Transcriptional Regulation of Proteus Mirabilis Pathogenesis in the Urinary Tract

Abstract

Urinary Tract Infections (UTIs) are common infections that are a significant burden in healthcare systems worldwide. A significant portion of UTIs are Catheter-Associated UTIs (CAUTIs), accounting for up to 40% of hospital acquired infections globally. Over 500,000 CAUTIs are reported annually in the US, generating $1.7 billion in healthcare costs each year. A major cause of CAUTI is Proteus mirabilis, an understudied Gram-negative pathogen noted for its ability to form urinary stones during infection. Stone formation is induced by urease production, a process regulated by UreR in a urea-dependent manner. The urease enzyme requires nickel as a co-factor, which is actively imported through two transport systems. Given that urea is highly abundant in human urine (~400 mM), I hypothesized that UreR may regulate other functions required to establish UTI such as nickel transport. To investigate this hypothesis, I used transcriptomic analysis to define the regulon of UreR and expanded the known targets of this regulator to include nickel transporters. I demonstrated that UreR directly binds the promoter of the ynt nickel transport operon and identified UreR as the first known regulator of nickel transport in P. mirabilis. Notably, the UreR-regulated urease is a nickel metalloenzyme. Thus, my work demonstrates that UreR specifically regulates genes required to produce mature urease, an essential virulence factor for P. mirabilis uropathogenesis. Using bioinformatics, I identified UreR-regulated urease loci in closely-related species. I also located two mobilized UreR-regulated urease loci that also encode the ynt transporter, implying that UreR regulation of nickel transport is a conserved regulatory relationship. Additionally, I sought to identify transcriptional regulators that orchestrate P. mirabilis pathogenesis in the urinary tract with a technique that leveraged the urease promoter as an inducible expression platform. Toward this end, I identified 217 putative transcriptional regulators in P. mirabilis type-strain HI4320, of which 109 have no associated gene name. However, preliminary work determined that the urease promoter exhibited activity in the absence of the inducer and required further development to produce a tightly-regulated platform for inducible gene expression. To facilitate this work, I generated a series of reporter constructs to assess the impact of specific promoter elements on the expression kinetics of the urease promoter. This work produced a suite of urea-inducible promoters that exhibit a variety of expression kinetics, resulting in a customizable gene expression platform that is suitable for applications in research and manufacturing. In addition to my studies of the urease promoter, my dissertation presents work characterizing two putative RTX toxins of P. mirabilis. Putative domains and repeats were identified using bioinformatics. Deletion of either toxin did not impact P. mirabilis pathogenesis in ascending UTI or bacteremia mouse models. However, there is some evidence that suggests these toxins may play a role in a polymicrobial context. Further work is required to elucidate the function of these RTX toxins. Overall, the work described in this dissertation expands our knowledge of transcriptional regulation of P. mirabilis urease while also advancing the hypothesis that urease is an important metabolic function for P. mirabilis. Additionally, I argue for the study of transcriptional regulators and their regulatory networks in a species-specific context. As we continue to study bacterial pathogens, we must look beyond traditional virulence genes and recognize that transcriptional regulators play integral roles in pathogenesis by enabling the expression of virulence genes in the appropriate context.