Thermosensing Mechanisms of the capsule regulator KvrA in Klebsiella pneumoniae

PROJECT SUMMARY This project will enhance our understanding of the fundamental biology of Klebsiella pneumoniae, an important human pathogen deemed an urgent threat by the CDC due to escalating antibiotic resistance rates. I seek to understand how a ubiquitous and opportunistic organism, one capable of colonizing many diverse environments, can alter its gene expression to suit its surroundings. Specifically, I am investigating the mechanism by which K. pneumoniae senses increased temperature upon infecting a host and increases expression of its polysaccharide coat in response. This project builds on my experience in bacteriology as well as the importance of physiological temperature differences in pathogenesis by focusing specifically on the known virulence regulator KvrA. KvrA regulates the amount of capsule produced by K. pneumoniae by competing for the same binding site as H-NS, a transcriptional silencer of capsule. Previous work has demonstrated that the translation of SlyA in Escherichia coli, homologous to KvrA in K. pneumoniae, increases in response to temperature. Yet, the mechanism by which the environmental signal of temperature is transduced to increase the translation of SlyA/KvrA, and the subsequent production of capsule is unknown. I hypothesize that temperature regulates the translation of KvrA in K. pneumoniae via an RNA thermometer (RNAT) mechanism and this sensing ability is essential for capsule expression and virulence within the host. RNATs are secondary structures located within the 5' untranslated region (UTR) of mRNAs that prevent translation from occurring at ambient temperatures but "melt" to free the Shine-Dalgarno (SD) sequence and allow translation at higher temperatures. Through a series of parallel experiments, I will determine whether the temperature regulation of K. pneumoniae KvrA occurs at the transcriptional or post-transcriptional level. GFP and luciferase reporter fusions will be constructed to test the region upstream of kvrA and determine if it acts as a temperature regulator independent of the kvrA coding sequence. In collaboration with Dr. Adrianus ("Jacco") Boon, an expert in RNA structural biology at Washington University in St. Louis, I will characterize the secondary structure of the 5' UTR of kvrA at both 37°C and 20°C using SHAPE-MaP. Alternative methods are available to investigate other non-RNAT thermoregulatory mechanisms of KvrA. A well-established murine model of pneumonia will be leveraged to evaluate the significance of this KvrA thermoregulatory mechanism in vivo. I will compare the virulence of strains with stabilizing mutations in the kvrA 5' UTR predicted to resist melting at 37°C to those with a wild-type kvrA 5' UTR. I will also evaluate the role of krvA in strains lacking H-NS, thus determining if KvrA's thermoregulatory effects on capsule are independent of H-NS. With the rise of antibiotic resistance, it is critical to find alternative methods for treating and preventing K. pneumoniae infection. This proposed work will define a critical mechanism of K. pneumoniae environmental sensing that may be amenable for therapeutic targeting.