Temperature-Regulated Microcolony Formation byBurkholderia pseudomalleiRequirespilAand Enhances Association with Cultured Human Cells

Abstract

Burkholderia pseudomallei is the causative agent of melioidosis, a potentially fatal disease that is endemic to Northern Australia and Southeast Asia and is acquired from soil or water. Adherence of B. pseudomallei 08 to cultured cells increases dramatically following prior growth at 30°C or less compared to that following prior growth at 37°C. Here, we show that this occurs almost entirely as the result of microcolony formation (bacterium-bacterium interactions) following growth at 27°C but not at 37°C, which considerably enhances bacterial association with eukaryotic cells. Further, we demonstrate that the type IVA pilin-encoding gene, pilA, is essential for microcolony development by B. pseudomallei 08, and thus optimum association with eukaryotic cells, but is not required for direct adherence (bacterium-cell interactions). In contrast, although the B. pseudomallei genome sequence strain, K96243, also contains transcriptionally active pilA, microcolony formation rarely occurs following growth at either 27°C or 37°C and cell association occurs significantly less than with strain 08. Analysis of pilA transcription in 08 identified that pilA is dramatically upregulated under microcolony-forming conditions, viz., growth at low temperature, and association with eukaryotic cells; the pattern of transcription of pilA in K96243 differed from that in 08. Our study also suggests that biofilm formation by B. pseudomallei 08 and K96243 on polyvinylchloride is not mediated by pilA. Adherence and microcolony formation, and pilA transcription, vary between strains, consistent with known genomic variation in B. pseudomallei, and these phenotypes may be relevant to colonization from the environment.

The Genome of Burkholderia cenocepacia J2315, an Epidemic Pathogen of Cystic Fibrosis Patients

“…Type IV pili have also been shown to play a role in the adherence of B. pseudomallei to eukaryotic cells. Microcolony formation is a key process in cell adherence-an ability reduced in pilA mutants (15). In P. aeruginosa, type IV pili have been shown to bind to human epithelial cells (53), as well as induce apoptosis (55).…”

Burkholderia pseudomallei sequencing identifies genomic clades with distinct recombination, accessory, and epigenetic profiles

“…For instance, one haplotype overrepresented in nonsynonymous SNPs occurred in the virulenceassociated TFP1 locus (BPSL0782-BPSL0783), within the pilA gene in ST51a strains. Notably, pilA plays a role in virulence yet its role in adherence and microcolony formation varies considerably in different Bp strains (Essex-Lopresti et al 2005;Boddey et al 2006). These findings suggest that haplotype variation may contribute to differences in Bp pathogenicity or survival in different strains.…”

“…Isogenic Bp mutants carrying a 12.9-kb deletion TFP8 were generated in a two-step process as previously described (see Supplemental Methods; Essex-Lopresti et al 2005;Boddey et al 2006). Virulence of wild-type and mutant Bp strains was assessed using an intranasal BALB/c mouse model (Essex-Lopresti et al 2005).…”

CdpA Is a Burkholderia pseudomallei Cyclic di-GMP Phosphodiesterase Involved in Autoaggregation, Flagellum Synthesis, Motility, Biofilm Formation, Cell Invasion, and Cytotoxicity

“…The B. pseudomallei cdpA mutant, which showed increased cell-to-cell adhesion, was in fact slightly less invasive on A549 cells than the wild type, possibly because formation of microcolonies might not be necessary for B. pseudomallei which normally infect lung epithelial cells via the respiratory route rather than the bloodstream. Data on the role of relevance of microcolony formation in colonization of eukaryotic pseudomallei can form microcolonies to enhance its colonization of eukaryotic cells, the process was not required by all strains and was also temperature dependent, occurring at 27°C but not 37°C (2). Given the pivotal roles of c-di-GMP-specific PDEs in the regulation of many bacterial behaviors, elucidation of the sensory and catalytic functions of CdpA in B. pseudomallei would contribute to deciphering the role of the cyclic di-GMP signaling in virulence and environmental adaptation of this important pathogen and also in the formation of biofilm, which is hypothesized to resemble the chronic form of melioidosis (11).…”