Alaullah Sheikh

Enterotoxigenic Escherichia coli Secretes a Highly Conserved Mucin-Degrading Metalloprotease To Effectively Engage Intestinal Epithelial Cells

ABSTRACT Enterotoxigenic 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.

EatA, an Immunogenic Protective Antigen of Enterotoxigenic Escherichia coli, Degrades Intestinal Mucin

ABSTRACT 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 (rEatAp) 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.

Transcriptional Modulation of Enterotoxigenic Escherichia coli Virulence Genes in Response to Epithelial Cell Interactions

ABSTRACT Enterotoxigenic Escherichia coli (ETEC) strains are a leading cause of morbidity and mortality due to diarrheal illness in developing countries. There is currently no effective vaccine against these important pathogens. Because genes modulated by pathogen-host interactions potentially encode putative vaccine targets, we investigated changes in gene expression and surface morphology of ETEC upon interaction with intestinal epithelial cells in vitro. Pan-genome microarrays, quantitative reverse transcriptase PCR (qRT-PCR), and transcriptional reporter fusions of selected promoters were used to study changes in ETEC transcriptomes. Flow cytometry, immunofluorescence microscopy, and scanning electron microscopy were used to investigate alterations in surface antigen expression and morphology following pathogen-host interactions. Following host cell contact, genes for motility, adhesion, toxin production, immunodominant peptides, and key regulatory molecules, including cyclic AMP (cAMP) receptor protein (CRP) and c-di-GMP, were substantially modulated. These changes were accompanied by visible changes in both ETEC architecture and the expression of surface antigens, including a novel highly conserved adhesin molecule, EaeH. The studies reported here suggest that pathogen-host interactions are finely orchestrated by ETEC and are characterized by coordinated responses involving the sequential deployment of multiple virulence molecules. Elucidation of the molecular details of these interactions could highlight novel strategies for development of vaccines for these important pathogens.

Natural Selection in a Bangladeshi Population from the Cholera-Endemic Ganges River Delta

Natural selection in a Bangladeshi population from the cholera-endemic Ganges River Delta has targeted genes associated with cholera resistance and an innate immunity pathway activated by Vibrio cholerae. Modern Lessons from an Ancient Disease A history of natural selection favoring resistance to an infectious disease should drive the emergence of underlying genetic variants that can be readily detected. In a new study, Karlsson et al. show this for cholera, an ancient, often fatal disease that likely exerted selection pressure on Bangladeshi populations living in the Ganges River Delta where cholera is endemic. The authors combine a selection scan with an association study of cholera susceptibility, and translate the resulting genetic discoveries into clinically relevant biology. They performed whole-genome scans of Bangladeshi families to identify 305 genomic regions of selection. These regions are highly enriched for potassium channel genes and genes in the NF-κB pathway, a master regulator of inflammation and immunity that is also involved in protecting the lining of the gut. They show, by comparing cholera-affected and healthy individuals, that top selected genes correlate with cholera susceptibility. These genes regulate an innate immune signaling pathway that is activated by Vibrio cholerae, the pathogen that causes cholera, and is repeatedly targeted by selection. This combined selection and association approach identifies genes not previously implicated in the cholera host response and highlights the role of innate immunity and intestinal homeostasis in disease pathogenesis. This approach of leveraging ancient history in genetic studies is applicable to many other ancient infectious diseases still circulating in the population today. 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 adenosine monophosphate–mediated chloride secretion and for components of the innate immune system involved in nuclear factor κB (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-κB/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.

Novel antigens for enterotoxigenic Escherichia coli vaccines

Enterotoxigenic Escherichia coli (ETEC) are the most common bacterial pathogens causing diarrhea in developing countries where they lead to hundreds of thousands of deaths, mostly in children. These organisms are a leading cause of diarrheal illness in travelers to endemic countries. ETEC pathogenesis, and consequently vaccine approaches, have largely focused on plasmid-encoded enterotoxins or fimbrial colonization factors. To date these approaches have not yielded a broadly protective vaccine. However, recent studies suggest that ETEC pathogenesis is more complex than previously appreciated and involves additional plasmid and chromosomally encoded virulence molecules that can be targeted in vaccines. Here, we review recent novel antigen discovery efforts, potential contribution of these proteins to the molecular pathogenesis of ETEC and protective immunity, and the potential implications for development of next generation vaccines for important pathogens. These proteins may help to improve the effectiveness of future vaccines by making them simpler and possibly broadly protective because of their conserved nature.

Examination of the Enterotoxigenic Escherichia coli Population Structure during Human Infection

ABSTRACT Enterotoxigenic E. coli (ETEC) can cause severe diarrhea and death in children in developing countries; however, bacterial diversity in natural infection is uncharacterized. In this study, we explored the natural population variation of ETEC from individuals with cholera-like diarrhea. Genomic sequencing and comparative analysis of multiple ETEC isolates from twelve cases of severe diarrhea demonstrated clonal populations in the majority of subjects (10/12). In contrast, a minority of individuals (2/12) yielded phylogenomically divergent ETEC isolates. Detailed examination revealed that isolates also differed in virulence factor content. These genomic data suggest that severe, cholera-like ETEC infections are largely caused by a clonal population of organisms within individual patients. Additionally, the isolation of similar clones from geographically and temporally dispersed cases with similar clinical presentations suggests that some isolates are particularly suited for virulence. The identification of multiple genomically diverse isolates with variable virulence factor profiles from a single subject highlights the dynamic nature of ETEC, as well as a potential weakness in the examination of cultures obtained from a single colony in clinical settings. These findings have implications for vaccine design and provide a framework for the study of population variation in other human pathogens. IMPORTANCE Enterotoxigenic Escherichia coli (ETEC) has been identified as one of the major causes of diarrheal diseases in children as well as travelers. It has been previously appreciated that this pathogenic variant of E. coli is diverse, both at the genomic level, as defined with multilocus sequence typing, and with regard to the presence or absence of virulence factors within clonal groups. Using whole-genome sequencing and comparative analysis, we identified and characterized diverse enterotoxigenic E. coli isolates from individual patients. In 17% of patients, we identified multiple distinct ETEC isolates, each with unique genomic features and in some cases diverse virulence factor profiles. These studies ascertained that any one person may be colonized by multiple pathogenic ETEC isolates, which may impact how we think about the development of vaccines and therapeutics against these organisms. Enterotoxigenic Escherichia coli (ETEC) has been identified as one of the major causes of diarrheal diseases in children as well as travelers. It has been previously appreciated that this pathogenic variant of E. coli is diverse, both at the genomic level, as defined with multilocus sequence typing, and with regard to the presence or absence of virulence factors within clonal groups. Using whole-genome sequencing and comparative analysis, we identified and characterized diverse enterotoxigenic E. coli isolates from individual patients. In 17% of patients, we identified multiple distinct ETEC isolates, each with unique genomic features and in some cases diverse virulence factor profiles. These studies ascertained that any one person may be colonized by multiple pathogenic ETEC isolates, which may impact how we think about the development of vaccines and therapeutics against these organisms.

Contribution of the Highly Conserved EaeH Surface Protein to Enterotoxigenic Escherichia coli Pathogenesis

ABSTRACT Enterotoxigenic Escherichia coli (ETEC) strains are among the most common causes of diarrheal illness worldwide. These pathogens disproportionately afflict children in developing countries, where they cause substantial morbidity and are responsible for hundreds of thousands of deaths each year. Although these organisms are important targets for enteric vaccines, most development efforts to date have centered on a subset of plasmid-encoded fimbrial adhesins known as colonization factors and heat-labile toxin (LT). Emerging data suggest that ETEC undergoes considerable changes in its surface architecture, sequentially deploying a number of putative adhesins during its interactions with the host. We demonstrate here that one putative highly conserved, chromosomally encoded adhesin, EaeH, engages the surfaces of intestinal epithelial cells and contributes to bacterial adhesion, LT delivery, and colonization of the small intestine.

Typhoid fever in young children in Bangladesh: Clinical findings, antibiotic susceptibility pattern and immune responses

Background Children bear a large burden of typhoid fever caused by Salmonella enterica serotype Typhi (S. Typhi) in endemic areas. However, immune responses and clinical findings in children are not well defined. Here, we describe clinical and immunological characteristics of young children with S. Typhi bacteremia, and antimicrobial susceptibility patterns of isolated strains. Methods As a marker of recent infection, we have previously characterized antibody-in-lymphocyte secretion (TPTest) during acute typhoid fever in adults. We similarly assessed membrane preparation (MP) IgA responses in young children at clinical presentation, and then 7-10 days and 21-28 days later. We also assessed plasma IgA, IgG and IgM responses and T cell proliferation responses to MP at these time points. We compared responses in young children (1-5 years) with those seen in older children (6-17 years), adults (18-59 years), and age-matched healthy controls. Principal Findings We found that, compared to age-matched controls patients in all age cohorts had significantly more MP-IgA responses in lymphocyte secretion at clinical presentation, and the values fell in all groups by late convalescence. Similarly, plasma IgA responses in patients were elevated at presentation compared to controls, with acute and convalescent IgA and IgG responses being highest in adults. T cell proliferative responses increased in all age cohorts by late convalescence. Clinical characteristics were similar in all age cohorts, although younger children were more likely to present with loss of appetite, less likely to complain of headache compared to older cohorts, and adults were more likely to have ingested antibiotics. Multi-drug resistant strains were present in approximately 15% of each age cohort, and 97% strains had resistance to nalidixic acid. Conclusions This study demonstrates that S. Typhi bacteremia is associated with comparable clinical courses, immunologic responses in various age cohorts, including in young children, and that TPTest can be used as marker of recent typhoid fever, even in young children.

Highly conserved type 1 pili promote enterotoxigenic E. coli pathogen-host interactions

Enterotoxigenic Escherichia coli (ETEC), defined by their elaboration of heat-labile (LT) and/or heat-stable (ST) enterotoxins, are a common cause of diarrheal illness in developing countries. Efficient delivery of these toxins requires ETEC to engage target host enterocytes. This engagement is accomplished using a variety of pathovar-specific and conserved E. coli adhesin molecules as well as plasmid encoded colonization factors. Some of these adhesins undergo significant transcriptional modulation as ETEC encounter intestinal epithelia, perhaps suggesting that they cooperatively facilitate interaction with the host. Among genes significantly upregulated on cell contact are those encoding type 1 pili. We therefore investigated the role played by these pili in facilitating ETEC adhesion, and toxin delivery to model intestinal epithelia. We demonstrate that type 1 pili, encoded in the E. coli core genome, play an essential role in ETEC virulence, acting in concert with plasmid-encoded pathovar specific colonization factor (CF) fimbriae to promote optimal bacterial adhesion to cultured intestinal epithelium (CIE) and to epithelial monolayers differentiated from human small intestinal stem cells. Type 1 pili are tipped with the FimH adhesin which recognizes mannose with stereochemical specificity. Thus, enhanced production of highly mannosylated proteins on intestinal epithelia promoted FimH-mediated ETEC adhesion, while conversely, interruption of FimH lectin-epithelial interactions with soluble mannose, anti-FimH antibodies or mutagenesis of fimH effectively blocked ETEC adhesion. Moreover, fimH mutants were significantly impaired in delivery of both heat-stable and heat-labile toxins to the target epithelial cells in vitro, and these mutants were substantially less virulent in rabbit ileal loop assays, a classical model of ETEC pathogenesis. Collectively, our data suggest that these highly conserved pili play an essential role in virulence of these diverse pathogens.

Resistance Pattern and Molecular Characterization of Enterotoxigenic Escherichia coli (ETEC) Strains Isolated in Bangladesh.

Background Enterotoxigenic Escherichia coli (ETEC) is a common cause of bacterial infection leading to acute watery diarrhea in infants and young children as well as in travellers to ETEC endemic countries. Ciprofloxacin is a broad-spectrum antimicrobial agent nowadays used for the treatment of diarrhea. This study aimed to characterize ciprofloxacin resistant ETEC strains isolated from diarrheal patients in Bangladesh. Methods A total of 8580 stool specimens from diarrheal patients attending the icddr,b Dhaka hospital was screened for ETEC between 2005 and 2009. PCR and Ganglioside GM1- Enzyme Linked Immuno sorbent Assay (ELISA) was used for detection of Heat labile (LT) and Heat stable (ST) toxins of ETEC. Antimicrobial susceptibilities for commonly used antibiotics and the minimum inhibitory concentration (MIC) of nalidixic acid, ciprofloxacin and azithromycin were examined. DNA sequencing of representative ciprofloxacin resistant strains was performed to analyze mutations of the quinolone resistance-determining region of gyrA, gyrB, parC and parE. PCR was used for the detection of qnr, a plasmid mediated ciprofloxacin resistance gene. Clonal variations among ciprofloxacin resistant (CipR) and ciprofloxacin susceptible (CipS) strains were determined by Pulsed-field gel electrophoresis (PFGE). Results Among 1067 (12%) ETEC isolates identified, 42% produced LT/ST, 28% ST and 30% LT alone. Forty nine percent (n = 523) of the ETEC strains expressed one or more of the 13 tested colonization factors (CFs) as determined by dot blot immunoassay. Antibiotic resistance of the ETEC strains was observed as follows: ampicillin 66%, azithromycin 27%, ciprofloxacin 27%, ceftriazone 13%, cotrimaxazole 46%, doxycycline 44%, erythromycin 96%, nalidixic acid 83%, norfloxacin 27%, streptomycin 48% and tetracycline 42%. Resistance to ciprofloxacin increased from 13% in 2005 to 34% in 2009. None of the strains was resistant to mecillinam. The MIC of the nalidixic acid and ciprofloxacin of representative CipR strains were 256 μg/ml and 32μg/ml respectively. A single mutation (Ser83-Leu) in gyrA was observed in the nalidixic acid resistant ETEC strains. In contrast, double mutation in gyrA (Ser83-Leu, Asp87-Asn) and a single mutation in parC (Glu84-Ly) were found in ciprofloxacin resistant strains. Mutation of gyrB was not found in either the nalidixic acid or ciprofloxacin resistant strains. None of the ciprofloxacin resistant strains was found to be positive for the qnr gene. Diverse clones were identified from all ciprofloxacin resistant strains by PFGE analysis in both CF positive and CF negative ETEC strains. Conclusion Emergence of ciprofloxacin resistant ETEC strains results in a major challenge in current treatment strategies of ETEC diarrhea.