posted on 2016-04-11, 11:15authored byToyosi 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.
History
Supervisor(s)
Rajakumar, Kumar; Galyov, Edouard
Date of award
2015-12-17
Author affiliation
Department of Infection, Immunity and Inflammation