Background information
• Antimicrobial peptides
• Intestinal epithelial cells regulate immune-cell function
• Virulence plasmid is essential forShigella pathogenesis
• Pathogenesis of Shigella
Antimicrobial peptides (AMPs)
• also called host defense peptides
• small peptides with broad spectrum antimicrobial activity against bacteria,
fungi, viruses
• ubiquitously expressed by epithelial cells throughout the GI tract
• usually positively charged (cationic peptides)
• play an important role in controlling the resident and transient bacterial
populations
E.g. of AMPs
Defensins
Cathelicidin
Histatins
Cathepsin G
Azurocidin
Latoferrin
Antimicrobial peptides (AMPs)
•
Human defensins
α- defensins (neutrophil granules)
β- defensins (HBD1, HBD2, HBD3, HBD4)
SP – Signal peptide
Intestinal epithelial cells regulate immune-cell function
• physical barrier
• immune barrier
Glycocalyx - polysaccharides that projects from cell surface
Defensin – a host defense peptide with antimicrobial activity
Virulence plasmid is essential forShigella pathogenesis
• Invasion plasmid antigens (Ipa )
• Membrane expression of Ipas (Mxi)
TTSS
• Surface presentation of invasion
plasmid antigens (Spa)
• Intracellular spread (IcsA or VirG)
• Type III Secretory System - TTSS
(encoded by Mxi and Spa)
Shigella
Pathogenesis of Shigella
Synopsis
In vitro studies –
• Human intestinal cell lines were infected with Shigella flexneri
• Observed suppression of transcription of genes mainly coding for
antimicrobial peptides, like β-defensin (e.g., hBD-3), in these cell lines
• MxiE (bacterial regulator) is responsible for such regulatory process
In vivo studies –
• Human intestinal xenotransplants were used as model, infected with
S.flexneri
• Confirmed = MxiE dependent system that allows Shigella to suppress
expression of antimicrobial peptides
• This helps Shigella to progress deeper into intestinal crypts, thereby causing
the disease
• Down-regulation of additional innate immunity genes (e.g., CCL20) leading to
compromised recruitment of DCs at infected area
Targeted survival strategy used by Shigella to survive in the
host by weakening the host immune system and thus surviving in
the intestine
Experimental Details
 Different strains of Shigella
• invasive wild-type strain M90T (virulent factors and TTSS system loaded)
• invasive mxiE mutant (impaired for the MxiE transcriptional activator regulating
expression of several virulence plasmid-encoded effectors, e.g., Osp and IpaH, but TTSS
functioning)
• non-invasive mxiD mutant (impaired for the MxiD protein, a component of the TTSS
required for its functionality )
• non-invasive plasmid-cured BS176 strain
 Antimicrobial factors gene expression
• ß defencins – HBD1, HBD2, HBD3
• cathelicidin – LL37
• CCL20 and its receptor CCR6
 TC7 and HT29 colonic epithelial cell lines, Human intersinal xenografts in SCID mice
Results – Figure 1 (A and B)
Virulent S. flexneri modulates expression of specific innate immune genes in vitro
M90T
BS176
mxiD
mxiE
Results – Figure 1 (C and D)
Virulent S. flexneri modulates expression of specific innate immune genes in vitro
S. flexneri is unable to significantly invade polarized and differentiated
TC7and HT29 epithelial cells
Gentamicin assay
Lactate de-hydrogenase assay
TC7
HT29
M90T was unable to significantly
invade cells
MOI = multiplicity of infection (100)
Cellular viability is same
Conclusion – Figure 1 (A to D)
• wt S. flexneri impairs the expression of specific innate immunity genes by
injecting virulent factors into epithelial cells through its TTSS, thereby
affecting the host immune system
• MxiE dependent effectors are responsible for the observed manipulation of
the host immune response through transcriptional damping
S. Flexneri >>>>> host innate immune parameter (e.g. defensins) >>>>> role of
bacteria’s MxiE and TTSS
Results – Figure 2
Virulent S. flexneri modulates expression of specific innate immune genes in vitro
TC7 cells stimulated by IL-1ß
Uninfected
M90T
BS176
mxiD
mxiE
Conclusion - S. flexneri manipulates the host innate immune response
through injection of the Mxi-E dependent effectors even in the cells
already expressing an inflammatory pattern
Results – Figure 3 (A)
Antimicrobial factors whose transcription is repressed by S. flexneri are those affecting
the highest bactericidal activity against the pathogen
Experimental read out = DiBAC fluorescent molecule binds to depolarized
membranes during bactericidal activity
TC7
M90T
BS176
mxiD
mxiE
DiBAC = bis-(1,3-dibutylbarbituric acid)-tetramethylene oxonol fluorescent molecule
Results – Figure 3 (B)
Antimicrobial factors whose transcription is repressed by S. flexneri are those affecting
the highest bactericidal activity against the pathogen
Experimental read out = DiBAC fluorescent molecule binds to depolarized
membranes during bactericidal activity
Listeria monocytogenes and L. innocua as control for experimental conditions
TC7
L. Monocytogeneses
L. innocua
Conclusions – Figure 3 (A and B)
Among the antimicrobial molecules tested in vitro, hBD-3 is the most active
antimicrobial factor for S. flexneri
From previous results - S. flexneri represses the transcription expression of
HBD-3 gene
Results – Figure 4 (A)
S. flexneri regulates expression of innate immunity genes in vivo
• Used human intestinal xenografts in SCID mice – chimerical structure,
combining essentially human intestinal epithelium and mouse vascular and immune
cells
• Essential tool for studying gene expression in human epithelial cells
• Focused on genes encoding chemokines, cytokines, and antimicrobial peptides
Results – Figure 4 (A)
Total 46 genes whose expression was
significantly modulated by S. flexneri
Of which 11 genes were less
transcribed upon infection with M90T
compared to mxiE or other strains
CCL3, CCL4, CCL20, CCL25, CCR12 –
Mainly produced by EC, are involved in
recruitment
and
activation
of
inflammatory and immune effector
cells, like – monocytes, DCs, T cells
IL-7, IL-18, IL-13RA1, IL-20RA
-Lymphocytes, IFN-γ
SOCS 3 (suppressor of cytokine
signaling) – upregulated in M90T
>>>>> involvement of Jak-Stat pathway
in suppressing HBD1 and HBD3
Results – Figure 4 (B)
S. flexneri regulates expression of innate immunity genes in vivo
Xenografts
M90T
BS176
mxiE
Conclusions – Figure 4 (A and B)
In vivo results show that S. flexneri
is able to manipulate the host innate
response through MxiE-dependent effectors by up- or down-regulating
expression of crucial innate immunity genes
Results – Figure 5 (A-F)
S. flexneri blocks antimicrobial factors expression in vivo
Immuno-histochemistry on infected human intestinal xenografts using antisera
to hBD1, hBD3, and CCL20
hBD1
hBD3
CCL20
M90T
Weak labeling
Weak production
mxiE
Massive luminal release
Strong production
Results – Figure S1 (A-F) – supplementary material
S. flexneri blocks antimicrobial factors expression in vivo
hBD1
hBD3
CCL20
uninfected
mxiD
Massive luminal release
Strong production
Conclusions – Figure 5 (A-F)
hBD1
M90T
mxiE
hBD3
hBD1
CCL20
hBD3
CCL20
uninfected
mxiD
Mxi-E dependent S flexneri effectors affect the production of hBD-1, hBD-3
and CCL20 molecules exhibiting antimicrobial activities, thereby weakening the
antimicrobial defense barrier at infected mucosal surfaces
Results – Figure 6 (A-C)
Blocking of antimicrobial factor expression correlates with deeper progression of
S. flexneri towards intestinal crypts
Bacterial counts in infected human intestinal xenografts using polyclonal
antibody to S. flexneri 5a LPS - Qualitative analysis
M90T
Diffusely distributed in the
mucus layer from the top of
the villi to the crypts
mxiE
mxiD
Localized to the top of villi,
trapped in luminal mucus
Results – Figure 6 (D)
Blocking of antimicrobial factor expression correlates with deeper progression of
S. flexneri towards intestinal crypts
Bacterial counts in infected human intestinal xenografts using polyclonal
antibody to S. flexneri 5a LPS - Quantitative analysis
M90T
mxiE
mxiD
Conclusions – Figure 6 (A-D)
Correlates the ability of S. flexneri to block antimicrobial factors expression,
and there capacity to progress deeply in intestinal crypts, at the early time
point of infection
Results – Figure 7 (A-D)
S. flexneri compromises recruitment of DCs to the lamina propria of infected tissues
Immuno-histochemistry of infected intestinal xenografts by monoclonal
biotinylated antibody to mouse CD11c
M90T
Restricted presence
of DCs in submucosa
And lamina propria
mxiE
mxiD
Massive recruitment of DCs into the
lamina propria and submucosal region
uninfected
Conclusions – Figure 7 (A-D)
Existence of a dedicated MxiE-dependent system allowing S. flexneri to
suppress expression of immune effectors, leading to compromised recruitment
of DCs to lamina propria of infected tissues.
Synopsis
In vitro studies –
• Human intestinal cell lines were infected with Shigella flexneri
• Observed suppression of transcription of genes mainly coding for
antimicrobial peptides, like β-defensin (e.g., hBD-3), in these cell lines
• MxiE (bacterial regulator) is responsible for such regulatory process
In vivo studies –
• Human intestinal xenotransplants were used as model, infected with
S.flexneri
• Confirmed = MxiE dependent system that allows Shigella to suppress
expression of antimicrobial peptides
• This helps Shigella to progress deeper into intestinal crypts, thereby causing
the disease
• Down-regulation of additional innate immunity genes (e.g., CCL20) leading to
compromised recruitment of DCs at infected area
Thanks for your attention !!!
Shigella
• gram negative bacteria belongs to family Enterobacteriaceae
• non-motile, rod shaped
• causes disease called Shigellosis – bacillary dycentry (bloody diarrhea)
• 4 species of Shigella – S. boydii, S. dycenteriae, S. flexneri, S. sonnei
• can be treated by antibiotics
• transmission via fecal-oral route
• a very small inoculum (only 10-200 organisms) is sufficient to cause infection
Infectious pathway for shigellosis
Activities of Shigella type III secretion system
(T3SS) effectors
Klotman et al. Nature Reviews Immunology 6, 447–456 (June 2006) | doi:10.1038/nri1860
Klotman et al. Nature Reviews Immunology 6, 447–456 (June 2006) | doi:10.1038/nri1860
Klotman et al. Nature Reviews Immunology 6, 447–456 (June 2006) | doi:10.1038/nri1860
A simplified model of membrane-ruffle production in response to the
stimulation of host cellular signalling by Shigella effectors.
Shigella movement within the host-cell cytoplasm requires actin
polymerization and microtubule degradation.
Shigella and the downregulation of the host
inflammatory response.