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Shigella spp. are Gram-negative enteric pathogens and the leading cause of bacterial dysentery worldwide. Since the discovery more than three decades ago that the large virulence plasmid of Shigella is essential for pathogenesis, our understanding of how the bacterium orchestrates inflammation and tissue destruction at the mucosal surface has been informed by studies employing the rabbit ileal loop model. Here, we outline how Phillippe Sansonetti, together with his co-workers and collaborators, exploited this model to provide a holistic view of how Shigella survives in the intestinal tract, traverses the intestinal epithelial barrier, and manipulates the host immune system to cause disease., (© 2019 John Wiley & Sons Ltd.)

Pathogenic enterobacteria face various oxygen (O 2 ) levels during intestinal colonization from the O 2 -deprived lumen to oxygenated tissues. Using Shigella flexneri as a model, we have previously demonstrated that epithelium invasion is promoted by O 2 in a type III secretion system-dependent manner. However, subsequent pathogen adaptation to tissue oxygenation modulation remained unknown. Assessing single-cell distribution, together with tissue oxygenation, we demonstrate here that the colonic mucosa O 2 is actively depleted by S. flexneri aerobic respiration-and not host neutrophils-during infection, leading to the formation of hypoxic foci of infection. This process is promoted by type III secretion system inactivation in infected tissues, favouring colonizers over explorers. We identify the molecular mechanisms supporting infectious hypoxia induction, and demonstrate here how enteropathogens optimize their colonization capacity in relation to their ability to manipulate tissue oxygenation during infection.

Intracellular trafficking pathways in eukaryotic cells are essential to maintain organelle identity and structure, and to regulate cell communication with its environment. Shigella flexneri invades and subverts the human colonic epithelium by the injection of virulence factors through a type 3 secretion system (T3SS). In this work, we report the multiple effects of two S. flexneri effectors, IpaJ and VirA, which target small GTPases of the Arf and Rab families, consequently inhibiting several intracellular trafficking pathways. IpaJ and VirA induce large-scale impairment of host protein secretion and block the recycling of surface receptors. Moreover, these two effectors decrease clathrin-dependent and -independent endocytosis. Therefore, S. flexneri infection induces a global blockage of host cell intracellular transport, affecting the exchange between cells and their external environment. The combined action of these effectors disorganizes the epithelial cell polarity, disturbs epithelial barrier integrity, promotes multiple invasion events, and enhances the pathogen capacity to penetrate into the colonic tissue in vivo., Competing Interests: The authors declare no conflict of interest.

The enteric bacterium and intracellular human pathogen Shigella causes hundreds of millions of cases of the diarrheal disease shigellosis per year worldwide. Shigella is acquired by ingestion of contaminated food or water; upon reaching the colon, the bacteria invade colonic epithelial cells, replicate intracellularly, spread to adjacent cells, and provoke an intense inflammatory response. There is no animal model that faithfully recapitulates human disease; thus, cultured cells have been used to model Shigella pathogenesis. However, the use of transformed cells in culture does not provide the same environment to the bacteria as the normal human intestinal epithelium. Recent advances in tissue culture now enable the cultivation of human intestinal enteroids (HIEs), which are derived from human intestinal stem cells, grown ex vivo , and then differentiated into "mini-intestines." Here, we demonstrate that HIEs can be used to model Shigella pathogenesis. We show that Shigella flexneri invades polarized HIE monolayers preferentially via the basolateral surface. After S. flexneri invades HIE monolayers, S. flexneri replicates within HIE cells and forms actin tails. S. flexneri also increases the expression of HIE proinflammatory signals and the amino acid transporter SLC7A5. Finally, we demonstrate that disruption of HIE tight junctions enables S. flexneri invasion via the apical surface., (Copyright © 2019 American Society for Microbiology.)

Shigella flexneri is an enteropathogenic bacterium responsible for approximately 100 million cases of severe dysentery each year. S. flexneri colonization of the human colonic epithelium is supported by direct spread from cell to cell, which relies on actin-based motility. We have recently uncovered that, in intestinal epithelial cells, S. flexneri actin-based motility is regulated by the Bruton's tyrosine kinase (Btk). Consequently, treatment with Ibrutinib, a specific Btk inhibitor currently used in the treatment of B-cell malignancies, effectively impaired S. flexneri spread from cell to cell. Thus, therapeutic intervention capitalizing on drugs interfering with host factors supporting the infection process may represent an effective alternative to treatments with antimicrobial compounds.

Upon infection with Shigella flexneri, epithelial cells release ATP through connexin hemichannels. However, the pathophysiological consequence and the regulation of this process are unclear. Here we showed that in intestinal epithelial cell ATP release was an early alert response to infection with enteric pathogens that eventually promoted inflammation of the gut. Shigella evolved to escape this inflammatory reaction by its type III secretion effector IpgD, which blocked hemichannels via the production of the lipid PtdIns5P. Infection with an ipgD mutant resulted in rapid hemichannel-dependent accumulation of extracellular ATP in vitro and in vivo, which preceded the onset of inflammation. At later stages of infection, ipgD-deficient Shigella caused strong intestinal inflammation owing to extracellular ATP. We therefore describe a new paradigm of host-pathogen interaction based on endogenous danger signaling and identify extracellular ATP as key regulator of mucosal inflammation during infection. Our data provide new angles of attack for the development of anti-inflammatory molecules., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Shigella flexneri is a Gram-negative intracellular pathogen that infects the intestinal epithelium and utilizes actin-based motility to spread from cell to cell. S. flexneri actin-based motility has been characterized in various cell lines, but studies in intestinal cells are limited. Here we characterized S. flexneri actin-based motility in HT-29 intestinal cells. In agreement with studies conducted in various cell lines, we showed that S. flexneri relies on neural Wiskott-Aldrich Syndrome protein (N-WASP) in HT-29 cells. We tested the potential role of various tyrosine kinases involved in N-WASP activation and uncovered a previously unappreciated role for Bruton's tyrosine kinase (Btk) in actin tail formation in intestinal cells. We showed that Btk depletion led to a decrease in N-WASP phosphorylation which affected N-WASP recruitment to the bacterial surface, decreased the number of bacteria displaying actin-based motility, and ultimately affected the efficiency of spread from cell to cell. Finally, we showed that the levels of N-WASP phosphorylation and Btk expression were increased in response to infection, which suggests that S. flexneri infection not only triggers the production of proinflammatory factors as previously described but also manipulates cellular processes required for dissemination in intestinal cells.

An understanding of the signaling mechanism(s) that regulate the differential expression of gastric mucin MUC5AC in colonic epithelial cells would contribute significantly to investigations of its role in colonic mucosa infected with the bacterial pathogen Shigella dysenteriae. Here we show that S. dysenteriae-Sinduced expression of interleukin-1β upregulates MUC2 expression and the differential expression of MUC5AC. Differential expression of MUC5AC involves crosstalk between interleukin-1β and Akt, whereby the trefoil factor family peptide TFF3 activates Akt by phosphorylation of EGFR. TFF3 also downregulates E-cadherin expression, causing accumulation of β-catenin in the cytosol. Phosphorylation of GSK-3β (inactivated) by activated Akt inhibits ubiquitylation of β-catenin, leading to its nuclear translocation, which then induces the expression of MUC5AC and cyclin D1. Accumulation of cyclin D1 alters the cell cycle, promoting cell survival and proliferation. Human colon HT29MTX cells, which overexpress MUC5AC, were resistant to adherence and invasion of S. dysenteriae when compared with other mucin-secreting HT29 cell types. Thus, during infection with S. dysenteriae, crosstalk between interleukin-1β and Akt wired by TFF3 induces expression of MUC5AC in colonic epithelial cells. Differentially expressed gastric MUC5AC aids in mucosal clearance of S. dysenteriae, inhibiting adherence and invasion of the pathogen to colonic epithelial cells, which protects the host.

Shigella is a major cause of morbidity and mortality for children in many developing countries. Emergence of antibiotic-resistance among Shigella demands the development of effective medicines. Antimicrobial peptides (AMPs) are expressed in phagocytes and at epithelial surfaces and are important effector molecules of innate immunity. We have found that pathogens are able to turn off the endogenous expression of AMPs, resulting in serious infections such as shigellosis. A therapeutic rationale to prevent microbial invasion would be to strengthen the epithelial line of defence through enhancing AMP expression. We have identified several inducers of AMP-production, including butyrate, phenylbutyrate and vitamin D, which have been investigated in animal models of shigellosis as well as in clinical trials. We believe that the conceptual framework presented here can be applied to additional clinical entities and that this novel approach can be an alternative or complement to traditional antibiotics in the future., (2010. Published by Elsevier Inc.)

Objective: To explore the effects of Huoxiang Zhengqi liquid (HXZQ) on enteric mucosal immune responses in mice with Bacillus dysenteriae and Salmonella typhimurium induced diarrhea (BSD)., Method: Mice were randomly divided into four groups with 10 mice in each group: control group (control), BSD group, Huoxiang Zhengqi liquid treated BSD groups at high dosage and low dosage (HXZQ high, HXZQ low). HXZQ was administrated from the day of diarrhea induction at dosage of 5.21 g kg(-1) and 0.52 g kg (-1) respectively. Peyer's patch and periphery lymphocytes were prepared for flow cytometry, and level of TNF-alpha in periphery and enteric tissue homogenate were determined with ELISA. Student's t-test was used for statistics., Result: Mice in BSD group started showing continuous diarrhea at the day of induction till the fourth day when the mice were sacrificed. Diarrhea in the mice of HXZQ high and low groups lasted for 36 and 54 h respectively. There were more CD4+ and CD8+ cells in periphery, less CD4+ cells in peyer's patch in BSD mice comparing to normal mice. In peyer's patch, there were more CD8+ cells in mice in HXZQ high and low groups and more CD4+ in mice in HXZQ high group. Higher level TNF-alpha in periphery and intestinal tissue homogenate in BSD group were observed. Mice in HXZQ high group showed the decreased level TNF-alpha in periphery and enteric tissue homogenate., Conclusion: The immune regulation on peyer's patch CD4+ and CD8+ cells and suppression on TNF-alpha level in enteric homogenate might partially explain the effect of HXZQ on improvement of BSD.

Shigella flexneri is a Gram-negative pathogen that invades and causes inflammatory destruction of the human colonic epithelium, thus leading to bloody diarrhea and dysentery. A type III secretion system that delivers effector proteins into target eukaryotic cells is largely responsible for cell and tissue invasion. However, the respective role of this invasive phenotype and of lipid A, the endotoxin of the Shigella LPS, in eliciting the inflammatory cascade that leads to rupture and destruction of the epithelial barrier, was unknown. We investigated whether genetic detoxification of lipid A would cause significant alteration in pathogenicity. We showed that S. flexneri has two functional msbB genes, one carried by the chromosome (msbB1) and the other by the virulence plasmid (msbB2), the products of which act in complement to produce full acyl-oxy-acylation of the myristate at the 3' position of the lipid A glucosamine disaccharide. A mutant in which both the msbB1 and msbB2 genes have been inactivated was impaired in its capacity to cause TNF-alpha production by human monocytes and to cause rupture and inflammatory destruction of the epithelial barrier in the rabbit ligated intestinal loop model of shigellosis, indicating that lipid A plays a significant role in aggravating inflammation that eventually destroys the intestinal barrier. In addition, neutralization of TNF-alpha during invasion by the wild-type strain strongly impaired its ability to cause rupture and inflammatory destruction of the epithelial lining, thus indicating that TNF-alpha is a major effector of epithelial destruction by Shigella.

Shigella species cause bacillary dysentery in humans by invading epithelial cells of the colonic mucosa leading to colonic epithelial cell destruction and inflammation. For further analysis of local gut inflammation, morphological changes and the potential involvement of mediators in regulatory mechanisms of cell activation and cell proliferation were studied immunohistochemically in rectal mucosal biopsies taken from patients during the acute phase of shigellosis and at convalescence. Rectal biopsies from 25 Shigella dysenteriae-1 and 10 Shigella flexneri-infected patients and from 40 controls were studied. The frequencies of proliferative cells (Ki67-positive cells), p53-immunostaining cells, and cells coexpressing Ki67 with CD3 or with p53 were analyzed. Immunostaining for the inducible nitric oxide synthase (iNOS) and the endothelial NOS was assessed. In addition, the frequencies of apoptotic cells and CD68+ cells that engulf apoptotic cells were assessed. By morphological grading, 20% of the patients had advanced inflammation (grade 3) in the acute phase; mild inflammation (grade 1) was seen in 37% of the patients at convalescence as well as in 10% of the controls. The findings in the present study suggest that in the acute phase of shigellosis inflammation is characterized by increased cell turnover in the lamina propria (LP) and the epithelium, increased iNOS expression in the surface epithelium, and apoptosis, which seems to be associated with LP macrophages. The findings also suggest that neither p53 nor iNOS are important factors for the induction of apoptosis in shigellosis. Expression of p53 may be related to early cell activation in crypt epithelium. Moreover, there is an indication of an active, low-level inflammatory process at convalescence. The results thus indicate that Shigella-induced inflammation is associated with a complex series of cellular reactions in the rectal gut mucosa which persist long after clinical symptoms have resolved.

Proliferative processes were studied using radioautography with 3H-thymidine at the height of the disease and in the early convalescence of the bacterial dysentery. The mucous membrane of the sigmoid colon of healthy persons undergoing rectoromanoscopy due to the epidemic reasons served as control. No pathologic changes were found in these healthy persons; the labeled nuclei index (LNI) was 5.75 +/- 0.4%, the label intensity (LI) characterizing the rate of DNA synthesis was 14.9 +/- 0.8. During the peak of bacterial dysentery LNI increased up to 12.9 +/- 1.3% and there was a three-fold increase of LI. Activation of the proliferative processes persisted in the period of an early recovery. LNI was 9.4 +/- 1.0%, LI was 2.6 times higher as compared to control.