Temperature-regulated microcolony formation by Burkholderia pseudomallei requires pilA and 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 degrees C or less compared to that following prior growth at 37 degrees C. Here, we show that this occurs almost entirely as the result of microcolony formation (bacterium-bacterium interactions) following growth at 27 degrees C but not at 37 degrees 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 degrees C or 37 degrees 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.

FIG. 1.

Microcolony formation by B. pseudomallei 08 but not K96243 is modulated by temperature and enhances association with ME-180 cells. (A) Mean numbers of ME-180-associated bacteria per cell ± standard errors of the means (association index) following growth at 27°C and 37°C. (B) Mean numbers of adherent microcolonies per field of view ± standard errors of the means (microcolony index) following growth at 27°C and 37°C. (C) Digital image of B. pseudomallei 08 adherence and microcolony formation (arrows) to ME-180 cells following growth at 27°C. Bar represents 10 μm. (D) Digital image of B. pseudomallei 08 adherence, with few microcolonies (arrow), to ME-180 cells following growth at 37°C. (E and F) Digital images of B. pseudomallei K96243 adherence to ME-180 cells without adherent microcolonies following growth at 27°C and 37°C, respectively. Data in panels A and B are pooled from triplicate experiments in which approximately equal inocula were delivered. *, P value of <0.001.

FIG. 2.

Temperature is a minor modulator of B. pseudomallei 08 adherence to ME-180 cells. Mean percentages of ME-180 cells with one or more adherent bacteria ± standard errors of the means (infection index) following growth of 08 or K96243 at 27°C or 37°C are shown. Data are pooled from triplicate experiments in which approximately equal inocula were delivered. *, P value of 0.012.

FIG. 3.

Role of pilA in microcolony formation and bacterial association with ME-180 cells. (A) Mean numbers of ME-180-associated bacteria per cell ± standard errors of the means (association index) following growth at 27°C and 37°C. (B) Mean numbers of adherent microcolonies per field of view ± standard errors of the means (microcolony index) following growth at 27°C and 37°C. Data (A and B) are pooled from triplicate experiments in which approximately equal inocula were delivered. *, P value of <0.004. (C) Digital image capturing B. pseudomallei 08 adherence, with microcolony formation (arrows), to ME-180 cells following growth at 27°C. Bar represents 10 μm. (D) Digital image capturing JAB1608 adherence, without microcolony formation, to ME-180 cells following growth at 27°C.

FIG. 4.

pilA is not required for adherence of B. pseudomallei 08 to ME-180 cells. Mean percentages of ME-180 cells with one or more adherent bacteria ± standard errors of the means (infection index) following growth at 27°C and 37°C are shown. Data are pooled from triplicate experiments in which approximately equal inocula were delivered.

FIG. 5.

Expression of pilA mRNA in B. pseudomallei 08 and K96243. Q-PCR was used to measure the expression of mRNA for pilA in B. pseudomallei 08 (A) and K96243 (B) grown at 27°C and 37°C with different media and under different conditions. Expression is reported as mean numbers of copies of pilA per copy of 16S rRNA ± standard errors of the means. Experiments using McCoy's 5a media were conducted at 37°C, with bacteria pregrown at 27°C or 37°C, as shown and in the presence or absence of ME-180 cells. Data are pooled from triplicate experiments. *, P value of <0.025.

FIG. 6.

Biofilm formation by B. pseudomallei 08 and K96243 is modulated by temperature and media and is pilA independent. Mean adjusted biofilm formation levels ± standard errors of the means for B. pseudomallei 08, K96243, and respective ΔpilA strains JAB1608 and JAB16 when grown at 27°C and 37°C in Luria broth (A) and M9 medium supplemented with Casamino Acids (B) are shown. Data are pooled from triplicate experiments. *, P value of 0.028.