FULLY FUNCTIONAL AND TIGHTLY REGULATED FLAGELLA ARE ESSENTIAL FOR COLONIZATION BY MAMMARY PATHOGENIC ESCHERICHIA COLI (MPEC) IN THE MURINE MASTITIS MODEL

Mammary pathogenic Escherichia coli (MPEC) is a leading cause of acute mastitis in dairy animals. Because coliform mastitis is considered to occur in an ascending manner, flagellum-mediated motility might be envisage to contribute to virulence by enabling MPEC to disseminate from the teat canal to the milk ducts and alveolar system. Nevertheless, the role of flagellum-mediated motility and chemotaxis in the pathogenesis of E. coli mastitis is currently unknown. In this work our objectives were to better understand the spatial distribution of MPEC bacteria and their effects in the mammary gland and the role of flagellum-mediated motility and chemotaxis in MPEC colonization and virulence. Using the murine mastitis model we compared mammary colonization and virulence of MPEC P4 and a series of specific P4 mutants defective in flagellum assembly, motility and chemotaxis constructed by specific deletions of the following genes;flhD, fliC, fliD, motA, and CheY. While both WT and mutants were able to elicit disease, all mutants were associated with reduced virulence, defective colonization and rapid clearing. Flagellum assembly and function are also tightly regulated by the flhDC locus in the bacterial genome. We next perturbed this regulation by constitutively expressing flhDC in the P4 and P4∆flhD strains. Interestingly, the hyper motile P4/pflhDC and the normal motile complemented P4∆flhD/pflhDC were virulent albeit colonization defective upon intra mammary challenge.

This data provide compelling evidence that fully functional flagellum motility, chemotaxis and their regulation are all essential for mammary colonization by MPEC P4 in the murine lactating mammary gland.