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 AMPsDefensinsCathelicidinHistatinsCathepsin GAzurocidinLatoferrin

Antimicrobial peptides (AMPs)

•

SP – Signal peptide

Human defensins α- defensins (neutrophil granules)β- defensins (HBD1, HBD2, HBD3, HBD4)

Intestinal epithelial cells regulate immune-cell function

• physical barrier• immune barrier

Glycocalyx - polysaccharides that projects from cell surfaceDefensin – a host defense peptide with antimicrobial activity

Virulence plasmid is essential forShigella pathogenesis

I have well equipped machinery

Shigella

TTSS

• Invasion plasmid antigens (Ipa )

• Membrane expression of Ipas (Mxi)

• Surface presentation of invasion plasmid antigens (Spa)

• Intracellular spread (IcsA or VirG)

• Type III Secretory System - TTSS (encoded by Mxi and Spa)

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

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

Experimental Details

Results – Figure 1 (A and B)Virulent S. flexneri modulates expression of specific innate immune genes in vitro

M90TBS176mxiDmxiE

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

MOI = multiplicity of infection (100)

TC7HT29

Gentamicin assay Lactate de-hydrogenase assay

Cellular viability is same M90T was unable to significantly invade cells

• 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

Conclusion – Figure 1 (A to D)

TC7 cells stimulated by IL-1ß

UninfectedM90TBS176mxiDmxiE

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 2Virulent S. flexneri modulates expression of specific innate immune genes in vitro

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

M90TBS176mxiDmxiE

TC7

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

L. MonocytogenesesL. innocua

TC7

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

M90TBS176mxiE

Xenografts

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

mxiE

Weak labeling

Massive luminal release

Strong production

Weak 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)

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

hBD1 hBD3 CCL20

M90T

mxiE

uninfected

mxiD

hBD1 hBD3 CCL20

Results – Figure 6 (A-C)Blocking of antimicrobial factor expression correlates with deeper progression ofS. flexneri towards intestinal crypts

Bacterial counts in infected human intestinal xenografts using polyclonal antibody to S. flexneri 5a LPS - Qualitative analysis

M90T mxiE mxiD

Diffusely distributed in the mucus layer from the top of the villi to the crypts

Localized to the top of villi, trapped in luminal mucus

Results – Figure 6 (D)Blocking of antimicrobial factor expression correlates with deeper progression ofS. flexneri towards intestinal crypts

Bacterial counts in infected human intestinal xenografts using polyclonal antibody to S. flexneri 5a LPS - Quantitative analysis

M90TmxiEmxiD

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 mxiE mxiD uninfected

Massive recruitment of DCs into the lamina propria and submucosal region

Restricted presence of DCs in submucosa And lamina propria

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

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Infectious pathway for shigellosis

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

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.