Union
council of health ministers presented a strategy for battling antibiotic resistance and
proposed a policy recommendation for use of antibiotics and for better surveillance of
antibiotic resistance (according to the homepage of The Swedish Institute for Infectious
Disease Control, online) In 2005, the World Health Assembly (WHA) of the World
Health Organization (WHO) presented the resolution “Improving the containment of
antimicrobial resistance” to counteract antibiotic resistance (according to their
homepage, online). A frightening example of the consequences of antimicrobial
resistance, have been reported from the Centers for Disease Control and Prevention
(CDC) in the United States. According to their statistics, almost 2 million patients in US
that get an infection following hospitalized each year and more than 70% of the bacteria
that cause hospital-acquired infections are resistant to at least one of the antibiotics most
commonly used for treatment (according to homepage of CDC, online).
Antibiotic treatment of C. pneumoniae infection C. pneumoniae is susceptible to antibiotics belonging to the classes of macrolides,
tetracyclines and rifamycines in vivo261
. However, Chlamydia can develop antibiotic
resistance in vitro and therefore antibiotic resistance may become a future clinical
problem262-265
.
Clinical experience has shown that C. pneumoniae infections can recur after short
course therapy, why high dose treatment for 14 days is recommended117
. In vitro,
inappropriate treatment of C. pneumoniae infection may induce an aberrant persistent
infection state, unresponsive to antimicrobial treatment 91
. The nature of C. pneumoniae
infection makes prevention difficult, and since the recommended first choice treatment
includes tetracyclins and macrolides the development of new anti-chlamydial strategies
remains an important aim to reduce the use of these valuable antibiotics.
••
34
Small molecules
Chemical genetics and small chemical molecules Genetic approaches have been used for decades to identify genes that regulate a
biological process of interest. Knocking out a gene of interest is possible in many
organisms today although Chlamydia and several other bacterial species lack tools for
genetic manipulations. Instead, a chemical genetic approach can be instrumental for
studying bacterial functions (reviewed in reference 266). This strategy uses small
organic molecules to reveal the specific macromolecules responsible for regulating
biological systems. First, an assay for the biological process of interest is developed,
secondly, the process is systematically inhibited with small molecules and finally the
component affected by each molecule is determined to reveal the proteins or genes
regulating the process266
. The small molecules directly alter protein function by binding
to their targets and either enhancing or inhibiting the function of the targets.
Small molecules as inhibitors of virulence When developing new antimicrobial drugs, reducing the risk of resistance is important.
While antibiotics target bacterial growth, an alternative approach would be to inhibit
bacterial virulence mechanisms. Antimicrobial resistance rarely develops in virulent
bacteria during treatment. Instead, resistance develops in the large number of non-
virulent bacteria of the resident commensal flora and spreads through horizontal gene
transfer to neighboring bacteria. Small molecule inhibitors have a high molecular
affinity, targeting a specific virulence-related factor and therefore are potentially less
likely to interact with non-virulent bacteria. Thus, resistance against virulence-specific
inhibitors is unlikely to develop and spread among the resident commensal flora.
Examples of potential applications targeting virulence include two-component signal
transduction systems, quorum sensing, biofilm formation and T3SS267-270
. For example,
virstatin blocks the transcriptional regulator ToxT in Vibrio cholerae, thereby inhibiting
the expression of virulence factors regulating toxin and pili assembly271
. Moreover, a
hydroxymate, termed LFI, inhibits the anthrax toxin in Bacillus anthracis272
. Another
example of small molecules as inhibitors of virulence is a class of compounds termed
pilicides that targets and inhibits the formation of virulence-associated pili of
uropathogenic E. coli270
.
Small Molecules and inhibition of T3SS
In an effort to identify compounds that target and inhibit T3S-induced virulence,
Elofsson and Wolf-Watz designed a high-throughput screen and examined a
commercial library of more than 9,000 compounds273
. Their approach was based on the
ability to monitor the expression of yopE of Y. pseudotuberculosis in a luciferase
35
reporter assay. The system identified 12 potential T3SS inhibitors. One of the hits,
compound INP0007 belonging to a class of acylated hydrazones of salicylaldehydes,
was later shown to specifically block YopH secretion in infected HeLa cells274
.
Recently, INP0010 and INP0403, belonging to the same class of compounds as
INP0007, have been shown to inhibit T3SS of S. enterica, in two independent
studies275,276
. In the work by Hudson et al., the authors demonstrated an inhibition of
secretion via the SPI-1 T3SS-related effectors as well as an inhibition of invasion into
HeLa cells. However, INP0007 and INP0403 had to be pre-incubated with the bacteria
to suppress T3SS-dependent responses in a bovine intestinal loop model. In other work
done by Rehn et al., the authors confirmed similar inhibitory effects on invasion of
epithelial MDCK cells and secretion of SPI-1 T3SS-related effector proteins.
Interestingly, one of the tested compounds, INP0010 affected intracellular S. enterica
proliferation in macrophage-like cells at concentrations of 40 µM. Another example of
small molecules as inhibitors of T3SS has been described in EPEC277
. The most
efficient compound, composed of a halogenated salicylaldehyde derivative condensed
with a 3-aminoacetophenone, inhibited expression of T3SS-associated genes named
esps (for E. coli-secreted proteins). Notably, this compound does not belong to the same
class of compounds as INP0007.
••
36
OBJECTIVES OF THIS THESIS
The general aim of this thesis was to investigate Chlamydia infection biology,
especially the T3SS.
The more specific objectives were as follows:
To investigate the virulence properties of Chlamydia spp. using chemical
genetics.
To investigate the effect of different T3S-inhibtors in the developmental cycle
of C. pneumoniae and C. trachomatis.
To obtain a valid method for monitoring initiation of transcription in C.
pneumoniae in the presence of a T3S-inhibtor.
To investigate the effect of C. pneumoniae infection on bone biology in mice.
To investigate whether C. pneumoniae can infect osteoblasts and characterize
the cytokine response.
37
RESULTS AND DISCUSSION
Paper I
There are two major reasons the employment of small molecules could have important
implications in Chlamydia biology:
1. There are no molecular biological approaches to accomplish necessary genetic
manipulation in the spp. of Chlamydia. Thus, blocking a function by addition of
chemicals enables studies of its function.
2. Small molecules may present a novel opportunity for therapeutic interventions in
Chlamydia. Small molecules have previously been shown to inhibit the T3SS in
Yersinia. Given the close homology between T3SSs in Yersinia and Chlamydia we
hypothesized that small molecules could inhibit the T3SS also in Chlamydia.
Accordingly, this might evaluate the essentiality of the T3SS in pathogenesis of
Chlamydia, e.g. molecular mechanisms involved in attachment, entry, proliferation and
differentiation.
Screening of T3SS inhibitors using Y. pseudotuberculosis T3SS In a previous study, INP0007, belonging to the class of acylated hydrazones of
salicylaldehydes, prevented Y. Pseudotuberculosis YopH translocation into HeLa
cells274
. However, INP0007 displays limited solubility in water and exhibited cytotoxic
properties when used at higher concentrations in our HEp-2 infection model (data not
shown). Therefore, our interest was shifted towards the more soluble analogue
INP0010. Thus, the inhibitory as well as cytotoxic effects of INP0010 had to be
determined first. For this purpose we applied the rapid screening assay of T3S-inhibitors
that measures both virulence inhibition of Yersinia infection in cell culture, as well as
compound cytotoxicity. INP0010 was not cytotoxic in the assay at concentrations as
high as 50 µM. Moreover, microscopic examinations revealed that cell proliferation and
morphology were unaltered in HEp-2 cells grown at 10 µM of INP0010 for up to 72 h
(data not shown). Thus, based on these data INP0010 was chosen as a suitable T3S-
inhibitor in our infection model. Moreover, in order to ensure that any observed effects
on Chlamydia are the result of T3SS blockage, we also included the compound
INP0406 which belongs to the same chemical class as INP0010, but is unable to inhibit
the T3SS of Yersinia.
••
38
INP0010 inhibits C. pneumoniae intraceullar propagation When INP0010 at 10 µM was added to C. pneumoniae infected cells, no chlamydial
inclusions could be observed. These results suggest an altered developmental cycle. To
further investigate the effect of INP0010 on bacterial proliferation, we performed a set
of control experiments:
1. Pre-treatment of the host cells with INP0010 prior to cell infection had no
effect on C. pneumoniae propagation.
2. Independent of when INP0010 was added during the early developmental cycle
(0-12 h), C. pneumoniae proliferation was inhibited.
3. A dose-dependent effect of INP0010 was demonstrated both with immuno-
fluorescence and quantitative real-time PCR at 48 h p.i., respectively.
4. Heat-inactivated C. pneumoniae did not proliferate and was present at similar
bacterial numbers as when INP0010 at 10 µM was added to the cell culture.
INP0010 shows diverse effect in C. trachomatis To our surprise, when HEp-2 cells were infected with C. trachomatis serovar L2 and
treated with INP0010 at concentrations ranging from 10 – 30 µM, no inhibitory effect
could be observed. Importantly, no inhibitory effect could be observed on either C.
pneumoniae- or C. trachomatis-infected cells treated with INP0406. Together, these
results suggest important differences in the T3SS of different species. However, the
underlying reason for this is not clear since the T3SSs in the two Chlamydia species are
likely to be more closely related to each other than to Y. pseudotuberculosis.
Interestingly, C. pneumoniae have a broader repertoire of postulated effector proteins
than C. trachomatis, suggesting differential biology, tropism and pathogenesis between
the two different spp. This is supported by the ability of C. pneumoniae to be more
invasive and survive in a broader range of host cell types than C. trachomatis 18
. This
suggests the possibility of using small molecules to inhibit different targets and
functions in Chlamydia.
INP0400 is active against both C. pneumoniae and C.
trachomatis. Intracellular replication of C. trachomatis was not affected by 30 µM of INP0010.
Therefore we expanded the search for putative T3S-inhibitors that could block
proliferation for both C. pneumoniae and C. trachomatis. Compound INP0400 inhibited
propagation of C. pneumoniae and C. trachomatis at 10 µM and demonstrated similar
inhibitory effect in Y. pseudotuberculosis, without cytotoxic effects. In conclusion, by
modifying the chemical structure(s) and screening each compound in our infection
39
model, it was possible to identify a novel compound which was active against both
chlamydial spp. This is in contrast to INP0010, which was selectively active against C.
pneumoniae. This indicates that the target for this class of compound shows minor
differences in the two Chlamydia spp. examined herein. Thus, different small molecules
made it possible to further characterise the differences in the T3SS in the different
Chlamydia spp. This strategy constitutes a good substitute for the lack of genetic
methods in these pathogens.
Treatment with INP0010 inhibits secretion of putative T3SS
effector proteins Inclusion membrane proteins IncA, IncB and IncC are predicted to be T3S-mediated
effector proteins of C. pneumoniae as they are secreted in heterlogous T3SS 239
. We
demonstrated by using immunofluorescence microscopy, that IncB and IncC was
localized to the chlamydial inclusion in close association with the bacteria. However,
no specific staining of IncB and IncC could be observed in the presence of INP0010 at
10µM. These results suggest of an inhibition in translocation of the predicted effector
proteins when INP0010 is present. This is later supported in paper IV.
••
40
Paper II
In paper I we identified INP0400 as an inhibitor of Chlamydia propagation. In paper II
we have further evaluated the action of INP0400 on C. trachomatis growth-mediated
processes, revealing the T3SS as essential for development and pathogenesis of C.
trachomatis.
INP0400 inhibits proliferation but not primary differentiation C. trachomatis-infected McCoy cells treated with INP0400 at 20 µM showed similar
inhibitory effects as that observed in HEp-2-infected cells (paper I). This was later
corroborated by another group, whereas INP400 at 20 µM inhibited growth of C.
trachomatis serotype D in McCoy and HeLa cells278
.
However, the process of re-differentiation from RBs-to-EBs is poorly investigated.
During the re-diffrentiation process, the expression of a number of late-cycle T3SS-
associated genes occurs. We demonstrated a dose-dependent reduction in inclusion size
and a concomitant reduction in the number of intracellular bacteria in infected cells
following INP0400-treatment, 30 h p.i. This is indicative of an inhibition of
proliferation, as RBs have already started to re-differentiate to infectious EBs, making
the visual inclusion larger in size at 30 h p.i. By electron microscopy we confirmed that
internalized EBs in the presence of INP400 converted to RBs. However, the
proliferation of RBs were inhibited in a dose-dependent manner, resulting in smaller
inclusion bodies containing just one or a few RBs. Accordingly, this implicates that the
T3SS might regulate the terminal differentiation of Chlamydia This is supported by the
work of Fields and co-workers279
.
INP400 inhibits secretion of putative effectors We further investigated whether INP0400 could interfere with secretion of T3S-effector
proteins. We could demonstrate that homotypic inclusion fusions were inhibited when
INP0400 was given at 8 h p.i, a time-point in the developmental cycle that precedes the
onset of IncA production87
. Accordingly, no detection of IncA-antibody signals was
seen in the non-fused inclusions. This proposes an inhibition of effector translocation
when INP0400 is present. This result is supported by the work by Wolf et al., who also
could not detect IncA in the inclusion membrane following INP0007-treatment of C.
trachomatis infected cells279
.
Moreover, IncG of C. trachomatis has been shown to interact with the eukaryotic
protein 14-3-3β89
. Accordingly, we demonstrated that treatment with INP0400, early in
establishment of infection in McCoy cells, prevented the host protein 14-3-3 from
localizing to the inclusion membrane. Therefore, it can be proposed that the T3S-
41
inhibition prevents translocation of IncG into the inclusion membrane. Interestingly, C.
pneumoniae do not have homologues to IncG nor undergo homotypic fusions resulting
in a single inclusion in cells that are multiply infected. This might support the idea of
two differentially regulated T3SS between C. pneumoniae and C. trachomatis, as
proposed in paper II.
INP0400 treatment during the late cycle promotes bacterial
dissociation from the inclusion membrane C. trachomatis RBs are typically found juxtaposed to the inner surface of the inclusion
membrane during mid-cycle development. However, it is unknown whether this
attachment requires effectors delivered by the chlamydial TTS system. Likewise, it is
not known whether RB association with the inclusion membrane is required for
bacterial multiplication and/or RB-to-EB transition. Peters et al., have predicted a model
for T3S contact-dependent development using a mathematical model, which predicts
that RBs detach from the inclusion membrane after secretion of T3S effectors, thus
T3SS become inactivated and late differentiation begins217,252
. It can be described as a
“switch button” turning the T3SS on-and-off. T3SS is on (active) when the bacteria are
in contact with the inclusion membrane and off (inactive) when terminal RB-to-EB
transition occurs. We showed that INP0400-treatment during mid-developmental cycle
induced a failure of the bacteria to localize to the inclusion membrane, instead leaving
free space between the inclusion membrane and the bacteria. Surprisingly, when
INP0400 was removed at the end of the developmental cycle and infected cells further
cultured for a short period (4 hours) in fresh culture media, the bacteria re-associated
with the inclusion membrane. This might support the “switch button” theory whereby
the presence of the T3S-inhibitor mimics the naturally occurring stage when RBs detach
from the inclusion membrane and begin differentiation.
••
42
Paper III
In paper III, we describe the inconvenience of using different internal gene-expression
controls for normalizing the expression of a target gene of interest. Instead, DNA should
be considered, since it is always present, stable and a varied DNA level directly reflects
proliferation of the bacteria. Moreover, INP0010 decreases initiation of transcription in
C. pneumoniae during the early developmental cycle as demonstrated by a novel
mathematical model.
DNA is preferable to RNA for normalization of gene expression In general, stable transcripts are used to normalize the expression of a target gene. This
can, however, be deceptive given:
1. Variation in transcript stability between control and target mRNAs.
2. Levels of mRNA can vary dependent of when it is essential in the
developmental cycle.
In this study, we monitored expression of T3SS-associated genes and genes encoding
proteins involved in RNA-polymerisation, heat shock responses and DNA replication.
Accordingly, some of the genes were presumed to be efficient control genes for
normalization, at 12 hours p.i. This window of time was suitable when the primary
differentiation takes place and replication is initiated. To test transcript stability, we
used rifampicin which inhibits de novo protein synthesis by binding to the RNA-
polymerase. Rifampicin was added to infected cells with or without INP0010 at two
distinct phases of the developmental cycle:
1. During early infection (0 hours p.i.) when represented as infectious EBs.
2. During early mid-developmental growth (12 hours p.i.) and when RBs are
present.
In general, transcripts of EBs were more stable, when compared to RB transcripts. In
contrary to the situation at 0 hours p.i., transcripts of rpoD, cdsS and incA could not be
detected after one hour of rifampicin treatment, at 12 h p.i. This suggests that these
transcripts are quickly turned-over, during the transition from EBs to RBs. Taken
together, target and control RNAs display a varied stability at different developmental
phases, both in the presence or absence of INP0010.
Relative gene expression is not necessarily correlated to proliferation since the
expression might fluctuate significantly throughout the developmental cycle. This
would give a relative target mRNA expression that follows the expression-pattern of the
control mRNA, rather than reflecting the bacterial proliferation. Accordingly, several
control and target mRNAs were induced at the early-mid developmental cycle (12 hours
43
p.i.) when compared to early infection (2 h p.i.). Consequently, using 16S, rpoA and
rpoD as controls for measuring the relative target gene-expression throughout the
developmental cycle, would result in a markedly reduced relative gene-expression at 12
hours p.i., compared to early infection. Although the numbers of bacteria remains
unaltered. Relative gene expression should be correlated both to the expression of the
target and the control mRNA as well as the degradation, when using RNA as control.
Moreover, the diverse stability between different/or the same RNA species could
depend on the growth phase and presence/absence of INP0010. Consequently, RNA
should be avoided as control for normalization against your target gene expression of
interest, instead, DNA should be used as a control.
INP0010 decreases initiation of transcription Determination of mRNA half-life is important to fully understand mechanisms involved
in gene regulation in response to INP0010 or by developmental signals. In addition, the
stability of mRNA may indicate how rapidly the translation of the encoded protein can
be shut down. Therefore, we have determined the relative transcription initiation
constant (k) using expression of C. pneumoniae genes and by introducing the relative
transcript amount and decay into a mathematical model. Using this model, we could
demonstrate that when INP0010 is present, initiation of transcription decreases. This
suggests that stabilized mRNA levels induced with INP0010, in theory, could increase
protein amount. This proposal is consistent with the work by Wolf et al., who
demonstrated accumulation of T3SS-associated substrates within INP0007-treated
RBs279
. However, further work is required to determine whether there is a correlation
between an increased chemical half-life and a more functional mRNA.
The developmental cycle is initiated as the metabolically inactive EBs are endocytosed
by the host cell, where they are tightly bound within a membranous vesicle. Within this
vesicle, unknown environmental signals trigger an initial round of chlamydial
transcription. Our results suggest that INP0010 in general decreases initiation of
transcription.
Expression of T3SS in the presence of INP0010 In paper I, we presented results indicating that expression of most T3SS-associated
genes were down-regulated after INP0010-treatment. However, these results were based
on normalisation against the ribosomal 16S RNA. We therefore refined previous results
and normalised the presence of mRNA against the native DNA. When normalised
against DNA, a reduction was detected for all RNAs tested in the presence of INP0010.
Thus, the inhibition of expression was not specific for T3SS-associated genes during
early developmental events. This indicates that INP0010 functions generally during
early developmental cycle and reduces expression of all, at least those tested herein,
genes by a hitherto unknown mechanism.
••
44
Paper IV
In paper IV, we suggest that INP0010 target the T3SS in C. pneumoniae infected cells
and thereby arrest the RB, either after primary differentiation or during terminal re-
differentiation. Moreover, INP0010 inhibited expression of T3S-genes necessary for the
exterior part of the T3S injectisome during the late developmental cycle. Consequently,
the effector protein IncB was not detected in the cytoplasm following INP0010
treatment. The effect of INP0010 on iron-responsive genes indicates a role for T3S in
iron acquisition. Accordingly, our results suggest a possibility for C. pneumoniae to
acquire iron via the intracellular trafficking pathway of endocytosed transferrin.
INP0010 inhibits early- and mid-developmental cycle
C. pneumoniae modulates its entry by rearranging the host cells cytoskeleton upon
binding. Other pathogens are known to use their T3SS to inject effector proteins upon
host cell contact. However, our uptake experiments demonstrated that INP0010 did not
interfere with C. pneumoniae internalisation, suggesting either a non-active T3S
apparatus or an independent T3S-mechanism during entry. Accordingly, INP0010
analogue in infection models of C. trachomatis L2 and serovar D also failed to interfere
with invasion278,279
. We have previously suggested (paper II) that INP400 inhibits
progression of C. trachomatis in either the mid-developmental phase when replication
of the RBs occurs or during the re-differentiation of RBs back into infectious EBs. In
addition, we could show that the metabolically inactive EB converts to an RB, but in
contrast to when C. trachomatis is INP400-treated, RB multiplication is totally inhibited
when INP0010 is present. Thus, INP0010 interferes with signals required for early RB
proliferation processes which are in agreement with the results presented in paper III,
demonstrating a general reduction of transcription.
Iron initiates proliferation but not terminal re-differentiation in
the presence of INP0010 Dual action of T3S-inhibitors has been suggested, whereby INPs are also capable of
chelating iron278
. Thus, this enabled us to use INP0010 to examine the T3SS and its
ability to regulate iron required for Chlamydia propagation. To be able to monitor
potential iron modulated process governed by the T3S, INP0010-effects were compared
with the effects achieved by the classical iron-chelator desferal (DAM). We showed that
Fe2+
could reverse the RB proliferation inhibitory effect of INP0010 when additional
Fe2+
was available during infection. However, reorganization of C. pneumoniae RBs
into EBs was inhibited as demonstrated by re-infection experiments and TEM. This
suggests an essential role of the T3S and iron in transducing an undefined signal for
terminal differentiation in C. pneumoniae. Moreover, expression of the putative iron-
45
binding proteins, YtgA and DcrA, increased when compared to wild-type infection,
supporting a function for the T3SS in iron acquisition. Furthermore, addition of iron-
saturated holo-transferrin to INP0010-treated C. pneumoniae-infected cells resulted in
100% inhibition of EB production. In contrast, Slepenkin et al. demonstrated 100%
recovery of C. trachomatis when holo-transferrin was added in the presence of
INP0341278
. This further suggests differences in T3SSs between the two Chlamydia spp.
It has previously been shown that the early endosome, containing transferrin is a likely
Chlamydia-source of iron95,97
. In addition, we could demonstrate when using DAM that
decreasing the host cellular pool of iron re-directed transferrin receptors to the inclusion
sites, presumably due to lack of iron-accessibility for Chlamydia. However, when
INP0010 was present, no accumulation of transferrin receptors was shown in close
contact to the inclusions, even though INPs has iron-chelating properties. Thus,
INP0010 could have blocked the T3S-secretion of effectors essential for host trafficking
pathway e.g. endosomal trafficking. Presumably, the Chlamydia could use the T3S-
injectisome to interfere with the early endosome to sequester iron deposited by
transferrin and this is might have been initiated by secretion of effector proteins for e.g.
redirection of the endosome.
INP0010 blocks secretion of IncB into the host cell cytoplasm We propose that IncB can be secreted into the host cell cytosol via a T3S iron-
dependent mechanism. Supplementation of ferrous iron was sufficient to re-establish the
expression of IncB in the bacteria, both at the transcriptional and translational levels.
However, it appears that secretion was affected when INP0010 was added in the
presence Fe2+
. Taken together, our results suggest that C. pneumoniae might use the
T3SS needle structure to interfere with the endosome to sequester iron deposited by
transferrin (summarised in Fig. 8).
••
46
FIGURE 8. Schematic illustration of C. pneumoniae development in the presence of the T3S-inhibtior
INP0010 at 10 µM. C. pneumoniae is endocytosed independently of INP0010 1. INP0010 arrests the RB after
primary differentiation 2. IncB secretion is inhibited in the presence of INP0010 3. Effector protein(s)
presumably interacts with the transferrin receptor trafficking pathway and/or early endosomes for iron
acquisition. RBs prolifierate in the presence of INP0010 and ferrous iron (insert). 4. Re-differentiation is
inhibited in the presence of INP0010 and ferrous iron.
47
Paper V
In paper 5, we have for the first time presented data showing generalized bone loss in C.
pneumoniae infection in mice. The infection was associated with increased levels of the
bone resorptive cytokines IL-6 and IL-1, as well as demonstrating an increased sub-
population of T-cells expressing RANKL on their cell surface. In addition, C.
pneumoniae established an infection in a human osteoblast cell line in vitro with similar
cytokine profiles as seen in vivo, supporting a causal linkage. Collectively, these data
may indicate a previously unknown pathologic role of C. pneumoniae in generalized
bone loss.
C. pneumoniae infection decreases bone mineral density in mice Mice were inoculated intranasal once a week for three consecutive weeks and bone
mineral density was measured before and 16 days p.i. Infected mice demonstrated a
significant reduction in bone mineral density of the distal femur and proximal tibia
when compared with sham infected mice. Importantly, there was no significant
difference in weight gain during the infection period when comparing sham infected and
infected mice. The infection was associated with increased levels of the bone resorptive
cytokines IL-6 and IL-1, as well as demonstrating an increased sub-population of T-
cells expressing RANKL. Taken together, these results suggest that C. pneumoniae
mediated induction of the innate immune system might modulate bone biology
processes in mice. This was further evaluated in vitro.
C. pneumoniae growth in hFOBs increases production of IL-6
and expression of RANKL In this paper we used the hFOB cell line that expresses the normal osteoblastic
phenotype as well as RANKL280,281
to study the effect of C. pneumoniae infection. The
infected hFOBs demonstrated significantly higher amounts of IL-6, both on mRNA and
protein level, when compared to non-infected cells. Moreover, the secretion increased in
a dose-dependent manner when the cells were infected. Interestingly, other studies have
demonstrated similar production of IL-6 at sites of bacterial infection in human S.
aureus-associated osteomyelitis175
. In addition, the increased production of IL-6, in our
study, was dependent of chlamydial protein synthesis as demonstrated by a set of
control experiments:
1. Supernatants collected from previously infected hFOBs could not induce
expression of IL-6.
••
48
2. Heat-inactivated C. pneumoniae did not induce secretion of IL-6. Thus, neither
LPS nor surface exposed glycoproteins mediates the IL-6 production.
3. The presence of non-proliferating C. trachomatis could not induce significant
cytokine secretion when compared to non-infected cells.
Additionally, IL-1 expression was up-regulated in hFOB cells in response to infection.
However, the levels of secreted IL-1 were not increased in the supernatant of infected
cells when compared to non-infected cells. This was further investigated and we found
that hFOBs do not secret high levels of IL-1 even when stimulated with pure LPS from
E. coli (data not shown). Finally, RANKL expression was 3-fold higher in infected
cells.
Interestingly, the inflammatory response of bone destruction resembles the pathogenesis
of atherosclerosis, postulated to be accelerated by C. pneumoniae infection. Therefore,
C. pneumoniae could be involved in processes of the vascular- and bone remodelling-
system (summarized in Fig. 9). Our novel results indicate that certain bacteria can
contribute to general bone loss, and this needs to be further investigated in humans.
FIGURE 9. Influence of C. pneumoniae on bone and vascular tissue.
49
CONCLUSIONS
The T3S-inhibtors INP0010 and INP0400 block the developmental cycle of C.
pneumoniae and C. trachomatis, without any cytotoxic effect, respectively.
INP0400 inhibits secretion of the T3S effector-protein IncA in C. trachomatis, which is
needed for inclusion fusion.
INP0400 targets the T3SS and provokes a bacterial dissociation from the inclusion
membrane presumed to mimic the natural occurrence of terminal differentiation
INP0010 decreases initiation of transcription in C. pneumoniae during the early mid-
developmental cycle as demonstrated by a novel calculation, useful for measurement of
transcription initiation in any intracellular pathogen.
INP0010 targets the T3SS of C. pneumoniae and thereby arrests RB proliferation as
well as RB to EB re-differentiation.
C. pneumoniae uses the transferrin receptor pathway as an iron-uptake system, and may
be regulated via the T3SS.
C. pneumoniae infection decreases bone mineral density in mice, and is associated with
increased production of IL-6, IL-1 and RANKL.
C. pneumoniae infection of human osteoblast-like cells in vitro increases production of
IL-6 and expression of IL-1 and RANKL.
••
50
ACKNOWLEDGEMENTS
Reflections of writing a thesis: Stayed without my family for weeks, crashed my car,
lived at IKSU without using any of their facilities, ate pasta daily, tried not to be
distracted even though my hockey team had their worse season ever… Suddenly I heard
an echo of Sven Bergström’s voice in my head; you are always so positive Lelle! I
almost forgot…
I like to acknowledge all the people at MolBiol and UmU who have helped me through
the years, without any ranking score. Sven, my co-supervisor (sounds strange to call co-
) who gave me the opportunity to be in his lab and for taking care of me. I have never
told you this before, but you have always remained me of one the greatest leaders in the
history of football– Peter Antonine! Peter, my supervisor, who has been a great
inspiration with lots of exciting ideas through the years. Anders, co-supervisor, for
always being so positive and open-minded in our projects. Åsa co-worker and co-
supervisor, you have been great to work with and helpful in all aspects. Johnny
(Hegerfors), co-worker, co-trainer, co-driver, co-babysitter but most of all, my beloved
friend. Hasse, for being an endless source of ideas and knowledge. J.J, a great
researcher and an even greater hockey-fan, Forza LHF! Mikael E., for being so helpful
and interested in the T3S project. Matt, for with a very short notice, reading my thesis –
You did a great job! Thanks!! Our collaborators at MTC, Sandra, Birgitta and
Staffan. Thanks for a great work with the PNAS paper. Göran K Hansson and co-
workers, for sharing knowledge concerning atherosclerosis. Ulf and co-worker Anita,
for sharing your enthusiasm in infections and bone biology. Patte, the one I have shared
most ideas and lab-time with through the years. You have become like a younger
brother to me. It has been a privilege to work with you and “stafettpinnen” is yours now.
Just remember to always have a plan B. Lenore for being incredible with the electron
microscope.
The Borrelia clan: Betty, or simply B, for being the best corrector and co-worker. I
don’t know how to pay you back but I will come up with something! Iggy for being a
great room-mate on tour and all your assistance in the office – Iggyyy!! Christer, is-it-
good-or-is-it-bad, Larsson, for being a joy of life. Coma, full with unknown talents, at
least for me You really kicked ass with the Finns! (Anna: TACK!). Marie A, a.k.a Ms
Bindefeldt, for always being so helpful, with everything. You are probably the most
pedagogic person I ever met – have seen you in action at course! Ingela for being the
coordinator per se. Your contribution is eternal. Elin, the best belly-dancer in groupSB.
You are a sunshine! Lisette, for spreading some Norrbottens-spirit in the group. You
are a true lab-inspiration! Marie B, for sharing your energy every morning, always a
smile! Former members of group SB; Yngve, Pinnen och Palle, you were all inspiring.
51
All my former students, who have been great colleagues and interested in our projects:
Nisse, Wolfie, Tobbe, Marco, Lisa, Tina and Susanne.
Marita, Ethel, Berith, and the rest of the coordinating-staff for always being so caring!
Kerstin at Medicine, you have been amazing helping me handling all papers. Lab
service, Agnetha and co-workers for doing the best job, for us and our students! Britt-
Inger and Jenny (51135) at Djuris, for taking care of our mice.
Ulle, for all nice conversions, Smut, Eddie, Jenny, Babsan – the funniest person ever.
Keep it up Babsan! Stina, Anna, Annika and Connie – thanks for the breakfasts!
Jempa for being DJ Roccos biggest fan and for having the nicest mum – love her food
and bakeries. Janne, Maria N, Sofia, Petra, Sara G, Lissandro, Jeanette, David,
Jocke, Linda J, Sara C, Sara R, Gosia, Monica, Katrin, Olena and Stefan for
supporting DJ Rocco. Krisse, Pelle and Mike for your kindness and support. Micke W,
still go strong! Näsorna, Mari, Vickan, Johan, Maria, Mats and the latin-lover
Geovanni for all laughs in tha Bunkern. Rutan, for your patience in the FACS room.
Till alla mina fotbollsvänner: GCK P84-86, MSK P87-90 . Många av er har blivit mina
vänner, men kom ihåg att jag fortfarande är CoachB! Fredde, Fuad, Andy, Babbe,
Zacke, Adam, Lasse, Nisse, Said, Solle, the Dunkels, Are, Nille, Jacke, Ragge,
Jeppe, Robban, Calle, Tobbe, Peter och alla ni andra! Håkan Arestav – keep up the
good work. Ettan väntar! Till fotbollsfamiljerna Stenman’s och Chennoufi’s. Ni är
väldigt betydelsefulla för mig och min familj, med den kärlek ni visat Elliot.
Till alla mina Rosviks-bröder med respektive familj: Pierre (still…), Sotar´n, Sniff,
Andreas, Micke, Fredde, Pelle L och Janne. Mina Pite-bröder med respektive familj:
Eng (TACK mannen!), Ros, Pelle B, Macce, Ibbe och OK/Q8-Kalle. Nitte, I´m your
biggest fan (TACK mannen!). Jag är evigt tacksam er vänskap. Fastän vi är utspridda i
landet så finns det ingen gårdag när vi träffas. Jag älskar er alla, och kommer alltid finns
där för er, det vet ni!
Elliots bästis Emma, du är en pärla!
Till Lisas föräldrar Birgitta och Tomas. Ni har varit fantastiska under denna tid. Jag
lovar att aldrig mer skriva en avhandling! Resterande ”Lundbergare” Sollan, Curthen,
Sofi, Anders och storkusinerna Theo och Vilmer – Tack för all kärlek ni ger oss!!
Till min far och bäste vän, Leslie Sr. Du har alltid uppmuntrat mig till att göra det jag
tror på. Mannen, jag tror Barcelona fortfarande har en chans! Lilian, min syster, mitt
stöd och min ögonsten. Du är bäst!!! Jag älskar er!
Dragamama, ovom knjigom zelim ti pokloniti svu moju ljubav i postovanje. Ti si mi
podarila zivot, tvoja ljubav ispunjava svaki trenutak u mom zivotu. Volim te i
obozavam najvise na svijetu. Tvoj sin Lelle.
Till sist vill jag tacka Lisa och Elliot som skrivit denna bok tillsammans med mig. Ni är
de käraste jag har och jag ser fram emot att leva resten av mitt liv med er. Jag älskar er!
••
52
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