Chlamydial biology and its associated virulence blockers

2014

http://informahealthcare.com/mbyISSN: 1040-841X (print), 1549-7828 (electronic)

Crit Rev Microbiol, 2014; 40(4): 313–328! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/1040841X.2012.726210

REVIEW ARTICLE

Chlamydial biology and its associated virulence blockers

Delphine S. Beeckman, Leentje De Puysseleyr*, Kristien De Puysseleyr*, and Daisy Vanrompay

Department of Molecular Biotechnology, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, B-9000 Ghent, Belgium

Abstract

Chlamydiales are obligate intracellular parasites of eukaryotic cells. They can be distinguishedfrom other Gram-negative bacteria through their characteristic developmental cycle, in additionto special biochemical and physical adaptations to subvert the eukaryotic host cell. The hostspectrum includes humans and other mammals, fish, birds, reptiles, insects and even amoeba,causing a plethora of diseases. The first part of this review focuses on the specific chlamydialinfection biology and metabolism. As resistance to classical antibiotics is emerging amongChlamydiae as well, the second part elaborates on specific compounds and tools to blockchlamydial virulence traits, such as adhesion and internalization, Type III secretion andmodulation of gene expression.

Keywords

Antibiotic resistance, chlamydia, infectionbiology, virulence, virulence blockers

History

Received 16 May 2012Revised 26 August 2012Accepted 29 August 2012

Introduction to Chlamydiaceae

Micro-organisms in the family of the Chlamydiaceae are

obligate intracellular pathogens of both mammals and birds.

The different species in this family infect many hosts, with

variable tissue tropism causing a multiplicity of acute and

chronic diseases, from sexually transmitted infertility, to

trachoma and respiratory and cardiovascular diseases.

Chlamydia (C.) trachomatis and Chlamydia (C.) pneumo-

niae are the most common chlamydial pathogens in humans,

whereas the other chlamydial species mainly occur in other

animals. In the so-called developed countries, C. trachomatis

is the causative agent of the most frequent sexually

transmitted disease (STD), leading to pelvic inflammatory

disease, infertility and possibly ectopic pregnancy. Moreover,

some C. trachomatis serovars are known to induce trachoma,

the leading cause of infectious blindness in developing

countries. Ten percent of the pneumonia and 5% of bronchitis

and sinusitis cases in adults is attributable to a C. pneumoniae

infection and chronic infection could contribute to athero-

sclerosis (Campbell & Kuo, 2004, Belland et al., 2004).

As proven pathogens of vertebrates, Chlamydiaceae cause

reproductive, respiratory, cardiovascular, gastrointestinal or

systemic disease, as well as conjunctivitis, arthritis and

encephalitis in both live stock, companion and wild animals

(Everett, 2000). Chlamydia psittaci primarily infects birds,

predominantly impacting on commercial turkey and duck

farms, but is also of public health importance due to its

zoonotic nature (Beeckman et al., 2009). Other highly

prevalent zoonotic species in animals include C. abortus

(mainly in sheep and goats), C. suis (endemic in pigs) and

C. felis (cats).

Due to spatial limitations and for reasons of clarity, the

description of the chlamydial infection biology and possible

ways to combat the infection will focus on aspects which are

important for their survival and are likely to be implicated in

virulence.

Chlamydial biology

Developmental forms

During the unique biphasic life cycle (detailed description in

section 0), at least two morphologically different structures

can be observed: the infectious elementary bodies or EBs and

the replicating reticulate bodies or RBs. An overview of the

most important discriminative characteristics between these

two developmental forms can be found in Table 1. In addition,

intermediate bodies (IBs) can be observed during the

maturation from EBs to RBs (Vanrompay et al., 1996).

Elementary bodies are usually small, spherical, electron

dense structures (Costerton et al., 1976; Longbottom &

Coulter, 2003), characterized by a dense, eccentric core of

condensed DNA and chromatin. The EB has a granular

appearance due to the presence of 70S ribosomes. A lipid

cytoplasmic membrane and a rigid outer membrane (both

~8 nm) with extensive disulphide bridging between cysteine

and methionine residues of ‘Major Outer Membrane Protein’

(MOMP) and other sulphur amino acid rich outer membrane

proteins surround the cytoplasm (Newhall & Jones, 1983).

This high level of cross-linking possibly compensates for the

low amounts of the usual cell wall strengthening substance

peptidoglycan in the outer membrane. In this way, the spore-

like EBs are osmotically more stable and less permeable than

RBs, which allows them to survive up to several months

outside the host cell (Longbottom & Coulter, 2003).

*These authors contributed equally to this work.Address for correspondence: Daisy Vanrompay, Department ofMolecular Biotechnology, Ghent University, Coupure Links 653,Ghent, B-9000, Belgium. E-mail: [email protected]

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With their 1:1 RNA:DNA ratio, EBs are thought to be

metabolically inert until attachment to a susceptible host cell

and subsequent internalization.

Following uptake by the host cell, the disulphide bonds

between the outer membrane proteins are reduced, resulting

in a more permeable outer membrane for the reticulate body

to facilitate nutrient uptake (Newhall & Jones, 1983). The

differentiation of EB to RB also involves an expansion,

resulting in a less electron dense cytoplasm in which the

nucleus can no longer be clearly distinguished. The bacteria

become transcriptionally more active and as a consequence,

the cytoplasm contains more RNA and ribosomes, required

for protein synthesis (Ward, 1988). The metabolically active

reticulate bodies are no longer infectious and replicate

intracellularly by binary fission.

During maturation from RBs back to EBs, morphologic-

ally intermediate bodies can be observed in the host cells. In

these IBs, with a diameter ranging from 0.3 to 1.0 mm, a

central electron dense core can be observed with radially

arranged individual nucleoid fibers surrounding the core. As

for EBs, IBs are capable of infecting host cells, at least in

vitro (Litwin et al., 1961; Costerton et al., 1976; Vanrompay

et al., 1996; Rockey & Matsumoto, 2000).

Both EBs and RBs bear rosette-like structures and

projections, located in separate patches, anchored in the

cytoplasmic membrane and extending through the outer

membrane. On the RBs, the patches delineate a zone of

close contact between the bacterium and the plasma mem-

brane derived inclusion membrane. Bavoil and Hsia (1998)

speculated that the projections are in fact functional Type III

secretion system (T3SSs), injecting chlamydial virulence

proteins into the host cell cytoplasm. Beeckman and

Vanrompay (2010a) provided a detailed description of the

T3SS. However, its role in several aspects of the chlamydial

developmental cycle will be briefly mentioned here as well.

Outer membrane composition

Chlamydiaceae are surrounded by two membranes, as is the

case for all Gram-negative bacteria: a cytoplasmic inner

membrane and an outer membrane, separated by a periplas-

mic space. The outer membrane of EBs predominantly

consists of phospholipids, lipids, lipopolysaccharides and

proteins. In contrast to other Gram-negative bacteria,

Chlamydiaceae do not (Barbour et al., 1982) or hardly

possess any muramic acid (Fox et al., 1990). Similar to other

Gram-negative bacteria, an important part of the chlamydial

cell wall is insoluble in sarcosyl, an ionic detergent.

This fraction normally consists of peptidoglycan, covalently

linked to lipoproteins. In Chlamydiaceae, however, only

negligible amounts of peptidoglycan are present (Moulder,

1993; Hatch, 1996), although part of their cell wall is also

insoluble in sarcosyl. Moreover, genes for peptidoglycan

synthesis are present in the genome. Nevertheless,

Chlamydiaceae are sensitive to penicillin and other antibiotics

that target peptidoglycan synthesis, known as the ‘‘chlamydial

anomaly’’ (Moulder, 1993). However, three penicillin binding

proteins (PBPs) have so far been described and each of them

binds to and is inhibited by b-lactam antibiotics (Barbour

et al., 1982; Moulder, 1993; Gump, 1996) and a peptidogly-

can-associated lipoprotein (Pal) was recently found in the

COMC, normally anchoring the outer membrane to peptido-

glycan (Liu et al., 2010).

The cell wall fraction insoluble in sarcosyl is called the

‘‘Chlamydia Outer Membrane Complex’’ (COMC) or cell

envelope, and predominantly consists of MOMP, the cysteine

rich proteins (CRP) Omp2 and Omp3, as well as the

polymorphic membrane proteins (pmps) (Hatch et al., 1984;

Sardinia et al., 1988; Stephens & Lammel, 2001). The outer

membrane is further composed of lipopolysaccharides, PorB,

Omp85, the heat shock proteins hsp60 and hsp70 and OprB.

Some of these components will now be discussed in further

detail.

The MOMP protein has a molecular weight of ~40 kDa,

covering 60% of the outer membrane in RBs and almost 100%

in EBs. It is rich in cysteine residues and is always present as

a trimer. After reduction of disulphide bonds, MOMP can

function as a porin, allowing nutrient uptake by the RB. The

MOMP protein contains four variable domains (VD1-VD4),

which comprise family, genus, species, subspecies (or biovar)

and serovar specific epitopes (Caldwell et al., 1981; Yuan

et al., 1989; Everett, 2000; Kim & DeMars, 2001). In

addition, MOMP has been described to function as an

adhesin, mediating nonspecific (electrostatic and hydropho-

bic) interactions with host cells (Su et al., 1990).

The second most important components of the COMC are

two cysteine rich and highly immunogenic proteins, Outer

membrane protein 2 (Omp2) and 3 (Omp3), and are

abundantly present in EBs but hardly in RBs. Transcription

and translation of the CRP genes is most probably develop-

mentally regulated, the proteins being re-synthesized late in

the growth cycle at the time of differentiation of RBs to EBs

(Newhall, 1987; Sardinia et al., 1988). Outer membrane

protein 2 is the larger of the two CRPs. It is highly

immunogenic, Chlamydiaceae specific and can be used as a

marker for chlamydial infections (Caldwell et al., 1981;

Table 1. Characteristics of chlamydial elementary and reticulate bodies.

Characteristic Elementary body Reticulate body References

Morphology Spherical Spherical Costerton et al., 1976; Longbottom and Coulter, 2003Diameter 0.2–0.3 mm 0.5–1.6 mmElectron density High LowInfectivity for the host High NoneRNA/DNA ratio 1:1 3:1 (more ribosomes)Metabolic activity Relatively inactive Active, binary fissionCell wall Rigid, cross-linked Permeable, fragile Newhall and Jones, 1983Projections (T3SSs) 11–20, small patch Up to 83, larger patch Matsumoto et al., 1976; Matsumoto, 1982a, 1982b

314 D. Beeckman et al. Crit Rev Microbiol, 2014; 40(4): 313–328

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Sardinia et al., 1988; Sanchez-Campillo et al., 1999). The

Omp2 of C. trachomatis LGV1 is surface exposed, containing

a heparin binding peptide and has been shown to function

as an adhesin (Stephens et al., 2001; Fadel & Eley, 2007;

Fadel & Eley, 2008). The smaller CRP is known as Omp3.

This lipoprotein is probably inserted into the outer membrane

by means of a signal peptide. Its gene sequence (omp3) is less

conserved within the Chlamydiaceae as compared to omp2

(Everett & Hatch, 1991).

The polymorphic membrane proteins or pmps were first

discovered by the group of Longbottom et al. (1996) at the

surface of C. abortus S26/3. Genome sequencing revealed

chlamydial pmp gene families with variable numbers in

different chlamydial species, representing 3–5% of the

genome (Stephens et al., 1998; Read et al., 2003; Thomson

et al., 2005; Harley et al., 2007). Homology searches,

structural comparisons and amino acid sequence analysis

strongly suggest that the pmps in fact belong to the family of

autotransporters. Phylogenetic analysis indicates that they fall

into six subtypes, implying at least six different roles, most

probably also in chlamydial virulence (Henderson & Lam,

2001). Their role in the growth and development of

Chlamydiaceae is still unclear.

Overview of the developmental cycle

Chlamydiaceae have a unique biphasic replication and

survival mechanism. As obligate intracellular bacteria, multi-

plication of metabolically active RBs can only take place in

an eukaryotic host cell, while EBs are adapted to survive in

hostile extracellular environments and can infect new host

cells. An overview of the different stages in the chlamydial

life cycle is presented in Figure 1.

The acute infection starts with the attachment of EBs to a

eukaryotic host cell, followed by internalization within tight,

endocytic vesicles, termed inclusions. Bacteria preferentially

attach near microvilli on the apical cell surface of the host

cell. As the membrane regions at the bases of the microvilli

actively transport extracellular materials into the cells,

attachment there might assist in rapid and efficient entry

(Escalante-Ochoa et al., 1998). In addition, attachment of C.

psittaci EBs is sometimes observed in association with

clathrin-coated pits (Vanrompay et al., 1996). The exact

nature of attachment and entry remains elusive as several

conflicting mechanisms, possibly occurring independently

from each other, have been described in the past, elegantly

summarized as ‘‘parasite specific endocytosis’’ (Byrne &

Moulder, 1978). The newly formed inclusions efficiently

avoid fusion with cellular lysosomes and subsequent acidifi-

cation, and for C. trachomatis, but not for C. pneumoniae or

C. psittaci, fusion of different vacuoles into a larger inclusion

can be observed (Ridderhof & Barnes, 1989; Rockey et al.,

1996; Vanrompay et al., 1996; Hackstadt et al., 1999).

Beginning at 2 h post infection, EBs start differentiating into

RBs, then migrate towards the periphery of the inclusion, to

start replication from 8 h post infection on. Simultaneously,

the surface of the inclusion membrane increases through

acquisition of host plasma proteins and lipids and hijacking of

Golgi-derived vesicles with sphingomyelins (Hackstadt et al.,

1996; Scidmore et al., 1996). In addition, the inclusion

membrane is actively modified through insertion of so-called

‘‘inclusion membrane proteins’’ or Incs (Rockey et al., 2002).

Active replication through binary fission continues until late

in the developmental cycle, when RBs, detached from the

inclusion membrane, revert into EBs again, which are then

stored in the lumen of the inclusion. Depending on the

species, EBs and some non-differentiated RBs are released

from the host cell at 24–72 h post infection through lysis or

reverse endocytosis.

Chlamydiaceae can also engage in a long-term relationship

with the host cell (at least in vitro), a phenomenon known as

persistence, in which no visible growth of the chlamydial

organisms can be observed. The normal developmental cycle

can be interrupted by a number of conditions and agents, such

as antibiotics, nutrient deprivation, or immune factors,

interferon-gamma (IFN-g) in particular (Mpiga &

Ravaoarinoro, 2006). This is generally accompanied by the

development of relatively small inclusions, enlarged pleio-

trophic RBs or persistent bodies (PBs) (Hogan et al., 2004).

Persistent bodies accumulate chromosomes, but genes for cell

division are no longer expressed (Byrne et al., 2001; Mathews

et al., 2001; Gerard et al., 2001). Once the stress-inducing

factor is removed, PBs revert to normal RBs, complete

the developmental cycle and generate infectious EBs.

Whether the different described in vitro persistence models

are relevant to the in vivo described chronic infections

remains to be seen.

Attachment: about glycosaminoglycans, adhesins andhost cell receptors

Attachment of EBs to the host cell surface is thought to

consist of at least two individual steps: an initial, reversible

electrostatic interaction with heparan sulphate-like glycosa-

minoglycans (GAGs) (Zhang & Stephens, 1992; Su et al.,

1996; Davis & Wyrick, 1997), followed by an irreversible,

temperature-dependent binding of a chlamydial ligand to an

unknown host cell receptor, inducing internalization (Carabeo

& Hackstadt, 2001; Fudyk et al., 2002). There has been much

debate on whether the involved GAGs are of chlamydial or

host cell origin. However, genome sequencing indicated that

no chlamydial genes coding for GAG biosynthesis are present,

so the GAGs must be of host cell origin. Given the differential

effects of GAG on the attachment and infectivity of different

Chlamydiaceae species (Zhang & Stephens, 1992; Su et al.,

1996; Rasmussen-Lathrop et al., 2000; Fadel & Eley, 2004), it

seems likely there are both GAG-dependent and GAG-

independent mechanisms operating at different stages of

chlamydial attachment.

The specific host cell receptors and chlamydial ligands

involved in the irreversible attachment of EBs to their host

cells are largely undefined. However, possible bacterial

ligands so far described include MOMP (Su et al., 1990),

Hsp70 (Raulston et al., 1993), OmcB (Ting et al., 1995;

Moelleken & Hegemann, 2008) and pmp21 (pmpD), pmp6

and pmp20 (Wehrl et al., 2004; Crane et al., 2006; Moelleken

& Hegemann, 2008). These pmps thought to be implicated in

bacterial adhesion, although they are not classified amongst

the adhesive trimeric autotransporters (Cotter et al., 2005).

Chlamydial T3SS translocon components (CopB, CopD and

DOI: 10.3109/1040841X.2012.726210 Chlamydial biology and virulence blockers 315

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LcrV) as well could possibly serve as adhesions (Watarai

et al., 1996; Skoudy et al., 2000).

Internalization

On lipid rafts and clathrin-coated pits

Based on electron microscopic studies, two major possible

mechanisms for entry are described. The first involves

sequential zipper-like microfilament dependent phagocytosis

induced by binding of chlamydial adhesins to host cell

receptors (Byrne & Moulder, 1978), while receptor-mediated

endocytosis into clathrin-coated pits, independent of

microfilaments, has been described as a second entry

mechanism (Hodinka et al., 1988; Vanrompay et al., 1996).

Zipper-like microfilament dependent entry is clathrin-

independent and most probably occurs through cholesterol-

rich lipid raft microdomains, as has been shown for some

chlamydial species and serovars (Stuart et al., 2003). These

microdomains are also known as lipid rafts and are

characterized by a high cholesterol and glycosphingolipid

content. Moreover, these rafts are intimately connected to the

actin cytoskeleton (Lillemeier et al., 2006) and function as

signaling platforms (Simons & Toomre, 2000) to control

endocytosis (Parton & Richards, 2003), intracellular vesicle

Figure 1. Schematic overview of the developmental cycle of Chlamydiaceae. Bacteria attach preferentially at the base of microvilli and then enter thehost cell through parasite specific endocytosis. Within the thus formed inclusion, avoiding fusion with host cell lysosomes, EBs transform into RBs.RBs proliferate at the boundaries of the inclusion by binary fission, until detachment from the inclusion membrane. RBs revert back to EBs and arestored in the lumen of the inclusion until liberation through lysis or reverse endocytosis.


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trafficking (Helms & Zurzolo, 2004) and activation of

immune response and apoptosis (Gombos et al., 2006).

Their unique composition might therefore be a trigger to

release bacterial effectors in close proximity to these signal-

ing hotspots. Disruption of the rafts through extraction of

plasma membrane cholesterol inhibited internalization

(but not attachment) of C. trachomatis L2, however, as

C. trachomatis serovars A, B, C and L2 and C. muridarum

MoPn internalization occurred independently from lipid rafts,

while C. trachomatis serovar K, C. psittaci, C. pneumoniae

and C. caviae did enter the host cell through raft domains

(Stuart et al., 2003). Different methods or adaptation of the

strains used to different laboratory culture protocols could

have contributed to these conflicting results. The fact that

entry via lipid rafts remodels the actin skeleton and raft-

derived endosomes do not enter the lysosomal degradation

pathway (Helms & Zurzolo, 2004), two key elements of the

chlamydial life cycle, strongly suggests that microfilament

dependent entry occurs through these lipid rafts. The exact

mechanism remains elusive, but could be as proposed in

Figure 2.

A second proposed entry mechanism is receptor-mediated

endocytosis by clathrin-coated pits, based on observed

association of chlamydial organisms with clathrin-coated

pits, and uptake into clathrin-coated vesicles for C. tracho-

matis, C. psittaci and C. caviae strains (Reynolds & Pearce,

1990). Hybiske and Stephens (2007) found clathrin and

its coordinate accessory factors required for entry of

C. trachomatis LGV serovar L2. Other studies demonstrate

that clathrin-coated vesicles are not absolutely required for

chlamydial internalization (Dautry-Varsat et al., 2005; Balana

et al., 2005). In addition, conventional clathrin pits, normally

used for the uptake of physiologically essential and large

molecules, measure only 100 nm in diameter, and thus are too

small to accommodate chlamydial EBs (average size 250 nm).

Nevertheless, clathrin seems to be associated with chlamydial

entry in some way, its importance however strongly linked to

the inoculation route (static or centrifuge assisted) and culture

conditions (Wyrick et al., 1989; Prain & Pearce, 1989;

Reynolds & Pearce, 1990).

Actin recruitment

Within minutes upon attachment of EBs to the host cell,

chlamydiae recruit actin to the site of invasion leading to the

formation of an actin-rich pedestal underneath the attachment

site. This recruitment of actin is transient, eventually leading

to the uptake of EBs into membrane-bound vesicles, as has

already been shown for C. trachomatis (Carabeo et al., 2002),

C. pneumoniae (Coombes & Mahony, 2002), C. caviae

(Subtil et al., 2004) and C. psittaci (Beeckman et al., 2007).

A protein involved in this process has recently been identified

and was termed Tarp for Translocated Actin-Recruiting

Phosphoprotein. Tarp is translocated into the cytoplasm of

the host cell in the early stages of invasion, using a T3SS, and

is spatially and temporarily associated with the recruitment

of actin at the site of internalization (Clifton et al., 2004). The

N-terminal region of C. trachomatis Tarp contains a number

of tyrosine-rich tandem repeats (approximately 50 residues in

length) that are phosphorylated inside the host cell, initiating

a signal transduction cascade by interacting with guanosine

nucleotide exchange factors and small GTPases (Lane et al.,

2008). Orthologs of Tarp are also present in all pathogenic

Chlamydiaceae species examined to date. Moreover, recent

Figure 2. Attachment and entry of chlamydial EBs. Chlamydiaceae interact with the host cell through reversible electrostatic interaction with heparinsulphate-like GAGs on the cell surface, followed by an irreversible interaction with unidentified host cell receptors, possibly associated withcholesterol-rich lipid raft microdomains. Next, host specific signal transduction pathways mediate internalization of the bacteria bv actin recruitmentand pedestal formation, possibly after injection ot T3SS effector proteins (e.g. Tarp). Infection leads to rapid phosphorylation of host cell proteins.Image reproduced from (Dautry-Varsat et al., 2005).


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functional studies concluded the C-terminal region of Tarp to

be involved in the actin recruitment and nucleation of actin

filaments (Jewett et al., 2006). A proline-rich domain (S625-

N650 in C. trachomatis L2) induces homo-oligomerization

of Tarp and, in conjunction with the actin-binding domain

(D726-S825), the ability to nucleate actin. Both domains

are conserved among chlamydial strains sequenced so far,

suggesting that Tarp-mediated actin polymerization is not

merely the result of a stable association between Tarp and

actin but is more complex, involving multiple domains of

Tarp (Jewett et al., 2006).

Another protein involved in actin recruitment upon

attachment, at least in C. trachomatis, is the host protein

ezrin, an ezrin-radixin-moesin family protein, colocalizing

with actin at the tips and crypts of microvilli, the site of

chlamydial attachment and entry, respectively. Initial ezrin

activation through threonine phosphorylation is ubiquitous

among chlamydiae. However, subsequent tyrosine phosphor-

ylation of this protein was only observed for an infection of

cells with C. trachomatis strains. This might relate to an

undefined species-specific mechanism of pathogen entry

that involves chlamydial specific ligand(s) and host cell

co-receptor usage (Swanson et al., 2007). The fact that ezrin

is known to interact with the cytoplasmic domain of several

receptors, including CD44 and members of the integrin

superfamily, strengthens the hypothesis that T3SS translocon

components like CopB, CopD and LcrV could function as

possible chlamydial adhesins (see above).

A third mechanism represents host-receptor mediated

initiation of actin recruitment upon chlamydial attachment

to a Platelet-derived growth factor-like receptor (PDGFRb),

as inhibition of PDGFRb by RNA interference or by PDGFRbneutralizing antibodies significantly reduces bacterial binding

(Elwell et al., 2008).

In conclusion, Chlamydiaceae most probably enter their

host cells by more than one pathway, the different steps of

which may overlap to some extent and a strict equilibrium

between these different pathways may well be controlled by

small GTPases.

Inhibition of the phagolysosomal fusion

In both professional and non-professional phagocytes, phago-

somes containing a pathogen normally fuse with lysosomes,

where after the resulting phagolysosomes produce acidic

hydrolases to eradicate the pathogen. However,

Chlamydiaceae have evolved a mechanism to efficiently

thwart phagolysosomal fusion. This particular inhibition is

restricted to the chlamydial inclusion vacuoles, since in mixed

infections with C. psittaci and Saccharomyces cerevisiae or

E. coli, vacuoles not containing Chlamydiae do fuse with

lysosomes, and neither EBs or RBs can protect the co-

infecting organism from degradation (Eissenberg & Wyrick,

1981). In addition, in vitro analyses show that not all

inclusions escape phagolysosomal fusion depending on the

host cell, chlamydial strain and the mode of chlamydial entry

(Moulder, 1991). The chlamydial inclusion is not merely an

endosomal inclusion, as shown by the absence of markers of

the plasma cell membrane, or markers for either the early or

late endosome or for lysosomes (Heinzen et al., 1996;

Scidmore et al., 1996; Taraska et al., 1996; Al-Younes

et al., 1999). In accordance with the absence of vacuolar H+

ATPase, no acidification of the inclusion lumen can be

observed, although the neutral pH of the vacuole can also

result from the activity of the Na+, K+-ATPase ion pumps

(Heinzen et al., 1996; Schramm et al., 1996; Grieshaber et al.,

2002). Many theories explaining the phagolysosomal inhib-

ition have been proposed and these are elegantly summarized

in a review by Escalante-Ochoa et al. (1998). Even when

chlamydial protein synthesis is blocked through the addition

of chloramphenicol, EB containing vesicles only slowly

acquire lysosomal characteristics. Based on these results, a

two-stage mechanism for chlamydial avoidance of lysosomal

fusion has been proposed: (i) an initial phase of delayed

maturation to lysosomes due to an intrinsic property of EBs

and (ii) an active modification of the inclusion membrane

requiring chlamydial protein synthesis (Scidmore et al.,

2003), including the synthesis of chlamydial inclusion

membrane proteins. This so-called intrinsic property of EBs

could well be the translocation of already produced Type III

secretion (T3S) effector proteins to escape fusion with

lysosomes (Hackstadt et al., 1997; Wyrick, 2000).

Proliferation

Bacterial division through binary fission takes place during

the RB stage. Genome sequencing indicated that

Chlamydiae lack an identifiable ftsZ (filamentation tempera-

ture sensitive mutant Z) ortholog, which encodes a key

protein in bacterial cell division and is highly conserved

among all other sequenced eubacteria. However, RBs

synthesize small amounts of peptidoglycan that may play a

role in bacterial cell division, perhaps by substituting for the

lack of FtsZ in the formation of nascent division septa

(Chopra et al., 1998) and reviewed in McCoy and Maurelli

(2006).

Type III secretion

The role of the Type III secretion system in the chlamydial

life cycle has partly been described above but will be, for

reasons of clarity, summarized here. Firstly, T3SS translocon

components may be involved in the irreversible attachment of

EBs, assuming that CopB functions as an adhesin, binds the

hyaluronan receptor CD44 in lipid rafts, thereby inducing

complete assembly of the T3SS translocon in the eukaryotic

membrane. This could then be followed by an interaction of

the CopB-CopD-LcrV complex with a nearby a5b1 integrin

receptor on the host cell (Watarai et al., 1996; Skoudy et al.,

2000). Chlamydiaceae also employ receptor-mediated uptake

by clathrin-coated pits. Possible ligands could include surface

exposed T3SS components, e.g. the outer membrane secretin

SctC or the needle protein SctF (Beeckman & Vanrompay,

2010b).

Translocation of the T3S effector protein Tarp into the host

cell within minutes following attachment induces actin

recruitment and the formation of pedestal-like structures

beneath the attached EB. In C. trachomatis at least two

accompanying signaling cascades (Arp2/3 dependent or not)

have already been described (Jewett et al., 2006; Lane et al.,

2008), but most likely, additional currently uncharacterized


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T3SEs, are implicated in other signaling pathways mediating

EB internalization.

As the infection progresses, inclusion membrane proteins

or Incs are being inserted, presumably through T3S, into the

chlamydial inclusion membrane. At least some of them

are believed to actively prevent fusion between the inclusion

and lysosomes (Hackstadt et al., 1997; Wyrick, 2000), thus

preventing acidification of the inclusion body and ensuring

chlamydial survival. Other Incs such as CT229, Cpn0585 or

IncA are believed to divert intracellular host cell trafficking

to the nascent inclusion to intercept nutrients and constitu-

ent molecules from normal eukaryotic trafficking

pathways (Rzomp et al., 2006; Cortes et al., 2007; Delevoye

et al., 2008).

The Chlamydia protein associating with death domains

(CADD), another T3S effector protein, may help in the

reprogramming of the host cell or modulate host cell

apoptosis via binding to the death domains of tumor

necrosis factor receptor (Stenner-Liewen et al., 2002;

Schwarzenbacher et al., 2004).

While proliferating, RBs are intimately connected to the

inclusion membrane through a well-delineated patch contain-

ing T3SSs. Because of restrictions in host cell size, some RBs

and therefore also the T3SSs lose contact with the inclusion

membrane, inactivating the T3SS. This induces the asyn-

chronous re-differentiation into EBs (Wilson et al., 2006;

Peters et al., 2007).

Nucleotide acquisition

Multiple findings in literature indicate that the

Chlamydiaceae behave as ‘‘energy parasites.’’ However,

genome sequencing revealed that Chlamydiae possess genes

allowing to produce their own ATP, probably by both the

glycolytic pathway and their truncated tricarboxylic acid

cycle (Iliffe-Lee & McClarty, 1999). Still, Chlamydiaceae

posses nucleotide transport proteins (NTTs), enabling them to

perform ATP-ADP counter-exchange and import of nucleo-

tides. Similarly, the genome of C. trachomatis contains two

genes coding for nucleoside phosphate transporters 1 and 2

(Npt1Ct and Npt2Ct), each performing a different type of

transport (Tjaden et al., 1999). Npt1Ct is an ATP-ADP

exchanger, able to function in ATP acquisition from the host

cytosol. On the contrary, Npt2Ct catalyses transport of H+ and

all four ribonucleoside triphosphates into the cell, and thus

provides for the net uptake of ribonucleoside triphosphates

required for anabolic reactions. The ribonucleoside triphos-

phate/H+ transporters of other Chlamydia spp. may be of

different specificity (Tjaden et al., 1999). Both transporters

are present in C. pneumonia (Kalman et al., 1999), and

also C. psittaci exhibits an ATP/ADP exchange (Hatch

et al., 1982).

Emerging antibiotic resistance in Chlamydiaceae

In most cases, chlamydial infections can easily be resolved by

treatment with antibiotics such as tetracycline derivatives and

macrolides (especially azithromycin). However, reports

regarding treatment failure have been described already

(Johnson and Spencer, 1983; Jones et al., 1990; Lefevre &

Lepargneur, 1998; Misyurina et al., 2004; Di Francesco

et al., 2008; Andersen & Rogers, 1998) and frequently

heterotypic resistance (resistance in which only a small

portion of the population displays the resistant phenotype) is

observed. In these cases, it is not always clear whether

persistence or actual antibiotic resistance is involved. Drug

resistance frequently arises through point mutations, altering

the expression or the functionality of the antibiotic target, or

following the insertion of resistance genes into the bacterial

genome. An excellent overview on mutations described in

Chlamydiae to acquire resistance against antibiotics can be

found in Sandoz and Rockey (2010). Until recently, it was

generally believed that the acquisition of antibiotic resistance

in Chlamydia spp. through lateral gene transfer from other

organisms was limited due to their obligate intracellular life

style. However, Dugan et al. (2004, 2007) were the first to

demonstrate stable tetracycline resistance in C. suis probably

occurs through horizontal gene transfer. This was followed by

studies of DeMars and colleagues (2007, 2008) demonstrating

in vitro lateral gene transfer and homologous recombination

between single antibiotic resistant C. trachomatis strains to

obtain doubly resistant isolates. Moreover, evidence exists

that antibiotic resistance genes can also be transferred

within and among chlamydial species, such as C. suis,

C. trachomatis and C. muridarum, and into clinical isolates

from human patients infected with C. trachomatis (Suchland

et al., 2009).

The supplementation of feeds with antibiotics (especially

tetracyclines) was widespread in the poultry, porcine and live

stock industry in order to promote growth and counter

bacterial infections (Sarmah et al., 2006; Castanon, 2007;

Moulin et al., 2008; BelVet-SAC, 2012). Considering the high

prevalence of tetracycline resistance in e.g. porcine C. suis

isolates (Schautteet et al., 2012), it is not unconceivable that

this also is or will be the case in other meat producing

industries, resulting in treatment difficulties and potentially

severe economic losses due to antibiotics resistance. Perhaps

more importantly, there is a potential risk for public health as

contact between tetracycline resistant and tetracycline sensi-

tive Chlamydia spp., in different settings such as farms,

veterinary clinics and slaughterhouses, may lead to transfer of

both the resistance genes and the resulting phenotype, which

could then be propagated and selected for in patients treated

with tetracycline. This would interfere with the treatment of

chlamydial infections, resulting in more severe complications

and a higher mortality rate. In order to combat pathogenic

bacteria which are untreatable using conventional antibiotics,

new means of therapy should be developed, thereby focusing

on traits which are indispensable for pathogenic characteris-

tics, the so-called virulence factors, rather than merely killing

the bacteria.

Blocking chlamydial virulence

Virulence blockers can be defined as compounds that

specifically target virulence determinants of pathogenic

bacteria, thereby preventing the bacteria to colonize the host

and allowing the host immune system to clear the infection.

As most of these blockers do not directly kill the bacteria –

they disarm rather than destroy – it is presumed that the

evolutionary pressure for the development of resistant strains


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is smaller than with classic antibiotics. Popular targets include

biofilm formation, bacterial toxins, specialized secretion

systems, organism-specific virulence gene expression or

cell-to-cell signaling, as Rasko and Sperandio (2010) ele-

gantly reviewed. For the purpose of this review, we will focus

on possible mechanisms and compounds that may efficiently

block different stages in the chlamydial life cycle.

Inhibition of adhesion

The very first interaction between bacteria and their host cell

is the process of adhesion to the cell membrane. Therefore, in

order to effectively prevent bacterial colonization of the host,

one could already prevent attachment of the pathogen to the

host cell membrane. For most bacteria adhesins such as

fimbriae (Type1 and 4 pili) or adhesive autotransporters

(see below) have been described. Assembly of these pili by

the chaperone/usher pathway can be effectively blocked by

treatment with so-called pilicides (Aberg & Almqvist, 2007).

However, the adhesion mechanism in chlamydial species

remains rather elusive, and pili do not seem to be involved.

Research should therefore focus on already characterized

chlamydial adhesins such as MOMP and the pmp-proteins.

Such adhesins could effectively be blocked by specific

antibodies, thereby neutralizing chlamydial infectivity and

reducing colonization by blocking chlamydial attachment to

epithelial cells. In this respect, it has been shown that

monoclonal antibodies against MOMP could neutralize

chlamydial infection in vitro (Peeling et al., 1984, Peterson

et al., 1991) and could provide a modest level of protection

against infection when administered passively to mice (Cotter

et al., 1995). Similarly, antibodies specific to PmpD of

C. trachomatis and C. pneumoniae and Pmp2 and 10 of

C. pneumoniae were shown to be neutralizing, at least in vitro

(Wehrl et al., 2004; Finco et al., 2005; Crane et al., 2006).

As described above, heparin sulphate-like glycosaminogly-

cans are also involved in the chlamydial attachment process.

Monoclonal antibodies specifically directed against heparan

sulphate specifically bind glycosaminoglycans localized to

the surface of C. trachomatis and C. pneumoniae and

effectively neutralize their infectivity (Rasmussen-Lathrop

et al., 2000).

However, evidence exists that chlamydial bacteria most

likely use different mechanisms of attachment to the host cell

(see above), rendering the development of a general anti-

adhesion therapy that would completely block chlamydial

attachment unlikely.

Inhibition of internalization

As described above, chlamydiae induce actin recruitment to

the site of infection, followed by a localized and temporary

nucleation, to facilitate uptake into host cell membrane-bound

vesicles. The most straightforward way to block internaliza-

tion would therefore be to interfere with this polymerization

by treatment with molecules such as cytochalasin, latrunculin,

phalloidin, taxol or colchicines (Peterson & Mitchison, 2002).

However, as actin is also implicated in other cellular functions

such as cell shape or cell migration, the side-effects of a

similar treatment would be considerable. One would therefore

have to focus on the process of endocytosis itself to prevent

chlamydiae from invading the host cells. Research could be

directed towards toxins used by pathogenic bacteria such as

Yersinia spp. to prevent phagocytosis. Especially proteins

such as Yersinia YopH and YopE and Pseudomonas

aeruginosa (P. aeruginosa) ExoS and ExoT, interacting with

small GTPases, which are also implicated in chlamydial

internalization, could be of interest. These proteins, which are

Type III secretion substrates, convert Rho family members in

an accelerated manner to their GDP-bound, inactive states and

thus inhibit endocytotic processes (Ernst, 2000).

Inhibition of the Type III secretion system

As described above, T3S is involved in different stages of the

chlamydial life cycle and mediates translocation of virulence

related effector proteins to the host cell cytoplasm (Beeckman

& Vanrompay, 2010b). Consequently, chlamydial disease

might be effectively treated by either blocking T3S or

inhibiting the interaction with the eukaryotic host. In recent

years, several studies describing small molecules specifically

inhibiting T3S have been published (Kauppi et al., 2003;

Keyser et al., 2008). These inhibitors have been identified

through mass screening of chemical libraries using whole-cell

reporter gene assays or ELISA to assess inhibition of T3S and

included salicylideneacylhydrazides, saliylanilides, sulfonyla-

minobenzanilides, salicylideneanilides, phenoxyacetamides,

thiazolidones and N-hydroxybenzimidazoles (Keyser et al.,

2008; Aiello et al., 2010; Escaich, 2010). In the Chlamydia

research community, research has predominantly focused on

the effects of acylated hydrazones of salicylaldehyddes

whereby host cell cytokine expression as well as chlamydial

growth and T3S gene expression, but not entry, were shown to

be affected at non- or low-cytotoxic concentrations (Muschiol

et al., 2006; Wolf et al., 2006; Bailey et al., 2007; Slepenkin

et al., 2007; Prantner & Nagarajan, 2009; Muschiol et al.,

2009; Chiliveru et al., 2010). Wang et al. (2011) identified

putative target proteins of the salicylideneacylhydrazides.

Those proteins are involved in regulation of T3SS gene

expression. In addition, the phenoxyacetamide MBX 1641 is

capable of inhibiting T3S translocation in C. trachomatis

infected Hep-2 cells (Aiello et al., 2010). Although the exact

mode of action has not yet been uncovered, it is very likely

that the conserved (structural) elements of the T3SS or its’

assembly are targeted, especially given the broad spectrum of

bacteria inhibited.

Another strategy is to screen for natural products inhibiting

bacterial T3S. Such components have been described,

including glycolipids (Linington et al., 2002, 2006) and

transferrins (Ochoa et al., 2003; Gomez et al., 2003; Ochoa &

Clearly, 2004; Yekta et al., 2010), of which lactoferrin and

ovotransferrin have proven their potential to inhibit chlamyd-

ial infections in vitro as well (Beeckman et al., 2007).

Moreover, ovotransferrin was shown to efficiently prevent

C. psittaci infection in experimentally infected SPF turkeys

(Van Droogenbroeck et al., 2008) and on a commercial turkey

farm (Van Droogenbroeck et al., 2011). Alternatively, the

chlamydial T3S can also be inhibited in a pure mechanical

manner. Several studies have been published describing

in vitro and in vivo blockage of T3S using antibodies directed

against the translocon adaptor protein LcrV and its analogues


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in other bacteria (Frank et al., 2002; Goure et al., 2005;

Philipovskiy et al., 2005; Gebus et al., 2008; Eisele &

Anderson, 2009; Markham et al., 2010; Van Blarcom et al.,

2010). Whether the LcrV protein is essential in the chlamyd-

ial internalization process as well could be studied while

infecting epithelial cells and/or macrophages in the presence

of anti-LcrV antibodies. If indeed anti-LcrV antibodies could

significantly inhibit C. psittaci internalization and subsequent

replication in vitro, one could test whether active immuniza-

tion with LcrV or passive immunization with anti-LcrV

antibodies could provide protection against C. psittaci

infections in vivo as well, (Mueller et al., 2008). Recently,

a chlamydial T3S effector protein (Tarp) was identified as a

novel immunodominant antigen in human antisera and

immunization with Tarp can induce protective immunity

against chlamydial infection and pathology in mice (Wang

et al., 2009). Information on other T3S effectors in

Chlamydiaceae is scarce, put potential targets for antibody-

mediated inhibition could include the Chlamydia protein

associated with Death Domains CADD, the serine-threonine

kinase Pkn5 or the macrophage infectivity potentiator MIP

(Beeckman & Vanrompay, 2010b).

Blocking bacterial proliferation

Although bacterial proliferation is no virulence determinant

sensu strictu, processes currently not targeted by classical

antibiotics could open possibilities for the generation of novel

antibacterials. Likewise, interest increases in the FtsZ protein

as therapeutic target in the antimicrobial research field. This

protein is essential for bacterial cell division and thus

targeting FtsZ would lead to disruption of cell division and

therefore bacterial infection (Awasthi et al., 2011). However,

as mentioned earlier, Chlamydiaceae do not possess a ftsZ-

ortholog. Nevertheless, some interesting alternative mechan-

ism to inhibit bacterial proliferation exist, such as the

limitation of Fe3+ availability, which is crucial in the bacterial

metabolism and biofilm formation (Raulston, 1997;

Cianciotto, 2007).

The Chlamydiaceae proliferate predominantly in epithelial

cells and macrophages (Vanrompay et al., 1995). The latter

play an important role in the clearance of aged and apoptotic

cells and are therefore continuously exposed to high intracel-

lular iron loads. Though the mode of Fe3+ scavenging from

the environment by Chlamydia and other intracellular bacteria

such as Legionella or Mycobacterium, is undefined, the

cytosolic iron pool is most likely the source.

Accordingly, depletion of cytosolic iron could limit the

growth of intracellular bacteria. This concept is demonstrated

by the incubation of C. psittaci – or L. pneumophila – infected

mouse macrophages with iron chelators deferriprone or

desferasirox which results in a reduced level of bacterial

infections (Paradkar et al., 2008). Both compounds, deferri-

prone and desferasirox, have previously been approved for

human use. They are membrane permeable as they can

remove iron from iron loaded macrophages (Paradkar et al.,

2008). This new generation of chelators has great therapeutic

potential for treatment of persistent bacterial infections.

An alternative strategy to limit intracellular Fe3+-levels is

the use of the ‘‘Trojan horse’’ transition metal gallium (Ga3+),

an ion chemically similar to iron. Unlike Fe3+, it does not

undergo redox reactions and thus cannot execute the cellular

functions of Fe3+ within the bacterial cell (Chitambar &

Narasimhan, 1991). Through competition with Fe3+, gallium

decreases thus bacterial iron uptake. Consequently, the iron

need of the bacteria is not fulfilled and bacterial growth is

inhibited. Furthermore, gallium proved to be effective both

in vitro and in vivo in treatment of Psuedomonas aeroginosa

infections in rabbit and mouse models (Kaneko et al., 2007;

Banin et al., 2008) and is already approved by the FDA for

use in large doses to treat hypercalcemia of malignancy

(Warrell & Bockman, 1989). All together, this hints gallium

as a promising treatment strategy in bacterial infections.

Nucleotide transporters

As mentioned above, Chlamydiaceae scavenge energy mol-

ecules from the host using nucleotide transport proteins.

These proteins not exclusively constitute bacterial mem-

branes, but are similarly essential to plant chloroplasts where

they participate in the import process of cytosolic ATP under

certain conditions (Winkler & Neuhaus, 1999; Linka et al.,

2003). Interestingly, the bacterial and plant transporters do not

exhibit structural similarity with mitochondrial and peroxi-

somal adenylate transporters, belonging to the mitochondrial

carrier (MC) family (Klingenberg, 1989; Saier, 2000;

Ren et al., 2004). Hence, NTTs are absent in mammalian

and human cells and thus represent an attractive target for the

development of highly specific anti-chlamydial drugs.

However, how the highly charged ATP molecules pass the

inclusion membrane to reach the bacteria is unknown so far,

as pores for passive diffusion are absent in the inclusion

membrane (Heinzen & Hackstadt, 1997).

As earlier stated, the genome of C. trachomatis and

C. pneumoniae contains genes that might encode enzymes of

the glycolytic pathway, pentose pathway and tricarboxylic

acid cycle (Kalman et al., 1999; Stephens et al., 1998).

Accordingly, this suggests chlamydial bacteria are able to

generate their own ATP through oxidative phosphorylation

and would not be strict auxotrophic. In the case of

C. trachomatis, however, strong upregulation of the ATP/

ADP anti-porter gene occurs early in the developmental life

cycle. Namely, uptake of ATP from the host cell would be

most relevant early in the infection process when the whole

complement of enzymes for ATP generation via glycolytic

and pentose pathway is not present yet, e.g. during initial

differentiation of elementary bodies to reticulate bodies

shortly after infection of the host cell. Moreover, the substrate

for oxidative phosphorylation is now limited in the host

cytosol. Under these circumstances, an alternative way for

energy generation is an advantage. Later on, only after

inclusion niche establishment, structural proteins and proteins

of intermediary metabolism are expressed. Once cell division

starts, the energy need rises and available energy generation

increases by the glycolytic and pentose pathways (Shaw et al.,

2000). As the transport proteins are not present in human

cells, and energy parasitism in the initial phase of the

infection process is crucial for the survival of Chlamydia,

blocking this transport could be a specific and efficient

strategy in controlling chlamydial infections. To our


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knowledge, there are until now no inhibitors of chlamydial

nucleotide transport identified.

Regulation of virulence gene expression

Quorum sensing

Though the concept of quorum sensing is commonly used by

bacteria, the exact molecular mechanism may differ among

species. At least six different QS pathways are identified so

far (Table 2) (Surette et al., 1999; Schauder et al., 2001; Chen

et al., 2002; Sperandio et al., 2003; Henke & Bassler, 2004;

Kendall et al., 2007; Higgins et al., 2007). A common

signaling pathway containing the membrane-bound QseC

histidine sensor kinase (Clarke et al., 2006) or its homo-

logues, is present in more than 25 important pathogens of

humans and plants. QseC perceives the bacterial quorum

sensing signal autoinducer 3 (AI-3) and/or host derived

adrenalin and/or noradrenalin. After binding of the signal,

QseC increases its autophosphorylation. The following phos-

phorylation cascade in the bacterial cell regulates the

expression of virulence genes (Sperandio et al., 2003;

Hughes & Sperandio, 2008).

The genome of C. trachomatis comprises two genes, ctcB

and ctcC, with protein sequence similarity to histidine kinase-

response regulator pairs of two-component systems. The latter

are a type of QS pathway, which play a role in stage-specific

gene expression, this could be e.g. in- and outside the host

cell, two completely different environments in the

Chlamydiaceae biphasic life cycle. Generally, the histidine

sensor kinase component in the bacterial membrane autopho-

sphorylates upon signal perception and subsequently phos-

phorylates and activates a response regulator, usually a

transcription factor, which binds to the promoter of a target

gene and initiates transcription upon activation. The sensor

kinase and response regulator pair form a genetic network

together with a range of downstream molecular factors. This

network controls a specific subset of genes, including

virulence genes (Novick, 2003; Lyon & Novick, 2004).

Two-component systems are a primary mechanism to

adapt to environmental conditions. Though little is known

about how transcriptional regulation in Chlamydiae, gene

expression and development are most likely controlled

through recognition of environmental cues or intracellular

conditions. Although many bacteria possess several systems

to adapt to diverse environmental changes, this is the only

complete two-component system identified in C. trachomatis.

Moreover, this ctcB-ctcC system proved to be functional as

it is capable of autophosphorylation and phosphotransfer

reactions (Koo & Stephens, 2003).

The CtcB and CtcC genes posses a late expression profile

and, accordingly, the corresponding proteins are present in

EBs, but not in RBs (Koo & Stephens, 2003). Most two-

component system components, however, are constitutively

expressed to adapt efficiently to a changing environment. The

late expression profile thus implies involvement in the control

of a subset of late genes participating in RB to EB transition.

Moreover, the sensor kinase CtcB possesses a redox sensing

domain (Koo & Stephens, 2003). This could sense the change

in redox state when EBs enter the host cells and disulfide-

linkage in the outer membrane proteins are reduced, which

results in a higher membrane flexibility and increased nutrient

uptake. Similarly, a decrease in energy sources or reducing

agents results in oxidation of sulfhydrylgroups, hindering RB

development and decelerating metabolic activity (Bavoil

et al., 1984; Hackstadt et al., 1985; Ward, 1988). To conclude,

CtcB and CtcC are developmentally late-expressed proteins

with redox sensing domain. This domain is most likely

involved in late gene activation, including the regulation of

RBs to EBs differentiation.

Quorum sensing inhibitors

Blocking QS is more and more considered as a viable

approach for developing therapeutics in the treatment of

bacterial infections.

The ideal QS inhibitor (QSI) is a chemically stable, low-

molecular mass molecule without toxic side-effect on the

bacterium or host, and chemically stable and resistant to

metabolization and disposal by the host. It should be specific

for the particular regulon and have a significant and similar

reduction in expression on all the QS regulon comprised

genes, however this is not always the case. The strength of an

inhibitor depends on the percentage of QS-controlled genes it

targets (Arevalo-Ferro et al., 2003; Hentzer et al., 2003;

Rasmussen et al., 2005b). QSIs fall roughly into three

categories according to the level of interruption of the

signalization: repressors of signal generation, disruptors of the

signals or signal molecules and inhibitors of the signal

perception. Alternatively, inhibitors are categorized into four

different classes: nonpeptide small molecules, peptides,

enzymes and antibodies (Pan & Ren, 2009).

To our knowledge, no chlamydial QSI compounds are

known yet. As there is evidence that Chlamydiaceae can sense

the redox-state of their environment, blockage or destruction

of receptor proteins could be an interesting strategy for

therapeutic purposes in this context. One method for receptor

blockage is the use of an analogue of the signal molecule.

More knowledge about the signal perception is needed to

Table 2. Quorum sensing pathways in bacteria.

Pathway Signal molecules Bacteria References

AHL (AI-1 pathway) AHLs Gram-negative Salmond et al., 1995; Ravn et al., 2001; Zavil’gel’skii andManukhov, 2001

4Qs pathway PQS and HHL Gram-negative Diggle et al., 2006AI-3 pathway AI-3 Gram-negative Sperandio et al., 2003; Kendall et al., 2007AI-2 pathway Two different forms Gram-negative and Gram-positives Surette et al., 1999; Schauder et al., 2001; Chen et al., 2002AIP pathway Oligopeptides Gram-positive McDowell et al., 2001CAI-1 Hydroyketones Gram-negative Henke and Bassler, 2004; Higgins et al., 2007


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explore this possibility. Generally, a synthetic library of signal

molecule derivatives is used to screen for inhibitors. However,

random compound libraries with natural and synthetic

compounds can evenly be used (Smith et al., 2003a, 2003b;

Suga & Smith; 2003). In both cases, a screening system and

further validation is necessary to be able to identify potential

inhibitors.

A valuable source for QSI compounds are other bacteria,

fungi and plants. These organisms have co-existed for

millions of years and some of them probably produce QSI

compounds, such as Penicillium species for example

(Rasmussen et al., 2005b). Examples of plants producing

QS inhibitors are garlic, carrot, soybean, tomato and many

more (Rasmussen et al., 2005a).

Beside the species specific inhibitors discussed above,

also broad spectrum inhibitors are already described in

literature. Most QS signals only appear in a small number

of species. However, certain signaling pathways are

common for a range of species, while they are not found

in the eukaryotic hosts. A high throughput screen of a

library of 150.000 small organic compounds identified the

lead structure LED209 (N-phenyl-4-[[(phenylamino) thiox-

omethyl]amino]-benzenesulphonamide). This non-toxic

compound has no effect on pathogen growth but blocks

binding of signaling molecules to QseC, thus preventing the

autophosphorylation of QseC and consequent activation of

virulence genes. LED209 was tested for inhibitory effect,

and showed both in vitro and in vivo a virulence decrease in

models of infection for several pathogens. Furthermore,

molecular concentrations showed a 10-fold reduction

compared to previously characterized virulence inhibitory

compounds (Rasko et al., 2008). LED209 can be considered

as the proof of concept that blocking inter-kingdom

chemical signaling is a viable strategy to develop novel

drugs to control bacterial infection. Unlike the LED209

compound mentioned above, most inhibitors show efficacy

in vitro, but have not been tested in vivo in animal models

yet. This partially explains why no QSI is at clinical stage

of drug development. Moreover, because most of these QSIs

are not in the clinical phase, no information on their

efficacy or toxicity in humans is available. Therefore, more

research in the field of quorum sensing and in vivo testing

is required in order to explore the potential, advantages and

limitations of QSIs as therapeutics in the control of

bacterial infections.

Conclusion

Antibiotic resistance has been reported in Chlamydia.

Virulence blockers could fulfill a role in future prevention

and/or treatment of Chlamydia infections, as they do not

directly inhibit the growth of pathogens, but rather target

virulence associated processes. Therefore, they are considered

to exert a lower selective pressure to develop resistance

compared to classic antibiotics. So far, only ovotransferrin,

the avian homologue of mammalian lactoferrin, has been

tested in an animal (turkeys) model and in veterinary clinical

trials. Ovotransferrin efficiently prevented C. psittaci respira-

tory disease in commercially raised broiler turkeys demon-

strating its potential for veterinary use.

To our knowledge, anti-virulence strategies for human

chlamydial infections have not been implemented in animal

models or human clinical trials. Nevertheless, promising

virulence blockers such as deferriprone and desferasirox are

already approved for human use.

Further in vivo testing of innovative candidate virulence

blockers as well as in vivo testing in animal models and

clinical trials is needed. Not only to assess the potential of

Chlamydia virulence blockers but also to study possible

limitations and safety.

Declaration of interest

The Research Foundation Flanders (FWO-Vlaanderen) is

acknowledged for providing a grant to Delphine S.A.

Beeckman. This study was funded by the Federal Public

Service of Health, Food Chain Safety and Environment

(convention RF-10/6234).

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Chlamydial biology and its associated virulence blockers

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