Intracellular persistence factors in urinary tract infections

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Persistent infections are often caused by bacterial pathogens that display in vitro antibiotic susceptibility, illustrating the intrinsic limitations of antibiotic therapy in the complex environment of the host. While variable patient factors contribute to antibiotic efficacy, fundamental principles of antibiotic persistence have been established. In vitro, antibiotic persistence is observed under ideal growth conditions in dormant subpopulations that tolerate antibiotics due to inactivity of antibiotic targets. In vivo, intracellular infection stages provide protection against many components of the immune system, while host pathogen interactions can promote antibiotic persistence. In my postdoctoral studies, my colleagues and I studied the adaptive evolution of multidrug- resistant Klebsiella pneumoniae and discovered global evolution of persistence in urinary tract infections, which involved an intrinsic ability of K. pneumoniae to persist in LAMP1 positive vacuoles of bladder epithelial cells and tolerate lethal concentrations of antibiotics. A follow-up investigation of intracellular antibiotic persistence in K. pneumoniae found that dormant, multidrug-tolerant bacteria occurred at higher frequency in the intracellular environment compared to the extracellular environment, in line with an increasing body of evidence linking host pathogen interactions to antibiotic tolerance. To target intracellular bacterial infections, we developed a high-throughput screening assay of intracellular survival and conducted a compound screen that led to the discovery of a host-targeting compound that induces intracellular antimicrobial activity in bladder cells and displays in vivo efficacy in a mouse model of latent UTI. I propose to further investigate the intrinsic ability of K. pneumoniae to persist intracellularly in LAMP1 positive vacuoles of bladder epithelial cells to gain a better understanding of the molecular basis of persistent urinary tract infections. Intracellular persistence factors will be identified in an arrayed transposon mutant library. Validation experiments and phenotypic characterization of genes with the strongest impact on intracellular survival will lead to a more detailed mechanistic investigation that focuses on one of the identified persistence factors. Protein interaction experiments will help to elucidate a pathway of intracellular persistence which could lead to the discovery of host pathogen interactions that promote intracellular survival. Moreover, the impact of a mutation that disrupts intracellular survival will be investigated in a mouse model of urinary tract infection which will provide an opportunity to study the role of intracellular infection stages in causing persistent infections.