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Research Associate Professor
6203 Marsico Hall

As a long-time member of Virginia Miller’s lab and recent member of Rita Tamayo’s lab, I split my time between labs doing research and mentoring students & staff.

Klebsiella projects in the Miller Lab

Klebsiella pneumoniae is a Gram-negative bacterial pathogen that has a remarkable ability to cause a wide range of human diseases. It is divided into two broad classes: Classical strains that are a notable problem in healthcare settings due to multidrug resistance, and hypervirulent (hv) strains that are drug sensitive, but able to establish disease in immunocompetent hosts. Alarmingly, there has been an increased frequency of clinical isolates that have both drug resistance and hv-associated genes. One such hv-associated gene is rmpA that encodes a transcriptional regulator required for maximal capsule gene (cps) expression and confers hypermucoviscosity (HMV). This link has resulted in the assumption that HMV is caused by elevated capsule production. However, we recently found that rmpA is part of a three-gene operon and that RmpA autoregulates the expression of the promoter driving expression of these genes, named rmpD and rmpC. We have shown that RmpC is a transcriptional regulator that is required for maximal capsule expression and RmpD is a small protein that is required for the HMV phenotype. This data, combined with our previous work, suggests a model in which the RmpA-associated phenotypes are largely due to RmpA activating the expression of rmpD to produce HMV and rmpC to stimulate cps expression. Importantly, this has indicated that HMV and capsule are separable phenotypes. We are currently employing a variety of approaches to investigate how RmpD and RmpC contribute to HMV, capsule and virulence.

Ongoing research goals are focused on two broad questions:

1) How does RmpD influence HMV? We hypothesize that RmpD acts by modifying the activity of another protein through direct interactions. We will screen for interacting partners, then begin to dissect the mechanism of action based on the putative function of the partner protein.

2) How does RmpC contribute to virulence? The partial virulence attenuation of the rmpC suggests RmpC regulates something other than capsule that contributes to the full virulence of K. pneumoniae. We have performed RNA-seq and determined the RmpC regulon contains nearly 500 genes. We will construct mutations in genes of interest from this list, narrowing in on ~3-4 pathways, to determine if these are required for virulence. Both in vivo and in vitro assays will be used.

Clostridioides difficile projects in the Tamayo Lab

C. difficile is a Gram-positive, obligate anaerobe that causes a variety of gastrointestinal complications. It is particularly problematic in health care settings where it is a significant source of nosocomial infections. C. difficile produces two toxins whose role in disease has been well-studied. Like many bacterial organisms, C. difficile undergoes phase variation at numerous loci, and this ability is likely to contribute to the ability of this pathogen to cause disease. The Tamayo Lab studies several of these loci and I am involved in one such project. The cmrRST genes encode an unusual phosphorelay signal transduction system, and it was found to contribute to a striking phenotypic switch. The promoter region contains a phase variable sequence that influences expression of cmrRST. When in the “on” orientation such that cmrRST are expressed, the colonies have a flat, rough morphology. When “off”, the colonies are round and glossy. Of note, the “Rough” morphotype is more virulent than the “Smooth” in a hamster model of infection. In an effort to better understand the differences between these morphotypes, a combination of computer modeling and RNA-seq analysis has led to predictions about the metabolic properties of these “Rough” and “Smooth” isolates. We are now testing these predictions by examining growth and metabolism of the bacteria in a variety of media to validate the computer modeling. We are hopeful that this combinatorial approach will lead to a better understanding of what elements lead to the establishment of C. difficile infection in mammals.

Kim Walker