The development of chemical inhibitors of Klebsiella pneumoniae biofilm formation

© 2017 Dr. Jason Wei Han TanKlebsiella pneumoniae is a Gram-negative, opportunistic pathogen that is responsible for nosocomial infections amongst immunocompromised patients. One of the putative key K. pneumoniae pathogenic mechanisms involves the formation of biofilms on implanted medical devices such as urinary catheter. Biofilms, or surface-associated bacteria embedded in a self-produced extracellular matrix, have been implicated in the virulence of many bacteria. Indeed, according to The National Institute of Health, bacterial biofilms account for approximately 80% of microbial infections, with the urinary tract being one of the most common areas for bacterial colonization. Due to the high pathogenicity of some multidrug-resistant K. pneumoniae strains, a better understanding of the K. pneumoniae virulence mechanism and strategies to prevent biofilm-related infections are required. K. pneumoniae biofilm formation is mediated through the production of type 3 fimbriae (hair-like protein structures anchored to the outside of the bacterium). The mrk genes encoding these fimbriae were shown to be under tight regulatory control by a c-di-GMP-dependent transcriptional activator, MrkH. However, knowledge of how MrkH expression is regulated in K. pneumoniae is not well understood. Given the important role of MrkH as a master regulator of K. pneumoniae biofilm formation, one of the studies reported in this thesis focused on the regulation of MrkH expression. Sequence analysis revealed a putative MrkH recognition sequence “MrkH box; TATCAA” located within the promoter region of the mrkHI operon. Mutational analysis, gel shift assay, and real-time quantitative PCR experiments demonstrated that MrkH binds to the cognate DNA binding site to increase its own gene expression. The data presented here suggested a model whereby the c-di-GMP-dependent MrkH recruited the RNA polymerase to its own promoter to activate mrkH expression. Increased production of MrkH activates type 3 fimbriae expression to promote K. pneumoniae biofilm formation. Subsequently, this study aimed at identifying chemical compounds that can interfere with the MrkH-mediated mrk transcriptional process to prevent K. pneumoniae biofilm formation. A total of 13,440 small compounds from a synthetic library were screened using an E. coli reporter system, in an assay designed to identify compounds that can reduce the transcription of the mrk promoter by MrkH. One compound, later named JT71, reduced K. pneumoniae mrkA promoter activity and biofilm formation by 50% without affecting cell viability. Western blot analysis, hemagglutination assays, and electron microscopy showed that JT71 significantly down regulated type 3 fimbriae expression. Real-time qPCR performed on K. pneumoniae grown in the presence of JT71 showed that the compound reduced transcription of both mrkA and mrkH. The studies on type 3 fimbriae were extended to access mrk binding through MrkD adhesin. A comparative model of the receptor-binding domain of K. pneumoniae MrkD adhesin was constructed based on crystallographic data of a homologous protein. The functional role of amino acid residues of MrkD to mediate biofilm formation was studied by site-directed mutagenesis and transmission electron microscopy. Interestingly, as opposed to previous studies suggesting that a functional MrkD is not required for abiotic biofilm formation, the research conducted and reported here showed that K. pneumoniae attachment to both type IV collagen-coated and polystyrene surfaces was MrkD dependent. An integrated structure-based in silico and in vitro high throughput screening assay for small molecule inhibitors of MrkD was utilized, leading to identification of two compounds (named 3086 and 3130) that inhibited biofilm formation by K. pneumoniae. Taken together, the research has resulted in a greater understanding of the mechanisms through which K. pneumoniae regulates the expression of the key protein MrkH, and provided important insights in the control of K. pneumoniae biofilm formation in the context of inhibiting the MrkH-mediated mrk transcriptional process. Finally, the functional role of MrkD adhesin was further characterized, and inhibitors predicted to interact with MrkD, based on mutagenesis and computer-docking analysis, were identified. Collectively, these findings demonstrated the utility of a conventional high throughput screening and a structure-based virtual screening approach for the discovery of chemical compounds to prevent K. pneumoniae biofilm formation