Our laboratory has had a long-standing interest in studying the molecular mechanisms of pathogenicity for Salmonella and pathogenic E. coli and applying this information toward the development of new therapeutic agents. Much of our work is on pathogenic E. coli (enterohemorrhagic E. coli [EHEC; O157] and enteropathogenic E. coli [EPEC]) and the related mouse pathogen Citrobacter rodentium, focusing on their type III secreted effectors and disease mechanisms in two relevant animal models. Diarrheagenic E. coli include at least five types of E. coli, each type possessing a particular set of virulence factors. Enteropathogenic E. coli (EPEC) is a key because of infantile diarrhea worldwide, posing a major endemic health threat to young children in developing countries. Enterohemorrhagic E. coli (EHEC) causes a more severe diarrhea than EPEC (enteric colitis) and, in 10 percent of cases, progresses to an often fatal kidney disease, hemolytic uremic syndrome (HUS). The most common EHEC serotype in North America is O157:H7. EHEC can contaminate meats at slaughter (hence the colloquial name "hamburger disease"), resulting in 50,000 cases per year and 500 deaths in the United States alone, making it a major priority for food and water safety. The basic virulence factors we are studying (the LEE [locus of enterocyte effacement] region) are strongly conserved between EPEC and the various serotypes of EHEC. C. rodentium is a naturally occurring mouse pathogen that encodes a set of virulence factors similar to EHEC and EPEC and is considered a good murine model to study disease processes.
We recently found that there are several non-LEE–encoded type III effectors in at least three new pathogenicity islands (PAIs), in addition to the six LEE effectors in the attaching and effacing (A/E) pathogens EHEC, enteropathogenic E. coli (EPEC), and C. rodentium. Previously, nearly all work on EHEC and EPEC pathogenesis was focused on the LEE region and the Shiga toxins. It is thought that the large number of type III effectors (several dozen) contribute to many aspects of the disease process, including altering the host innate response and affecting normal cellular processes such as cytoskeletal function. However, for most effectors the specific cognate host-binding protein and host pathways affected are poorly understood. It is believed that A/E pathogens encode a significant repertoire of both LEE and non-LEE–encoded type III secreted effectors that directly alter host cellular functions, collectively contributing to disease in a coordinated manner. Research in this area focuses on identifying effectors, identifying their host cognate partners, studying their mechanisms of action, and addressing their individual contributions to the colonization and disease process. The murine infection model is also being used to probe the role of these effectors in disease, including bacterial colony counts, histological analysis, and colonic hyperplasia as read-outs of disease. Several other studies are ongoing to define various structural and functional components of the type III system, given its importance to many significant Gram-negative pathogens. Such studies probe the mechanisms of assembly for this complex (23-component) membrane complex, investigate various regulatory mechanisms that control translocon and effector secretion, and seek to identify anddefine interactions between specific type III components. A close collaboration with a crystallography laboratory has provided much structural information about several type III components; by using this information to guide biological studies, much can be learned about type III apparatus structure and function.
Various genetically altered mice can be used to probe the role of the host innate response in the infectious outcome. Access to many genetically modified animals enables us to study the contribution of specific innate responses to infections. It is apparent that the host response contributes to the infectious outcome and these animals permit a more in-depth analysis of the mechanisms involved.
To complement these studies, we have begun to probe the role of the microbiota (normal flora) during infections. For example, we studied the effect of murine infection with C. rodentium on the microbiota. We found that the number of flora is significantly decreased during infection but that, surprisingly, there are major shifts in the phyla distribution of the microbiota. The numbers of pathogen only reached a minor proportion of the total microbes, rather than overwhelming the population, as we had predicted. We used chemical inflammatory mediators and mice that had hyper (iNOS KO) and hypoinflammatory (Il-10 KO) effects to demonstrate that the shifts in population are due to inflammation, rather than to a specific pathogen. These shifts are also more permissive for growth of non-pathogenic E. coli (closely related to Citrobacter). This led to the intriguing hypothesis that the pathogen intentionally triggers inflammation to shift flora to create a more favorable environment for the pathogen. When the acquired immune system engages, it eliminates the pathogen, and the microbiota reset to their normal distribution. Further studies are ongoing, using specific C. rodentium mutants in defined virulence factors to probe the role of virulence factors on the microbiota distribution. To further define the contribution of the microbiota, specific antibiotic treatments are being used to specifically perturb the microbiota prior to infection. In addition, specific mutants in various murine innate and acquired immune responses can be used to follow the role of the host response on the microbiota during infection.
By probing the contribution of pathogen, microbiota, and host immune response, one can obtain a more complete understanding of the infectious process. Collectively, these approaches will further define the complex molecular interactions that occur between these significant human pathogens and their hosts and provide important information about the molecular mechanisms of the diseases that they cause.
Last updated August 2009
