Research Article Molecular Characterization of Putative Virulence Determinants in Burkholderia pseudomallei

Copyright © 2014 Suat Moi Puah et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Gram-negative saprophyte Burkholderia pseudomallei is the causative agent of melioidosis, an infectious disease which is endemic in Southeast Asia and northern Australia. This bacterium possesses many virulence factors which are thought to contribute to its survival and pathogenicity. Using a virulent clinical isolate of B. pseudomallei and an attenuated strain of the same B. pseudomallei isolate, 6 genes BPSL2033, BP1026B I2784, BP1026B I2780, BURPS1106A A0094, BURPS1106A 1131, and BURPS1710A 1419 were identified earlier by PCR-based subtractive hybridization. These genes were extensively characterized at the molecular level, together with an additional gene BPSL3147 that had been identified by other investigators. Through a reverse genetic approach, single-gene knockout mutants were successfully constructed by using site-specific insertion mutagenesis and were confirmed by PCR. BPSL2033::Km and BURPS1710A 1419::Kmmutants showed reduced rates of survival inside macrophage RAW 264.7 cells and also low levels of virulence in the nematode infection model. BPSL2033::Km demonstrated weak statistical significance