2015OBASANJOTAPhD.pdf (16.89 MB)
Fimbrial systems in Klebsiella: An investigation of genetic variants of type 1 fimbriae
thesisposted on 2016-04-11, 11:15 authored by Toyosi Aduke Obasanjo
K. pneumoniae associated genomic island KpGI-5 was found to possess a novel type 1-like gene cluster dubbed fim2. Despite extensive studies on fim2, certain issues regarding the operon remain unresolved. In this project, the transcriptional analysis suggested controlled expression of fim2 during bacterial growth phase. In vivo assay using G. mellonella killing assay did not show any statistically significant in vivo role for fim2, but suggested a contribution to immunogenic properties of the bacteria. To ascertain fimbriae forming capabilities of fim2, a hybrid fimbriae of fim and fim2 was constructed such that fimA was replaced with fim2A on the fim on pJTOOL-7 plasmid. This plasmid was transformed into afimbriate E. coli HB101 (HB101/pFim-HYA:2A). fim2A was also replaced with fimA on fim2 (HB101/pFim2-HY2A:A) as well as fim2H replaced with fimH (HB101/pFim- HYH:2H). Transmission electron microscopy of HB101/pFim-HYA:2A and HB101/pFim2-HY2A:A showed evidence of fimbrial structures on the bacterial surface, confirming that fim2A bears similar function with fimA and hence, confers fimbrial forming capabilities. The replacement of adhesin fimH with fim2H did not show mannose sensitive agglutination of erythrocytes characteristic of fimH. Other Klebsiella strains were screened for the presence of fim and fim2 genes. Of the strains that tested positive for fim2, 3 unique strains sp15, sp25 and sp28 tested negative for typical fim genes. PCR mapping and sequence analysis done in this project showed that this operon bears high similarity to fim but contains higher synonymous nucleotide substitutions than normally found among Klebsiella fim and harbours a unique fimK homolog. Analyses suggest microevolution of fim to fim3 in these strains. Ambiguity surrounding species classification was clarified using multiple locus sequence typing (MLST) method. In vitro assays and Galleria killing assay did not show statistical significant differences between fim in K. pneumoniae KR116 and fim3 in sp28. This thesis reports first possibility of microevoution of fim gene cluster within K. pneumoniae and also confirms the fimbriae coding potential for novel fim variant fim2.
Supervisor(s)Rajakumar, Kumar; Galyov, Edouard
Date of award2015-12-17
Author affiliationDepartment of Infection, Immunity and Inflammation
Awarding institutionUniversity of Leicester