Escherichia coli and Proteus mirabilis are important urinary tract pathogens and the most common causes of
catheter-associated urinary tract infections (CAUTIs). CAUTIs are one of the most common health care-
associated infections and contribute to nearly double the mortality rate of catheterized compared to non-
catheterized patients. CAUTIs are frequently polymicrobial and inter-species interactions contribute to the
development of catheter biofilms and increase the risk of sepsis. Polymicrobial environments are shaped by both
cooperative and competitive interactions between species. However, specific interactions between different
urinary tract colonizers currently remain poorly defined. Characterizations of competitive interactions is
additionally hampered by the fact that a large number of competition systems remain undiscovered. Our overall
goal is to study the role of competition systems for successful colonization of Enterobacteriaceae in the urinary
tract. This proposal specifically aims to identify and further characterize a novel inter-species competition system
employed by Proteus mirabilis to kill competitor species. We showed that clinical and commensal P. mirabilis
strains quickly and drastically (2-million-fold) reduced viability of E. coli and other Enterobacteriaceae
during co-culture. Cell-free P. mirabilis culture supernatant alone did not reduce viability of target cells. Killing
instead required direct contact between cells. However, a heat-labile component in stationary phase P. mirabilis
supernatant was sufficient to induce killing, suggesting that the system is likely regulated via quorum sensing.
Killing also occurred on solid surfaces, where P. mirabilis was able to penetrate and kill all cells in established
microcolonies of E. coli. This highlights a potential role of the killing system in mixed biofilms, which is an
important component of urinary catheter colonization and persistence. The only known contact-dependent
competition system in P. mirabilis is the Type 6 Secretion System (T6SS), but a mutant deficient in this system
still killed E. coli. We thus hypothesize that P. mirabilis employs a novel contact-dependent system to kill E.
coli cells. The objective of this study is to identify the killing system and its regulation, define the mechanism of
action, and elucidate the role of the killing system for competition of P. mirabilis with E. coli during polymicrobial
infections in vivo. In Aim 1, we will use a transposon library of P. mirabilis and a bioluminescence-based assay
to screen for P. mirabilis mutants unable to kill E. coli and to identify genes coding for effector molecules, delivery
system and regulatory molecules of the killing system. We will also test the hypothesis that quorum sensing
molecules regulate the activity of the system and investigate the mechanism of how E. coli cells die after contact
with P. mirabilis. In Aim 2 we will assess the role of the P. mirabilis killing system during polymicrobial infection.
We will first test the ability of P. mirabilis to kill E. coli during biofilm formation on urinary catheters and then
assess colonization of catheters and dissemination to peripheral organs for P. mirabilis and E. coli in single and
polymicrobial infections using a CAUTI mouse model.