Shigella induces epigenetic reprogramming of zebrafish neutrophils.

Trained immunity is a long-term memory of innate immune cells, generating an improved response upon reinfection. Shigella is an important human pathogen and inflammatory paradigm for which there is no effective vaccine. Using zebrafish larvae, we demonstrate that after Shigella training, neutrophils are more efficient at bacterial clearance. We observe that Shigella-induced protection is nonspecific and has differences with training by BCG and ß-glucan. Analysis of histone ChIP-seq on trained neutrophils revealed that Shigella training deposits the active H3K4me3 mark on promoter regions of 1612 genes, dramatically changing the epigenetic landscape of neutrophils toward enhanced microbial recognition and mitochondrial ROS production. Last, we demonstrate that mitochondrial ROS plays a key role in enhanced antimicrobial activity of trained neutrophils. It is envisioned that signals and mechanisms we discover here can be used in other vertebrates, including humans, to suggest new therapeutic strategies involving neutrophils to control bacterial infection.

High-Throughput CRISPR Screens To Dissect Macrophage-Shigella Interactions.

Shigellosis causes most diarrheal deaths worldwide, particularly affecting children. Shigella invades and replicates in the epithelium of the large intestine, eliciting inflammation and tissue destruction. To understand how Shigella rewires macrophages prior to epithelium invasion, we performed genome-wide and focused secondary CRISPR knockout and CRISPR interference (CRISPRi) screens in Shigella flexneri-infected human monocytic THP-1 cells. Knockdown of the Toll-like receptor 1/2 signaling pathway significantly reduced proinflammatory cytokine and chemokine production, enhanced host cell survival, and controlled intracellular pathogen growth. Knockdown of the enzymatic component of the mitochondrial pyruvate dehydrogenase complex enhanced THP-1 cell survival. Small-molecule inhibitors blocking key components of these pathways had similar effects; these were validated with human monocyte-derived macrophages, which closely mimic the in vivo physiological state of macrophages postinfection. High-throughput CRISPR screens can elucidate how S. flexneri triggers inflammation and redirects host pyruvate catabolism for energy acquisition before killing macrophages, pointing to new shigellosis therapies. IMPORTANCE Treatment for shigellosis is becoming increasingly difficult as resistance to antibiotics becomes more prevalent. One way to prevent this significant public health problem from developing into a full-blown crisis is to approach shigellosis intervention from the point of view of the host. So far, little is known about the specific biological pathways that might be modulated in macrophages, sentinel cells of the innate immune system, to strengthen the response to Shigella infection. In this work, we conducted CRISPR screens to comprehensively decipher the complexity of macrophage-Shigella interactions and to discover new potential therapeutic interventions against Shigella flexneri infection. Our work highlights systematic genetic perturbation strategies to provide direct causal evidence showing how intracellular pathogens manipulate innate immune cells.

Shigella flexneri subverts host polarized exocytosis to enhance cell-to-cell spread.

Shigella flexneri is a gram-negative bacterial pathogen that causes dysentery. Critical for disease is the ability of Shigella to use an actin-based motility (ABM) process to spread between cells of the colonic epithelium. ABM transports bacteria to the periphery of host cells, allowing the formation of plasma membrane protrusions that mediate spread to adjacent cells. Here we demonstrate that efficient protrusion formation and cell-to-cell spread of Shigella involves bacterial stimulation of host polarized exocytosis. Using an exocytic probe, we found that exocytosis is locally upregulated in bacterial protrusions in a manner that depends on the Shigella type III secretion system. Experiments involving RNA interference (RNAi) indicate that efficient bacterial protrusion formation and spread require the exocyst, a mammalian multi-protein complex known to mediate polarized exocytosis. In addition, the exocyst component Exo70 and the exocyst regulator RalA were recruited to Shigella protrusions, suggesting that bacteria manipulate exocyst function. Importantly, RNAi-mediated depletion of exocyst proteins or RalA reduced the frequency of protrusion formation and also the lengths of protrusions, demonstrating that the exocyst controls both the initiation and elongation of protrusions. Collectively, our results reveal that Shigella co-opts the exocyst complex to disseminate efficiently in host cell monolayers.

Shigella ubiquitin ligase IpaH7.8 targets gasdermin D for degradation to prevent pyroptosis and enable infection.

The pore-forming protein gasdermin D (GSDMD) executes lytic cell death called pyroptosis to eliminate the replicative niche of intracellular pathogens. Evolution favors pathogens that circumvent this host defense mechanism. Here, we show that the Shigella ubiquitin ligase IpaH7.8 functions as an inhibitor of GSDMD. Shigella is an enteroinvasive bacterium that causes hemorrhagic gastroenteritis in primates, but not rodents. IpaH7.8 contributes to species specificity by ubiquitinating human, but not mouse, GSDMD and targeting it for proteasomal degradation. Accordingly, infection of human epithelial cells with IpaH7.8-deficient Shigella flexneri results in increased GSDMD-dependent cell death compared with wild type. Consistent with pyroptosis contributing to murine disease resistance, eliminating GSDMD from NLRC4-deficient mice, which are already sensitized to oral infection with Shigella flexneri, leads to further enhanced bacterial replication and increased disease severity. This work highlights a species-specific pathogen arms race focused on maintenance of host cell viability.

Biofilm Formation and Virulence of Shigella flexneri Are Modulated by pH of Gastrointestinal Tract.

Shigella infection remains a public health problem in much of the world. Classic models of Shigella pathogenesis suggest that microfold epithelial cells in the small intestine are the preferred initial site of invasion. However, recent evidence supports an alternative model in which Shigella primarily infects a much wider range of epithelial cells that reside primarily in the colon. Here, we investigated whether the luminal pH difference between the small intestine and the colon could provide evidence in support of either model of Shigella flexneri pathogenesis. Because virulence factors culminating in cellular invasion are linked to biofilms in S. flexneri, we examined the effect of pH on the ability of S. flexneri to form and maintain adherent biofilms induced by deoxycholate. We showed that a basic pH (as expected in the small intestine) inhibited formation of biofilms and dispersed preassembled mature biofilms, while an acidic pH (similar to the colonic environment) did not permit either of these effects. To further elucidate this phenomenon at the molecular level, we probed the transcriptomes of biofilms and S. flexneri grown under different pH conditions. We identified specific amino acid (cysteine and arginine) metabolic pathways that were enriched in the bacteria that formed the biofilms but decreased when the pH increased. We then utilized a type III secretion system reporter strain to show that increasing pH reduced deoxycholate-induced virulence of S. flexneri in a dose-dependent manner. Taken together, these experiments support a model in which Shigella infection is favored in the colon because of the local pH differences in these organs.

Behavior of Salmonella Enteritidis and Shigella flexneri during induction and recovery of the viable but nonculturable state.

Bacteria may enter into a viable but nonculturable (VBNC) state as a response to stresses, such as those found in food processing. Cells in the VBNC state lose the ability to grow in a conventional culture medium but man recover culturability. The viability, culturability and intracellular reactive oxygen species (ROS) of Salmonella Enteritidis and Shigella flexneri were evaluated under stress conditions to induce a VBNC state. Cells were maintained under nutritional, osmotic and cold stresses (long-term induction) in Butterfield's phosphate solution plus 1.2 M of NaCl at 4°C and under nutritional and oxidative stresses (short-term induction) in 10 mM of H2O2. Culture media, recovery agents, sterilization methods of media and incubation temperature, were combined and applied to recover the culturability of the VBNC cells. Salmonella entered in the VBNC state after 135 days under long-term induction, while Shigella maintained culturability after 240 days. Under short-term induction, Salmonella and Shigella lose culturability after 135 and 240 min, respectively. Flow cytometric analysis revealed viable cells and intracellular ROS in both species in VBNC. It was not possible to recover the culturability of VBNC cells using the 42 combinations of different factors.

Pathogenic ubiquitination of GSDMB inhibits NK cell bactericidal functions.

Gasdermin B (GSDMB) belongs to a large family of pore-forming cytolysins that execute inflammatory cell death programs. While genetic studies have linked GSDMB polymorphisms to human disease, its function in the immunological response to pathogens remains poorly understood. Here, we report a dynamic host-pathogen conflict between GSDMB and the IpaH7.8 effector protein secreted by enteroinvasive Shigella flexneri. We show that IpaH7.8 ubiquitinates and targets GSDMB for 26S proteasome destruction. This virulence strategy protects Shigella from the bacteriocidic activity of natural killer cells by suppressing granzyme-A-mediated activation of GSDMB. In contrast to the canonical function of most gasdermin family members, GSDMB does not inhibit Shigella by lysing host cells. Rather, it exhibits direct microbiocidal activity through recognition of phospholipids found on Gram-negative bacterial membranes. These findings place GSDMB as a central executioner of intracellular bacterial killing and reveal a mechanism employed by pathogens to counteract this host defense system.

Ferulic Acid Inactivates Shigella flexneri through Cell Membrane Destructieon, Biofilm Retardation, and Altered Gene Expression.

Antibiotic resistance and capacity for biofilm formation of Shigella flexneri render previous prevention and control strategies minimally effective. Ferulic acid (FA) has been demonstrated to be useful due to its application in foods as an alternative natural preservative. However, information regarding the S. flexneri phenotype and molecular responses to FA exposure is limited. The present study investigated the effects of FA on S. flexneri planktonic growth and biofilm formation. The results demonstrated that the cell membrane of S. flexneri in planktonic growth mode exhibited irreversible destruction after FA exposure, as characterized by decreased cell viability, leakage of cytoplasmic constituents, accelerated adenosine triphosphate (ATP) consumption, cell membrane depolarization, and cellular morphological changes. FA significantly inhibited S. flexneri adhesion and biofilm formation at a working concentration (1/8 MIC) that almost did not inhibit planktonic growth. Transcriptomics profiling showed that the exposure to a subinhibitory concentration of FA dramatically altered gene expression in the S. flexneri biofilm, as a total of 169 differentially expressed genes (DEGs) were upregulated and 533 DEGs were downregulated, compared to the intact biofilm. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis showed that the DEGs were mainly involved in pathways of ribosomes, ABC transporters, and the citrate cycle. Furthermore, we show that FA altered the transcription of S. flexneri genes associated with adhesion, transcriptional regulation, and the synthesis and transport of extracellular polymeric substances that contribute to biofilm formation. These data provide novel insights into S. flexneri behavioral responses to FA exposure and suggest that FA could effectively constrain S. flexneri and its biofilm formation.

Antibiofilm Activity of Lactobacillus plantarum 12 Exopolysaccharides against Shigella flexneri.

In developing countries, Shigella flexneri is the most common enteric pathogen causing bacillary dysentery. Biofilm formation by S. flexneri can cause the emergence of antibiotic-resistant strains, which poses serious threats to food safety and human health. In this study, the effects of Lactobacillus plantarum 12 exopolysaccharides (L-EPSs) and S. flexneri exopolysaccharides (S-EPSs) on S. flexneri CMCC51574 biofilm formation were investigated. The results showed that L-EPS could decrease polysaccharide production in the extracellular polymeric matrix of S. flexneri and inhibit biofilm formation by S. flexneri L-EPS could decrease the minimum biofilm elimination concentration (MBEC) of antibiotics against S. flexneri biofilm and inhibit S. flexneri adhesion to and invasion into HT-29 cell monolayers, which might be ascribed to S. flexneri biofilm disturbance by L-EPS. In contrast, S-EPS exhibited the opposite effects compared to L-EPS. The monosaccharide composition analysis showed that L-EPS was composed of mannose, glucuronic acid, galactosamine, glucose, galactose, and xylose, with the molar ratio of 32.26:0.99:1.79:5.63:0.05:4.07, while S-EPS was composed of mannose, glucuronic acid, galactosamine, glucose, and galactose, with the molar ratio of 25.43:2.28:7.13:5.35. L-EPS was separated into the neutral polysaccharide L-EPS 1-1 and the acidic polysaccharide L-EPS 2-1 by ion-exchange chromatography and gel chromatography. L-EPS 2-1 exerted higher antibiofilm activity than L-EPS 1-1. The antibiofilm activity of L-EPS might be associated with its structure.IMPORTANCES. flexneri is a widespread foodborne pathogen causing food contamination and responsible for food poisoning outbreaks related to various foods in developing countries. Not only has biofilm formation by S. flexneri been difficult to eliminate, but it has also increased the drug resistance of the strain. In the present study, it was demonstrated that L-EPSs secreted by Lactobacillus plantrum 12 could inhibit S. flexneri biofilm formation on, adhesion to, and invasion into HT-29 cells. Also, L-EPSs could decrease the minimum biofilm elimination concentration (MBEC) of the antibiotics used against S. flexneri biofilm. Therefore, L-EPSs were shown to be bioactive macromolecules with the potential ability to act against S. flexneri infections.

Transcytosis subversion by M cell-to-enterocyte spread promotes Shigella flexneri and Listeria monocytogenes intracellular bacterial dissemination.

Microfold (M) cell host-pathogen interaction studies would benefit from the visual analysis of dynamic cellular and microbial interplays. We adapted a human in vitro M cell model to physiological bacterial infections, expression of fluorescent localization reporters and long-term three-dimensional time-lapse microscopy. This approach allows following key steps of M cell infection dynamics at subcellular resolution, from the apical onset to basolateral epithelial dissemination. We focused on the intracellular pathogen Shigella flexneri, classically reported to transcytose through M cells to initiate bacillary dysentery in humans, while eliciting poorly protective immune responses. Our workflow was critical to reveal that S. flexneri develops a bimodal lifestyle within M cells leading to rapid transcytosis or delayed vacuolar rupture, followed by direct actin motility-based propagation to neighboring enterocytes. Moreover, we show that Listeria monocytogenes, another intracellular pathogen sharing a tropism for M cells, disseminates in a similar manner and evades M cell transcytosis completely. We established that actin-based M cell-to-enterocyte spread is the major dissemination pathway for both pathogens and avoids their exposure to basolateral compartments in our system. Our results challenge the notion that intracellular pathogens are readily transcytosed by M cells to inductive immune compartments in vivo, providing a potential mechanism for their ability to evade adaptive immunity.

The vacuole guard hypothesis: how intravacuolar pathogens fight to maintain the integrity of their beloved home.

Intravacuolar bacterial pathogens establish intracellular niches by constructing membrane-encompassed compartments. The vacuoles surrounding the bacteria are remarkably stable, facilitating microbial replication and preventing exposure to host cytoplasmically localized innate immune sensing mechanisms. To maintain integrity of the membrane compartment, the pathogen is armed with defensive weapons that prevent loss of vacuole integrity and potential exposure to host innate signaling. In some cases, the microbial components that maintain vacuolar integrity have been identified, but the basis for why the compartment degrades in their absence is unclear. In this review, we point out that lessons from the microbial-programmed degradation of the vacuole by the cytoplasmically localized Shigella flexneri provide crucial insights into how degradation of pathogen vacuoles occurs. We propose that in the absence of bacterial-encoded guard proteins, aberrant trafficking of host membrane-associated components results in a dysfunctional pathogen compartment. As a consequence, the vacuole is poisoned and replication is terminated.

Shigella flexneri: an emerging pathogen.

Shigella flexneri is a leading etiologic agent of diarrhea in low socioeconomic countries. Notably, various serotypes in S. flexneri are reported from different regions of the world. The precise approximations of illness and death owing to shigellosis are missing in low socioeconomic countries, although it is widespread in different regions. The inadequate statistics available reveal S. flexneri to be a significant food and waterborne pathogen. All over the world, different antibiotic-resistant strains of S. flexneri serotypes have been emerged especially multidrug-resistant strains. Recently, increased resistance was observed in cephalosporins (3rd generation), azithromycin, and fluoroquinolones. There is a need for a continuous surveillance study on antibiotic resistance that will be helpful in the update of the antibiogram. The shigellosis burden can be reduced by adopting preventive measures like delivery of safe drinking water, suitable sanitation, and development of an effective and inexpensive multivalent vaccine. This review attempts to provide the recent findings of S. flexneri related to epidemiology and the emergence of multidrug resistance.

Functional screenings reveal different requirements for host microRNAs in Salmonella and Shigella infection.

MicroRNAs (miRNAs) are increasingly recognized for their role in infection by bacterial pathogens, although the effect of each individual miRNA remains largely unknown. Here, we used a comparative genome-wide microscopy-based functional screening approach to identify miRNAs controlling infection by two bacterial pathogens-Salmonella enterica serovar Typhimurium and Shigella flexneri. Despite the similarities between these pathogens, we found infections to be controlled by largely non-overlapping subsets of miRNAs, seemingly reflecting different requirements prompted by their distinct intracellular lifestyles. By characterizing a small subset of miRNAs chosen among the strongest inhibitors of Shigella infection, we discovered that miR-3668, miR-4732-5p and miR-6073 exert a selective effect on Shigella infection by impairing bacterial actin-based motility by downregulating N-WASP. Additionally, by identifying let-7i-3p miRNA as a strong inhibitor of Salmonella replication and performing in-depth analysis of its mechanisms of action, we showed that this miRNA specifically inhibits Salmonella infection via modulation of endolysosomal trafficking and the vacuolar environment by targeting the host RGS2 protein. These findings illustrate two paradigms underlying miRNA-mediated regulation of bacterial infection, acting as part of the host response to infection, or as part of bacterial strategies to modulate the host environment and favour pathogenesis.

Cytosolic Gram-negative bacteria prevent apoptosis by inhibition of effector caspases through lipopolysaccharide.

The cytosolic appearance and propagation of bacteria cause overwhelming cellular stress responses that induce apoptosis under normal conditions. Therefore, successful bacterial colonization depends on the ability of intracellular pathogens to block apoptosis and to safeguard bacterial replicative niches. Here, we show that the cytosolic Gram-negative bacterium Shigella flexneri stalls apoptosis by inhibiting effector caspase activity. Our data identified lipopolysaccharide (LPS) as a bona fide effector caspase inhibitor that directly binds caspases by involving its O-antigen (O Ag) moiety. Bacterial strains that lacked the O Ag or failed to replicate within the cytosol were incapable of blocking apoptosis and exhibited reduced virulence in a murine model of bacterial infection. Our findings demonstrate how Shigella inhibits pro-apoptotic caspase activity, effectively delays coordinated host-cell demise and supports its intracellular propagation. Next to the recently discovered pro-inflammatory role of cytosolic LPS, our data reveal a distinct mode of LPS action that, through the disruption of the early coordinated non-lytic cell death response, ultimately supports the inflammatory breakdown of infected cells at later time points.

The interferon stimulated gene viperin, restricts Shigella. flexneri in vitro.

The role of interferon and interferon stimulated genes (ISG) in limiting bacterial infection is controversial, and the role of individual ISGs in the control of the bacterial life-cycle is limited. Viperin, is a broad acting anti-viral ISGs, which restricts multiple viral pathogens with diverse mechanisms. Viperin is upregulated early in some bacterial infections, and using the intracellular bacterial pathogen, S. flexneri, we have shown for the first time that viperin inhibits the intracellular bacterial life cycle. S. flexneri replication in cultured cells induced a predominantly type I interferon response, with an early increase in viperin expression. Ectopic expression of viperin limited S. flexneri cellular numbers by as much as 80% at 5hrs post invasion, with similar results also obtained for the intracellular pathogen, Listeria monocytogenes. Analysis of viperins functional domains required for anti-bacterial activity revealed the importance of both viperin's N-terminal, and its radical SAM enzymatic function. Live imaging of S. flexneri revealed impeded entry into viperin expressing cells, which corresponded to a loss of cellular cholesterol. This data further defines viperin's multi-functional role, to include the ability to limit intracellular bacteria; and highlights the role of ISGs and the type I IFN response in the control of bacterial pathogens.

Mapping of Shigella flexneri's tissue distribution and type III secretion apparatus activity during infection of the large intestine of guinea pigs.

Shigella spp. are bacterial pathogens that invade the human colonic mucosa using a type III secretion apparatus (T3SA), a proteinaceous device activated upon contact with host cells. Active T3SAs translocate proteins that carve the intracellular niche of Shigella spp. Nevertheless, the activation state of the T3SA has not been addressed in vivo. Here, we used a green fluorescent protein transcription-based secretion activity reporter (TSAR) to provide a spatio-temporal description of S. flexneri T3SAs activity in the colon of Guinea pigs. First, we observed that early mucus release is triggered in the vicinity of luminal bacteria with inactive T3SA. Subsequent mucosal invasion showed bacteria with active T3SA associated with the brush border, eventually penetrating into epithelial cells. From 2 to 8 h post-challenge, the infection foci expanded, and these intracellular bacteria displayed homogeneously high-secreting activity, while extracellular foci within the lamina propria featured bacteria with low secretion activity. We also found evidence that within lamina propria macrophages, bacteria reside in vacuoles instead of accessing the cytosol. Finally, bacteria were cleared from tissues between 8 and 24 h post-challenge, highlighting the hit-and-run colonization strategy of Shigella. This study demonstrates how genetically encoded reporters can contribute to deciphering pathogenesis in vivo.

IpaD-loaded N-trimethyl Chitosan Nanoparticles Can Efficiently Protect Guinea Pigs against Shigella flexneri.

BACKGROUND: Shigella flexneri is a pathogen responsible for shigellosis around the world, especially in developing countries. Many immunogenic antigens have been introduced as candidate vaccines against Shigella, including N-terminal region of IpaD antigen (NIpaD). OBJECTIVE: To evaluate the efficiency of O-metylated free trimethyl chitosan nanoparticles (TMC NPs) in the oral delivery of NIpaD. METHODS: TMC was synthesized by a two-step method from high molecular weight chitosan. The recombinant NIpaD protein was used as the immunogen. The protein was overexpressed in E. coli BL21 (DE3) and characterized by gel electrophoresis. The NIpaD-loaded TMC NPs were synthesized by ionic gelation method and were characterized by electron microscopy. NPs were orally administered to guinea pigs and specific humoral and mucosal immune responses were assessed by serum IgG and secretory IgA, respectively. The protectivity of the formulation was assessed by keratoconjunctivitis (Sereny) test. RESULTS: The immunized guinea pigs showed a significant raise in rNIpaD-specific serum IgG and faecal IgA titers. Specific secretory IgA was detected in eye-washes. Sereny test results showed that immunized animals vaccinated with IpaD-loaded TMC NPS tolerated the wild type of Shigella flexneri 2a in Sereny test. However, in the group immunized with NIpaD antigen and non-immunized group, no increase was observed in antibody titer against NIpaD. These animals were infected following the challenge with Shigella flexneri 2a (p<0.0152). CONCLUSION: The recombinant rNIpaD formulated with TMC obtained from high molecular weight chitosan, can be considered as a mucosal vaccine against Shigella flexneri through oral route.

WIPF2 promotes Shigella flexneri actin-based motility and cell-to-cell spread.

Shigella flexneri is an intracellular pathogen that disseminates in colonic epithelial cells through actin-based motility and formation of membrane protrusions at cell-cell contacts, that project into adjacent cells and resolve into vacuoles, from which the pathogen escapes, thereby achieving cell-to-cell spread. Actin nucleation at the bacterial pole relies on the recruitment of the nucleation-promoting factor N-WASP, which activates the actin nucleator ARP2/3. In cells, the vast majority of N-WASP exists as a complex with WIP. The involvement of WIP in N-WASP-dependent actin-based motility of various pathogens, including vaccinia virus and S. flexneri, has been highly controversial. Here, we show that WIPF2 was the only WIP family member expressed in the human colonic epithelial cell line HT-29, and its depletion impaired S. flexneri dissemination. WIPF2 depletion increased the number of cytosolic bacteria lacking actin tails (non-motile) and decreased the velocity of motile bacteria. This correlated with a decrease in the recruitment of N-WASP to the bacterial pole, and among N-WASP-positive bacteria, a decrease in actin tail-positive bacteria, suggesting that WIPF2 is required for N-WASP recruitment and activation at the bacterial pole. In addition, when motile bacteria formed protrusions, WIPF2 depletion decreased the number of membrane protrusions that successfully resolved into vacuoles.

The Role of the Type III Secretion System in the Intracellular Lifestyle of Enteric Pathogens.

Several pathogens have evolved to infect host cells from within, which requires subversion of many host intracellular processes. In the case of Gram-negative pathogenic bacteria, adaptation to an intracellular life cycle relies largely on the activity of type III secretion systems (T3SSs), an apparatus used to deliver effector proteins into the host cell, from where these effectors regulate important cellular functions such as vesicular trafficking, cytoskeleton reorganization, and the innate immune response. Each bacterium is equipped with a unique suite of these T3SS effectors, which aid in the development of an individual intracellular lifestyle for their respective pathogens. Some bacteria adapt to reside and propagate within a customized vacuole, while others establish a replicative niche in the host cytosol. In this article, we review the mechanisms by which T3SS effectors contribute to these different lifestyles. To illustrate the formation of a vacuolar and a cytosolic lifestyle, we discuss the intracellular habitats of the enteric pathogens Salmonella enterica serovar Typhimurium and Shigella flexneri, respectively. These represent well-characterized systems that function as informative models to contribute to our understanding of T3SS-dependent subversion of intracellular processes. Additionally, we present Vibrio parahaemolyticus, another enteric Gram-negative pathogen, as an emerging model for future studies of the cytosolic lifestyle.