c Enterotoxigenic Escherichia coli (ETEC) is a major cause of morbidity and mortality due to infectious diarrhea in developing countries for which there is presently no effective vaccine. A central challenge in ETEC vaccinology has been the identification of conserved surface antigens to formulate a broadly protective vaccine. Here, we demonstrate that EatA, an immunogenic secreted serine protease of ETEC, contributes to virulence by degrading MUC2, the major protein present in the small intestinal mucous layer, and that removal of this barrier in vitro accelerates toxin access to the enterocyte surface. In addition, we demonstrate that vaccination with the recombinant secreted passenger domain of EatA (rEatA p ) elicits high titers of antibody and is protective against intestinal infection with ETEC. These findings may have significant implications for development of both subunit and live-attenuated vaccines against ETEC and other enteric pathogens, including Shigella flexneri, that express similar proteins.

dEnterotoxigenic Escherichia coli (ETEC) is a leading cause of death due to diarrheal illness among young children in developing countries, and there is currently no effective vaccine. Many elements of ETEC pathogenesis are still poorly defined. Here we demonstrate that YghJ, a secreted ETEC antigen identified in immunoproteomic studies using convalescent patient sera, is required for efficient access to small intestinal enterocytes and for the optimal delivery of heat-labile toxin (LT). Furthermore, YghJ is a highly conserved metalloprotease that influences intestinal colonization of ETEC by degrading the major mucins in the small intestine, MUC2 and MUC3. Genes encoding YghJ and its cognate type II secretion system (T2SS), which also secretes LT, are highly conserved in ETEC and exist in other enteric pathogens, including other diarrheagenic E. coli and Vibrio cholerae bacteria, suggesting that this mucin-degrading enzyme may represent a shared virulence feature of these important pathogens.

Many currently available diagnostic tests for typhoid fever lack sensitivity and/or specificity, especially in areas of the world where the disease is endemic. In order to identify a diagnostic test that better correlates with typhoid fever, we evaluated immune responses to Salmonella enterica serovar Typhi (serovar Typhi) in individuals with suspected typhoid fever in Dhaka, Bangladesh. We enrolled 112 individuals with suspected typhoid fever, cultured day 0 blood for serovar Typhi organisms, and performed Widal assays on days 0, 5, and 20. We harvested peripheral blood lymphocytes and analyzed antibody levels in supernatants collected on days 0, 5, and 20 (using an antibody-in-lymphocyte-supernatant [ALS] assay), as well as in plasma on these days. We measured ALS reactivity to a serovar Typhi membrane preparation (MP), a formalin-inactivated whole-cell preparation, and serovar Typhi lipopolysaccharide. We measured responses in healthy Bangladeshi, as well as in Bangladeshi febrile patients with confirmed dengue fever or leptospirosis. We categorized suspected typhoid fever individuals into different groups (groups I to V) based on blood culture results, Widal titer, and clinical features. Responses to MP antigen in the immunoglobulin A isotype were detectable at the time of presentation in the plasma of 81% of patients. The ALS assay, however, tested positive in all patients with documented or highly suspicious typhoid, suggesting that such a response could be the basis of improved diagnostic pointof-care-assay for serovar Typhi infection. It can be important for use in epidemiological studies, as well as in difficult cases involving fevers of unknown origin.Salmonella enterica serovar Typhi (serovar Typhi) is the cause of typhoid fever, an illness that affects over 20,000,000 individuals worldwide each year, killing over 200, 000 (5, 8, 16). The largest burden of typhoid fever is borne by impoverished individuals in resource-poor areas of the world. Serovar Typhi is a human-restricted invasive enteric pathogen which, after ingestion, crosses the intestinal mucosa, is taken up by gutassociated lymphoreticular tissues, and enters the systemic circulation. Both mucosal and systemic host immune responses are stimulated after infection. Serovar Typhi is an intracellular pathogen, and antibody and cell-mediated immune responses occur after infection or immunization with live oral attenuated typhoid vaccines (10,25,34).Diagnostic tests for typhoid fever often lack sensitivity and/or specificity, especially in areas of the world that are endemic for typhoid fever, where clinically distinguishing typhoid fever from other febrile illnesses is difficult (5, 17, 39). Microbiologic culturing of blood is approximately 30 to 70% sensitive, with the highest sensitivity being associated with an absence of prior use of antibiotics and the culturing of larger volumes of blood, features that complicate this mode of diagnosis in young children (5,6,8,36). Microbiologic culturing of bone marrow aspirates is more sensitive than bl...

As an ancient disease with high fatality, cholera has likely exerted strong selective pressure on affected human populations. We performed a genome-wide study of natural selection in a population from the Ganges River Delta, the historic geographic epicenter of cholera. We identified 305 candidate selected regions using the Composite of Multiple Signals (CMS) method. The regions were enriched for potassium channel genes involved in cyclic AMP-mediated chloride secretion and for components of the innate immune system involved in NF-κB signaling. We demonstrate that a number of these strongly selected genes are associated with cholera susceptibility in two separate cohorts. We further identify repeated examples of selection and association in an NF-kB / inflammasome-dependent pathway that is activated in vitro by Vibrio cholerae. Our findings shed light on the genetic basis of cholera resistance in a population from the Ganges River Delta and present a promising approach for identifying genetic factors influencing susceptibility to infectious diseases.

BackgroundEnterotoxigenic Escherichia coli (ETEC) are common causes of diarrheal morbidity and mortality in developing countries for which there is currently no vaccine. Heterogeneity in classical ETEC antigens known as colonization factors (CFs) and poor efficacy of toxoid-based approaches to date have impeded development of a broadly protective ETEC vaccine, prompting searches for novel molecular targets.MethodologyUsing a variety of molecular methods, we examined a large collection of ETEC isolates for production of two secreted plasmid-encoded pathotype-specific antigens, the EtpA extracellular adhesin, and EatA, a mucin-degrading serine protease; and two chromosomally-encoded molecules, the YghJ metalloprotease and the EaeH adhesin, that are not specific to the ETEC pathovar, but which have been implicated in ETEC pathogenesis. ELISA assays were also performed on control and convalescent sera to characterize the immune response to these antigens. Finally, mice were immunized with recombinant EtpA (rEtpA), and a protease deficient version of the secreted EatA passenger domain (rEatApH134R) to examine the feasibility of combining these molecules in a subunit vaccine approach.Principal FindingsEtpA and EatA were secreted by more than half of all ETEC, distributed over diverse phylogenetic lineages belonging to multiple CF groups, and exhibited surprisingly little sequence variation. Both chromosomally-encoded molecules were also identified in a wide variety of ETEC strains and YghJ was secreted by 89% of isolates. Antibodies against both the ETEC pathovar-specific and conserved E. coli antigens were present in significantly higher titers in convalescent samples from subjects with ETEC infection than controls suggesting that each of these antigens is produced and recognized during infection. Finally, co-immunization of mice with rEtpA and rEatApH134R offered significant protection against ETEC infection.ConclusionsCollectively, these data suggest that novel antigens could significantly complement current approaches and foster improved strategies for development of broadly protective ETEC vaccines.