Showing papers by "B. Brett Finlay published in 2010"

Gut Microbiota in Health and Disease

Abstract: Gut microbiota is an assortment of microorganisms inhabiting the length and width of the mammalian gastrointestinal tract. The composition of this microbial community is host specific, evolving throughout an individual's lifetime and susceptible to both exogenous and endogenous modifications. Recent renewed interest in the structure and function of this "organ" has illuminated its central position in health and disease. The microbiota is intimately involved in numerous aspects of normal host physiology, from nutritional status to behavior and stress response. Additionally, they can be a central or a contributing cause of many diseases, affecting both near and far organ systems. The overall balance in the composition of the gut microbial community, as well as the presence or absence of key species capable of effecting specific responses, is important in ensuring homeostasis or lack thereof at the intestinal mucosa and beyond. The mechanisms through which microbiota exerts its beneficial or detrimental influences remain largely undefined, but include elaboration of signaling molecules and recognition of bacterial epitopes by both intestinal epithelial and mucosal immune cells. The advances in modeling and analysis of gut microbiota will further our knowledge of their role in health and disease, allowing customization of existing and future therapeutic and prophylactic modalities.

Molecular mechanisms of Escherichia coli pathogenicity

Abstract: Escherichia coli is a remarkable and diverse organism. This normally harmless commensal needs only to acquire a combination of mobile genetic elements to become a highly adapted pathogen capable of causing a range of diseases, from gastroenteritis to extraintestinal infections of the urinary tract, bloodstream and central nervous system. The worldwide burden of these diseases is staggering, with hundreds of millions of people affected annually. Eight E. coli pathovars have been well characterized, and each uses a large arsenal of virulence factors to subvert host cellular functions to potentiate its virulence. In this Review, we focus on the recent advances in our understanding of the different pathogenic mechanisms that are used by various E. coli pathovars and how they cause disease in humans.

Muc2 Protects against Lethal Infectious Colitis by Disassociating Pathogenic and Commensal Bacteria from the Colonic Mucosa

Abstract: Despite recent advances in our understanding of the pathogenesis of attaching and effacing (A/E) Escherichia coli infections, the mechanisms by which the host defends against these microbes are unclear. The goal of this study was to determine the role of goblet cell-derived Muc2, the major intestinal secretory mucin and primary component of the mucus layer, in host protection against A/E pathogens. To assess the role of Muc2 during A/E bacterial infections, we inoculated Muc2 deficient (Muc2−/−) mice with Citrobacter rodentium, a murine A/E pathogen related to diarrheagenic A/E E. coli. Unlike wildtype (WT) mice, infected Muc2−/− mice exhibited rapid weight loss and suffered up to 90% mortality. Stool plating demonstrated 10–100 fold greater C. rodentium burdens in Muc2−/− vs. WT mice, most of which were found to be loosely adherent to the colonic mucosa. Histology of Muc2−/− mice revealed ulceration in the colon amid focal bacterial microcolonies. Metabolic labeling of secreted mucins in the large intestine demonstrated that mucin secretion was markedly increased in WT mice during infection compared to uninfected controls, suggesting that the host uses increased mucin release to flush pathogens from the mucosal surface. Muc2 also impacted host-commensal interactions during infection, as FISH analysis revealed C. rodentium microcolonies contained numerous commensal microbes, which was not observed in WT mice. Orally administered FITC-Dextran and FISH staining showed significantly worsened intestinal barrier disruption in Muc2−/− vs. WT mice, with overt pathogen and commensal translocation into the Muc2−/− colonic mucosa. Interestingly, commensal depletion enhanced C. rodentium colonization of Muc2−/− mice, although colonic pathology was not significantly altered. In conclusion, Muc2 production is critical for host protection during A/E bacterial infections, by limiting overall pathogen and commensal numbers associated with the colonic mucosal surface. Such actions limit tissue damage and translocation of pathogenic and commensal bacteria across the epithelium.

Quorum sensing in bacterial virulence

Abstract: Bacteria communicate through the production of diffusible signal molecules termed autoinducers. The molecules are produced at basal levels and accumulate during growth. Once a critical concentration has been reached, autoinducers can activate or repress a number of target genes. Because the control of gene expression by autoinducers is cell-density-dependent, this phenomenon has been called quorum sensing. Quorum sensing controls virulence gene expression in numerous micro-organisms. In some cases, this phenomenon has proven relevant for bacterial virulence in vivo. In this article, we provide a few examples to illustrate how quorum sensing can act to control bacterial virulence in a multitude of ways. Several classes of autoinducers have been described to date and we present examples of how each of the major types of autoinducer can be involved in bacterial virulence. As quorum sensing controls virulence, it has been considered an attractive target for the development of new therapeutic strategies. We discuss some of the new strategies to combat bacterial virulence based on the inhibition of bacterial quorum sensing systems.

The future of mucosal immunology: studying an integrated system-wide organ

Abstract: Over the next 10 years, it will be important to shift the focus of mucosal immunology research to make further advances. Examination of the mucosal immune system as a global organ, rather than as a group of individual components, will identify and characterize relationships between mucosal sites.

The pathogenic E. coli type III effector EspZ interacts with host CD98 and facilitates host cell prosurvival signalling

Abstract: Enterohaemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC respectively) are diarrhoeal pathogens that cause the formation of attaching and effacing (A/E) lesions on infected host cells. These pathogens encode a type III secretion system (T3SS) used to inject effector proteins directly into host cells, an essential requirement for virulence. In this study, we identified a function for the type III secreted effector EspZ. Infection with EPEC DeltaespZ caused increased cytotoxicity in HeLa and MDCK cells compared with wild-type EPEC, and expressing espZ in cells abrogated this effect. Using yeast two-hybrid, proteomics, immunofluorescence and co-immunoprecipitation, it was demonstrated that EspZ interacts with the host protein CD98, which contributes to protection against EPEC-mediated cytotoxicity. EspZ enhanced phosphorylation of focal adhesion kinase (FAK) and AKT during infection with EPEC, but CD98 only appeared to facilitate FAK phosphorylation. This study provides evidence that EspZ and CD98 promote host cell survival mechanisms involving FAK during A/E pathogen infection.

Should the Human Microbiome Be Considered When Developing Vaccines

Abstract: The human microbiome, especially in the intestinal tract has received increased attention in the past few years due to its importance in numerous biological processes. Recent advances in DNA sequencing technology and analysis now allow us to better determine global differences in the composition of the gut microbial population, and ask questions about its role in health and disease. Thus far, roles of these commensal bacteria on nutrient acquisition, vitamin production, and intestinal development have been identified [1]. Furthermore, resistance or susceptibility to a number of diseases, including inflammatory bowel disease, obesity, enteric infections, and most recently ectopic diseases, have been linked to the intestinal microbiota [1], [2]. Data on the mechanisms through which the intestinal microbiota impacts host immune development have also begun to emerge [2]. The impact of the intestinal microbiota on host physiology is undeniable, and experiments using germ-free, mono-, and poly-colonized mice have addressed many aspects of the microbiota's influence on the mammalian immune system. Despite all the increased attention on the interface between the microbiota and host immune responses, it is still unclear whether these commensal bacteria affect the efficacy of vaccines. Due to its impact in the development of immune function, both in the gut and other organs, it is reasonable to consider that the intestinal microbiota will significantly affect how individuals respond to vaccine antigens [3], [4]. For example, segmented filamentous bacteria present in the intestinal microbiota have been shown to induce maturation of intestinal T cell adaptive functions [5]. Moreover, it has been shown that the intestinal microbiota exerts a profound effect on the metabolism of certain drugs and toxins [1], [6], and this may also indicate that oral vaccines could be differentially processed by the body depending on variations in microbial communities between individuals. Hence, the microbiota could be an underappreciated yet important player to consider in the development of vaccines, and also may help explain some of the discrepancies observed in vaccine efficacy in different populations around the world. Clinical trials testing the efficacy of oral vaccines against polio, rotavirus, and cholera have showed a lower immunogenicity of these vaccines in individuals from developing countries when compared to individuals from the developed world [7]–[11]. Clinical trials for a killed oral cholera vaccine in Swedish and Nicaraguan children have also shown blunted antibody responses in Nicaraguan children compared to Swedish children [11]. In a study testing a live cholera oral vaccine, Lagos and colleagues [12] demonstrated that excessive bacterial growth in the small intestine of children in less developed countries might contribute to the low antibody response to the vaccine. Different vaccine strains of Shigella flexneri also showed differential protection on individuals from developing countries. In a study testing Bangladeshi adults and children, no significant immune response to this vaccine was mounted, although the same antigen was reactogenic in North American individuals [13]. Altogether, these data highlight that individuals from different parts of the world can mount different immune responses to the same vaccine. Several hypotheses that may explain this phenomenon exist. For instance, socioeconomic conditions, nutritional status, host genetics, and earlier exposure to related microorganisms are some of the aspects that could contribute to the disparity in the vaccine efficacies in different populations. However, one poorly explored possibility is that the composition of the intestinal microbiota of these individuals may also be a determining factor of vaccine efficacy. In a way analogous to the hygiene hypothesis [14], which states that reduced exposure to microorganisms at an early age may lead to increased susceptibility to allergies, it is possible that the gut microbiota of individuals with increased exposure to microorganisms (and therefore antigens) make them more tolerant to vaccination, being unable to mount a proper response compared to individuals living in better socioeconomic conditions. Discerning the effects of genetic and environmental factors on vaccine efficacy is a challenging task. Large clinical trials involving individuals from different areas of the world will likely be required to shed light on whether the blunt immune responses to some of the oral vaccines mentioned herein are a consequence of genetic factors or environmental variations, such as the gut microbial community. Studies involving immigrant volunteers could be useful in addressing this issue by providing a clear distinction between the effects of genetics and the environment. Although this is still an open question, data in the literature suggest a more direct link between the intestinal microbiota composition and the development of immune responses to certain vaccine antigens. For instance, the use of antibiotics in chickens has been shown to increase the antibody response following immunization [15]. Because antibiotic treatment will have profound effects on the intestinal microbiota, it is tempting to hypothesize that the microbial populations of these animals are important players in their immunological response to the vaccine antigens. Furthermore, certain probiotic strains have been shown to enhance antibody responses to oral vaccines against rotavirus [16], Salmonella [17], polio [18], and cholera [19] in human volunteers, and this effect was observed after a short period (1–5 weeks) of probiotic treatment. The positive effect of probiotics on immune responses was also seen in parenterally administered vaccines against diphtheria, tetanus, Haemophilus influenzae type B, and hepatitis B [20]–[22] in infants after a 6-month period. Because of the number of licensed oral-administered human vaccines available is limited, studies on how the intestinal microbiota affect parenterally administered human vaccines would have a more significant impact on human health. However, in all studies cited above, there was no long-term follow-up on the enhanced effects of the probiotics on vaccine efficacy. Additionally, more detailed studies on the establishment of the probiotic strains within the resident microbiota will be required to establish minimal doses and treatment regimens, important aspects that need to be addressed if the microbiota is to be considered in vaccine development in the future. It has also been suggested that prebiotics, which are compounds that can enhance the proliferation of certain commensals, can enhance the efficacy of oral vaccines. Recently, a well-studied fructo-oligosaccharide prebiotic has been shown to improve the efficacy of a vaccine against Salmonella infection [23]. In this study, administration of the prebiotic prior to vaccination improved host responses and rates of protection against infection in mice. Unfortunately, the authors were unable to show significant changes in microbiota composition, possibly due to the lack of detailed analyses. In another study, Vos et al. [24] showed that a prebiotic mixture containing galacto- and fructo-oligosaccharides enhanced systemic adaptive immune responses in a murine influenza vaccination model. In this case, increased proportions of certain members of the microbiota could be observed, suggesting a role for the microbial community in the increased host immune response. Although some studies indicate that the microbiota may play an important role in vaccine efficacy, this area of research is still in its infancy. For instance, the mechanisms involved in the pro- and prebiotic enhancement of vaccine efficacy mentioned above are largely unknown. Nevertheless, current knowledge of the effect of the intestinal microbiota on the development of not only local but also systemic immune functions provides a direct link between commensal populations in the intestine and immune responses to vaccine antigens [3], [4]. We now have the tools to study and take advantage of what the microbiota has to offer in order to enhance host responses to vaccines, with the use of probiotics or prebiotics as adjuvants. Studies using animal models with defined intestinal microbial communities can be helpful to evaluate the effect of intestinal commensals on the immune response to vaccines. However, animal models can only partially elucidate this issue and, although cumbersome, studies in human volunteers will be essential in defining the effect of commensals in vaccine efficacy. We suggest that the study of the relationships between individual commensal populations of humans and responses to vaccines will be instrumental in our quest to improve general vaccine development. By taking into consideration the microbial populations of geographically diverse groups of individuals, we may be able to develop better-targeted vaccines that will improve protection against multiple pathogens.

EseG, an Effector of the Type III Secretion System of Edwardsiella tarda, Triggers Microtubule Destabilization

Abstract: Edwardsiella tarda is a Gram-negative enteric pathogen that causes hemorrhagic septicemia in fish and both gastrointestinal and extraintestinal infections in humans. A type III secretion system (T3SS) was recently shown to contribute to pathogenesis, since deletions of various T3SS genes increased the 50% lethal dose (LD50) by about 1 log unit in the blue gourami infection model. In this study, we report EseG as the first identified effector protein of T3SS. EseG shares partial homology with two Salmonella T3SS effectors (SseG and SseF) over a conserved domain (amino acid residues 142 to 192). The secretion of EseG is dependent on a functional T3SS and, in particular, requires the chaperone EscB. Experiments using TEM-1 beta-lactamase as a fluorescence-based reporter showed that EseG was translocated into HeLa cells at 35 degrees C. Fractionation of infected HeLa cells demonstrated that EseG was localized to the host membrane fraction after translocation. EseG is able to disassemble microtubule structures when overexpressed in mammalian cells. This phenotype may require a conserved motif of EseG (EseG(142-192)), since truncated versions of EseG devoid of this motif lose their ability to cause microtubule destabilization. By demonstrating the function of EseG, our study contributes to the understanding of E. tarda pathogenesis. Moreover, the approach established in this study to identify type III effectors can be used to identify and characterize more type III and possible type VI effectors in Edwardsiella.

Salmonella SPI-1-mediated neutrophil recruitment during enteric colitis is associated with reduction and alteration in intestinal microbiota.

Abstract: Gastrointestinal infections involve an interactive tripartite relationship between the invading pathogen, the host, and the host's resident intestinal microbiota. To characterize the host inflammatory response and microbiota alterations during enteric salmonellosis, C57BL/6 mice were pre-treated with a low dose of streptomycin (LD model) and then infected with S. typhimurium strains, including mutants in the two Type III secretion systems, SPI-1 and SPI-2 (invAmut and ssaRmut, respectively). Cecal colonization and inflammation in the LD model were evaluated to assess infection success and progression, and compared to the traditional high dose (HD) model. Perturbations to the microbial community in the LD model were assessed via evaluation of total microbial numbers, the proportion of intestinal γ-Proteobacteria and tRFLP analysis. In the LD model, consistently high colonization by the parental strain (WT) and invAmut S. typhimurium was associated with significant intestinal pathology. However, microbial community profiles were more similar both in numbers and composition between mice infected with the mutant strains, than with the WT strain. Consequently, significant infection-induced inflammation did not always produce similar microbiota perturbations. Large numbers of luminal neutrophils were observed in the ceca of WT-infected, but not in invAmut or ssaRmut infected mice. Neutrophils were thus implicated as a potential mediator of microbiota perturbations during WT enteric salmonellosis. These studies offer a new model of S. typhimurium-induced intestinal disease that retains the three participants of the disease process and further defines the role of virulence factors, the host microbiota, and inflammation in S. typhimurium-induced intestinal disease.

Functional analysis, overexpression, and kinetic characterization of pyruvate kinase from methicillin-resistant Staphylococcus aureus.

Abstract: Novel antimicrobial targets are urgently needed to overcome rising antibiotic resistance of important human pathogens including methicillin-resistant Staphylococcus aureus (MRSA). Here we report the essentiality and kinetic properties of MRSA pyruvate kinase (PK). Targetron-mediated gene disruption demonstrated PK is essential for S. aureus growth and survival, suggesting that this protein may be a potential drug target. The presence of the pfk (6-phosphofructokinase)-pyk operon in MRSA252, and the nonessential nature of PFK shown by targetron, further emphasized the essential role of PK in cell viability. The importance of PK in bacterial growth was confirmed by showing that its enzymatic activity peaked during the logarithmic phase of S. aureus growth. PK from Staphylococcus and several other species of bacteria have an extra C-terminal domain (CT) containing a phosphoenolpyruvate (PEP) binding motif. To elucidate the possible structure and function of this sequence, the quaternary structures and kinetic properties of the full-length MRSA PK and truncated MRSA PK lacking the CT domain were characterized. Our results showed that (1) MRSA PK is an allosteric enzyme with homotetramer architecture activated by AMP or ribose 5-phosphate (R5P), but not by fructose 1,6-bisphosphate (FBP), which suggests a different mode of allosteric regulation when compared with human isozymes, (2) the CT domain is not required for the tetramerization of the enzyme; homotetramerization occurred in a truncated PK lacking the domain, (3) truncated enzyme exhibited high affinity toward both PEP and ADP and exhibited hyperbolic kinetics toward PEP in the presence of activators (AMP and R5P) consistent with kinetic properties of full-length enzyme, indicating that the CT domain is not required for substrate binding or allosteric regulation observed in the holoenzyme, (4) the kinetic efficiency (k(cat)/S(0.5)) of truncated enzyme was decreased by 24- and 16-fold, in ligand-free state, toward PEP and ADP, respectively, but was restored by 3-fold in AMP-bound state, suggesting that the sequence containing the CT domain (Gly(473)-Leu(585)) plays a substantial role in enzyme activity and comformational stability, and (5) full-length MRSA PK activity was stimulated at low concentrations of ATP (e.g., 1 mM) and inhibited by inorganic phosphate and high concentrations of FBP (10 mM) and ATP (e.g., >2.5 mM), whereas for truncated enzyme, stimulation at low concentrations of ATP was lost. These findings suggest that the CT domain is involved in maintaining the specificity of allosteric regulation of MRSA PK by AMP, R5P, and ATP. The CT extension also encodes a protein domain with homology to enzyme I of the Escherichia coli sugar-PTS system, suggesting that MRSA PK may also exert an important regulatory role in sugar transport metabolism. These findings yield new insights into MRSA PK function and mode of allosteric regulation which may aid in the development of clinically important drugs targeting this enzyme and further define the role of the extra C-terminal domain in modulating the enzyme's activity.

Metabolomics: towards understanding host–microbe interactions

Abstract: Metabolomics employs an array of analytical techniques, including high-resolution nuclear magnetic resonance spectroscopy and mass spectrometry, to simultaneously analyze hundreds to thousands of small-molecule metabolites in biological samples. In conjunction with chemoinformatics and bioinformatics tools, metabolomics enables comprehensive characterization of the metabolic phenotypes (metabotypes) of the human, and other mammalian, hosts that have co-evolved with a large number of diverse commensal microbes, especially in the intestinal tract. Correlation of the metabotypes with the microbial profiles derived from culture-independent molecular techniques is increasingly helping to decipher inherent and intimate host–microbe relationships. This integrated, systems biology approach is improving our understanding of the molecular mechanisms underlying multilevel host–microbe interactions, and promises to elucidate the etiologies of human disorders resulting from unfavorable human–microbial associations, in...

Gap junction hemichannels contribute to the generation of diarrhoea during infectious enteric disease.

Abstract: Objective: The attaching and effacing (A/E) pathogens enterohemorrhagic E. coli , enteropathogenic E. coli and C. rodentium colonize intestinal tracts, attach to enterocytes, collapse infected cell microvilli and alter numerous host cell processes during infection. Enterocyte alterations result in numerous small molecules being released from host cells that likely contribute to diarrheal phenotypes observed during these infections. One possible route for small molecules to be released from intestinal cells may be through functional gap junction hemichannels. Here we examine the involvement of these hemichannels during the diarrheal disease caused by A/E pathogens in vivo . Design: Mice were infected with the diarrhea-causing murine A/E pathogen C. rodentium for 7 days. Connexin43 (Cx43) protein levels and immunolocalization in the colon were initially used to determine alterations during A/E bacterial infections in vivo . Connexin mimetic peptides and connexin permeable tracer molecules were used to gage the presence and function of unpaired connexin hemichannels. The role of Cx43 in diarrhea generation was assessed by comparing infections of wild-type mice to Cx43 mutant mice and determining the water abundance in the colonic luminal material. Results: We demonstrate that Cx43 protein levels are increased in colonocytes during in vivo A/E bacterial infections, resulting in functionally open connexon hemichannels in apical membranes of infected cells. Moreover, infected Cx43 +/- mice do not suffer from diarrheal disease. Conclusions: This study provides the first evidence that functional connexon hemichannels can occur in the intestine and are a novel molecular mechanism of water release during infectious diarrhea.

Salmonella enterica serovar Typhimurium-induced placental inflammation and not bacterial burden correlates with pathology and fatal maternal disease.

Abstract: Food-borne infections caused by Salmonella enterica species are increasing globally, and pregnancy poses a high risk. Pregnant mice rapidly succumb to S. enterica serovar Typhimurium infection. To determine the mechanisms involved, we addressed the role of inflammation and bacterial burden in causing placental and systemic disease. In vitro, choriocarcinoma cells were a highly conducive niche for intracellular S. Typhimurium proliferation. While infection of mice with S. Typhimurium wild-type (WT) and mutant (ΔaroA and ΔinvA) strains led to profound pathogen proliferation and massive burden within placental cells, only the virulent WT S. Typhimurium infection evoked total fetal loss and adverse host outcome. This correlated with substantial placental expression of granulocyte colony-stimulating factor (G-CSF), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF-α) and increased serum inflammatory cytokines/chemokines, such as G-CSF, IL-6, CCL1, and KC, evoked by WT S. Typhimurium infection. In contrast, infection with high doses of S. Typhimurium ΔaroA, despite causing massive placental infection, resulted in reduced inflammatory cellular and cytokine response. While S. Typhimurium WT bacteria were dispersed in large numbers across all regions of the placenta, including the deeper labyrinth trophoblast, S. Typhimurium ΔaroA bacteria localized primarily to the decidua. This correlated with the widespread placental necrosis accompanied by neutrophil infiltration evoked by the S. Typhimurium WT bacteria. Thus, the ability of Salmonella to localize to deeper layers of the placenta and the nature of inflammation triggered by the pathogen, rather than bacterial burden, profoundly influenced placental integrity and host survival.

The role of the immune system in regulating the microbiota

Abstract: A diverse population of bacteria, archaea and fungi, collectively known as the microbiota, abounds within the gastrointestinal tract of the mammalian host. This microbial population makes many important contributions to host physiology through inter-kingdom signalling and by providing nutrients that have both local and systemic effects. In a healthy state the overall host-microbial interaction is symbiotic; however, a growing number of diseases have been associated with a dysregulated microbiota. To avoid these consequences, the host exerts substantial effort to maintain proper regulation of the microbiota with respect to localization and composition. Although important to maintaining microbial balance, the host immune response can also be the cause of a disrupted microbiota, contributing to disease severity. Here, we discuss the role of the host in both maintaining and disrupting a balanced gastrointestinal microbiota.

Inhibition of Salmonella host cell invasion by dimethyl sulfide

Abstract: We show that dimethyl sulfoxide (DMSO) inhibits Salmonella hilA expression and that this inhibition is stronger under anaerobiosis. Because DMSO can be reduced to dimethyl sulfide (DMS) during anaerobic growth, we hypothesized that DMS was responsible for hilA inhibition. Indeed, DMS strongly inhibited the expression of hilA and multiple Salmonella pathogenicity island 1 (SPI-1)-associated genes as well as the invasion of cultured epithelial cells. Because DMSO and DMS are widespread in nature, we hypothesize that this phenomenon may contribute to environmental sensing by Salmonella.

Proteomics as a probe of microbial pathogenesis and its molecular boundaries

Abstract: Proteomic technology offers an unprecedented systematic approach to investigate the protein complement of any organism. The field of microbial pathogenesis has greatly benefited from other systems approaches, and the application of proteomics to the study of infectious agents is beginning to emerge. Such applications include unambiguously identifying complete virulence factor inventories, studying the response of both host and pathogen to the infection process and elucidating mechanistic actions of virulence factors as they interface with host cells. This review will highlight examples where proteomic studies have contributed to our understanding of pathogenesis in these areas, with an emphasis on pathogens that employ type III and type IV secretion systems. In addition, we will discuss areas where proteomics may help shape further investigation and discovery in this field.

Defensins keep the peace too.

Abstract: The mammalian intestine contains a large number of commensal bacterial strains. New work suggests that antimicrobial peptides used for defense against pathogenic bacteria are also used to adjust the balance among bacterial populations and to control intestinal homeostasis.

Role for CD2AP and Other Endocytosis-Associated Proteins in Enteropathogenic Escherichia coli Pedestal Formation

Abstract: Enteropathogenic Escherichia coli (EPEC) strains are extracellular pathogens that generate actin-rich structures (pedestals) beneath the adherent bacteria as part of their virulence strategy. Pedestals are hallmarks of EPEC infections, and their efficient formation in vitro routinely requires phosphorylation of the EPEC effector protein Tir at tyrosine 474 (Y474). This phosphorylation results in the recruitment and direct attachment of the host adaptor protein Nck to Tir at Y474, which is utilized for actin nucleation through a downstream N-WASP-Arp2/3-based mechanism. Recently, the endocytic protein clathrin was demonstrated to be involved in EPEC pedestal formation. Here we examine the organization of clathrin in pedestals and report that CD2AP, an endocytosis-associated and cortactin-binding protein, is a novel and important component of EPEC pedestal formation that also utilizes Y474 phosphorylation of EPEC Tir. We also demonstrate the successive recruitment of Nck and then clathrin prior to actin polymerization at pedestals during the Nck-dependent pathway of pedestal formation. This study further demonstrates that endocytic proteins are key components of EPEC pedestals and suggests a novel endocytosis subversion strategy employed by these extracellular bacteria.

The role of Tir, EspA, and NleB in the colonization of cattle by Shiga toxin producing Escherichia coli O26:H11.

Abstract: Shiga toxin producing Escherichia coli (STEC) O26:H11 is an enteric pathogen capable of causing severe hemorrhagic colitis that can lead to hemolytic uremic syndrome. This organism is able to colon...

CD34 mediates intestinal inflammation in Salmonella-infected mice.

Abstract: Summary CD34 is a highly glycosylated sialomucin expressed on a variety of cells, ranging from vascular endothelial cells to haematopoietic stem cells Depending on its glycosylation state, CD34 has been shown to promote or inhibit cell adhesion and migration; however, a functional role for CD34 in the gut has not been determined Using a model of Salmonella-induced gastroenteritis, we investigated the role of CD34 in the context of infection Upon oral infection, the number of CD34+ cells detected in the submucosa, vascular endothelium and lamina propria significantly increased in S Typhimurium-infected C57Bl/6 mice The pathology of S Typhimurium-infected C57Bl/6 mice was characterized by recruitment of neutrophils to the site of inflammation, submucosal oedema and crypt destruction In contrast, Cd34−/− mice showed a delayed pathology, a defect in inflammatory cell migration into the intestinal tissue and enhanced survival Importantly, this was not due to a lack of chemotactic signals in Cd34−/− mice as these mice had either similar or significantly higher levels of pro-inflammatory cytokines and chemokines post infection when compared with infected C57/Bl6 control mice In summary, we demonstrate a novel role for CD34 in enhancing migration of inflammatory cells and thereby exacerbating host-mediated immunopathology in the intestine of S Typhimurium-infected mice

Breaking the Stereotype: Virulence Factor–Mediated Protection of Host Cells in Bacterial Pathogenesis

Abstract: Bacterial pathogens have evolved extraordinary mechanisms to efficiently infect host organisms. A majority of these pathogens do so by delivering virulence factors into host cells, which act to dampen host defenses or utilize the host as a niche for replication. Although regulation of virulence factor expression by bacterial pathogens is a well known pathogenic mechanism [1], the concept of host-protective virulence factors is emerging. Recently, several strategies by which pathogens appear to be attenuating their own lethality towards host cells have been documented, suggesting that increased hostility and damage of host cells is not necessarily beneficial to the pathogen. Virulence is often defined as the ability of a pathogen to inflict damage on host cells, and the following discussion addresses the concept that increased virulence is not always beneficial to the pathogen, and moderating it to preserve host cells is a mechanism several pathogens use as part of their overall pathogenic strategy. This strategy is well known for obligate intracellular pathogens, but has become an emerging theme in extracellular and facultative intracellular bacteria. Yersinia spp., Shigella flexneri, Helicobacter pylori, and diarrheagenic Escherichia coli are well known for their ability to kill host cells. For Yersinia, death of infected macrophages dampens cytokine release and enables the pathogen to propagate with minimal challenges from the immune system [2]. Two recent studies suggest that cytotoxicity caused by Yersinia species is tightly regulated. Yersinia pestis, the etiologic agent of plague, and gastroenteritis-inducing Yersinia pseudotuberculosis and Yersinia enterocolitica all encode a cytotoxic virulence factor called YopJ/P (YopJ in the two former species and YopP in the latter), which are translocated into infected cells via a type III secretion system (T3SS) [2], [3]. Altering the cytotoxicity of Y. pseudotuberculosis affects its virulence. Decreased secretion of YopJ was shown to enhance Y. pseudotuberculosis pathogenesis in vivo [4]. Similarly for Y. pestis, enhanced cytotoxicity results in decreased incidence of pneumonic plague in vivo [5]. Tight regulation of cytotoxicity by pathogenic Yersinia is an efficient virulence strategy. Increased apoptosis of infected immune cells decreases production of proinflammatory cytokines; however, some inflammation at the early stages of infection is thought to facilitate tissue damage necessary for movement of bacteria and infected cells to other sites of replication within the host [4]. Enteropathogenic Escherichia coli, enterohaemorrhagic E. coli (EPEC and EHEC, respectively), and Citrobacter rodentium are attaching and effacing (A/E) pathogens that cause severe diarrheagenic disease [6]. The ability of A/E pathogens to kill intestinal epithelial cells has been well documented [7]–[14]. The type III secreted (T3S) effector EspF has a role in host cell death by causing mitochondrial-dependent apoptosis [11], [15]. We recently found that the T3S effector EspZ modulates cytotoxicity towards host cells. An EPEC espZ mutant (ΔespZ) caused enhanced cytotoxicity in host cells when compared to the wild-type strain [16], which was surprising since the ΔespZ strain is severely attenuated for virulence in vivo [17]. EspZ acts in part through the host transmembrane glycoprotein CD98 to activate focal adhesion kinase (FAK)-based survival pathways (Figure 1) [16]. Others found that the T3S effector NleH also dampens apoptosis of EPEC-infected cells, but via interaction with a Bcl-2-related protein involved in the mitochondrial death pathway (Figure 1) [18]. Unlike EspZ, NleH is not essential for EPEC colonization and only moderately impacts on A/E pathogen disease in vivo [19], [20]; however, there are likely other host-protective virulence factors that act redundantly to NleH during EPEC infection. Figure 1 Strategies evolved by bacterial pathogens to restrain virulence. H. pylori causes apoptosis of infected gastric epithelial cells [21]. Apoptosis induction by H. pylori has been linked to a secreted toxin called VacA, which induces cytochrome c release from mitochondria (Figure 1) [22]. Recently, it was determined that VacA-mediated apoptosis is counteracted by a type IV secreted (T4S) protein called CagA by both blocking pinocytosis of VacA and inhibiting VacA-mediated cytochrome c release from mitochondria [23] (Figure 1). Interestingly, loss of CagA in a VacA+ H. pylori strain decreases bacterial colonization and the incidence of gastric hyperplasia, adenocarcinoma, and inflammation [24]. Similar to the aforementioned pathogens, H. pylori has evolved a delicate interplay between host-protective and -detrimental virulence factors that are able to fine-tune virulence while promoting their propagation. S. flexneri, the etiologic agent of bacilliary dysentery, causes death of infected macrophages and epithelial cells [25]. Despite this, several host-protective strategies are employed by S. flexneri. The T3S effector OspE was recently found to enhance adhesion of infected host cells to the underlying extracellular matrix [26]. Whether OspE activates host cell survival pathways directly is unknown; however, its interaction with integrin-linked kinase inhibits sloughing of infected cells into the intestinal lumen [26], consequently preventing anoikis of Shigella-infected cells. An ospE mutant does not colonize as efficiently as wild-type S. flexneri in vivo; thus, OspE may enhance colonization by preventing premature release of infected cells [26]. Epithelial cells succumb to S. flexneri infection via necrotic cell death, which functions to release intracellular bacteria and enhance inflammation [25]. Interestingly, survival pathways involving Rip2/IKKβ/NFκB are activated early during infection, followed by mitochondrial dysfunction and necrotic cell death (Figure 1) [25]. The early expression of pro-survival genes may enable S. flexneri to postpone cell death in a similar manner to EPEC, thus ensuring greater bacterial load prior to dissemination. The mechanism(s) by which S. flexneri enhances NFκB-mediated pro-survival signals are unknown. All of the above pathogens have evolved strategies to attenuate their own host-damaging virulence factors. In many of these scenarios, removal of host-protective mediators actually reduces pathogenicity of the bacteria. The observation that EPEC encodes a host-protective virulence factor that is essential for its pathogenesis suggests that protecting host cells may be a key to the pathogenic strategies of other bacterial pathogens. The concept of host-protective virulence factors is only just emerging, and we believe host-protective virulence factors will become more apparent in other pathogenic strategies and may become interesting targets to combat bacterial disease. Importantly, virulence phenotypes that appear counterintuitive should not be ignored. Future studies into pathogenic mechanisms of virulent bacteria will likely reveal important roles for effectors or regulatory mechanisms that help the host cell and promote bacterial pathogenesis.

The art of bacterial warfare.

Abstract: The article reports on research into pathogenic bacteria, focusing on the evolutionary relationships between bacterial behaviors and toxins, and the immune systems of their hosts. Examples discussed include the mechanisms by which microorganisms such as Escherichia coli and Helicobacter pylori cause damage in humans. INSETS: Outwitting the Guards;Targeting Bacterial Weapons.

Impaired innate immune response and enhanced pathology during Citrobacter rodentium infection in mice lacking functional P-selectin.

Abstract: The selectin family of adhesion molecules mediates recruitment of immune cells to sites of inflammation which is critical for host resistance against infection. To characterize the role of selectins in host defence against Citrobacter rodentium infection, wild-type (WT) mice and mice lacking P-selectin glycoprotein ligand-1 (PSGL-1), P-, E- and L-selectin were infected using a Citrobacter-induced colitis model. Infected mice lacking PSGL-1 or P-selectin showed a more pronounced morbidity associated with higher bacterial load, elevated IL-12 p70, TNF-alpha, IFN-gamma, MCP-1 and IL-6 production, more severe inflammation and surprisingly higher leucocyte infiltration in the guts than WT control. Recruitment of neutrophils and macrophages and caecal inflammation were drastically reduced in infected P-selectin knockout mice receiving blocking monoclonal antibodies to ICAM-1 or LFA-1, indicating that these adhesion molecules may compensate for the loss of selectins in leucocyte recruitment. Furthermore, the adaptive immune response in mice lacking PSGL-1 or P-selectin remained functional since these infected mice were capable of eradicating the bacteria and being protected upon re-challenge with C. rodentium. These data demonstrate a definitive phenotypic impairment of innate response in mice lacking PSGL-1 or P-selectin, and suggest that these adhesion molecules are important in host innate immune response against Citrobacter infection.

Robust TLR4-induced gene expression patterns are not an accurate indicator of human immunity

Abstract: Background: Activation of Toll-like receptors (TLRs) is widely accepted as an essential event for defence against infection. Many TLRs utilize a common signalling pathway that relies on activation of the kinase IRAK4 and the transcription factor NFB for the rapid expression of immunity genes. Methods: 21 K DNA microarray technology was used to evaluate LPS-induced (TLR4) gene responses in blood monocytes from a child with an IRAK4-deficiency. In vitro responsiveness to LPS was confirmed by real-time PCR and ELISA and compared to the clinical predisposition of the child and IRAK4-deficient mice to Gram negative infection. Results: We demonstrated that the vast majority of LPS-responsive genes in IRAK4-deficient monocytes were greatly suppressed, an observation that is consistent with the described role for IRAK4 as an essential component of TLR4 signalling. The severely impaired response to LPS, however, is inconsistent with a remarkably low incidence of Gram negative infections observed in this child and other children with IRAK4-deficiency. This unpredicted clinical phenotype was validated by demonstrating that IRAK4-deficient mice had a similar resistance to infection with Gram negative S. typhimurium as wildtype mice. A number of immunity genes, such as chemokines, were expressed at normal levels in human IRAK4-deficient monocytes, indicating that particular IRAK4-independent elements within the repertoire of TLR4-induced responses are expressed. Conclusions: Sufficient defence to Gram negative immunity does not require IRAK4 or a robust, ‘classic’ inflammatory and immune response.