B. pseudomallei has been shown to persist intracellularly in melioidosis patients until reactivated by decreasing immunocompetence. We have shown by transmission electron microscopy the internalization of B. pseudomallei by human macrophages via conventional phagocytosis enclosed within membrane-bound vacuoles or phagosomes. Ferritin labeled lysosomes provided evidence of phagosome-lysosome fusion. Ingested bacilli were designated as "intact" or "damaged" on the basis of their ultrastructural features. An intact bacterium was seen with low electron opaque central nuclear region surrounded by dense bacterial cytoplasm, bounded externally by bacterial plasma membrane and cell wall. In contrast, B. pseudomallei were considered damaged when seen with cavitation within the central nuclear region, separation of bacterial cytoplasm from the cell wall, herniation of cytoplasmic contents and lamination of bacterial cell wall and its surrounding electron transparent zone. Our observations indicate that the microbicidal mechanism(s) in B. pseudomallei-infected macrophages failed to ensure complete clearance of the organism and this failure probably facilitates intracellular persistence and proliferation, and this may be one of the survival strategies adopted by this organism.
The occurrence of latency and relapse in human melioidosis suggests adaptations by Burkholderia pseudomallei that help to avoid the human immune response. Ruthenium red-stained preparations of bacterial cultures viewed by electron microscopy revealed three morphologically distinct variants; one with a very marked and another with a less electron-dense layer surrounding the cell wall, and a third variety devoid of such a structure. This structure may be attributable to a layer of polysaccharide, suggesting the presence of a glycocalyx that may aid in the survival of the organism during latency.
Burkholderia pseudomallei is an opportunistic pathogen and the causative agent of melioidosis. It is able to adapt to harsh environments and can live intracellularly in its infected hosts. In this study, identification of transcriptional factors that associate with the β' subunit (RpoC) of RNA polymerase was performed. The N-terminal region of this subunit is known to trigger promoter melting when associated with a sigma factor. A pull-down assay using histidine-tagged B. pseudomallei RpoC N-terminal region as bait showed that a hypothetical protein BPSS1356 was one of the proteins bound. This hypothetical protein is conserved in all B. pseudomallei strains and present only in the Burkholderia genus. A BPSS1356 deletion mutant was generated to investigate its biological function. The mutant strain exhibited reduced biofilm formation and a lower cell density during the stationary phase of growth in LB medium. Electron microscopic analysis revealed that the ΔBPSS1356 mutant cells had a shrunken cytoplasm indicative of cell plasmolysis and a rougher surface when compared to the wild type. An RNA microarray result showed that a total of 63 genes were transcriptionally affected by the BPSS1356 deletion with fold change values of higher than 4. The expression of a group of genes encoding membrane located transporters was concurrently down-regulated in ΔBPSS1356 mutant. Amongst the affected genes, the putative ion transportation genes were the most severely suppressed. Deprivation of BPSS1356 also down-regulated the transcriptions of genes for the arginine deiminase system, glycerol metabolism, type III secretion system cluster 2, cytochrome bd oxidase and arsenic resistance. It is therefore obvious that BPSS1356 plays a multiple regulatory roles on many genes.
In Burkholderia pseudomallei, the pathogen that causes melioidosis, the gene cluster encoding the capsular polysaccharide, is located on chromosome 1. Among the 19 capsular genes in this cluster, wzm has not been thoroughly studied. To study the function of wzm, we generated a deletion mutant and compared it with the wild-type strain. The mutant produced less biofilm in minimal media and was more sensitive to desiccation and oxidative stress compared with the wild-type strain, indicating that wzm is involved in biofilm formation and membrane integrity. Scanning electron microscopy showed that the bacterial cells of the mutant strain have more defined surfaces with indentations, whereas cells of the wild-type strain do not.