Identification of the Outer Membrane Immunoproteome of the Uropathogen Proteus mirabilis: Insights into Virulence and Potential Vaccine Candidates.

Abstract

Proteus mirabilis, a gram negative bacterium, represents a common cause of complicated urinary tract infections (UTIs) in catheterized patients or those with functional or anatomical abnormalities of the urinary tract. To systematically identify surface-exposed antigens, proteins in the outer membrane fraction of bacteria were separated by 2D gel electrophoresis and subjected to Western blotting with sera from mice experimentally infected with P. mirabilis. Proteins recognized by sera were identified by mass spectrometry. Thirty-seven antigens (including 24 outer membrane proteins) to which a humoral response had been mounted were identified; these antigens are presumably expressed during infection and therefore represent potential virulence factors. Six representative antigens were selected for further study. Of these antigens, three played no apparent role in pathogenesis, as strains with isogenic mutations were not attenuated in the mouse model of ascending UTI: a putative secreted 5’-nucleotidase (PMI0047), RafY (PMI0288), and FadL (PMI1810). However, two putative iron acquisition proteins, PMI0842 and PMI2596, both contribute to fitness in the urinary tract. The sixth antigen, ZnuB (PMI1150), was annotated as the inner membrane component of the high affinity zinc (Zn2+) transport system ZnuACB. Components of this system have been shown to contribute to virulence in other pathogens; therefore, the role of ZnuACB in P. mirabilis was investigated by constructing a strain with an insertionally interrupted copy of znuC. The znuC::kan mutant was more sensitive to zinc limitation than wild type, was outcompeted by wild type in minimal medium, displayed reduced swimming and swarming motility, and produced less flaA transcript and flagellin protein. Production of flagellin and swarming motility were restored by complementation with znuCB in trans. Swarming motility was also restored by the addition of Zn2+ to agar prior to inoculation. ZnuC offers a competitive advantage during urinary tract infection. Since we demonstrated a role for PMI0842, PMI2596, and ZnuC in UTI, we hypothesize that there is limited iron and zinc present in the urinary tract and that P. mirabilis must scavenge these ions to colonize and persist in the host.