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Characterisation of Burkholderia pseudomallei colony morphology variants by microarray

ABSTRACT: 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.

ORGANISM(S): Burkholderia pseudomallei Burkholderia pseudomallei K96243

PROVIDER: GSE87827 | GEO | 2016/10/12

SECONDARY ACCESSION(S): PRJNA347822

REPOSITORIES: GEO

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Project description:Background Small colony variants (SCVs) are slow-growing bacteria, which often show increased resistance to antibiotics and cause latent or recurrent infections. It is therefore important to understand the mechanisms at the basis of this phenotypic switch. Methodology/Principal findings One SCV (termed PAO-SCV) was isolated, showing high resistance to gentamicin and to the cephalosporine cefotaxime. PAO-SCV was prone to reversion as evidenced by emergence of large colonies with a frequency of 10-5 on media without antibiotics while it was stably maintained in presence of gentamicin. PAO-SCV showed a delayed growth, defective motility, and strongly reduced levels of the quorum sensing Pseudomonas quinolone signal (PQS). Whole genome expression analysis further suggested a multi-layered antibiotic resistance mechanism, including simultaneous over-expression of two drug efflux pumps (MexAB-OprM, MexXY-OprM), the LPS modification operon arnBCADTEF, and the PhoP-PhoQ two-component system. Conversely, the genes for the synthesis of PQS were strongly down-regulated in PAOSCV. Finally, genomic analysis revealed the presence of mutations in phoP and phoQ genes as well as in the mexZ gene encoding a repressor of the mexXY and mexABoprM genes. Only one mutation occurred only in REV, at nucleotide 1020 of the tufA gene, a paralog of tufB, both encoding the elongation factor Tu, causing a change of the rarely used aspartic acid codon GAU to the more common GAC, possibly causing an increase of tufA mRNA translation. High expression of phoP and phoQ was confirmed for the SCV variant while the revertant showed expression levels reduced to wild-type levels. Conclusions By combining data coming from phenotypic, gene expression and proteome analysis, we could demonstrate that resistance to aminoglycosides in one SCV mutant is multifactorial including overexpression of efflux mechanisms, LPS modification and is accompanied by a drastic down-regulation of the Pseudomonas quinolone signal quorum sensing system. We used microarrays to study changes in gene expression during early and late stationary phase of SCV and WT strains. SCV and WT cultures were grown in triplicate until early (24h) and late (48h) stationary phase. RNA was extracted, labelled and hybridised on Affymetrix P. aeruginosa expression microarrays.

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Characterisation of Burkholderia pseudomallei colony morphology variants by microarray

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