Roles of Putative Lipoproteins and Pathogenic Mechanisms of Klebsiella pneumoniae




Casillas, Jose

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There has been a significant rise in infections due to Klebsiella pneumoniae carbapenemase (KpC) producing bacteria with limited treatment options. In addition, hypervirulent strains of Klebsiella pneumoniae (hvKp) are also emerging with a dire need to develop strategies for prevention and treatment of infections in a variety of clinical settings. The most notable factors involved in determining the virulence of infection have been capsules, lipopolysaccharides, fimbriae, and siderophores. However, due to a significant amount of heterogeneity within KpC strains, not every one of these factors have similar contributions towards the virulence of all clinical strains. There is dire need to identify novel virulence factors produced by KpC and to characterize their role within infections among healthy and immunocompromised patients. Moreover, novel treatments that target either the bacterium or the host will be a significant advancement for treating patients infected with KpC. In this study we pursued two major goals. The first goal was the characterization of a set of 8 putative lipoproteins of Kp whose functions or roles in inducing an immune response are unknown. All 8 lipoproteins are conserved to various degrees. Genes encoding these lipoproteins of interest were amplified from genomic DNA of Kp K1 strain by PCR and the amplicons cloned into plasmid vectors pMAL-2C or pET23a for generating recombinant proteins fused with either maltose binding protein or 6X Histidine-Tag, respectively. The plasmids were transformed into E.coli Rosetta expression hosts and levels of expression were analyzed by SDS-PAGE and immunoblot analysis. Purification was performed by Ni-affinity chromatography for purifying proteins with 6X Histidine-Tag and the proteins were confirmed to be in the elution fractions via SDS-PAGE analysis. Among these recombinant proteins, KUF 71592, a putative lipoprotein, was characterized by a broad set of biochemical and biophysical methods including lipoprotein extraction and Proteinase K treatment. As part of the second goal, we determined if the thioredoxin system could serve as a target for novel strategies to prevent or treat KpC infection. We tested wild type and mutant strains of Kp lacking components of the Thioredoxin system. We also determined the growth rate of TrxA mutants in the presence of various oxidative stressors and observed the levels to which these genes are expressed under oxidative stress conditions. Each of these systems represent the possibility of development of novel tools that could aid in treatment of Kp infections.


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Molecular Microbiology and Immunology