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Temperature-Regulated Microcolony Formation by Burkholderia pseudomallei Requires pilA and Enhances Association with Cultured Human Cells

Justin A. Boddey,^1, Cameron P. Flegg,¹ Chris J. Day,² Ifor R. Beacham,^1* and Ian R. Peak¹

Institute for Glycomics,¹ School of Medical Science, Griffith University, Gold Coast, Queensland, Australia²

Received 6 April 2006/ Returned for modification 2 June 2006/ Accepted 28 June 2006

	ABSTRACT

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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.

	INTRODUCTION

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Burkholderia pseudomallei is a gram-negative, saprophytic bacterium that causes a variety of serious and potentially fatal diseases, known as melioidoses, in mammals. Melioidosis is acquired by inhalation, inoculation, or ingestion of B. pseudomallei from soil, dust, or water, and mammal-mammal transmission is very rare (10, 12, 32, 52). Melioidosis is endemic to tropical Australia and Southeast Asia, particularly Thailand and Singapore; however, the disease is considered to be emerging worldwide (9, 13). Melioidosis has a spectrum of clinical presentations that can mimic many other diseases and can thus be misdiagnosed (17, 32). Melioidosis may be acute, chronic, or subclinical (23, 25) and can affect essentially any organ, with lungs, liver, and spleen most commonly involved (32). Melioidosis is difficult to treat and results in up to 50% mortality in Thailand (51) and 19% mortality in Australia (11). B. pseudomallei is listed as a category B bioterrorism agent by the Centers for Disease Control and Prevention (53), and there is currently no vaccine against melioidosis.

Adherence to and colonization of host surfaces are essential first steps in the establishment of bacterial infections (19). Nonintimate adherence to a host cell is often mediated by pili, whereas more-intimate interactions are usually established by nonpilus adhesins (20, 26). A number of studies have investigated B. pseudomallei adherence to eukaryotic cells (1, 6, 18, 22, 27, 28, 45), and the pilA gene, putatively encoding a type IVA pilin, is necessary for adherence of B. pseudomallei K96243 to eukaryotic cells in vitro and contributes to complete virulence in animal models of disease (18).

Type IV pili can facilitate bacterium-bacterium interactions that result in microcolonies and/or biofilms (8). Microcolonies and biofilms are both important in pathogenesis as they increase bacterial numbers and can enhance protection from host defenses and antibiotics (7, 8, 21, 42). While B. pseudomallei can form microcolonies and biofilms both in vitro and in vivo (6, 31, 33, 44, 48-50), the molecular basis for these phenotypes is currently unknown.

This paper describes the comparative analysis of adherence, microcolony formation, and biofilm development by B. pseudomallei strains 08 and K96243 and identifies differences in these phenotypes and in their regulation between strains. Furthermore, we show that pilA in strain 08 is upregulated at lower temperatures and in the presence of eukaryotic cells and is essential for microcolony formation, which thereby enhances bacterial association with eukaryotic cells.

	MATERIALS AND METHODS

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Bacterial strains, plasmids, and growth conditions. The bacterial strains and plasmids used in this study are described in Table 1. Bacteria were grown at 27°C or 37°C on LB agar or in Luria broth at 200 rpm overnight. Antibiotics were added to the following final concentrations: chloramphenicol, 50 µg/ml (Escherichia coli) and 100 µg/ml (B. pseudomallei); gentamicin, 15 µg/ml; and streptomycin, 100 µg/ml.

Assay for bacterial adherence or association with eukaryotic cells. Our previous work with various cell lines determined that B. pseudomallei 08 adheres most to ME-180 cells (6), and this was also observed for K96243 (J. Boddey, unpublished data). ME-180 cells were therefore used in this study; they were grown to 95 to 99% confluence in McCoy's 5a medium supplemented with 10% heat-inactivated fetal calf serum in 24-well culture plates (Greiner BioOne) containing 13-mm coverslips (Nalge Nunc International). Two milliliters of overnight-grown bacterial starter cultures (37°C; 200 rpm) was diluted 1:100 into fresh Luria broth and grown with shaking for 18 h at 27°C or 37°C. Bacteria were seeded into triplicate wells at a multiplicity of infection of 25:1, spun at 198 x g for 2 min, and incubated at 37°C in 5% CO₂ for 2 h. Nonadherent bacteria were removed by four washes with phosphate-buffered saline. Samples were fixed with methanol at 4°C for 2 h, stained with Giemsa (pH 6.7; Sigma-Aldrich), and mounted onto glass slides (First Brand) with cover glasses (Marienfeld, Germany). Slides were ordered in a manner unknown to the observer ("blinded") and viewed under oil immersion. Five digital images per coverslip of cell-associated bacteria were captured (three coverslips per strain per experiment), and the following parameters were enumerated (43): the mean association index (the number of bacteria associated with one cell); the mean infection index (the number of cells with at least one adherent bacterium); and the mean microcolony index (the number of adherent microcolonies per field of view). The five greatest numbers of cell-associated bacteria per field of view were used to derive the association index. All cells that were entirely visible (i.e., not partially out of view) were included in the derivation of the infection index. Adherent microcolonies were arbitrarily defined as 10 or more continuous bacterium-bacterium interactions, and these were used to derive the microcolony index. The data for each parameter were pooled from triplicate experiments, averaged, and subjected to appropriate statistical analyses (see below), and finally, the strains were revealed ("unblinded"). It may be noted that only the infection index reflects adherence (direct bacterium-cell interaction), whereas the association index and the microcolony index reflect the formation of microcolonies.

pilA mRNA expression analysis by Q-PCR. For analysis of pilA mRNA expression in the presence or absence of eukaryotic cells, bacteria from overnight cultures (27°C or 37°C in Luria broth; 200 rpm) were seeded at a multiplicity of infection of 25:1 into T-25 flasks containing McCoy's 5a medium supplemented with 10% fetal calf serum, with or without ME-180 cells (95 to 99% confluence). Cultures were incubated for 3 h at 37°C in 5% CO₂. Cell monolayers were washed four times to remove nonadherent bacteria, and cells and associated bacteria were lysed with 4 M guanidium isothiocyanate-1% lauryl sarcosine solution. Media were collected from flasks lacking ME-180 cells and briefly centrifuged at 10,000 x g, and bacteria were resuspended in 4 M guanidium isothiocyanate-1% lauryl sarcosine solution. For analysis of pilA mRNA expression during growth in Luria broth or on LB agar, bacteria were grown for 18 h at 27°C or 37°C. Liquid cultures were briefly centrifuged at 10,000 x g, and bacterial pellets were resuspended in 4 M guanidium isothiocyanate-1% lauryl sarcosine solution. Bacterial cultures grown on agar were resuspended directly in 4 M guanidium isothiocyanate, 1% lauryl sarcosine solution. Following cell lysis, total RNA and first-strand cDNA were prepared as described previously (14). Quantitative real-time PCRs (Q-PCRs) were performed at an annealing temperature of 59°C or 62°C in 1x SYBR green I supermix (Bio-Rad). Primer concentrations were 5 µmol for 16S RNA (4) and 7.5 µmol for pilA expression with the oligonucleotide primer pair JAB16RTF (5'-GCCTATCAGGATTATCTCGC-3') and JAB16RTR (5'-CACCAGCACGAGCGTATT-3'). The PCR mixtures were cycled as follows: 96°C for 30 s, 59°C or 62°C (for 16S and pilA, respectively) for 45 s, and 72°C for 1 minute, followed by a melt curve ranging from 59 to 96°C, with data collected for 5 s at each half-degree-Celsius change. Copy numbers were calculated from standard curves of 10 to 10⁹ copies of PCR product.

Biofilm assay. The biofilm assay was adapted from reference 33. Nonsterile 96-well polyvinyl chloride culture plates (Falcon 3911 Microtest III, Becton Dickinson Labware) were sterilized with 70% ethanol and air dried in a sterile class II biosafety cabinet immediately prior to use. Luria broth (100 µl per well) was added followed by 1 µl of bacterial culture that had been grown at 37°C overnight at 200 rpm. Wells were covered and incubated without shaking for 18 h at 37°C. Thereafter, 1 µl from each well (mixed) was transferred into triplicate wells containing 100 µl fresh Luria broth or M9 medium (supplemented with 0.5% Casamino Acids [Sigma-Aldrich Co.]) and plates were incubated without shaking at 27°C or 37°C for 18 h. The supernatant was carefully removed, and wells were stained with 150 µl 1% crystal violet (Biomerieux) for 30 min at room temperature. The stain was removed, and the wells were washed twice with 175 µl sterile water. Crystal violet stain was solubilized by the addition of 175 µl dimethyl sulfoxide (Ajax Chemicals) to each well, and the absorbance at 595 nm (A₅₉₅) of the solution was measured in a Wallac Victor³ plate reader (Perkin Elmer). The absorbance was adjusted by subtracting the A₅₉₅ for wells that contained media but no bacteria, following the addition of dimethyl sulfoxide, from the overall A₅₉₅ for wells that contained bacteria. Each strain was investigated with three wells per experiment, over three independent experiments.

Statistical analyses. Statistical analyses were undertaken by using the independent-sample two-tailed t test. The Mann-Whitney test was applied to data that were not normally distributed. All t tests were performed with GraphPad Prism 4 for Windows and had a significance threshold () of 0.05.

	RESULTS

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Microcolony formation by B. pseudomallei 08 but not K96243 enhances association with eukaryotic cells and is modulated by temperature. We previously reported that adherence of B. pseudomallei 08 increases dramatically following prior growth at 30°C or less compared to that following prior growth at 37°C (6). Furthermore, adherent microcolonies were observed with growth at or below 30°C prior to assay but were rarely observed following growth at 37°C (6). We have further investigated the adherence phenotype of B. pseudomallei 08 and compared it with that of K96243 by determining the mean number of bacteria associated with one cell (association index), the mean number of cells with at least one adherent bacterium (the infection index; see below), and the derivation of the mean number of microcolonies per field of view (the microcolony index; see reference 43 and Materials and Methods). Only the infection index reflects adherence alone, as opposed to microcolony formation.

We observed that temperature drastically modulates the association of strain 08 with eukaryotic cells; a 3.4-fold increase in the association index was observed when 08 was grown at 27°C prior to assay compared to what was observed with growth at 37°C (Fig. 1A, C, and D). However, temperature did not influence K96243 association with cells, which was very low relative to that for 08 (a 5.9-fold difference was observed between 27°C-grown cultures; P < 0.001) (Fig. 1A).

View larger version (103K): [in this window] [in a new window]   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.

Temperature plays a minor role in modulating direct adherence of B. pseudomallei 08 but not K96243 to eukaryotic cells. As temperature regulates microcolony formation (bacterium-bacterium interactions) by B. pseudomallei 08 and K96243, we investigated whether temperature also affects adherence (bacterium-cell interactions) of these strains by determining the mean infection index (number of cells with at least one adherent bacterium).

B. pseudomallei 08 grown at 27°C prior to assay had an infection index 1.1-fold (14.2%) greater than 08 grown at 37°C (Fig. 2), indicating a minor, though statistically significant, increase in adherence to ME-180 cells. In contrast, growth temperature did not influence the infection index of K96243 (Fig. 2).

View larger version (8K): [in this window] [in a new window]   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.

View larger version (68K): [in this window] [in a new window]   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.

View larger version (9K): [in this window] [in a new window]   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.

pilA mRNA expression was measured by using Q-PCR; after overnight growth at 37°C in Luria Broth, expression was relatively low, as was expression 3 h after transfer to McCoy's 5a medium at 37°C with or without ME-180 cells (Fig. 5A). pilA mRNA expression after overnight growth at 27°C in Luria broth, and following 3 h in McCoy's 5a medium at 37°C, was also relatively low but increased dramatically after transfer to McCoy's 5a medium at 37°C when ME-180 cells were also present (32.5-fold increase) (Fig. 5).

View larger version (7K): [in this window] [in a new window]   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.

We conclude that both low growth temperature and the presence of either a solid substratum or cultured cells at 37°C are effective in eliciting pilA expression in strain 08.

In contrast, pilA expression was not temperature regulated in strain K96243 (P = 0.189 in the presence of ME-180 cells) (Fig. 5B). However, like 08, ME-180 cells elicited the most pilA expression by K96243; irrespective of growth temperature, pilA expression by K96243 in the presence of ME-180 cells was statistically equivalent to levels observed for 08 in the presence of ME-180 cells, when grown at 27°C prior to exposure (Fig. 5A, B).

Temperature and media modulate biofilm formation by B. pseudomallei 08 and K96243. It has been reported that biofilm formation by B. pseudomallei varies depending on growth media (33). Given that temperature is a regulatory stimulus for microcolony formation and for pilA expression in B. pseudomallei 08, we investigated whether temperature, in addition to media, was involved in regulating biofilm formation by B. pseudomallei strains 08 and K96243.

Biofilm formation was greater at 27°C than at 37°C for both B. pseudomallei 08 and K96243 when grown in Luria broth (P < 0.001) (Fig. 6A); however, this was not the case in minimal medium in which biofilm formation was significantly increased at 37°C for K96243 but not for 08 (Fig. 6B). Biofilm formation at 37°C was increased in M9 medium relative to that in Luria broth, but this was reversed when bacteria were grown at 27°C (Fig. 6).

View larger version (9K): [in this window] [in a new window]   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 pilA 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.

	DISCUSSION

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It is apparent from this and previous work (6) that temperature is a key regulatory signal for microcolony formation and, to a lesser extent, direct adherence of B. pseudomallei 08 to eukaryotic cells. However, it is also apparent that colonization of a eukaryotic monolayer by B. pseudomallei can involve two types of cellular interactions, depending on the strain: bacterium-cell interactions (adherence) and bacterium-bacterium interactions (microcolonies), the latter dramatically increasing the number of bacteria in association with cells. Given that microcolonies were infrequently formed when bacteria were grown at 37°C prior to assay and that the infection index (adherence) was essentially unchanged despite growth temperature, microcolonies clearly do not directly enhance the number of eukaryotic cells with adherent bacteria; rather, they enhance the number of bacteria associated with a cell via bacterium-bacterium interactions. For other pathogens, such as enteropathogenic Escherichia coli, Vibrio cholerae, Bartonella henselae, and Neisseria meningitidis, microcolony formation or "bacterial aggregation" is considered important for colonization in vivo or in in vitro models (3, 16, 29, 36), and this may also be the case for B. pseudomallei.

Since microcolonies of B. pseudomallei 08 form only at 37°C (during an adherence assay) following prior growth at 27°C, we propose that the environmental niche is an important component of microcolony formation by B. pseudomallei and for transmission to a host, since mammal-mammal transmission (37°C) is very rare and the majority of B. pseudomallei infections are acquired from soil and/or water (12, 32). Furthermore, the ability of B. pseudomallei 08 to form microcolonies may be relevant to virulence in vivo since microcolony formation in the lungs of infected humans and animals has been reported (49). However, the ability of strain K96243 to cause serious disease, despite forming very few microcolonies in vitro, suggests either that microcolony formation by K96243 occurs under conditions other than those used in this study or that microcolonies are not essential for virulence in vivo.

We have shown that pilA is essential for microcolony formation by B. pseudomallei 08, which significantly enhances the number of bacteria associated with a cell. Indeed, in the case of B. henselae, the aggregated bacteria are engulfed and internalized as a specific structure, the "invasome" (16). Type IV pili also mediate microcolony formation by Pseudomonas aeruginosa (35, 39), enteropathogenic Escherichia coli (3, 46), V. cholerae (29), and N. meningitidis (36). Our results indicate that the presence of ME-180 cells or media surrounding ME-180 cells or, alternatively, contact with a solid surface are important signals for pilA mRNA expression in 08 and that temperature preconditioning is essential for optimal pilA expression. These conditions for pilA mRNA expression correlate with those required for adherent microcolony formation. Since K96243 rarely forms microcolonies but requires pilA for adherence to eukaryotic cells in vitro (18), it appears that pilA has different roles, and is differentially regulated, in 08 and K96243. Our evidence does indeed suggest that the regulation of pilA in K96243 is different from that in 08: in particular, in K96243, pilA does not appear to be upregulated by low temperature when grown either in Luria broth or on LB agar. However, pilA is strongly expressed by K96243 in the presence of eukaryotic cells, following growth at either 27°C or 37°C, pointing to other explanations for the relative lack of microcolony formation under these conditions. It is possible that molecules in addition to PilA are required for microcolony formation and that these are differentially present/regulated between 08 and K96243. The regulation of pilA in 08 and K96243 clearly merits further investigation with respect to both the effect of temperature and the effect of agar or the presence of ME-180 cells. It may be noted that contact regulation of gene expression in bacteria is not unprecedented (15, 34).

Biofilms are important in bacterial pathogenesis as they generate a physiologically heterogeneous population of organisms and the biofilm matrix can protect bacteria from host defenses and antibiotics (7, 21, 42). Therefore, biofilms are often associated with chronic infection (21). Melioidosis is often a chronic disease, and the ability for B. pseudomallei to generate biofilms both in vitro and in vivo is well documented (33, 44), though the molecular basis is poorly understood.

Although microcolonies and biofilms may be mediated by the same pilus in other bacteria (30), this does not seem to be the case for strain 08. Firstly, pilA is not required for biofilm formation by 08 (or K96243). Indeed, under one condition, namely, minimal medium at 37°C, deletion of pilA increased biofilm formation. Secondly, K96243 rarely formed adherent microcolonies but was able to generate biofilms at least to the same extent as 08. We therefore conclude that the formation of biofilms by B. pseudomallei does not involve pilA and that the formation of microcolonies and biofilms by B. pseudomallei 08 involves two separate processes requiring different molecules.

Phenotypic and genotypic heterogeneity between strains, including differences between strains 08 and K96243 (24), is extensive in the microbial world and is well documented for B. pseudomallei (2, 22, 40, 44, 47). In this study, we report differences in microcolony formation, pilA expression, and biofilm formation between strains 08 and K96243; although the molecular basis for these differences is yet to be further delineated, they emphasize the need to consider virulence in B. pseudomallei to be due to a collection of factors distributed unequally between significantly different strains. Clearly, as many strains as possible should be systematically studied for their adherence properties. Also, potential adhesins other than type IVA pili should be investigated (18, 24). These tasks should be aided by the availability of genome sequence data for 10 additional B. pseudomallei isolates (52).

	ACKNOWLEDGMENTS

 
J.A.B. and C.J.D. were recipients of Australian Postgraduate Awards. This work was supported by the National Health and Medical Research Council, Australia.

	FOOTNOTES

 
* Corresponding author. Mailing address: School of Medical Science, Griffith University, Gold Coast Campus, PMB50, Gold Coast Mail Centre, Queensland 9726, Australia. Phone: 61 7 5552 8185. Fax: 011 61 7 5552 8908. E-mail: i.beacham{at}griffith.edu.au.

Editor: V. J. DiRita

Present address: The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia.

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