Christina S. Faherty

Staying alive: bacterial inhibition of apoptosis during infection

The ability of bacterial pathogens to inhibit apoptosis in eukaryotic cells during infection is an emerging theme in the study of bacterial pathogenesis. Prevention of apoptosis provides a survival advantage because it enables the bacteria to replicate inside host cells. Bacterial pathogens have evolved several ways to prevent apoptosis by protecting the mitochondria and preventing cytochrome c release, by activating cell survival pathways, or by preventing caspase activation. This review summarizes the most recent work on bacterial anti-apoptotic strategies and suggests new research that is necessary to advance the field.

Shigella flexneri type III secretion system effectors OspB and OspF target the nucleus to downregulate the host inflammatory response via interactions with retinoblastoma protein

OspF, OspG and IpaH9.8 are type III secretion system (T3SS) effectors of Shigella flexneri that downregulate the host innate immune response. OspF modifies mitogen‐activated protein kinase pathways and polymorphonuclear leucocyte transepithelial migration associated with Shigella invasion. OspF also localizes in the nucleus to mediate chromatin remodelling, resulting in reduced transcription of inflammatory cytokines. We now report that OspB can be added to the set of S. flexneri T3SS effectors required to modulate the innate immune response. T84 cells infected with a ΔospB mutant resulted in reduced polymorphonuclear leucocyte transepithelial migration and mitogen‐activated protein kinase signalling. Tagged versions of OspB localized with endosomes and the nucleus. Further, T84 cells infected with the ΔospB mutant showed increased levels of secreted IL‐8 compared with wild‐type infected cells. Both GST–OspB and GST–OspF coprecipitated retinoblastoma protein from host cell lysates. Because ΔospB and ΔospF mutants share similar phenotypes, and OspB and OspF share a host binding partner, we propose that OspB and OspF facilitate the remodelling of chromatin via interactions with retinoblastoma protein, resulting in diminished inflammatory cytokine production. The requirement of multiple T3SS effectors to modulate the innate immune response correlates to the complexity of the human immune system.

Serine protease autotransporters from Shigella flexneri and pathogenic Escherichia coli target a broad range of leukocyte glycoproteins

The serine protease autotransporters of Enterobacteriaceae (SPATEs) are secreted by pathogenic Gram-negative bacteria through the autotransporter pathway. We previously classified SPATE proteins into two classes: cytotoxic (class 1) and noncytotoxic (class 2). Here, we show that Pic, a class 2 SPATE protein produced by Shigella flexneri 2a, uropathogenic and enteroaggregative Escherichia coli strains, targets a broad range of human leukocyte adhesion proteins. Substrate specificity was restricted to glycoproteins rich in O-linked glycans, including CD43, CD44, CD45, CD93, CD162 (PSGL-1; P-selectin glycoprotein ligand 1), and the surface-attached chemokine fractalkine, all implicated in leukocyte trafficking, migration, and inflammation. N-terminal sequencing of proteolytic products revealed Pic (protease involved in colonization) cleavage sites to occur before Thr or Ser residues. The purified carbohydrate sLewis-X implied in inflammation and malignancy inhibited cleavage of PSGL-1 by Pic. Exposure of human leukocytes to purified Pic resulted in polymorphonuclear cell activation, but impaired chemotaxis and transmigration; Pic-treated T cells underwent programmed cell death. We also show that the Pic-related protease Tsh/Hbp, implicated in extraintestinal infections, exhibited a spectrum of substrates similar to those cleaved by Pic. In the guinea pig keratoconjunctivitis model, a Shigella pic mutant induced greater inflammation than its parent strain. We suggest that the class-2 SPATEs represent unique immune-modulating bacterial virulence factors.

Spa15 of Shigella flexneri Is Secreted through the Type III Secretion System and Prevents Staurosporine-Induced Apoptosis

ABSTRACT Shigella flexneri is a gram-negative, facultative intracellular pathogen that invades the colonic epithelium and causes bacillary dysentery. We previously demonstrated that S. flexneri inhibits staurosporine-induced apoptosis in infected epithelial cells and that a ΔmxiE mutant is unable to inhibit apoptosis. Therefore, we hypothesized that an MxiE-regulated gene was responsible for protection of epithelial cells from apoptosis. Analysis of all MxiE-regulated genes yielded no mutants that lacked the ability to prevent apoptosis. Spa15, which is defined as a type III secretion system chaperone, was analyzed since it associates with MxiE. A Δspa15 mutant was unable to prevent staurosporine-induced apoptosis. C-terminal hemagglutinin-tagged spa15 was secreted by S. flexneri within 2 h in the Congo red secretion assay, and secretion was dependent on the type III secretion system. Spa15 was also secreted by Shigella in infected epithelial cells, as verified by immunofluorescence analysis. Spa15 secretion was decreased in the ΔmxiE mutant, which demonstrates why this mutant is unable to prevent staurosporine-induced apoptosis. Our data are the first to show that Spa15 is secreted in a type III secretion system-dependent fashion, and the absence of Spa15 in the Δspa15 mutant results in the loss of protection from staurosporine-induced apoptosis in epithelial cells. Thus, Spa15 contributes to the intracellular survival of Shigella by blocking apoptosis in the infected host cell.

Shigella flexneri effectors OspE1 and OspE2 mediate induced adherence to the colonic epithelium following bile salts exposure

Shigella flexneri is a Gram‐negative pathogen that invades the colonic epithelium. While invasion has been thoroughly investigated, it is unknown how Shigella first attaches to the epithelium. Previous literature suggests that Shigella utilizes adhesins that are induced by environmental signals, including bile salts, encountered in the small intestine prior to invasion. We hypothesized that bile would induce adherence factors to facilitate attachment to colonic epithelial cells. To test our hypothesis, S. flexneri strain 2457T was subcultured in media containing bile salts, and the ability of the bacteria to adhere to the apical surface of polarized T84 epithelial cells was measured. We observed a significant increase in adherence, which was absent in a virulence plasmid‐cured strain and a type‐III secretion system mutant. Microarray expression analysis indicated that the ospE1/ospE2 genes were induced in the presence of bile, and bile‐induced adherence was lost in a ΔospE1/ΔospE2 mutant. Further studies demonstrated that the OspE1/OspE2 proteins were localized to the bacterial outer membrane following exposure to bile salts. The data presented are the first demonstration that the OspE1/OspE2 proteins promote initial adherence to the intestinal epithelium. The adhesins required for Shigella attachment to the colonic epithelium may serve as ideal targets for vaccine development.

Chromosomal and Plasmid-Encoded Factors of Shigella flexneri Induce Secretogenic Activity Ex Vivo

Shigella flexneri is a Gram-negative, facultative intracellular pathogen that causes millions of cases of watery or bloody diarrhea annually, resulting in significant global mortality. Watery diarrhea is thought to arise in the jejunum, and subsequent bloody diarrhea occurs as a result of invasion of the colonic epithelium. Previous literature has demonstrated that Shigella encodes enterotoxins, both chromosomally and on the 220 kilobase virulence plasmid. The Shigella Enterotoxins 1 and 2 (ShET1 and ShET2) have been shown to increase water accumulation in the rabbit ileal loop model. In addition, these toxins increase the short circuit current in rabbit tissue mounted in Ussing chambers, which is a model for the ion exchange that occurs during watery diarrhea. In this study, we sought to validate the use of mouse jejunum in Ussing chamber as an alternative, more versatile model to study bacterial pathogenesis. In the process, we also identified enterotoxins in addition to ShET1 and ShET2 encoded by S. flexneri. Through analysis of proteins secreted from wildtype bacteria and various deletion mutants, we have identified four factors responsible for enterotoxin activity: ShET1 and Pic, which are encoded on the chromosome; ShET2 (encoded by sen or ospD3), which requires the type-III secretion system for secretion; and SepA, an additional factor encoded on the virulence plasmid. The use of mouse jejunum serves as a reliable and reproducible model to identify the enterotoxins elaborated by enteric bacteria. Moreover, the identification of all Shigella proteins responsible for enterotoxin activity is vital to our understanding of Shigella pathogenicity and to our success in developing safe and effective vaccine candidates.

Survival of the Fittest: How Bacterial Pathogens Utilize Bile To Enhance Infection

SUMMARY Bacterial pathogens have coevolved with humans in order to efficiently infect, replicate within, and be transmitted to new hosts to ensure survival and a continual infection cycle. For enteric pathogens, the ability to adapt to numerous host factors under the harsh conditions of the gastrointestinal tract is critical for establishing infection. One such host factor readily encountered by enteric bacteria is bile, an innately antimicrobial detergent-like compound essential for digestion and nutrient absorption. Not only have enteric pathogens evolved to resist the bactericidal conditions of bile, but these bacteria also utilize bile as a signal to enhance virulence regulation for efficient infection. This review provides a comprehensive and up-to-date analysis of bile-related research with enteric pathogens. From common responses to the unique expression of specific virulence factors, each pathogen has overcome significant challenges to establish infection in the gastrointestinal tract. Utilization of bile as a signal to modulate virulence factor expression has led to important insights for our understanding of virulence mechanisms for many pathogens. Further research on enteric pathogens exposed to this in vivo signal will benefit therapeutic and vaccine development and ultimately enhance our success at combating such elite pathogens.

Microarray analysis of Shigella flexneri-infected epithelial cells identifies host factors important for apoptosis inhibition

BackgroundShigella flexneri inhibits apoptosis in infected epithelial cells. In order to understand the pro-survival effects induced by the bacteria, we utilized apoptosis-specific microarrays to analyze the changes in eukaryotic gene expression in both infected and uninfected cells in the presence and absence of staurosporine, a chemical inducer of the intrinsic pathway of apoptosis. The goal of this research was to identify host factors that contribute to apoptosis inhibition in infected cells.ResultsThe microarray analysis revealed distinct expression profiles in uninfected and infected cells, and these changes were altered in the presence of staurosporine. These profiles allowed us to make comparisons between the treatment groups. Compared to uninfected cells, Shigella- infected epithelial cells, both in the presence and absence of staurosporine, showed significant induced expression of JUN, several members of the inhibitor of apoptosis gene family, nuclear factor κB and related genes, genes involving tumor protein 53 and the retinoblastoma protein, and surprisingly, genes important for the inhibition of the extrinsic pathway of apoptosis. We confirmed the microarray results for a selection of genes using in situ hybridization analysis.ConclusionInfection of epithelial cells with S. flexneri induces a pro-survival state in the cell that results in apoptosis inhibition in the presence and absence of staurosporine. The bacteria may target these host factors directly while some induced genes may represent downstream effects due to the presence of the bacteria. Our results indicate that the bacteria block apoptosis at multiple checkpoints along both pathways so that even if a cell fails to prevent apoptosis at an early step, Shigella will block apoptosis at the level of caspase-3. Apoptosis inhibition is most likely vital to the survival of the bacteria in vivo. Future characterization of these host factors is required to fully understand how S. flexneri inhibits apoptosis in epithelial cells.

Analysis of Shigella flexneri Resistance, Biofilm Formation, and Transcriptional Profile in Response to Bile Salts

ABSTRACT The Shigella species cause millions of cases of watery or bloody diarrhea each year, mostly in children in developing countries. While many aspects of Shigella colonic cell invasion are known, crucial gaps in knowledge regarding how the bacteria survive, transit, and regulate gene expression prior to infection remain. In this study, we define mechanisms of resistance to bile salts and build on previous research highlighting induced virulence in Shigella flexneri strain 2457T following exposure to bile salts. Typical growth patterns were observed within the physiological range of bile salts; however, growth was inhibited at higher concentrations. Interestingly, extended periods of exposure to bile salts led to biofilm formation, a conserved phenotype that we observed among members of the Enterobacteriaceae. Characterization of S. flexneri 2457T biofilms determined that both bile salts and glucose were required for formation, dispersion was dependent upon bile salts depletion, and recovered bacteria displayed induced adherence to HT-29 cells. RNA-sequencing analysis verified an important bile salt transcriptional profile in S. flexneri 2457T, including induced drug resistance and virulence gene expression. Finally, functional mutagenesis identified the importance of the AcrAB efflux pump and lipopolysaccharide O-antigen synthesis for bile salt resistance. Our data demonstrate that S. flexneri 2457T employs multiple mechanisms to survive exposure to bile salts, which may have important implications for multidrug resistance. Furthermore, our work confirms that bile salts are important physiological signals to activate S. flexneri 2457T virulence. This work provides insights into how exposure to bile likely regulates Shigella survival and virulence during host transit and subsequent colonic infection.

Intestinal P-glycoprotein exports endocannabinoids to prevent inflammation and maintain homeostasis

Neutrophil influx into the intestinal lumen is a critical response to infectious agents, but is also associated with severe intestinal damage observed in idiopathic inflammatory bowel disease. The chemoattractant hepoxilin A3, an eicosanoid secreted from intestinal epithelial cells by the apically restricted efflux pump multidrug resistance protein 2 (MRP2), mediates this neutrophil influx. Information about a possible counterbalance pathway that could signal the lack of or resolution of an apical inflammatory signal, however, has yet to be described. We now report a system with such hallmarks. Specifically, we identify endocannabinoids as the first known endogenous substrates of the apically restricted multidrug resistance transporter P-glycoprotein (P-gp) and reveal a mechanism, which we believe is novel, for endocannabinoid secretion into the intestinal lumen. Knockdown or inhibition of P-gp reduced luminal secretion levels of N-acyl ethanolamine–type endocannabinoids, which correlated with increased neutrophil transmigration in vitro and in vivo. Additionally, loss of CB2, the peripheral cannabinoid receptor, led to increased pathology and neutrophil influx in models of acute intestinal inflammation. These results define a key role for epithelial cells in balancing the constitutive secretion of antiinflammatory lipids with the stimulated secretion of proinflammatory lipids via surface efflux pumps in order to control neutrophil infiltration into the intestinal lumen and maintain homeostasis in the healthy intestine.