Investigating virulence functions of mastitis-associated Extraintestinal pathogenic Escherichia coli relevant to human disease

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Abstract/Project Summary Extraintestinal Escherichia coli (ExPEC) are bacteria that live in the intestines of mammals and cause life- threatening illnesses when they infect other tissues. The societal burden due to this group of pathogens is vast and growing, in part because of increasing antibiotic resistance and a lack of vaccination options. Urosepsis, pneumonia and neonatal meningitis are human diseases caused by these strains. Our long-term goal is to understand the molecular mechanisms of pathogenesis of ExPEC that circulate in animals and humans. Many animals are susceptible to these pathogens and represent reservoirs for human infection. The central hypothesis of this application, based on substantial preliminary data, is that some mastitis strains of bovine origin also have the potential to cause disease in multiple hosts including humans. This is a novel finding, based on evidence that includes genetic similarities with human ExPEC lineages, virulence in Galleria mellonella infections, resistance to human serum, and the ability to cause sepsis and urinary tract infections in mice. The objective of this work is to characterize virulence factors that allow some mastitis-associated strains to infect multiple hosts and cause extraintestinal disease. A genome-wide screen for mastitis strain M12 virulence factors led to the discovery of a cluster of capsule biosynthesis genes encoding a Group 3 capsule. Group 3 capsules are present in many ExPEC strains, but their roles are not well defined. An unencapsulated mutant strain was unable to infect spleens or kidneys of mice. Additional mastitis-associated strains in our collection also encode Group 3 capsules, which may promote virulence by helping these bacteria avoid neutrophil phagocytosis. Neutrophils are critically important for defense against many bacterial pathogens including ExPEC, but we do not fully comprehend the mechanisms whereby ExPEC resist being engulfed or killed by these cells. The objectives of this proposal will be accomplished with three specific aims: (1) Estimate the fraction of mastitis-associated E. coli that can cause disease in established models of human ExPEC infection. We will utilize novel DNA barcoding strategies to measure competitive fitness of multiple strains in these experiments. (2) Characterize the role of Group 3 capsules found in mastitis-associated strains in conditions relevant to human disease. We will test whether these capsules promote resistance to killing by neutrophils or serum and during experimental infections. (3) Identify all of the genes that are needed for production of the M12 Group 3 capsule, including those both inside and outside the capsule locus. Since building the capsule is a critical virulence function, this could identify targets for new therapies. This proposal is innovative because it is based on the novel concept that mastitis-associated strains are potential human pathogens. It is significant because it will provide greater understanding of the molecular basis for ExPEC virulence in multiple hosts, and the relationships between ExPEC found in different environments. These findings may inform new ways to prevent disease caused by these bacteria and improve public health.