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Re-evaluation of a Neonatal Mouse Model of Infection With Enterotoxigenic Escherichia coli.

ABSTRACT: Enterotoxigenic E. coli (ETEC) is a common cause of diarrhea in children in low- and middle-income countries, and in travelers to these countries. ETEC is also an important cause of morbidity and premature mortality in piglets, calves, goat kids and lambs. The major virulence determinants of ETEC are enterotoxins and colonization factors, which enable the pathogen to colonize the small intestine and deliver enterotoxins, such as the heat-stable enterotoxins, STp and STh, to epithelial cells. Because most ETEC strains are host-specific, there are few convenient animal models to investigate the pathogenesis of ETEC infections or to evaluate specific anti-ETEC interventions, such as drugs and vaccines. An exception is ETEC strains bearing F41 pili, which mediate intestinal colonization of various young animals, including neonatal mice, to cause disease and in some cases death. In this study, we used the archetypal F41-producing bovine ETEC strain, B41 (O101:NM; K99, F41, STp) to validate and further explore the contribution of F41 and STp to bacterial virulence. By using targeted gene deletion and trans-complementation studies, augmented by whole genome sequencing, and in vitro and animal studies of virulence, we established that F41 mediates colonization of the mouse intestine and is essential for bacterial virulence. In addition, we showed for the first time that STp is as important as F41 for virulence. Together, these findings validate the use of neonatal mice to study the pathogenesis of F41-bearing ETEC and to investigate possible specific anti-ETEC interventions including vaccines that target heat-stable enterotoxins.

SUBMITTER: Carroll CJ

PROVIDER: S-EPMC8013722 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:UnlabelledEnterotoxigenic 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.ImportanceEnterotoxigenic 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.

Project description:Enterotoxigenic Escherichia coli (ETEC) is a major diarrheal pathogen in developing countries, where it accounts for millions of infections and hundreds of thousands of deaths annually. While vaccine development to prevent diarrheal illness due to ETEC is feasible, extensive effort is needed to identify conserved antigenic targets. Pathogenic Escherichia coli, including ETEC, use the autotransporter (AT) secretion mechanism to export virulence factors. AT proteins are comprised of a highly conserved carboxy terminal outer membrane beta barrel and a surface-exposed amino terminal passenger domain. Recent immunoproteomic studies suggesting that multiple autotransporter passenger domains are recognized during ETEC infection prompted the present studies.Available ETEC genomes were examined to identify AT coding sequences present in pathogenic isolates, but not in the commensal E. coli HS strain. Passenger domains of the corresponding autotransporters were cloned and expressed as recombinant antigens, and the immune response to these proteins was then examined using convalescent sera from patients and experimentally infected mice.Potential AT genes shared by ETEC strains, but absent in the E. coli commensal HS strain were identified. Recombinant passenger domains derived from autotransporters, including Ag43 and an AT designated pAT, were recognized by antibodies from mice following intestinal challenge with H10407, and both Ag43 and pAT were identified on the surface of ETEC by flow cytometry. Likewise, convalescent sera from patients with ETEC diarrhea recognized Ag43 and pAT, suggesting that these proteins are expressed during both experimental and naturally occurring ETEC infections and that they are immunogenic. Vaccination of mice with recombinant passenger domains from either pAT or Ag43 afforded protection against intestinal colonization with ETEC.Passenger domains of conserved autotransporter proteins could contribute to protective immune responses that develop following infection with ETEC, and these antigens consequently represent potential targets to explore in vaccine development.

Project description:An infection model with enterotoxigenic Escherichia coli (ETEC) harboring the F4 fimbriae can be used to assess the impacts that various challenges associated with weaning (e.g., dietary, psychological, environmental) have on the expression of postweaning diarrhea. The objective of this study was to develop a novel inoculation method for administering an ETEC culture that would induce a higher proportion of ETEC-F4 diarrhea, in pigs that genetically showed ETEC-F4 susceptibility or resistance. The study was designed as a factorial arrangement of treatments with the factors being 1) partially susceptible or resistant to ETEC-F4 based on genetic testing, and 2) 4 challenge treatments, being a) a conventional liquid broth method using a drenching gun [Positive control (PC)], b) a Syringe method, c) a Capsule method, and d) Negative control [pigs not challenged (NC)]. At 21 ± 3 d of age (mean ± SEM), 48 male castrate pigs (Large White × Landrace) weighing approximately 7.0 ± 1.18 kg were allocated to 4 treatment groups in 2 replicate pens (6 pigs per pen). Initial ETEC-F4 susceptibility was based on a DNA marker test and each treatment group had 9 partially susceptible and 3 resistant pigs. On days 7 and 8 after weaning, pigs were challenged with ETEC (serotype O149:K88; toxins LT1, ST1, ST2, and EAST). On each inoculation day the PC pigs were orally dosed with 9 mL 7.12 × 109 colony-forming unit (CFU), the Syringe pigs with 0.8 mL 6.72 × 109 CFU, the Capsule pigs were orally administered 2 capsules containing 0.8 mL 3.28 × 109 CFU, and the NC pigs 1 mL of phosphate-buffered saline (PBS) solution. Approximately 72 h after infection, 44, 22, 78, and 0% of partially susceptible pigs in the PC, the Syringe, the Capsule, and the NC group had developed ETEC-F4 diarrhea (P = 0.007). Partially susceptible pigs had a higher diarrhea index (DI) compared to resistant pigs (31.5 vs. 4.8, P < 0.001). The NC group had a lower DI compared to the PC and Capsule pigs (3.9, 38.1, and 40.3, respectively, P < 0.005). Following infection, genetically resistant pigs in the Capsule group had a DI of zero and the partially susceptible pigs had a DI of 55.6 (P = 0.014). This study showed that genetically screening pigs and using a Capsule to deliver ETEC-F4 can increase cases of diarrhea and the efficiency of the challenge model. Taken together, these methods have the potential to reduce the number of pigs needed in future experimental infection studies.

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Re-evaluation of a Neonatal Mouse Model of Infection With Enterotoxigenic Escherichia coli.

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