Characterisation of Burkholderia pseudomallei colony morphology variants by microarray

Burkholderia pseudomallei  is the causative agent of melioidosis which is endemic to Southeast Asia and Northern Australia. It is a Gram-negative soil and water bacterium that represents a potential bioterrorism threat. Colony morphology variation is a remarkable feature in primary clinical cultures of B. pseudomallei. Differences in expression of several potential virulence and survival genes were believed to be associated with B. pseudomallei colony morphology variants. Microarrray approach was used to investigate alterations of the global B. pseudomallei transcriptome profile at the mid-logarithmic phase of growth, among the wild type (WT) and small colony variant (SCV) of B. pseudomallei pre- and post-exposed to human lung epithelial cells, A549. Generally, SCV pre- and post-exposed have lower metabolic requirements and consume lesser energy than WT pre- and post-exposed to A549; however, both WT and SCV may limit their metabolic activity during the infection of A549 cells and this is indicated by the down-regulation of genes implicated in metabolism of amino acids, carbohydrate, lipid, and other amino acids, and biodegradation of xenobiotics. On the other hand, many well-known virulence and survival factors including T3SS, T6SS, fimbriae, capsular polysaccharides, drug resistance and stress response were up-regulated in both WT and SCV pre- and post-exposed to A549 cells. Several virulence factors expressed at the mid-logarithmic phase of growth. Microarray analysis on the different morphotypes demonstrated the essential difference in bacterial response associated with virulence and survival pre- and post-exposed to A549 cells. The Agilent-019078 Custom B. pseudomallei 8X15K microarray chip (Agilent, GEO reference GPL13233), designed by Chieng S et al., (2012) (Chieng et al., 2012) was used as the platform to inspect the changes in the gene expression of B. pseudomallei WT and SCV morphotypes pre- and post-exposed to A549 human lung epithelial cells, (Figure 2.7). Four biological replicates were used for each set of the experiments. The chip was designed for the analysis of 5,721 non-cross-hybridising ORFs of B. pseudomallei K96243. The 5,721 probe sequences were randomly replicated and distributed in a microarray of 15,000 probes to fit Agilent 8 × 15 K microarray format. Figure 2.7 shows the microarray experimental design scheme of the gene expression studies. The quality control data obtained confirmed that all the arrays on the microarray chip met the expected quality control, however one replicate among the 4 replicates have been excluded for the following samples (SCV pre-exposed, WT post-exposed and SCV post-exposed).