Identification ofBurkholderia pseudomalleiGenes Required for the Intracellular Life Cycle and In Vivo Virulence

Abstract

The bacterial pathogen Burkholderia pseudomallei invades host cells, escapes from endocytic vesicles, multiplies intracellularly, and induces the formation of actin tails and membrane protrusions, leading to direct cell-to-cell spreading. This study was aimed at the identification of B. pseudomallei genes responsible for the different steps of this intracellular life cycle. B. pseudomallei transposon mutants were screened for a reduced ability to form plaques on PtK2 cell monolayers as a result of reduced intercellular spreading. Nine plaque assay mutants with insertions in different open reading frames were selected for further studies. One mutant defective in a hypothetical protein encoded within the Bsa type III secretion system gene cluster was found to be unable to escape from endocytic vesicles after invasion but still multiplied within the vacuoles. Another mutant with a defect in a putative exported protein reached the cytoplasm but exhibited impaired actin tail formation in addition to a severe intracellular growth defect. In four mutants, the transposon had inserted into genes involved in either purine, histidine, or p-aminobenzoate biosynthesis, suggesting that these pathways are essential for intracellular growth. Three mutants with reduced plaque formation were shown to have gene defects in a putative cytidyltransferase, a putative lipoate-protein ligase B, and a hypothetical protein. All nine mutants proved to be significantly attenuated in a murine model of infection, with some mutants being essentially avirulent. In conclusion, we have identified a number of novel major B. pseudomallei virulence genes which are essential for the intracellular life cycle of this pathogen.

In vivo expression technology identifies a type VI secretion system locus in Burkholderia pseudomallei that is induced upon invasion of macrophages

“…BPSS1509 corresponds to the tssK orthologue in the macrophage-inducible tss-5 unit. Our observation that inactivation of tssH-5 does not significantly affect the ability of B. pseudomallei to survive in macrophages is in agreement with the observation of Pilatz et al (2006) regarding the normal growth phenotype of the tssK-5 mutant in macrophages. Moreover, in the three co-integrate strains that we isolated in which pGSTp was integrated in the tss-5 unit, it is likely that expression of the tss genes located downstream of the integrated vector were also disrupted (as the genomic fragments cloned into pGSTp were too small to contain the predicted promoter region).…”

“…2). T3SS-3 has been shown to be important for growth of B. pseudomallei within macrophage-like cells and for escape from endocytic vacuoles, and one of the associated effector proteins, BopE (BPSS1525), is required for efficient invasion of epithelial cells (Stevens et al, 2002(Stevens et al, , 2003Ribot & Ulrich, 2006;Pilatz et al, 2006). T3SS-3 is also required for maximum virulence of B. pseudomallei in murine and hamster models of infection (Stevens et al, 2004;Warawa & Woods, 2005).…”

“…Pertinently, in a screen for transposon mutants of B. pseudomallei that exhibited a decreased ability to plaque on PtK2 cell monolayers, one of the mutants obtained carried an insertion in BPSS1509, encoding a hypothetical protein (Pilatz et al, 2006). This mutant was shown to be less virulent than the wild-type strain in a mouse model of infection (Pilatz et al, 2006).…”

“…This mutant was shown to be less virulent than the wild-type strain in a mouse model of infection (Pilatz et al, 2006). BPSS1509 corresponds to the tssK orthologue in the macrophage-inducible tss-5 unit.…”

Development of Signature-Tagged Mutagenesis in Burkholderia pseudomallei To Identify Genes Important in Survival and Pathogenesis

“…A recent study by Pilatz et al demonstrated that a mutant with an insertion in BPSL2825 was severely attenuated in the i.n. BALB/c mouse model; this mutant was also shown to have a defect in iron acquisition and a reduced ability to grow intracellularly (31). In total, putative auxotrophs of five different compounds were identified during this study.…”