Please use this identifier to cite or link to this item: http://scholarbank.nus.edu.sg/handle/10635/28210
Title: GGDEF-EAL proteins of Burkholderia pseudomallei
Authors: LEE HWEE SIANG
Keywords: GGDEF-EAL, B. pesudomallei
Issue Date: 20-May-2009
Source: LEE HWEE SIANG (2009-05-20). GGDEF-EAL proteins of Burkholderia pseudomallei. ScholarBank@NUS Repository.
Abstract: The Gram-negative bacillus Burkholderia pseudomallei, is the causative agent of melioidosis. Its potential threat as a bioterrorism weapon, compounded by the high morbidity and mortality rates of melioidosis, has necessitated the continual research on the pathogenesis of this bacterium. Recently, a novel guanosine signaling nucleotide, cyclic diguanylic acid (c-di-GMP), has gathered much scientific interest. This ubiquitous second messenger is found in almost all sequenced branches of the phylogenetic tree of bacteria and shown to be involved in the regulation of adaptive bacterial functions such as motility, biofilm formation and virulence. In bacteria, the diguanylate cyclase and phosphodiesterase activities of proteins containing the highly conserved GGDEF and EAL domains regulate the intracellular levels of c-di-GMP. The B. pseudomallei genome encodes 16 putative proteins containing the GGDEF-EAL domains. This study focused on two such proteins, CdpA and BPSS0805, which were expressed in both mid-log phase and stationary phase. A comparison of the intracellular c-di-GMP in the gene knockout mutants with that of wild-type B. pseudomallei KHW revealed an 8-fold higher c-di-GMP level in the cdpA knockout mutant (KHWcdpA::Tet). The levels of intra cellular c-di-GMP was restored in cdpA complemented mutant, KHWcdpA::Tet/pUCP28T-cdpA and cdpA overexpression strain resulted in a 40% decrease in intracellular c-di-GMP levels. Taken together, these results suggested that CdpA most likely functions as a c-di-GMP phosphodiesterase in vivo. Phenotypic characterization of the mutants revealed that cdpA controlled the synthesis of flagella, cell length and swimming motility. Mutation in cdpA also increased cellulose synthesis, cell aggregation and enhanced biofilm formation. The cdpA mutant was also significantly attenuated cell invasion and cytotoxicity, thus providing preliminary evidence that high intracellular level of c-di-GMP could inhibit B. pseudomallei virulence. In contrast, BPSS0805 mutant (KHWBPSS0805::Tet) did not significantly alter the level of intracellular c-di-GMP. This mutant also yielded little observable differences in swimming motility, bacteria morphology, cell aggregation, cellulose production and cell invasion and cytotoxicity assays when compared to the wild-type B. pseudomallei, thus suggesting functional redundancy of BPSS0805 in vivo.
URI: http://scholarbank.nus.edu.sg/handle/10635/28210
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