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Qazvin university of
Medical Science, Fall 1392
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Listeria monocytogenes is a Gram-positive pathogenic bacterium that has adapted to various environments, from soils and food products to the intestinal tract and intracellular compartments of diverse animal species and humans.
The aerobic
Non–spore-forming
Catalse Positive
The organisms are motile at room temperature (25 ̊C) but less so at 37 °C
L. monocytogenes exhibits weak β-hemolysis when grown on sheep blood agar plates.
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L. monocytogenes is a facultative intracellular pathogen
that can live both inside and outside its host.
To infect its mammalian host and to cause the most severe
pathologies, L. monocytogenes is able to cross :
The intestinal
Blood-brain and
Maternofetal barriers
Crossing the host barriers involves bacterial invasion and
survival within a large variety of normally nonphagocytic
cells
It is therefore crucial to understand how L. monocytogenes
induces its own uptake by host cells.
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Listeriosis manifests itself through flu-like symptoms
and can lead to
diarrhea,
meningitis,
encephalitis,
meningoencephalitis and
Stillbirths
In humans, it primarily infects immunocompromised
individuals like
pregnant women,
neonates, and
the elderly
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Listeriosis is a severe foodborne disease characterized by bacteremia and meningoencephalitis in individuals with impaired cell-mediated immunity, Including :
Neonates,
Pregnant woman,
Elderly persons, and
Immunosuppressed patients.
The potential of L. monocytogenes to cause disease correlates with its capacity to survive within macrophages, to invade nonphagocytic cells and replicate therein and also to cross the intestinal, the blood–brain, and the fetoplacental barriers.
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The incidence of listeriosis is rather low, compared to
other common foodborne pathogens such as
Campylobacter species, Salmonella species, Shigella
species, andVibrio species.
However
The outcome is much more severe and often fatal.
In fact, it represents one of the most deadly bacterial
infections due to its high mean mortality rate of 20%–
30%, despite early antibiotic treatment.
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Internalins Proteins (ex : InL A, InL B ,....)
Listeriolysin O (LLO)
Phospholipases
Act A
The Virulence Genes (prfA, PlcA, hly, mpl,...)
PI-PLC
PC-PLC
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There exist two principle mechanisms by which L.
monocytogenes can enter into the host through the
intestinal mucosa.
The first route is direct invasion of the enterocytes
lining the absorptive epithelium of the
microvilli, leading to infection of the intestinal cells
This entry mechanism occurs only in humans and some
susceptible animals.
The second entry pathway is translocation across the
M-cells of Peyer’s patches.
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Internalin (InlA), and InlB were the first surface proteins of L.monocytogenes identified to promote host cell invasion.
They have an amino-terminal leucine-rich repeat domain(LRR) formed by tandem repeats of 20-22 amino acids.
The LRR regions are structurally and functionally important to theninternalization of LM.
LRRs provide versatile recognition units for protein-protein interactions and protein activation in a variety of prokaryotic and eukaryotic proteins.
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L. monocytogenes genome encodes 22 additional proteins
containing LRRs that form the internalin family.
The amino-terminal LRRs are followed by a conserved
inter-repeat domain (IR)
The carboxy-terminal LPXTG motif, which mediates
covalent binding to the cell wall peptidoglycan, is present
in 19 members including the internalin prototype, InlA
One member, InlB, is bound to the lipoteichoic acids on
the bacterial cell wall by electrostatic interactions
involving carboxy-terminal glycine/tryptophan-rich
(GW) modules.
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InlA, an 800-amino-acid protein, is responsible for
bacterial entry into epithelial cells.
InlA specifically interacts with E-cadherin.
E-cadherin: a member of the cadherin superfamily of
calcium-dependent cell adhesion molecules.
E-cadherin is involved in the formation of adherens
junctions in polarized epithelial cells of different tissues.
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The carboxyl terminal of E-cadherin directly interacts
with the intracellular β-catenin.
α-catenin, in turn, binds to β-catenin and interacts with
actin This interaction leads to the formation of a fusion
molecule consisting of the ectodomains of the E-
cadherin and the actin binding site of the α-catenin
which eventually leads to LM entry.
Furthermore, myosin VIIA and its ligand vezatin
together function as the molecular motor in the
internalization of Listeria.
When myosin VIIA binds vezatin, coupled with an actin
polymerization process, it provides the tension
necessary for bacterial internalization.
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InlB, a 630-amino-acid protein, promotes
L. monocytogenes entry into a large variety of mammalian cells including :
Epithelial cells
Endothelial cells
Hepatocytes
Fibroblasts
The hepatocyte growth factor receptor (Met/HGF-R) has
been identified as the major ligand for InlB responsible for
L. monocytogenes entry into non-phagocytic cells, and for
cell scattering and membrane ruffling induced by soluble
InlB
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Met belongs to the family of receptor tyrosine kinases
(RTKs), one of the largest and most important families of
transmembrane signaling receptors expressed by a large
variety of cells.
Met plays crucial roles in :
Organ morphogenesis,
Cell proliferation,
Cell migration and differentiation,
And also in cell growth and invasion during metastasis
in cancer cells
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InlB functionally mimics HGF, the natural Met
ligand, through the binding of its LRRs.
As in the case of the InlA/E-cadherin
interaction, InlB interaction with Met is species
specific: InlB interacts with human and mouse
Met, but does not recognize the guinea-pig or rabbit
receptor.
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Listeriolysin O (LLO), the major and first identified
virulence factor of L. monocytogenes is a member of the
cholesterol-dependent cytolysin (CDC) family of toxins.
These toxins are produced by numerous Gram-positive
bacterial pathogens including :
Streptococcus pneumonia
Bacillus anthracis.
Among these pathogens only L. monocytogenes is known
to infect nonphagocytic cells and LLO, secreted by L.
monocytogenes, is required for bacterial escape from
endocytic vacuoles.
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LLO binds to the host plasma membrane as a monomer
and then forms oligomers composed of up to 50 subunits
that inserts into the plasma membrane forming pores of
about 200e 300 A° diameter.
hly, one of the many genes activated during
infection, leads to the production of Listeriolysin O
(LLO).
Unlike the other CDCs, pore formation by LLO is more
efficient at low pH facilitating the disruption of endocytic
membranes following acidification of the vacuoles.
This is a crucial function for cellular invasion of both
phagocytic and nonphagocytic cells.
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LM also uses phospholipases C to aid in the escape from the vacuole.
Two specific phospholipases (PLCS) are used.
One is the phosphatidylinostiol-specific PLC (PI-PLC), and
The other is more general, phosphatidylinostiol-specific (PC-PLC).
The role of the PI-PLC secreted by LM is to catalyze the production of inositol phosphate and diacylglycerol (DAG) through cleavage of the membrane lipid PI.
DAG then has the ability to activate protein kinase C (PKC).
There are four types of PKCs, but the PKC of the host is shown to be linked with the PI-PLC signaling cascade.
The PKC has been shown to facilitate the permeation of the phagosomal membrane before the bacteria escape.
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In summary, the secretion of PLCs during listerial
infection has several effects on the host cell.
One of these effects has been shown to :
1) Increase the permeation of phagosomal membrane.
2) The activation of PKC through PI-PLC facilitates the
escape of the bacterium.
3) The decreased affinity of L. monocytogenes for the
glycan linker of the GPI-anchored protein due to the
lack or absence of the Vb-strand also increases the
ability for the bacterium to escape during infection.
4) PC-PLC leads to the activation of NF-kB, which allows
the bacteria to exploit the host cell machinery.
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The bacterial surface protein ActA is a major virulence
factor of L. monocytogenes that enables bacterial
propulsion in the cytosol leading to the invasion of yet
uninfected neighboring cells by a process called cell-to-
cell spreading.
Host cell interaction could be mediated by the amino-
terminal region of ActA that has several clusters of
positively charged amino-acids that could bind heparan
sulfate proteoglycans.
The precise mechanism involved in ActA -mediated
invasion remains to be elucidated.
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After infection within the gastrointestinal tract
immediate immune responses are essential for the
control of pathogens, such as L. monocytogenes.
Activation of the innate immune system is triggered
when pathogen-associated molecular patterns (PAMPs)
engage pattern recognition receptors (PRRs) on
intestinal epithelial cells (IECs).
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Typical PAMPs include bacterial carbohydrates, such as
Lipopolysaccharide (LPS)
Mannose
Nucleic acids (both DNA and RNA)
Peptidoglycan components
Lipoteichoic acids, and probably many other
molecules, and are able to trigger the innate immune
response.
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Innate immunity to L. monocytogenes is primarily
mediated by two types of pattern recognition receptors:
The Toll-like receptors (TLRs)
The nucleotide-binding oligomerization domain
(NOD)-like receptors (NLRs).
In addition, there is some experimental evidence for
the involvement of scavenger receptors and a TLR-9
independent cytosolic sensor system for bacterial DNA
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Upon recognition of the presence of microbes
through sensing pathogen-associated molecular
patterns.
TLRs can bind any of the 4 known activating
adaptors:
Myeloid differentiating factor-88 (MyD88)
MyD88 adapter-like (Mal)
TIR domain-containing adapterinducing IFN-ɣ(TRIF)
TRIF-related adapter molecule (TRAM)
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MyD88 appears to be the key adaptor
molecule, because it is required for signaling by all
TLRs with only one exception: TLR3 uses TRIF.
The binding of the activating adaptors results in the
subsequent recruitment of IL-1R, associated kinases
(IRAKs) and downstream activation of transcription
factors including NF-κB and IFN regulatory factor 3
(IRF3), which in turn induces the proinflammatory
cytokines and type I IFNs.
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Toll-like receptor 2 (TLR2) can interact with several
specific ligands, including :
Bacterial lipoproteins
Lipoteichoic
Acids of Gram-positive bacteria such as L.
monocytogenes
Yeast zymosan
TLR2 can form heterodimers with TLR1 and
TLR6, thereby improving the recognition of the target
lipoteichoic acids.
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TLR2 is expressed on the cell surface of intestinal
epithelial cells and its activation by commensal
bacteria is thought to play an important role in the
maintenance of the integrity of the intestinal epithelial
barrier.
TLR2 is also expressed within
phagolysosomes, thus, L. monocytogenes cells that
have escaped into the host cell cytoplasm were not
detected by TLR2.
The importance of TLR2 signaling for early protection
against L. monocytogenes is, however, inconclusive.
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Toll-like receptor 5 (TLR5) can bind to a protein motif
common to the flagellin protein making up the flagella
from many bacteria, such as L. monocytogenes.
TLR5 activation induces NF-κB and stimulates TNF
production, suggesting that TLR5 may serve as a
general alarm system, when the gastrointestinal barrier
is compromised by a broad spectrum of motile bacteria.
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Toll-like receptor 9 (TLR9) recognizes the CpG motifs
present in bacterial DNA. In immune cells, TLR9 is
exclusively localized in the endosomes.
In the intestine, TLR9 was shown to be located on
both, the apical and the basolateral surface of IECs.
Upon activation of TLR9, IκBα is degraded and NF-κB
is activated, again resulting in a proinflammatory
response.
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In conclusion, the available experimental evidence
suggests that TLR2 is the most relevant TLR for
recognition of L. monocytogenes cells However, as
IECs show.
TLR2 commensal ligand-induced activation, TLR2 is
also considered to play an important role in maintaining
the integrity of the intestinal epithelial barrier.
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The NLRs are critical for mucosal innate immunity as
sensors of microbial components and cell injury in the
cytoplasm.
Both NOD1 and NOD2 are important for the innate
immune response against L. monocytogenes, because
they represent intracellular sensors of bacterial
peptidoglycan components that are thought to enter
cells by endocytosis through clathrin-coated pits.
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NOD1 is ubiquitously expressed in adult human
Tissues.
NOD2 is expressed only in leukocytes, DCs, and
epithelial cells.
Activation of NOD1 and NOD2 results in
the translocation of NF-κB and mitogen-activated protein
kinase into the nucleus, to up-regulate the transcription of
proinflammatory genes and mediate antibacterial effects
by the up-regulation of another group of small
antibacterial peptides, the defensins.
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NOD1 recognizes a diaminopimelic acid-containing
dipeptide or tripeptide molecule generated by lysozyme
action on the peptidoglycan of many Gram-negative and
Gram-positive bacteria, including L. monocytogenes.
NOD2 is activated by muramyl dipeptide
(MDP), which is another degradation product of the
peptidoglycan produced by lysozyme and other
(bacterial) peptidoglycan hydrolases.
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DCs respond to different pathogens and initiate the appropriate type of T cell response needed to control the infection.
In response to L. monocytogenes infection, DCs are critical in priming the T cell response, since mice depleted of DCs are unable to generate a CD8 T cell response.
Due to the primarily intracellular localization of L. monocytogenes, CD4 and CD8 T cells mediate most of the adaptive immune response, and are crucial for long-termimmunity after initial L. monocytogenes infection.
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Almost any cell type that harbors L. monocytogenes in the cytoplasm can process the proteins secreted from the pathogen, by degradation and subsequent loading on MHC class I molecules, in order to present them on the cell surface to CD8 T cells.
Only professional antigen presenting cells (APCs) can present antigens derived from lysosomal degradation via the MHC class II pathway to CD4 T cells.
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The CD8 T cells mediate the anti-Listeria immunity by
two synergistic mechanisms:
first:
by secretion of IFN-γ to activate macrophages;
Secondly:
by lysis of infected cells via perforin and
granzymes, leading to the exposure of intracellular
bacteria to the activated macrophages
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IFN-γ is known to be essential for host resistance to
intracellular pathogens such as L. monocytogenes, as it
mediates the activation of resting macrophages that
more efficiently restricts the multiplication of
intracellular pathogens and promotes long-term
protective cellular immunity.
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L. monocytogenes induces a strong T-helper type 1
response and, similar to CD8 T cells, CD4 T cells
also secrete IFN-γ.
The strong CD8 and CD4 T cell responses results in a
stable population of memory T cells specific for L.
monocytogenes
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In the intestine, NKT cells (lymphocytes expressing
both NK and T cell markers) play an important role in
the control of early infection with L. monocytogenes.
In general, adaptive immune responses in the intestine
are characterized by high numbers of IgA producing
plasma cells, regulatory T cells, and IL-17 producing T
cells whose development is closely linked to factors
produced by PRRs expressing IECs, DCs, and
macrophages.
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