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1,2Bacteriology Division1 and Toxinology/Aerobiology Division2, United States Army Medical Research Institute of Infectious Diseases (USAMRIID), 1425 Porter Street, Fort Detrick, MD 21702-5011, USA 3Microbiology, Dstl, CBS Porton Down, Salisbury SP4 0JQ, UK 4Orion Genomics, Center for Emerging Technologies, 4041 Forest Park, St. Louis, MO 63108, USA
Correspondence Ricky L. Ulrich Ricky.Ulrich{at}AMEDD.ARMY.MIL
Received March 4, 2004
Accepted June 25, 2004
Burkholderia pseudomallei is the causative agent of human and animal melioidosis. The role of quorum sensing (QS) in the in vivo pathogenicity of B. pseudomallei via inhalational exposure of BALB/c mice and intraperitoneal challenge of Syrian hamsters has not been reported. This investigation demonstrates that B. pseudomallei encodes a minimum of three luxI and five luxR homologues that are involved in animal pathogenicity. Mass spectrometry analysis of culture supernatants revealed that wild-type B. pseudomallei and the luxI mutants synthesized numerous signalling molecules, including N-octanoyl-homoserine lactone, N-decanoyl-homoserine lactone, N-(3-hydroxyoctanoyl)-L-homoserine lactone, N-(3-hydroxydecanoyl)-L-homoserine lactone and N-(3-oxotetradecanoyl)-L-homoserine lactone, which was further confirmed by heterologous expression of the B. pseudomallei luxI alleles in Escherichia coli. Mutagenesis of the B. pseudomallei QS system increased the time to death and reduced organ colonization of aerosolized BALB/c mice. Further, intraperitoneal challenge of Syrian hamsters with the B. pseudomallei QS mutants resulted in a significant increase in the LD50. Using semi-quantitative plate assays, preliminary analysis suggests that QS does not affect lipase, protease and phospholipase C biosynthesis/secretion in B. pseudomallei. The findings of the investigation demonstrate that B. pseudomallei encodes multiple luxIR genes, and disruption of the QS alleles reduces animal pathogenicity, but does not affect exoproduct secretion.
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INTRODUCTION |
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 TOP INTRODUCTION METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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Quorum sensing (QS) is a cell-density-dependent communication system utilized by Gram-negative bacteria that incorporates N-acyl-homoserine lactones (AHLs) for the coordination of gene expression (Fuqua et al., 1994). LuxI proteins are responsible for AHL biosynthesis while LuxR transcriptional regulators, following association with their cognate AHL(s), mediate gene repression or expression (Schuster et al., 2003; Wagner et al., 2003). Functional QS networks have been identified in numerous Gram-negative bacterial pathogens and have been shown to both positively and negatively regulate various putative virulence factors in addition to contributing to bacterial pathogenicity in animal models (Baldwin et al., 2004; Brint & Ohman, 1995; Chapon-Herve et al., 1997; Gambello et al., 1993; Gotschlich et al., 2001; Latifi et al., 1995, 1996; Lewenza & Sokol, 2001; Lewenza et al., 1999; Ochsner & Reiser, 1995; Ochsner et al., 1994; Passador et al., 1993; Pearson et al., 1997, 2000; Schuster et al., 2003; Sokol et al., 2003; Valade et al., 2004; Wu et al., 2001).
Currently, no effective human or animal vaccine is available against melioidosis, and with the possibility of B. pseudomallei weaponization, investigations focusing on vaccine development against this infectious Burkholderia species are important. To determine if QS is involved in the in vivo pathogenicity of B. pseudomallei, each luxIR homologue was mutated and the derivative strains were analysed in BALB/c mice and hamster models of infection.
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METHODS |
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TOPINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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Gene identification and gene amplification.
Primers for generation of the B. pseudomallei disruption cassettes (merodiploids) were designed using the B. pseudomallei K96243 luxIR sequences identified in silico (http://www.sanger.ac.uk/). Primer sequences for each B. pseudomallei luxIR gene are listed in Table 2 while PCR cycling parameters, mutant construction and confirmation have been described elsewhere (Ulrich & DeShazer, 2004).
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AHL characterization.
AHLs were purified from culture supernatants of B. pseudomallei and each luxI QS mutant as described by Shaw et al. (1997). Approximately 500 nl of each extract were injected onto a CAPLC capillary liquid chromatograph (Waters Corporation) fitted with an Aquasil C18 HPLC column (10 cm x 75 µm) (New Objective) operating at a flow rate of 500 nl min–1. A gradient elution was employed starting at 100 % A (2 % acetonitrile/0.1 % formic acid) and ending at 100 % B (80 % acetonitrile/0.1 % formic acid) in 30 min. A voltage of 2.1 kV was applied to the column effluent entering the nanoelectrospray source attached to a Q-TOF-2 mass spectrometer (Micromass). The source temperature was 125 °C and a cone voltage of 18 V was applied. Argon [1 mbar (102 Pa) nominal pressure] was used as the collision gas with an energy setting of 15 V. The results obtained with MS (scanning from m/z 160 to 330 in 1.5 s) were acquired using data-directed analysis software (Waters Corporation). Ions meeting selected intensity and charge state criteria were further characterized by MS/MS. Precursor ions yielding a fragmentation ion at m/z 102, representing the lactone ring of AHL signalling molecules, were recorded and the (M+H)+ was determined. Fragmentation ions of MS/MS spectra containing an ion at m/z of 102 were compared to the fragmentation mass spectra of the corresponding AHL standard when possible. If a precursor ion with an (M+H)+ not equal to any of the AHL standards yielded an MS/MS spectrum containing an ion at m/z of 102, the mass spectra were further analysed for the presence of ions characteristic of acyl side chains containing substitutions that lose water molecule(s) following collisional-induced dissociation. AHL accumulation patterns were analysed using the bioreporter strain Agrobacterium tumefaciens NTL4 incorporating soft agar TLC overlays (Fuqua & Winans, 1996).
Inhalational challenge of female BALB/c mice (20 animals for wild-type B. pseudomallei and each QS mutant), LD50 evaluations and bacterial organ load determination were performed as previously described (Roy et al., 2003; Ulrich & DeShazer, 2004).
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RESULTS |
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TOPINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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B. pseudomallei synthesizes numerous AHL signalling molecules
Culture supernatants from wild-type B. pseudomallei contained N-octanoyl-homoserine lactone (C8-HSL), N- decanoyl-homoserine lactone (C10-HSL) and N-(3-hydroxyoctanoyl)-L-homoserine lactone (3-hydroxy-C8-HSL) (Table 3). Culture extracts from RJ10 (pmlI1–) contained the signalling molecules C8-HSL, 3-hydroxy-C8-HSL and N-(3-hydroxydecanoyl)-L-homoserine lactone (3-hydroxy-C10-HSL) (Table 3). In contrast, MS/MS analysis of culture supernatants from RJ11 (bpmI3–) revealed the presence of N-(3-oxotetradecanoyl)-L-homoserine lactone (3-oxo-C14-HSL) (Table 3). The relevant fragmentation ions for wild-type B. pseudomallei and each QS mutant are summarized in Table 3. Surprisingly, individual mutagenesis of the B. pseudomallei luxI genes had little effect on AHL accumulation. To determine if overlapping signalling molecules were being synthesized by the B. pseudomallei LuxI proteins, each luxI gene was cloned into the broad host range expression vector pBHR1, transformed into E. coli, and AHL accumulation was monitored in E. coli. Interestingly, and with the exception of 3-oxo-C14-HSL, which was not synthesized by PmlI1, the B. pseudomallei LuxI proteins when expressed in E. coli directed the biosynthesis of each AHL detected in wild-type culture supernatants (Table 3). The synthetic standards for the substituted (modified acyl side chains) AHLs identified in this study were not analysed; however, the spectra for these moieties are analogous to the MS results of a previous report (Shaw et al., 1997). It should be noted that mutagenesis of bpmI2 was unsuccessful.
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Mutagenesis of the B. pseudomallei QS network increases animal time to death following inhalational exposure
To determine if disruption of the B. pseudomallei QS system reduced virulence in an inhalation BALB/c animal model, mortality was monitored for 39 days post-exposure (p.e.). Female BALB/c mice (10 for time to death analysis and 10 for organ load enumeration) were challenged with approximately 1 x 104 c.f.u. (10 LD50s) of wild-type B. pseudomallei and each QS mutant. For mice aerosolized with wild-type B. pseudomallei, animal death (entire experimental group) occurred 5 days p.e. (Fig. 3a). In contrast, an increase in the time to death of animals challenged with the B. pseudomallei QS mutants was observed following inhalational exposure (Fig. 3a). The most notable reduction was obtained by disruption of the bpmI3 gene (RJ11) in which 7 out of 10 animals survived challenge 39 days p.e. (data not shown). The surviving 7 mice challenged with RJ11 (bpmI3–) exhibited no clinical symptoms but were found to be chronically infected (high bacterial loads within the lungs, liver and spleen).
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Disruption of the B. pseudomallei luxIR alleles reduces organ colonization
To determine if QS was involved in the ability of B. pseudomallei to initiate infection within the lungs following inhalational challenge, animals were sacrificed over a 4-day period and the bacterial loads were determined. Mutagenesis of the B. pseudomallei QS system reduced lung colonization for each mutant, in particular for RJ11 (bpmI3–) and RJ13 (bpmR2–), over the 4-day experimental course (Fig. 3b). Wild-type B. pseudomallei proliferated in the lungs of aerosolized animals and peaked 3 days p.e. with a concentration of 1.6 x 108 c.f.u. (g lung)–1 (Fig. 3b). At day 2, lungs of mice infected with RJ10 (pmlI1–) and RJ11 (bmpI3–) contained bacterial loads of 1.7 x 103 and 1.0 x 104 c.f.u. (g tissue)–1, respectively, compared to the lungs of animals exposed to wild-type B. pseudomallei, which harboured 1.2 x 106 c.f.u. (g lung)–1 (Fig. 3b). With the exception of RJ10 (pmlI1–) and RJ13 (bpmR2–), bacterial proliferation for the B. pseudomallei QS mutants peaked in the lungs of mice 3 days p.e. (Fig. 3b). At 4 days post-aerosolization, a substantial increase in lung colonization from non-detectable levels (3 days p.e.) was observed for RJ10 (pmlI1–; 1.7 x 106 c.f.u. g–1) and RJ13 (bpmR2–; 3.4 x 106 c.f.u. g–1) (Fig. 3b). In contrast, at day 4 a decrease in the bacterial burden within the lungs of mice aerosolized with RJ11 (bpmI3–; 2.2 x 104 c.f.u. g–1), RJ12 (pmlR1–; 3.4 x 104 c.f.u. g–1) and RJ15 (bpmR5–; 3.6 x 105 c.f.u. g–1) was observed compared to wild-type B. pseudomallei (3.4 x 107 c.f.u. g–1) (Fig. 3b).
To analyse whether the B. pseudomallei QS system affected dissemination to the peripheral organs, the bacterial loads within the spleen and liver of BALB/c mice were analysed following challenge. Wild-type B. pseudomallei was first detected in the spleen on day 3 and reached a maximum organ load of 3.0 x 108 c.f.u. (g tissue)–1 (Fig. 3c). Despite the similar doses inhaled for each strain tested in this study, RJ13 (bpmR2–) and RJ14 (bpmR3–) spread to the spleen more rapidly in contrast to wild-type B. pseudomallei (Fig. 3c). Further, RJ13 (bpmR2–) continued to multiply in the spleen of challenged animals over the 4-day experimental period and reached a maximum bacterial load of 6.1 x 107 c.f.u. g–1 (4 days p.e.). At 3 days p.e., RJ10 (pmlI1–), RJ11 (bpmI3–), RJ12 (pmlR1–), RJ13 (bpmR2–) and RJ15 (bpmR5–) were isolated from the spleens of aerosolized mice at concentrations varying from 7.1 x 103 (RJ11; bpmI3–) to 1.4 x 105 (RJ15; bpmR5–) c.f.u. (g tissue)–1 compared to 3.0 x 108 c.f.u. g–1 obtained for wild-type B. pseudomallei (Fig. 3c). Interestingly, 4 days p.e., the spleens of animals exposed to RJ12 (pmlR1–), RJ14 (bpmR3–) and RJ15 (bpmR5–) contained no recoverable QS mutants (Fig. 3c).
Disruption of the B. pseudomallei QS system did not cause a significant reduction in liver colonization. With the exception of RJ11 (bpmI3–), which was not detectable in livers of exposed mice until day 3 (1.9 x 103 c.f.u. g–1), wild-type B. pseudomallei and the remaining QS mutants were isolated at day 2 with bacterial loads ranging from 3.2 x 102 (RJ13, bpmR2–; and RJ14, bpmR3–) to 1.9 x 103 c.f.u. (g liver tissue)–1 (RJ12; pmlR1–) (Fig. 3d). Of the B. pseudomallei QS mutants tested, disruption of the bpmI3 allele (RJ11), a luxI homologue, caused the greatest reduction in liver proliferation (Fig. 3d).
The B. pseudomallei QS network is required for Syrian hamster pathogenicity
Incorporating a Syrian hamster model of infection, and to further confirm the reduced virulence observed in our inhalational BALB/c model, the LD50 for wild-type B. pseudomallei and each QS mutant was determined. The LD50 for wild-type B. pseudomallei, determined 3 days p.e., was found to be < 6 c.f.u. (Table 5). For the B. pseudomallei QS mutants, the most pronounced increases in LD50 were observed for RJ11 (bpmI3–; >1080 c.f.u.), RJ12 (pmlR1–; 674 c.f.u.), RJ14 (bpmR3–; >727 c.f.u.) and RJ15 (bpmR5–; >870 c.f.u.). Given the extreme sensitivity of Syrian hamsters to B. pseudomallei this animal model was chosen for complementation of the B. pseudomallei luxI mutants. As expected, parental phenotypes were restored by heterologous expression of pmlI1 and bpmI3 in RJ10 and RJ11 (Table 5).
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DISCUSSION |
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TOPINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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The structural organization of the B. pseudomallei DD503 and B. pseudomallei 008 pmlIR alleles was found to be analogous (Fig. 1a). However, and interestingly, our in silico search of the B. pseudomallei K96243 genome revealed the presence of several additional luxIR genes that are involved in the pathogenicity of B. pseudomallei (Figs 1a, 3). Incorporating BLASTX homology searches (Table 4) and CLUSTAL W nucleotide sequence alignments (Fig. 2), in addition to animal testing of the numerous luxIR mutants (Table 5 and Fig. 3), we demonstrate that B. pseudomallei encodes a complex QS network that is required for virulence. Interestingly, Burkholderia mallei, hypothesized to be a clone of B. pseudomallei that is an obligate mammalian pathogen (DeShazer & Waag, 2004; Godoy et al., 2003), does not encode the bpmIR2 genes which suggest these QS alleles may be involved in the environmental survival of B. pseudomallei and B. thailandensis.
Two AHL bioreporter assays were initially employed to characterize the signalling molecules produced by B. pseudomallei, and include A. tumefaciens A136 and A. tumefaciens NTL4 (Fuqua & Winans, 1996). Consistent with other members of the Burkholderia genus, we demonstrate that B. pseudomallei and each QS mutant synthesize a variety of AHL signalling molecules (Table 3) (Conway & Greenberg, 2002; Gotschlich et al., 2001; Lewenza et al., 1999; McKenney et al., 1995). However, to our knowledge, the detection of 3-oxo-C14-HSL is unique to B. pseudomallei (Table 3). As with B. pseudomallei, we have also identified 3-hydroxy-C8-HSL in culture supernatants from a B. mallei luxI mutant (unpublished data). Interestingly, and given the genetic relatedness between B. pseudomallei and B. thailandensis, especially with regard to the nucleotide similarity of their luxIR alleles, the primary signalling molecules produced by B. thailandensis are N-hexanoyl-homoserine lactone, C8-HSL and C10-HSL (Ulrich et al., 2004). The observation that B. pseudomallei synthesizes multiple signalling molecules is further supported by AHL analysis of E. coli supernatants from strains (RJ18–RJ20) heterologously expressing the B. pseudomallei luxI genes. Table 3 shows that E. coli expressing the pmlI1, bpmI2 or bpmI3 alleles is capable of directing the synthesis of each AHL identified in culture extracts from B. pseudomallei. Interestingly, and with the exception of pmlI1, which is not involved in 3-oxo-C14-HSL production, it appears that each B. pseudomallei luxI can synthesize overlapping signalling molecules, which may partially explain the appearance of additional AHLs in our luxI mutant backgrounds. Though preliminary, it seems that the product(s) of the BpmI3 protein has a regulatory role on the transcription of bpmI2. This hypothesis is further supported by the failure of RJ18 (E. coli expressing the B. pseudomallei pmlI1 gene) to produce 3-oxo-C14-HSL and the ability of RJ11 (bpmI3–) to accumulate this signalling molecule (Table 3). Additional experiments will have to be performed that would involve the generation of multi luxI deletion strains of B. pseudomallei in conjunction with gene fusion assays. The identification of C10-HSL in this investigation is consistent with a recent study that demonstrated that B. pseudomallei 008 produces C10-HSL (Valade et al., 2004). However, it is curious that the latter report failed to detect the accumulation of the additional AHLs (four total) identified in this investigation despite using three AHL bacterial reporter strains. In fact, and as depicted in Table 3, our pmlI1 mutant (RJ10) was capable of biosynthesizing C8-HSL, 3-hydroxy-C8-HSL and 3-hydroxy-C10-HSL. Though unlikely, it is possible that B. pseudomallei 008 is genetically diverse from B. pseudomallei K96243 with regard to the number of encoded luxI genes. Further, it may be that our approach for AHL detection (MS on crude culture extracts) is more sensitive than using bacterial reporter assays. Though we did not achieve adequate separation for MS detection of the B. pseudomallei AHLs using TLC, multiple signalling molecules were visualized when incorporating TLC overlays using the A. tumefaciens A136 and A. tumefaciens NTL4 bioreporter strains (data not shown). It is likely the additional AHL signalling molecules detected in our pmlI1– (RJ10) and bpmI3– (RJ11) backgrounds are being synthesized by the BpmI2 LuxI homologue, especially 3-oxo-C14-HSL in RJ11. This hypothesis is further supported by the E. coli AHL accumulation profiles when expressing the B. pseudomallei luxI alleles (Table 3).
Opportunistic pathogens have been shown to utilize QS to regulate virulence factors needed for in vivo pathogenicity (Donabedian, 2003; Hardman et al., 1998; Rumbaugh et al., 1999). It has recently been demonstrated that the B. cepacia cepIR and B. pseudomallei pmlIR QS networks are required for pathogenicity in murine animal models of infection (Baldwin et al., 2004; Valade et al., 2004). Consistent with the latter investigation, our findings also suggest that the pmlI luxI gene is involved in the in vivo pathogenicity of B. pseudomallei in an inhalational murine model (Fig. 3). This study has also identified an additional six luxIR homologues encoded by B. pseudomallei and has shown that disruption of these alleles significantly reduces virulence in BALB/c mice and Syrian golden hamster models. While preliminary, our findings suggest that QS in B. pseudomallei may be regulating organ-specific (i.e. virulence determinants needed for lung vs liver colonization) virulence factor(s). This hypothesis is further supported by the notable liver organ trophism for each of the B. pseudomallei QS mutants (Fig. 3d). Curiously, RJ10 (pmlI1–) and RJ11 (bpmI3–) culture supernatants contained various AHLs but these strains exhibited reduced virulence phenotypes (Fig. 3 and Table 5). It is conceivable that the relative timing of biosynthesis and concentration of the signalling molecule(s) play a role in the pathogenicity of B. pseudomallei. Our disruption cassettes were not designed to generate in-frame mutations which could possibly induce polar affects on downstream genes. Our in silico analysis of the genes located upstream and downstream (6 kb segments) from the B. pseudomallei luxIR alleles did not reveal the presence of any potential genes associated with pathogenicity, which implies that the reduction in virulence observed by mutagenesis of the B. pseudomallei QS system is not the result of polar mutations (Fig. 1a). Surprisingly, and especially considering the extreme sensitivity of Syrian hamsters to B. pseudomallei, mutagenesis of the B. pseudomallei QS system caused a greater reduction in hamster pathogenicity compared to the inhalational BALB/c mouse model, which suggests that QS may regulate differential virulence factors needed for mouse versus hamster pathogenicity. Further studies are needed to address these preliminary findings and will likely involve comparative (mice vs hamster) in vivo gene expression analysis utilizing whole-genome DNA microarrays.
Several virulence factors have been identified in B. pseudomallei and include capsule (Reckseidler et al., 2001), LPS (DeShazer et al., 1998) and type III secretion systems (Stevens et al., 2002). Reckseidler et al. (2001) demonstrated that a B. pseudomallei capsule mutant had an LD50 of 3.5 x 105 c.f.u. compared to < 10 c.f.u. for wild-type B. pseudomallei. DeShazer et al. (1998) showed that B. pseudomallei LPS is required for serum resistance and that an LPS mutant (SRM117) was attenuated in three animal models. Further, in Syrian hamsters, the LD50 values for wild-type B. pseudomallei and SRM117 were < 5 c.f.u. and 62 c.f.u., 2 x 103 c.f.u. and 2 x 104 c.f.u. in guinea pigs, and 2 x 104 c.f.u. and >5 x 106 c.f.u. in infant diabetic rats, respectively (DeShazer et al., 1998). Disruption of the B. pseudomallei secretory apparatus for protease, lipase and phospholipase C (PLC) delivery had a marginal effect on the pathogenicity of B. pseudomallei in a hamster model (DeShazer et al., 1999). Though semi-quantitative plate assays (measuring zone radius) were used in this study (DeShazer et al., 1999), and results may vary between strains of B. pseudomallei, each of the B. pseudomallei QS mutants tested in this investigation were not altered in protease, lipase or PLC production, suggesting that QS in B. pseudomallei DD503 has no regulatory effect on these potential virulence factors (data not shown). These findings are not consistent with a previous report that demonstrated that the B. pseudomallei 008 QS network negatively regulated the MprA protease, and are likely a result of the approach (EnzChek protease kit vs plate assays) used to analyse enzymic activity. Current studies are under way to determine if capsule biosynthesis and the type III secretion system(s) encoded by B. pseudomallei are regulated by QS. Interestingly, mutagenesis of the B. thailandensis btaIR2 (homologues of the B. pseudomallei bpmIR2) genes resulted in a hyper-lipolytic phenotype, i.e. QS negatively regulates the factor(s) needed for this enzyme activity (Ulrich et al., 2004). Also, inactivation of the B. thailandensis luxIR alleles induced the hyper-haemolysis of sheep erythrocytes and altered both swarming and twitching motility (Ulrich et al., 2004). Given the genetic and biochemical relatedness between B. thailandensis and B. pseudomallei, it is surprising that analogous phenotypes were not obtained by disruption of the B. pseudomallei QS system.
The B. pseudomallei QS network represents one of the most complex AHL-based intra-species communication systems identified in an opportunistic Gram-negative bacterial pathogen. By insertionally inactivating the B. pseudomallei QS network the results of this study demonstrate that QS is involved in the in vivo pathogenicity of B. pseudomallei. Considering the complexity of this QS system, additional studies will be needed to identify the virulence factor(s) controlled by this cell-density-dependent communication network required for mouse and hamster pathogenicity. We are currently employing whole-genome DNA microarrays in attempts to gain insight into this complex Gram-negative bacterial communication system.
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ACKNOWLEDGEMENTS |
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TOPINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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All research was conducted in compliance with the Animal Welfare Act and other federal statutes and regulations relating to animals and experiments involving animals and adheres to principles stated in the Guide for the Care and Use of Laboratory Animals, National Research Council, 1996. The facility where this research was conducted is fully accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International.
Opinions, interpretations, conclusions and recommendations are those of the authors and are not necessarily endorsed by the US Army.
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TOPINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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) indicate the mutated genes analysed in this work. Attempts to create mutations in the B. pseudomallei bpmI2 and bpmR4 genes were unsuccessful. (b) Confirmation that B. mallei ATCC 23344 lacks the bpmIR2 locus. Small internal gene fragments corresponding to the B. pseudomallei, B. mallei and B. thailandensis QS genes were PCR amplified with the primer pairs listed in 
) show invariant amino acids found within LuxIR proteins encoded by Gram-negative bacterial species. Sequence alignments were performed using MegAlign (DNASTAR, Madison, WI, USA) with the CLUSTAL W setting.