Primary diagnostic cultures from patients with melioidosis demonstrate variation in colony morphology of the causative organism, Burkholderia pseudomallei. Variable morphology is associated with changes in the expression of a range of putative virulence factors. This study investigated the effect of B. pseudomallei colony variation on survival in the human macrophage cell line U937 and under laboratory conditions simulating conditions within the macrophage milieu. Isogenic colony morphology types II and III were generated from 5 parental type I B. pseudomallei isolates using nutritional limitation. Survival of types II and III were compared with type I for all assays. Type I showed greater survival and replication following uptake by human macrophages compared to types II or III, while uptake of type III alone was associated with colony morphology switching. Specific morphotypes were associated with survival in the presence of H2O2 and antimicrobial peptide LL-37, but were not associated with susceptibility to acid, acidified sodium nitrite, or resistance to lysozyme, lactoferrin, human neutrophil peptide-1 or human beta defensin-2. Incubation under anaerobic condition was a strong driver for switching of type III to an alternative morphotype. Colony switching may be associated with a fitness advantage and survival within macrophages, and may be important for bacterial persistence in vivo.
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Burkholderia pseudomallei is an environmental Gram-negative bacterium that causes a severe and often fatal disease called melioidosis. This is an important cause of sepsis in Southeast Asia and northern Australia, a geographic distribution that mirrors the presence of B. pseudomallei in the environment (Cheng & Currie, 2005). Melioidosis may develop following bacterial inoculation or inhalation and occurs most often in people with regular contact with contaminated soil and water (Cheng & Currie, 2005). Clinical manifestations of melioidosis are highly variable and range from fulminant septicemia to mild localized infection. The overall mortality rate is 40% in northeast Thailand (rising to 90% in patients with severe sepsis) and 20% in northern Australia (Cheng & Currie, 2005; Wiersinga et al., 2006).
A major feature of melioidosis is that bacterial eradication is difficult to achieve. Fever clearance time is often prolonged (median 8 days), antimicrobial therapy is required for 12-20 weeks, and relapse occurs in around 10% of patients despite an appropriate course of antimicrobial therapy (Chaowagul et al., 1993; Currie et al., 2000). The basis for persistence in the infected human host is unknown, although several observations made to date may be relevant to the clinical behavior of this organism (Wiersinga et al., 2006; Adler et al., 2009). B. pseudomallei can resist the action of antibactericidal substances including complement and antimicrobial peptides in human serum (Egan & Gordon, 1996; DeShazer et al., 1998; Wikraiphat et al., 2009). B. pseudomallei can also survive after uptake by a range of phagocytic and non-phagocytic cells. Macrophages have several strategies to control bacterial infection, including bacterial killing following uptake through the action of reactive oxygen and reactive nitrogen compounds, antimicrobial peptides and lysozomal enzymes. Despite this, B. pseudomallei can invade and replicate in primary human macrophages (Puthucheary & Nathan, 2006; Charoensap et al., 2009; Wikraiphat et al., 2009).
Bacterial survival under adverse and rapidly changing environmental circumstances is likely to be facilitated by phenotypic adaptability and plasticity. We have reported previously that the colony morphology appearance of B. pseudomallei can undergo a process of reversible switching in response to starvation and other environmental challenges in vitro, and that this was associated with a spectrum of phenotypic changes including alteration in the expression of biofilm production, bacterial aggregation, and protease and lipase productions (Chantratita et al., 2007). In vitro models suggest that switching of morphotype leads to a significant increase in intracellular replication fitness after uptake by human epithelial cells and mouse macrophages. Switching between morphotypes has also been observed in an experimental mouse model, and 8% of primary clinical cultures associated with infection by a single strain were found to contain more than one morphotype (Chantratita et al., 2007). We postulated that colony morphology switching might represent a mechanism by which B. pseudomallei can adapt within the macrophage and persist in vivo.
Here, we report the findings of study in which we evaluated the interactions of three isogenic morphotypes with human macrophages and a range of laboratory conditions that aimed to simulate one or more conditions within the macrophage milieu.
Materials and Methods
Bacterial isolates and isolation of isogenic morphotypes
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Five B. pseudomallei isolates were examined in this study. Isolates 153, 164 and the reference isolate K96243 were cultured from cases of human melioidosis in Thailand, and isolates B3 and B4 were cultured from uncultivated land in northeast Thailand (Chantratita et al., 2008). The colony morphology of all five parental isolates was type I, and isogenic types II and III were generated from each using nutritional limitation (Chantratita et al., 2007). Briefly, a single colony of type I on Ashdown agar was inoculated into 3 mL of trypticase soy broth (TSB) and incubated at 37 oC in air for 21 days. Bacterial culture was diluted and spread plated onto Ashdown agar. Morphotypes were identified using a morphotyping algorithm (Chantratita et al., 2007).
Growth curve analysis
Growth curves were performed for the 3 isogenic morphotypes of each of the 5 B. pseudomallei isolates. A colony of B. pseudomallei was suspended in sterile phosphate buffered saline (PBS). The bacterial suspension was adjusted to an optical density (OD) at 600 nm of 0.15 and diluted 100 times. One hundred microlitres of bacterial suspension was added to 10 mL of TSB and incubated at 37 oC in air with shaking at 200 rpm for 28 h. At 2 h intervals, 100 mL of bacterial culture was removed, serially diluted 10-fold in PBS, and the bacterial count determined by plating on Ashdown agar in duplicate and performing a colony count following incubation at 37 oC in air for 4 days. Doubling time was calculated.
Cell line and culture conditions
Human monocyte-like cell line U937 (ATCC CRL-1593.2) originating from a histiocytic lymphoma was maintained in RPMI 1640 (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (PAA Laboratories), 100 units mL-1 of penicillin and 100 mg mL-1 of streptomycin (Invitrogen) and cultured at 37 oC in a 5% CO2 humidified incubator (Harada et al., 2007). Before exposure to B. pseudomallei, 1 Ã- 105 U937 cells per well were transferred to a 24 well-tissue culture plate (BD Falcon) and activated by the addition of 50 ng mL-1 of phorbol 12-myristate 13-acetate (PMA) (Sigma) over 2 days. The medium was then replaced with 1 mL of fresh medium without PMA and incubated for 1 day. Following washing 3 times with 1 mL of Hank's balance salt solution (HBSS) (Sigma), 1 mL of fresh medium was gently added to the differentiated macrophages (Harada et al., 2007).
Interaction of B. pseudomallei isogenic morphotypes with human macrophages
The interaction assay was performed as previously described (Chantratita et al., 2007). B. pseudomallei from an overnight culture on Ashdown agar was suspended in PBS, the bacterial concentration adjusted using OD at 600 nm and then diluted in PBS and inoculated into wells containing differentiated U937 cells to obtain an MOI of approximately 25 bacteria per cell. The MOI was verified by colony counting on Ashdown agar. Infected U937 cells were incubated at 37 oC in 5% CO2 for 2 h. Non-adherent bacteria were removed by washing gently 3 times with 1 mL of PBS. The U937 cells were lysed with 1 mL of 0.1% Triton X-100 (Sigma), and the cell lysates serially diluted in PBS and spread plated on Ashdown agar to obtain the bacterial count. Colony morphology was observed (Chantratita et al., 2007). The percentage of bacteria that were cell-associated was calculated by (number of associated bacteria Â´ 100)/number of bacteria in the inoculum. The experiment was performed in duplicate for 2 independent experiments.
Intracellular survival and multiplication of B. pseudomallei in human macrophages were determined at a series of time points following the initial co-culture described above of differentiated U937 with B. pseudomallei for 2 h. Following removal of extracellular bacteria and washing 3 times with PBS, medium containing 250 mg of kanamycin mL-1 (Invitrogen) was added and incubated for a further 2 h (4 h time point). New medium containing 20 mg of kanamycin mL-1 was then added to inhibit overgrowth by any remaining extracellular bacteria at further time points. Intracellular bacteria were determined at 4, 6 and 8 h after initial inoculation. Infected cells were washed, lysed and plated as above. Intracellular survival and multiplication of B. pseudomallei based on counts from cell lysates were presented. Percent intracellular bacteria was calculated by (number of intracellular bacteria at 4h) Ã- 100/number of bacteria in the inoculum. Percent intracellular replication was calculated by (number of intracellular bacteria at 6 or 8 h Ã- 100)/number of intracellular bacteria at 4 h. The experiment was performed in duplicate for 2 independent experiments.
Growth in acid conditions
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B. pseudomallei from an overnight culture on Ashdown agar was suspended in PBS and adjusted using OD at 600 nm to a concentration of 1 Â´ 106 CFU mL-1 in PBS. Thirty microlitres of bacterial suspension was inoculated into 3 mL of Luria-Bertani (LB) broth at a pH 4.0, 4.5 or 5.0. Growth in LB broth at pH 7.0 was used as a control. The culture was incubated at 37 oC in air with shaking at 200 rpm. At 1, 3, 6, 12 and 24 h time intervals, the culture was aliquoted and viability and growth determined by serial dilution and plating on Ashdown agar.
Susceptibility of B. pseudomallei to reactive oxygen intermediates (ROI)
The sensitivity of B. pseudomallei to reactive oxygen intermediates was determined by growth on oxidant agar plates and in broth containing H2O2. Assays on agar plates were performed as described previously (Loprasert et al., 2002), with some modifications. Briefly, an overnight culture of B. pseudomallei harvested from Ashdown agar was suspended in PBS and the bacterial concentration adjusted using OD at 600 nm. A serial dilution of the inoculum was spread plated onto Ashdown agar to confirm the bacterial count and colony morphology. Ten microlitres of serial dilutions of bacteria in PBS were spotted onto LB agar containing 0, 170, 310, 625, 1250 and 2500 ÂµM H2O2. Colony counts were performed after incubation at 37 oC in air for 24 h. The number of colonies on plates containing H2O2 was compared with that on control plates and presented as bacterial survival (%). The assay was performed for 4 independent experiments.
Sensitivity to killing by hydrogen peroxide was further examined in LB broth. An overnight culture of B. pseudomallei on Ashdown agar was suspended in PBS and adjusted to approximately 1 Â´ 108 CFU mL-1. Ten microlitres of bacterial suspension was added into 1 mL of LB broth containing two-fold decreasing concentrations of H2O2 ranging from 500 to 31.25 mM. The mixtures were statically incubated at 37 oC in air for 24 h and then the viable count and colony morphotype were determined by serial dilution and plating on Ashdown agar. The experiment was performed for 2 independent experiments.
Susceptibility of B. pseudomallei to reactive nitrogen intermediates (RNI)
B. pseudomallei from an overnight culture on Ashdown agar was suspended in PBS and the bacterial concentration adjusted using OD at 600 nm. Thirty microlitres of bacterial suspension was added into 3 mL of two-fold decreasing concentrations of sodium nitrite (ranging from 10 to 0.1 mM) in LB broth at pH 5.0. The mixture was incubated at 37 oC in air with shaking at 200 rpm and viable bacteria were determined at 6 h by serial dilution and plating on Ashdown agar. The number of viable bacteria in the presence of NaNO2 was compared with the number of bacteria in the inoculum and presented as bacterial survival (%). The experiment was performed in duplicate for 2 independent experiments.
Susceptibility of B. pseudomallei to lysozyme and lactoferrin
B. pseudomallei cultured overnight on Ashdown agar was harvested and suspended in 10 mM Tris-HCl buffer pH 5.0 (Callewaert et al., 2008). The bacterial suspension was adjusted to a concentration of 1 Ã- 107 CFU mL-1. Fifty microlitres of bacterial suspension was added to an equal volume of 400 mg mL-1 chicken egg white lysozyme (48,000 U mg protein-1) (Sigma) to obtain a final concentration of 200 mg mL-1. The mixture was incubated at 37 oC in air for 24 h, after which 10 ml of 10-fold serial dilutions were dropped on Ashdown agar. Sensitivity to lysozyme was also tested in the presence of 3 mg mL-1 of lactoferrin (Sigma) in a separate experiment (Callewaert et al., 2008). E. coli strain HB101 was tested in parallel as a control.
Susceptibility to human a-defensin and b-defensin
B. pseudomallei was tested for resistance to HNP-1 and HBD-2 (Peptide international) as described previously (Jones et al., 1996), with the exception that HNP-1 was used at twice the dose. E. coli strain HB101 was tested in parallel as a control. Briefly, B. pseudomallei or E. coli strain HB101 colonies were washed and suspended in 1 mM sodium phosphate buffer pH 7.4 containing 1% TSB (Jones et al., 1996). The bacterial suspension was adjusted to a concentration of 1 Ã- 107 CFU mL-1. Twenty microlitres of bacterial suspension was mixed with an equal volume of HNP-1 200 mg mL-1 or HBD-2 to obtain a final concentration of 100 mg mL-1 antimicrobial peptide and incubated at 37 oC in air for 3 h. The viable bacterial count was determined by dropping a 10-fold serial dilution on Ashdown agar.
Susceptibility to antimicrobial activity of human cathelicidin
B. pseudomallei susceptibility to cathelicidin LL-37 was tested using a microdilution method (Chen et al., 2005; den Hertog et al., 2005). LL-37 was kindly provided by Dr. Suwimol Taweechaisupapong, Department of Oral Diagnosis, Faculty of Dentistry, Khon Kaen University and Dr. Jan G.M. Bolscher, Department of Oral Biochemistry, Van der Boechorststraat, Amsterdam, The Netherlands. A loop of bacteria was washed 3 times in 1 mM potassium phosphate buffer (PPB) pH 7.4 and suspended in the same buffer. The bacterial suspension was adjusted to a concentration of 1 Ã- 107 CFU mL-1. Fifty microlitres of suspension was added into wells containing 50 Âµl of a 2-fold serial dilution of human cathelicidin in PPB (to obtain a final concentration of 3.125-100 mM), The mixture was incubated at 37 oC in air for 6 h and viability of bacteria was determined by plating a 10-fold serial dilution on Ashdown agar.
Growth in low oxygen and anaerobic conditions
An overnight culture of B. pseudomallei on Ashdown agar was suspended in PBS and adjusted to a concentration of 1 Ã- 108 CFU mL-1. The bacterial suspension was 10-fold serially diluted and 100 mL spread plated on Ashdown agar to obtain approximately 100 colonies per plate. Three sets of plates were prepared per strain and incubated separately at 37 oC in 3 conditions: (1) in air for 4 days (control); (2) in an GasPak EZ Campy Pouch System to produce an atmosphere containing approximately 5-15% oxygen (BD) for 2 weeks; or (3) in an anaerobic jar (Oxoid) with an O2 absorber (AnaeroPack; MGC) for 2 weeks and then re-exposed to air at 37 oC for 4 days. The number and colony morphology of B. pseudomallei were examined.
Colony morphology switching
Colony morphology switching of B. pseudomallei was determined following uptake and intracellular replication in macrophages, and during survival assays following exposure to H2O2, RNI, LL-37, low oxygen and anaerobic conditions. The colony morphology type was defined after plating to single colonies and incubation on Ashdown agar, and compared with the starting morphology type. Morphotype switching was presented as the proportion (%) of alternative morphotypes in relation to the total colonies present. Results are only presented for those assays where morphotype switching was different from that for control plates exposed to the same conditions but in the absence of bactericidal substances. Assays of resistance to HNP-1, HBD-2, lysozyme and lactoferrin using employed a drop method to assess bacterial survival and colony morphology could not be accurately delineated.
Statistical analysis was performed using the statistical program STATA version 10.1. Log transformation of continuous dependent variables was performed as appropriate. Nested repeated measures ANOVA was used to test continuous dependent variables between 3 isogenic morphotypes. A difference between 3 morphotypes was considered to be statistically significant when the P value was less than or equal to 0.05, after which pairwise comparisons were performed between each morphotype. All P values for pairwise analyses were corrected using the Benjamini-Hochberg method for multiple comparisons (Benjamini & Hochberg, 1995).
Growth curve analysis of isogenic morphotypes
Different growth rates may affect the number of intracellular bacteria following uptake by host cells. Thus, prior to observation of intracellular replication in macrophages, extracellular growth of B. pseudomallei was compared between 3 isogenic morphotypes cultured in TSB. Using a starting inoculum of 1 Ã- 104 CFU mL-1, log and stationary phase occurred at 2 h and 12 h, respectively, for all 3 morphotypes. There was no difference in doubling time between 3 isogenic morphotypes (P = 0.14) with an average doubling time of 40.2, 39.2 and 38.3 minutes for types I, II and III, respectively. Types I and II did not demonstrate colony morphology variation over time, but for type III 1-13% of colonies on the 28 h culture plate (the range reflecting variation between isolates) demonstrated switching to type I (isolates K96243, 164, B3 and B4) or to type II (isolate 153).
Replication of B. pseudomallei isogenic morphotypes in macrophages
Evaluation of the initial B. pseudomallei-macrophage cell interaction demonstrated that 3.0% of the bacterial inoculum (range 1.2-8.0% for different isolates) was associated with macrophages at 2 h. There was no difference in this value between 3 isogenic morphotypes (P = 0.08). Following removal of extracellular bacteria and incubation for a further 2 h, only 1.5% of the bacterial inoculum (range 0.4-3.4% for different isolates) was recovered. There was no difference in this value between 3 isogenic morphotypes (P = 0.07).
Intracellular replication of B. pseudomallei between 4 and 8 h was defined in relation to the 4 h time point which was used as the reference count. Type I demonstrated a significantly higher rate of intracellular replication than either type II or III (Figure 1A). Intracellular replication of type I at 8 h was 2.0 (95%CI 1.5-2.6, P = 0.004) times higher than that of type II, and 1.9 (95%CI 1.4-2.5, P = 0.004) times higher than that of type III.
Observation of morphotype switching by plating intracellular bacteria on Ashdown agar at each time point demonstrated that types I and II were stable after uptake by macrophages. As shown in Figure 1B, switching of type III increased over time after uptake such that by the 8 h time point, between 35-99% of the agar plate colonies (the range representing differences between isolates) had switched to type I (isolates K96243, 164, B3 and B4) or to type II (isolate 153).
Susceptibility of isogenic morphotypes to acid
To examine the effect of acid, growth of 3 isogenic morphotypes at pH 4.0, 4.5, 5.0 and 7.0 were compared at 5 time points over 24 h of incubation. All three isogenic morphotypes grew well at pH 4.5, 5.0 and pH 7.0, with no difference observed in growth between the different morphotypes (P > 0.10 for all time points). When cultured in LB broth at pH 4.0, all bacteria died within 12 h incubation.
Susceptibility of isogenic morphotypes to reactive oxygen intermediates (ROI)
The susceptibility of 3 morphotypes to ROI was initially examined on LB agar plates containing a range of H2O2 concentrations (0, 170, 310, 625, 1,250 and 2,500 ÂµM). As B. pseudomallei did not grow on plates with H2O2 at a concentration higher than 625 ÂµM, the percentage of viable bacteria were enumerated using agar plates with 625 ÂµM H2O2 compared to those on plates without H2O2. This demonstrated a difference in bacterial survival between the three isogenic morphotypes (P < 0.001). Percentage survival of type I was 3.8 (95%CI 2.9-5.0, P < 0.001) times higher than that for type II, and was 5.2 (95%CI 4.0-6.8, P < 0.001) times higher than that for type III (Figure 2A).
Further examination was undertaken of the susceptibility of the 3 morphotypes with various concentrations of H2O2 in LB broth. No bacteria survived in 500 ÂµM and 250 ÂµM. H2O2. In 125 Âµm H2O2, type I of all 5 isolates survived. By contrast, all five type III and 4 type II isolates (the exception being type II derived from isolate 164) did not survive in the same conditions. This confirmed a higher resistance to H2O2 of parental type I compared to types II and III. A difference was also observed between three isogenic morphotypes in 62.5 ÂµM H2O2 (P < 0.001). Bacterial growth of type I was 1.5 (95%CI 1.1-2.0, P = 0.02) times higher than that for type II, and was 2.7 (95%CI 2.0-3.7, P < 0.001) times higher than that for type III.
Observation of morphotype switching by plating bacteria from broth culture containing 62.5 ÂµM H2O2 onto Ashdown agar at 24 h demonstrated that types I and II did not change in colony morphology. In contrast, there was increasing switching of type III over time of incubation, ranging between 21-53% of the plate colonies for different isolates. Type III of 4 isolates (K96243, 164, B3 and B4) switched to type I, and one isolate (153) switched from type III to type II.
Susceptibility of isogenic morphotypes to reactive nitrogen intermediates (RNI)
Susceptibility of B. pseudomallei to RNI was observed following 6 h exposure to various concentrations of NaNO2 ranging between 0.1 to 10 mM in acidified pH 5.0 in LB broth. Killing of B. pseudomallei was observed for a concentration of 2 mM NaNO2. This concentration was used to observe the susceptibility of 3 isogenic morphotypes. Percent survival of type I, II and III were 43.8%, 43.7% and 40.1%, respectively, with no difference observed between the three morphotypes (P > 0.10).
Susceptibility of isogenic morphotypes to lysozyme and lactoferrin
Treatment with 200 Âµg mL-1 lysozyme at pH 5.0 did not inhibit the growth of 3 isogenic morphotypes of B. pseudomallei, while this concentration could suppress growth of E. coli from 4.9 Ã- 106 CFU mL-1 (the starting inoculum) to 425 CFU mL-1. Susceptibility was further examined in the presence of 3 mg mL-1 lactoferrin. A kinetic study over time demonstrated that lactoferrin alone could kill an entire E. coli inoculum of 1 Ã- 106 CFU mL-1 within 3 h at pH 5.0. The same treatment did not affect the survival of B. pseudomallei. Adding 200 lysozyme Âµg mL-1 with lactoferrin did not enhance the killing efficacy of E. coli and had no effect on B. pseudomallei. There was no difference in growth between the three morphotypes (P > 0.10).
Susceptibility of isogenic morphotypes to antimicrobial peptides
Macrophages produce several antimicrobial peptides (Agerberth et al., 2000; Duits et al., 2002). We examined the susceptibility of isogenic morphotypes to HNP1, HBD-2 and cathelicidin LL-37, three of the main human antimicrobial peptides. The results demonstrated that 100 Âµg mL-1 HNP-1 and 100 Âµg mL-1 HBD-2 did not inhibit the growth of 3 isogenic morphotypes of any of the B. pseudomallei isolates.
In a pilot experiment with a range of LL-37 concentrations and exposure times, we found that LL-37 reduced the B. pseudomallei count at a concentration of 6.25 ÂµM at 6 h. This condition killed 100% of a starting inoculum of 4.6 Ã- 106 CFU mL-1 E. coli control and caused a 75.7 to 99.8% reduction of B. pseudomallei for different isolates. A difference in bacterial survival was observed between the three isogenic morphotypes (P < 0.001). Survival of type I was 1.5 (95%CI 1.1-2.2, P = 0.02) times higher than that for type II, but was 3.7 (95%CI 2.6-5.3, P < 0.001) times lower than that for type III (Figure 2B.
Growth in low oxygen concentrations
Low oxygen concentration may limit the intracellular growth of aerobic bacteria within the host (Rustad et al, 2008). We examined the survival of 3 isogenic morphotypes and determined whether morphotype switching occurred in response to different oxygen concentrations during incubation on Ashdown agar at 37 oC. B. pseudomallei survived in 5-15% oxygen concentration for 14 days, with an average colony count of 95% (range 72-109% for different isolates and morphotypes) compared to control plates incubated in air for 4 days (Table 1). There was no difference in the survival pattern between 3 isogenic morphotypes (P > 0.10). B. pseudomallei colonies were not visible on Ashdown agar after incubation in an anaerobic chamber for 2 weeks, but colonies were visible at 48 h after the plates were reincubated at 37 oC in air, and colony counts were performed after incubation for 4 days. The percentage of bacteria recovered was not different between three morphotypes (P > 0.10). There was no change in morphotype observed for type I or II inocula. However, for the type III inoculum, between 15-100% of the total number of colonies on the plate (the range reflecting variation between isolates) switched to type I or II. The pattern of morphotype switching was similar to as observed previously, with four isolates switching from type III to type I (K96243, 164, B3 and B4), and one isolate switching to II (153) (Table 1).
Bacterial adaptation is essential for survival in the human host. Our previous paper reported a process of B. pseudomallei colony morphology switching that occurred during human melioidosis, and in an animal model, mouse macrophage cell line J774A.1, human lung epithelial cell line A549, and under starvation conditions in vitro. The differences in colony morphology were associated with variable phenotypes involving biofilm production, flagella production, motility, aggregation, and protease and lipase enzymes production (Chantratita et al., 2007). In this study, we investigated whether the variable phenotype associated with different morphotypes resulted in a survival fitness or disadvantage during interactions with macrophages and after exposure to factors that simulate the macrophage milieu.
Comparison of the initial interaction between the human macrophage cell line U937 and 3 isogenic morphotypes of B. pseudomallei failed to demonstrate a difference between the morphotypes, although heterogeneity in intracellular survival/growth was observed. Despite a comparable rate of extracellular growth between isogenic morphotypes, types II and III appeared to persist but not to replicate well after uptake by macrophages. Taken together with our previous observation that type II can persist in an animal model (Chantratita et al., 2007), it is possible to speculate that type II may be important in persistence in the infected host. Type III was not capable of multiplication after uptake by macrophages as type I, and was associated with a change in morphotype. This suggests that type III has a fitness disadvantage under these circumstances. A possible explanation for this is that type III does not appear to produce biofilm (Chantratita, et al., 2007). This idea is supported by previous work using a B. pseudomallei mutant defective in biofilm, suggested that biofilm production is associated with the ability to survive in primary human macrophages (Wikraiphat et al., 2009). Other surface-associated components such as lipopolysaccharide (LPS) may also be involved. LPS has been shown to be associated with virulence and resistant to killing in primary human macrophages and in the serum (DeShazer et al., 1998; Wikraiphat et al., 2009). The association between LPS, capsule expressions and colony morphotype warrants further investigation.
Several components of the innate immune system are efficient in killing organisms within human macrophages (Radtke & O'Riordan, 2006). The most important of these are the antimicrobial peptides and nitric oxide (NO), the superoxide anion (O2-), and hydrogen peroxide (H2O2), all of which are directly toxic to bacteria. Reactive oxygen species generated by the phagocyte NADPH oxidase have an essential role in the control of B. pseudomallei infection in C57BL/6 bone marrow derived macrophages (Breitbach et al., 2006). Type I of all 5 B. pseudomallei isolates tested here had the greatest resistance to H2O2, followed by types II and III, respectively, suggesting that type I has the greatest potential to scavenge or degrade H2O2 molecules. This may explain the finding that type I had the highest replication after uptake by the macrophage cell line. Type III switched to type I or II during culture in medium containing H2O2. This suggested that type III had a survival disadvantage in the presence of oxidative stress from ROI. This may explain the finding that uptake of type III by macrophages was associated with colony morphology switching. The differential expressions of antioxidant components between isogenic morphotypes need further investigation.
Colony morphology differences did not influence resistance to RNI. B. pseudomallei is protected from RNI by the production of alkyl hydroperoxide reductase (AhpC) protein and depends on OxyR regulator and a compensatory KatG expression (Loprasert et al., 2003). This bacterium is able to repress inducible nitric oxide synthase (iNOS) by activating the expression of two negative regulators, a suppressor of cytokine signaling 3(SOC3) and cytokione-inducible src homology2-containing protein (CIS) (Ekchariyawat et al., 2005). These mechanisms may not be associated with colony morphology variability.
B. pseudomallei survive in the phagolysosome (Puthucheary & Nathan, 2006) which are acidified environments containing lysozymes, proteins and antimicrobial peptides that destroy pathogen. There was no difference in growth for the 3 isogenic morphotypes of B. pseudomallei derived from all five isolates at all pH levels tested above 4.0, but a pH of 4.0 was universally bactericidal, suggesting that morphotype switching did not provide a survival advantage against acid conditions.
All morphotypes of B. pseudomallei were highly resistance to lysozyme and lactoferrin. Lysozyme functions to dissolve cell walls of some Gram-positive bacteria. Lactoferrin is a competitor that works by binding essential nutrients, iron and preventing their uptake by the bacteria. Possible mechanisms for resistance to these factors of B. pseudomallei are the presence of capsule and LPS (Wikraiphat et al, 2009) and lysozyme inhibitors. Blast analysis demonstrated that B. pseudomallei K96243 genome contains BPSL1057 and BPSL1113 with homology to Ivy and MliC, lysozyme inhibitors present in other Gram-negative bacteria (Deckers et al., 2004; Callewaert et al., 2008). B. pseudomallei may produce these lysozyme inhibitors and survive against lysozyme and lactoferrin which are independently associated with variation in colony morphology.
Antimicrobial peptides (AMPs) are efficient at killing a broad range of organisms. They distribute in a variety tissues, neutrophils and macrophages (Agerberth et al., 2000; Duits et al., 2002). All 3 isogenic B. pseudomallei morphotypes resisted to Î±-defensin HNP-1 and Î²-defensin HBD-2, but were susceptible to LL-37. Type III was more resistant than type I or II to LL-37. These isogenic morphotypes may express different level of surface components such as capsule and O-polysaccharide that interfere AMP binding. These two components have been reported previously to be involved with resistant to antimicrobial agents in B. pseudomallei (Kanthawong et al, 2009; Wikraiphat et al, 2009). Additional mechanisms that might be associated with variable levels of diminished AMP activity isogenic morphotypes are efflux pump activity and proteolytic degradation (Peschel et al., 2006).
Another feature of bacterial survival during the establishment of persistent infection in the host is adaptation to hypoxia in the host microenvironment (Rustad et al, 2008). This study demonstrated that all 3 isogenic morphotypes were able to tolerate a low oxygen concentration and anaerobic conditions for at least two weeks. Type III switching to either type I or II occurred during recovery from anaerobic incubation. This is consistent with the previous finding that type III does not form a pellicle on the surface of broth culture exposed to the air, a characteristic of B. pseudomallei type I and II (Chantratita et al., 2007). The fact that types I and II were stable following anaerobic incubation suggests that they are tolerant of fluctuations in oxygen concentration.
These results provide evidence that colony morphology variation represents heterogeneous phenotypes of B. pseudomallei with different fitness advantages to interact, survive and replicate in the presence of anti-bactericidal substances within human macrophages. Under host pressure, this adaptability may be a key process leading to either quiescence in the macrophage or invasion or spreading to other cells. The molecular mechanism of morphotype switching and differential transcriptional patterns require further investigation. Understanding this process may help to identify a therapeutic target for B. pseudomallei eradication.
We are grateful to Dr. Suwimol Taweechaisupapong and Dr. Jan G.M. Bolscher for providing LL-37, to Dr. Sue Lee for statistical advice and to Mrs. Vanaporn Wuthiekanun for providing B. pseudomallei isolates. We thank staff at the Mahidol-Oxford Tropical Medicine Research Unit for their assistance and support. S.T was supported by a Siriraj Graduate Thesis Scholarship, Thailand. N.C. was supported by a Wellcome Trust Career Development award in Public Health and Tropical Medicine, UK, and a Thailand Research Fund award, Thailand. This work was funded by the Wellcome Trust, UK.