Genetic and Functional Characterisation of a Novel Klebsiella Pneumoniae Genomic Island Harbouring an Accessory Chaperone/Usher Fimbrial Operon

Strain-specific Klebsiella pneumoniae virulence determinants have been described but almost exclusively for hypervirulent liver abscess-associated strains. Island-tagging and fosmid-based marker rescue were used to capture and sequence KpGI-5, a novel genomic island integrated into the met56 tRNA gene of K. pneumoniae KR116. This 14.0 kb island exhibited a genome-anomalous G+C content, possessed near-perfect 46 bp direct repeats, and encoded a putative γ1-chaperone/usher fimbrial cluster (fim2). This island was shown to belong to a previously unknown KpGI-5-like island family and was hypothesized to have undergone substantial reductive evolution. Transcriptional analysis demonstrated expression of three fim2 genes and suggested that the eight-gene fim2A-fim2K cluster comprised an operon. In vivo models of urinary tract and lung infection, and large intestinal colonization, in addition to in vitro assays were used to examine the role of fim2 in pathogenesis by comparing KR2107, a streptomycin-resistant derivative of KR116, to three isogenic mutants (Δfim, Δfim2 and ΔfimΔfim2) constructed using optimized allelic exchange techniques. Although no statistically significant in vivo role for fim2 was demonstrable, liver and kidney CFU counts for lung and urinary tract infection models, respectively, hinted at an ordered gradation of virulence as fimbrial clusters were lost: KR2107 (most virulent), KR2107Δfim2, KR2107Δfim and KR2107ΔfimΔfim2 (least virulent). Despite using several methods the putative Fim2 fimbriae could not be visualised. Furthermore, the fim2-encoded putative phosphodiesterase Fim2K was determined to alter several c-di-GMP-dependent phenotypes, including biofilm formation and exopolysaccharide production. Additionally, fim2 was present in 14 % of Klebsiella strains studied and appeared intact in the majority of fim2-positive strains. Given the above findings, fim2 may confer a niche-specific evolutionary advantage, potentially related to its ability to encourage dissemination. This thesis is the first study of fim2 and KpGI-5-like islands and will aid further investigations into the full impact of these loci on the lifestyle of Klebsiella species.