Identification of Burkholderia mallei and Burkholderia pseudomallei adhesins for human respiratory epithelial cells
- Rachel Balder†1,
- Serena Lipski2,
- John J Lazarus2,
- William Grose1, 2,
- Ronald M Wooten2,
- Robert J Hogan1,
- Donald E Woods3 and
- Eric R Lafontaine†1Email author
© Balder et al; licensee BioMed Central Ltd. 2010
Received: 7 August 2010
Accepted: 28 September 2010
Published: 28 September 2010
Burkholderia pseudomallei and Burkholderia mallei cause the diseases melioidosis and glanders, respectively. A well-studied aspect of pathogenesis by these closely-related bacteria is their ability to invade and multiply within eukaryotic cells. In contrast, the means by which B. pseudomallei and B. mallei adhere to cells are poorly defined. The purpose of this study was to identify adherence factors expressed by these organisms.
Comparative sequence analyses identified a gene product in the published genome of B. mallei strain ATCC23344 (locus # BMAA0649) that resembles the well-characterized Yersinia enterocolitica autotransporter adhesin YadA. The gene encoding this B. mallei protein, designated boaA, was expressed in Escherichia coli and shown to significantly increase adherence to human epithelial cell lines, specifically HEp2 (laryngeal cells) and A549 (type II pneumocytes), as well as to cultures of normal human bronchial epithelium (NHBE). Consistent with these findings, disruption of the boaA gene in B. mallei ATCC23344 reduced adherence to all three cell types by ~50%. The genomes of the B. pseudomallei strains K96243 and DD503 were also found to contain boaA and inactivation of the gene in DD503 considerably decreased binding to monolayers of HEp2 and A549 cells and to NHBE cultures.
A second YadA-like gene product highly similar to BoaA (65% identity) was identified in the published genomic sequence of B. pseudomallei strain K96243 (locus # BPSL1705). The gene specifying this protein, termed boaB, appears to be B. pseudomallei-specific. Quantitative attachment assays demonstrated that recombinant E. coli expressing BoaB displayed greater binding to A549 pneumocytes, HEp2 cells and NHBE cultures. Moreover, a boaB mutant of B. pseudomallei DD503 showed decreased adherence to these respiratory cells. Additionally, a B. pseudomallei strain lacking expression of both boaA and boaB was impaired in its ability to thrive inside J774A.1 murine macrophages, suggesting a possible role for these proteins in survival within professional phagocytic cells.
The boaA and boaB genes specify adhesins that mediate adherence to epithelial cells of the human respiratory tract. The boaA gene product is shared by B. pseudomallei and B. mallei whereas BoaB appears to be a B. pseudomallei-specific adherence factor.
Burkholderia pseudomallei is a Gram-negative bacterium readily recovered from the water and wet soils of endemic areas bordering the equator, particularly Southeast Asia and Northern Australia [1–9]. The organism is a motile, aerobic bacillus that can survive environmental extremes as well as the bactericidal activities of complement [10–12], defensins [13–15], and phagocytes [1, 2, 16–18]. The genome of the B. pseudomallei isolate K96243 has been published by the Wellcome Trust Sanger Institute and was shown to consist of two chromosomes of 4.1 and 3.2 Mbp . Burkholderia mallei is a non-motile, host-adapted clone of B. pseudomallei that does not persist outside of its equine host and is endemic to certain parts of Asia, Africa, the Middle East and South America [8, 9, 20–25]. The genomic sequence of the B. mallei strain ATCC23344 has been published by TIGR  and is smaller (2 chromosomes of 3.5 and 2.3 Mbp) than that of B. pseudomallei K96243. B. mallei ATCC23344 was found to specify a large number of mobile DNA elements that have contributed to extensive deletions and rearrangements relative to the genome of B. pseudomallei K96243. Despite these differences, the genes shared by the two isolates have an average identity of 99% at the nucleotide level [19, 26]. The genomic sequence of several B. pseudomallei and B. mallei isolates are also publicly available through the NCBI genomic BLAST service (http://www.ncbi.nlm.nih.gov/sutils/genom_table.cgi), which provides a wealth of resources to study these organisms.
B. pseudomallei causes the human disease melioidosis, which is notoriously difficult to diagnose. Clinical manifestations vary greatly and may present as flu-like symptoms, benign pneumonitis, acute and chronic pneumonia, or fulminating septicemia. Infection occurs via inhalation of contaminated aerosol particles or through skin abrasions, and the risk of contracting the disease is proportional to the concentration of B. pseudomallei in soil and water. In endemic areas, heavy rainfalls result in a shift from percutaneous inoculation to inhalation as the primary mode of infection, which leads to a more severe illness. Melioidosis commonly affects the lungs and is characterized by the spread of bacteria to various internal organs including the spleen and liver. Many patients become bacteremic and the mortality rate is high (19-51%) despite aggressive antimicrobial therapy [1–9]. B. pseudomallei is refractory to most antibiotics and resistance mechanisms include efflux pumps and β-lactamases [27–36]. The recommended treatment entails the use of ceftazidime, carbapenems, TMP-SMZ, chloramphenicol and/or Augmentin for several weeks. Response to treatment is slow and eradication of B. pseudomallei is difficult to achieve, resulting in recrudescence [1, 37–39].
B. mallei causes the zoonosis glanders, which primarily affects solipeds [8, 9, 20–25]. In humans, infection occurs by contact with infected animals via the cutaneous or respiratory route. The clinical manifestations of the disease include febrile pneumonia associated with necrosis of the tracheobronchial tree or pustular skin lesions and the development of abscesses. Most patients become bacteremic and B. mallei disseminates to the liver and spleen where it rapidly causes necrosis. Even with antibiotic treatment, the mortality rate for human glanders is 50% and the basis for this high mortality rate is not understood, though B. mallei has been shown to be resistant to complement-mediated killing , macrophages  and antimicrobials [32, 42].
One key aspect of pathogenesis by B. mallei and B. pseudomallei is their ability to invade and multiply within a variety of eukaryotic cells, where bacteria are shielded from the host humoral immune response and antibiotics. Once internalized, B. mallei and B. pseudomallei escape from endocytic vacuoles and enter the cytoplasm of infected cells where they multiply. The organisms subsequently spread to neighboring cells through a process involving the formation of actin tails and membrane protrusions that push bacteria from one cell to another. This intracellular lifestyle is crucial to virulence and has been a focus of research efforts aimed at understanding pathogenesis by B. mallei and B. pseudomallei[2, 9, 16–18, 22, 41, 43–49]. Several gene products, such as BimA, type 3 secretion system effectors, and type 6 secretion proteins, have been shown to play key roles in this process. By contrast, the mechanisms used by these organisms to adhere to eukaryotic cells are poorly defined. Adherence is an essential step of pathogenesis by most infectious agents because it is necessary for colonizing a new host [50–52]. Moreover, B. pseudomallei and B. mallei are facultative intracellular pathogens that gain access to the interior of target cells. Though not always a prerequisite for this process, bacterial adherence is a widespread strategy that precedes and promotes invasion [50–52]. Thus far, only the B. pseudomallei flagellum  and type 4 pilus  have been implicated in adherence and their exact roles remain to be elucidated. The present study reports the identification of B. pseudomallei and B. mallei gene products that mediate adherence to epithelial cells derived from the human respiratory tract, thus relevant to the aerosol route of infection by these organisms.
Identification of a gene shared by B. mallei and B. pseudomallei that encodes a potential autotransporter adhesin
Characteristicsa of boaA and boaB genes and their encoded products
GenBank accession #
Potential signal sequence
Percent identity shared by boaA and boaB gene products
BoaA (Bm ATCC23344)
BoaA (Bm NCTC10247)
BoaA (Bp K96243)
BoaA (Bp DD503)
BoaA (Bp 1710b)
BoaB (Bp K96243)
BoaB (Bp DD503)
BoaB (Bp 1710b)
BoaA (Bm ATCC23344)
BoaA (Bm NCTC10247)
BoaA (Bp K96243)
BoaA (Bp DD503)
BoaA (Bp 1710b)
BoaB (Bp K96243)
BoaB (Bp DD503)
BoaB (Bp 1710b)
Identification of a B. pseudomallei-specific gene encoding a putative autotransporter adhesin that resembles BoaA
The boaB gene was sequenced from B. pseudomallei DD503 and was predicted to encode a protein that is 96.7% identical to BoaB of B. pseudomallei K96243. Database searches using NCBI genomic BLAST revealed that the genomes of at least 10 more B. pseudomallei strains contain the gene. Overall, the BoaB proteins are highly-conserved (90-99% identity) and characteristics of the ORF from selected strains are shown in Tables 1 and 2 and Fig 2 for comparison purposes. Importantly, database searches also revealed that none of the B. mallei isolates available through the NCBI genomic BLAST service have a boaB gene. Taken together, these results indicate that BoaB is a highly-conserved B. pseudomallei-specific molecule.
Expression of the Burkholderia BoaA and BoaB proteins in E. coli
In addition to showing that BoaA and BoaB are associated with the OM by protein separation and western blot, we used immunofluorescent labeling of non-permeabilized E. coli cells to demonstrate their display on the bacterial surface. As depicted in Fig 3C, E. coli harboring pSLboaA and pSLboaB are labeled by the α-BoaA and α-BoaB Abs, respectively, while recombinant bacteria carrying the control plasmid pCC1.3 are not. Staining of nucleic acids with the fluorescent dye DAPI verified that comparable numbers of bacterial cells were examined (Fig 3C). Quantitative attachment assays revealed that E. coli expressing BoaB attach to HEp2 (laryngeal) and A549 (type II pneumocytes) epithelial cell lines at levels 18- and 68-fold greater than bacteria carrying pCC1.3, respectively (Fig 3D). In addition, BoaB expression was found to increase adherence to differentiated primary cultures of normal human bronchial epithelium (NHBE). Under the growth conditions used, NHBE cultures form a pseudostratified epithelium with tight junctions containing both ciliated and non-ciliated cells. This epithelium exhibits transepithelial resistance, mucus secretion, mucociliary activity, and an apical surface not submerged in tissue culture medium, thus representing an environment that is similar to the airway lumen in vivo[67–69]. Expression of the B. mallei ATCC23344 BoaA protein on the surface of E. coli also substantially increased adherence to monolayers of A549 and HEp2 cells and to NHBE cultures. Taken together, these data demonstrate that BoaA and BoaB are OM proteins mediating adherence to epithelial cells of the human respiratory tract.
B. pseudomallei and B. mallei are facultative intracellular organisms that can invade, survive and replicate in a variety of eukaryotic cells. Moreover, autotransporter adhesins often specify additional biological functions such as invasion , biofilm formation , survival within host cells  and intracellular motility . For these reasons, we measured the ability of E. coli expressing BoaA and BoaB to invade epithelial cells as well as their ability to survive within murine macrophages. We also measured the ability of these recombinant strains to form biofilms on the plastic support of tissue culture plates using a crystal violet-based assay. The results of these experiments indicated that neither BoaA nor BoaB substantially increase invasion of epithelial cells, phagocytosis of recombinant bacteria by J774A.1 murine macrophages, survival inside these immune cells, or biofilm formation (data not shown).
Construction and characterization of Burkholderia mutant strains
Autotransporters are involved in various biological traits of Gram-negative bacteria including invasion , serum resistance [56, 73], phospholipolysis [76, 77], cytotoxicity , adherence [61, 79], biofilm formation [71, 80], survival within eukaryotic cells  and intracellular motility . These proteins share an N-terminal extracellular passenger domain that specifies the biological activity of the autotransporter and a C-terminus containing several β-strands, which tether the molecule to the OM. Based on the structure of this membrane-anchoring domain, autotransporters can be classified as conventional (contain 12 β-strands) or trimeric (contain 4 β-strands) . One of the best characterized trimeric autotransporters is the Y. enterocolitica adhesin YadA. This protein, along with structurally-related adherence proteins such as M. catarrhalis Hag and H. influenzae Hia, are often referred to as oligomeric coiled-coil adhesins (Oca) .
Tiyawisutsri and colleagues previously reported that the published genomic sequences of B. pseudomallei K96243 and B. mallei ATCC23344 contain several ORFs encoding putative trimeric autotransporters . Of these, only BimA (i.e. B. pseudomallei and B. mallei locus tag numbers BPSS1492 and BMAA0749, respectively) has been functionally characterized and shown to be required for actin-based motility of the organisms inside eukaryotic cells [16, 17]. In the present study, we identified the boaA ORF based on similarities to the Oca proteins Y. enterocolitica YadA and M. catarrhalis Hag. Specifically, we searched the genome of B. mallei ATCC23344 for gene products specifying N-terminal AIG β-roll motifs, a transporter module containing 4 β-strands, and a YadA-like C-terminal domain (PF03895). We demonstrated that when expressed by E. coli, boaA increases adherence to the human epithelial cell lines HEp2 (laryngeal cells) and A549 (type II pneumocytes) grown as monolayers in submerged cultures. Though these cell types are relevant to the aerosol route of infection by B. mallei and B. pseudomallei, they lack important features of the airway mucosa such as cilia and mucociliary activity. The ciliated cells of the respiratory tract and other mucosal membranes keep secretions moving and contribute to preventing colonization by pathogens. For these reasons, we also measured the adherence of E. coli expressing BoaA to cultures of normal human bronchial epithelium (NHBE) grown in an air-liquid interface system. These cultures mimic the structure and function of the airway mucosa more accurately as they are fully differentiated, form a pseudostratified epithelium with tight junctions, contain ciliated and mucus-producing goblet cells, and exhibit mucociliary activity [67–69]. Quantitative attachment assays utilizing this culture system revealed that BoaA expression increases adherence to NHBE cultures (Fig 3D).
In addition to showing that BoaA specifies adhesive properties when expressed in the heterologous genetic background of E. coli, we determined that disruption of the boaA gene in the genome of B. mallei ATCC23344 reduces adherence of the organism to monolayers of HEp2 and A549 cells and to NHBE cultures, therefore substantiating the function of BoaA as an adhesin. Database searches using the NCBI genomic BLAST service identified boaA in several B. pseudomallei and B. mallei isolates and we demonstrated that inactivation of boaA in the B. pseudomallei strain DD503 also decreases attachment to HEp2 laryngeal cells, A549 pneumocytes, and NHBE cultures. Together, our data indicate that BoaA is an adhesin common to B. mallei and B. pseudomallei and mediates adherence to host cells relevant to pathogenesis by the organisms. These findings are consistent with the recent inclusion of BoaA (i.e. B. mallei ATCC23344 and B. pseudomallei K96243 locus tag numbers BMAA0649 and BPSS0796, respectively) in the virulome of B. mallei and B. pseudomallei, which consists of a set of 650 putative virulence genes that are shared by B. pseudomallei and B. mallei but are not present in five closely-related non-pathogenic Burkholderia species .
Comparative genomic analyses revealed that several B. pseudomallei isolates possess a second Oca-like gene product highly similar to BoaA, which we termed BoaB. The C-terminus of BoaB is strikingly similar to that of BoaA (Fig 2) and the predicted passenger domains of the molecules contain numerous matching serine-rich SLST motifs (Fig 1). The proteins are also functionally related as they mediate adherence to the same types of host cells (Fig 3D and 5). Therefore, it is tempting to speculate that boaA and boaB are the result of gene duplication. This hypothesis would be consistent with the genomic organization of the genes. In B. pseudomallei strains K96243, 1710b, 1655, 576 and MSHR346, the boaB gene is located on chromosome 1 while boaA is on chromosome 2. Moreover, the boaB gene in all these isolates is preceded by two ORFs specifying an invertase and a transposase. These genes may be the remnants of mobile genetic elements possibly involved in gene duplication. Database searches also revealed that B. mallei isolates do not possess a boaB gene, which was likely lost during evolution of the organism into a host-adapted pathogen. Interestingly, the closely-related bacterium Burkholderia thailandensis has been reported by others to bind poorly to epithelial cells . This organism exhibits high genomic similarities to B. pseudomallei and B. mallei and, like B. pseudomallei, is a natural inhabitant of the tropical soil environment. However, B. thailandensis is not considered pathogenic to humans or higher animals [84–87]. This difference in virulence can be attributed to the fact that B. thailandensis does not produce a capsule  and lacks the 650 genes comprising the aforementioned virulome of B. mallei and B. pseudomallei. Analysis of the published genome of the B. thailandensis strain E264  indicated that it contains neither the boaA nor the boaB gene.
B. pseudomallei DD503 and B. mallei ATCC23344 do not produce detectable amounts of the BoaA and BoaB proteins under the conditions tested. These results are consistent with qRT-PCR experiments demonstrating that the organisms express very low levels of the boa genes relative to the Burkholderia recA control (Fig 4). Similar observations were made by Druar and colleagues while studying expression of the Burkholderia Type 3 Secretion System-3 (T3SS-3) proteins BipB and BipD . These proteins were not detected in lysates of B. pseudomallei or B. mallei grown under different conditions, even though the antibodies used in their western blot experiments recognized recombinant forms of BipB and BipD. The authors concluded that these two T3SS-3 molecules must be expressed in detectable amounts only under very specific in vitro conditions . Using a gfp reporter strain, Burtnick et al recently showed that the B. mallei Type 6 Secretion System-1 (T6SS-1) gene tssE is not expressed at detectable levels when bacteria are grown in LSLB or tissue culture medium, but is expressed upon phagocytosis of the organisms by murine macrophages . The protein preparations tested in our studies were obtained from bacteria cultured on LSLB agar plates at 37°C, conditions which may not be optimal for expression of the BoaA and BoaB proteins. Additionally, Chantratita and colleagues reported that growth of B. pseudomallei under various conditions triggers a complex adaptive process altering the expression of surface molecules . This process, termed phenotypic plasticity, was correlated with changes in the morphology of B. pseudomallei colonies grown on agar plates and appears to modulate the environmental fitness, as well as virulence, of the organism. Given their surface location and likely role in virulence (i.e. adherence to host cells), it is possible that BoaA and BoaB are subject to phenotypic plasticity and are expressed in detectable amounts only under very specific in vitro conditions. In concordance, the reduced adherence phenotype of the boaA and boaB mutant strains suggests increased level of expression of the genes when Burkholderia is incubated with epithelial cells. However, efforts to detect protein expression under these conditions (i.e. immunofluorescence, immunoprecipitation) have been unsuccessful. Of further note, studies have shown that sera from horses infected with B. mallei and sera from melioidosis patients contain antibodies reacting with BoaA (i.e. B. mallei ATCC23344 locus tag number BMAA0649)  and with BoaB (i.e. B. pseudomallei K96243 locus tag number BPLS1705), respectively, which indicates expression of the autotransporters in vivo. Determining the conditions and mechanisms that modulate expression of the Boa adhesins, and their influence on the binding of B. pseudomallei and B. mallei to host surfaces, represent key areas for future study.
Disruption of boaA and boaB in the B. pseudomallei double mutant strain DD503.boaA.boaB was found to have a significant effect on the growth of the organism within murine macrophages (Fig 6B). At present, it is not clear whether BoaA and BoaB play a direct role in intracellular replication. It is possible that the absence of both Boa proteins in the OM of DD503.boaA.boaB affects the proper surface display of another molecule involved in this phenotypic trait. One candidate is LPS, as this molecule was previously shown to play an important role in the ability of B. pseudomallei to grow inside host cells [93, 94]. B. pseudomallei produces multiple T3SS and T6SS that are involved in the intracellular lifestyle of the organism. These specialized secretion apparatuses are used to inject bacterial effector proteins inside host cells where they exert cytopathic effects or manipulate signaling pathways. One important step in this process is the proper docking of bacteria to the host cell to deliver the effectors. Given their roles in adherence, it is possible that the lack of expression of the boaA and boaB gene products interferes with the delivery of T3SS and/or T6SS cell-altering effectors, which in turn reduces the intracellular fitness of the double mutant strain DD503.boaA.boaB. The Yersinia pestis OM adhesin Ail was recently shown to affect delivery of Yop effector proteins to HEp2 cells and macrophages in such a manner . Alternatively, the reduced intracellular growth of the double boaA boaB mutant may be due to a greater sensitivity to immune effectors produced by the macrophages. The molecular basis for this phenotype is currently being investigated.
The present study reports the identification of B. pseudomallei and B. mallei gene products mediating adherence to epithelial cells. Because of their classification as select agents, there is currently a shortage of tools for genetic studies in B. pseudomallei and B. mallei (i.e. paucity of acceptable antibiotic markers, lack of low copy plasmids suitable for expressing surface proteins), which precluded us from complementing mutants. Our ability to express BoaA and BoaB in E. coli, however, conclusively demonstrates that the proteins directly mediate binding to epithelial cells. These results, along with our analyses of the mutant strains, clearly establish that these molecules participate in adherence by B. pseudomallei and B. mallei. Adherence is an essential step in pathogenesis by most infectious agents because it is necessary for colonization and precedes invasion of host cells by intracellular pathogens. Thus, continued investigation of BoaA and BoaB will yield important information regarding the biology and virulence of these organisms.
Strains, plasmids, tissue culture cell lines and growth conditions
Strains and plasmids
Parental strain; polymyxin BR zeocinS kanamycinS streptomycinR
Isogenic boaA mutant strain of DD503; polymyxin BR zeocinR kanamycinS streptomycinR
Isogenic boaB mutant strain of DD503; polymyxin BR zeocinR kanamycinS streptomycinR
Isogenic boaA boaB double mutant strain of DD503; polymyxin BR zeocinR kanamycinR streptomycinS
Wild-type strain; polymyxin BR zeocinS kanamycinS
Isogenic boaA mutant strain of ATCC23344; polymyxin BR zeocinR kanamycinS
Strain used for conjugational transfer of suicide plasmids from E. coli to B. pseudomallei or B. mallei
Cloning vector; chloramphenicol resistant (CmR)
Mobilizable suicide plasmid; kanamycinR and ampicillinR
pCC1-based plasmid control, does not confer adherence; CmR
pCC1 containing the B. mallei ATCC23344 boaA gene; CmR
pSLboaA in which a zeocinR marker was introduced near the middle of the boaA gene; CmR and zeocinR
pKAS46 containing the insert from pSLboaAZEO; zeocinR , ampicillinR and kanamycinR
pCC1 containing the B. pseudomallei DD503 boaB gene; CmR
pSLboaB in which a zeocinR marker was introduced near the middle of the boaB gene; CmR and zeocinR
pKAS46 containing the insert from pSLboaBZEO; zeocinR , ampicillinR and kanamycinR
pKAS46 containing a 0.8-kb insert which corresponds to a region located within the 5' end of the B. pseudomallei DD503 boaB ORF; ampicillinR and kanamycinR
pKASboaB5' in which the ampicillinR marker was removed; ampicillinS and kanamycinR
Source of the zeocinR marker; ampicillinR and zeocinR
E. coli was cultured using LSLB containing 15 μg/ml chloramphenicol, 50 μg/ml Kan or 50 μg/ml zeocin, where indicated. For preparation of plasmid DNA, extraction of Sarkosyl-insoluble outer membrane proteins, RNA isolation, immunofluorescence labeling, as well as for adherence, invasion and macrophage assays, recombinant E. coli strains were grown in LSLB supplemented with the EPICENTRE® Biotechnologies CopyControl™ Induction Solution as previously reported .
The epithelial cell lines HEp2 (human laryngeal epithelium; ATCC CCL-23) and A549 (type II alveolar lung epithelium; ATCC CCL85) were cultured as outlined by others  and the murine macrophage cell line J774A.1 (ATCC TIB-67) was grown in DMEM medium (Mediatech, Inc) supplemented with 10% fetal bovine serum (Invitrogen™) at 37°C and in the presence of 7.5% CO2. Normal human bronchial epithelium (LONZA) were expanded, cryopreserved and cultured in an air-liquid interface system as previously described [67–69]. Normal human bronchial epithelium (NHBE) were grown on Transwell permeable inserts (Corning) and their apical surfaces were exposed to air for a minimum of 3 weeks prior to use in biological assays to ensure proper cellular differentiation and the development of functional cilia.
Recombinant DNA methodology
Standard molecular biology techniques were performed as described elsewhere . Genomic DNA was isolated using the Invitrogen™ Easy-DNA™ kit. Plasmid DNA was obtained with the QIAprep Spin Miniprep Kit (Qiagen). The Failsafe™ PCR System (EPICENTRE® Biotechnologies) was used to amplify the 5.5-kb boaA gene of B. mallei ATCC23344 with primers P1 (5'-TCA GAT GAA CCG CGT TTC CGT ATC-3') and P2 (5'-ACT CAT ACG GCT CGC GCA TAA A-3'). This amplicon was cloned in the vector pCC1™ using the CopyControl™ PCR Cloning Kit (EPICENTRE® Biotechnologies), yielding the plasmid pSLboaA (Table 3). The 5.4-kb boaA gene of B. pseudomallei DD503 was amplified with P3 (5'-GCT TGC CGC ACG CAA TGG CT-3') and P4 (5'-ATG GCG AGC GCG AAA CAT GGA AA-3') and the purified PCR product was used as a template in sequencing reactions. The 5.9-kb boaB gene of B. pseudomallei DD503 was generated with the Failsafe™ PCR system using P5 (5'-TCC ATA AAT TCC CGG CGC TTG TTG-3') and P6 (5'-TGT CTC GAC ATC AGC GGT TCA CTT-3'), sequenced, and then cloned in pCC1™ as described above, yielding the plasmid pSLboaB (Table 3). Of note, the inserts of plasmids pSLboaA and pSLboaB were sequenced to verify that PCR did not introduce mutations resulting in amino acid (aa) substitutions in the boaA and boaB gene products.
Construction of boaA isogenic mutant strains of B. mallei and B. pseudomallei
A 0.45-kb zeocinR cassette was introduced into a unique NheI site located near the middle of the boaA ORF in pSLboaA. The resulting construct, designated pSLboaAZEO, was digested with BamHI and a 6-kb fragment corresponding to the boaA ORF interrupted by the zeocinR marker was excised from an agarose gel, purified with the High Pure PCR Product Purification Kit (Roche Applied Science), and treated with the EPICENTRE® Biotechnologies End-It™ DNA End Repair Kit. This blunt DNA fragment was then subcloned into the EcoRV site of the suicide vector pKAS46. The resulting plasmid, pKASboaAZEO, was introduced into the E. coli strain S17 by electroporation and subsequently transferred into B. mallei ATCC23344 or B. pseudomallei DD503 by conjugation as reported by others .
Upon conjugation, B. pseudomallei colonies were first selected for resistance to PmB (to prevent growth of E. coli S17) and zeocin (to select strains containing the disrupted copy of boaA in their genome). These putative mutants were then tested for their sensitivity to kanamycin and resistance to streptomycin, which identified strains that did not contain the suicide vector pKAS46 integrated in their genome. Lastly, these PmBR zeocinR KanS SmR conjugants were screened by PCR using Platinum®Pfx DNA Polymerase (Invitrogen™) with the primers P7 (5'-TTG AGC ACG ACC AAC AGC AAC GTC-3') and P8 (5'-CCA ATG CGG TCG AAT GAT TGC C-3'), which led to the identification of the mutant strain DD503.boaA. These primers yielded a PCR product of 1.3-kb in B. pseudomallei DD503 and a larger amplicon of 1.8-kb in the mutant. The primers P9 (5'-TAT CGC AAG GTT TGG AAC AAG GCG-3') and P10 (5'-ACG CCG AAT ACC CTT GAT AGC TG-3') were also used to further confirm gene replacement in the B. pseudomallei mutant strain. These primers amplified DNA fragments of 5-kb in the parent strain DD503 and of 5.5-kb in the isogenic boaA mutant. After the conjugative transfer of plasmid pKASboaAZEO into the B. mallei strain ATCC23344, colonies shown to be PmBR, zeocinR and KanS were screened by PCR with P7 and P8 as described above to identify the mutant strain ATCC23344.boaA. Of note, the boaA genes of both isogenic mutant strains DD503.boaA and ATCC23344.boaA were amplified and sequenced in their entirety to verify proper allelic exchange and successful disruption of boaA.
Construction of a boaB B. pseudomallei isogenic mutant strain
The plasmid pSLboaB was digested with NheI to remove a 162-bp fragment internal to the boaB ORF, treated with the End-It™ DNA End Repair Kit and ligated with the 0.45-kb zeocinR marker to yield the construct pSLboaBZEO. This plasmid was digested with BamHI and a 6.2-kb fragment, which corresponds to the boaB ORF disrupted with the zeocinR cassette, was purified from agarose gel slices, subcloned into the suicide plasmid pKAS46 and introduced into B. pseudomallei DD503 by conjugation as described above. Conjugants shown to be PmBR zeocinR KanS SmR were screened by PCR using Platinum®Pfx DNA Polymerase (Invitrogen™) with primers P11 (5'-AGG TGG CGAC TCA AAT AGA ACC GT-3') and P12 (5'-GTT CGT GTT GTT GGC TAC GGC AAT-3') to identify the mutant strain DD503.boaB. These primers amplified a PCR product of 1.7-kb in B. pseudomallei DD503 and of 2.0-kb in the mutant. The primers P13 (5'-AGG TGG CGA CTC AAA TAG AAC CGT-3') and P10 were also used to further confirm gene replacement in the B. pseudomallei mutant strain. These primers generated amplicons of 5.2-kb and 5.5-kb in strains DD503 and DD503.boaB, respectively. Additionally, the boaB gene of DD503.boaB was amplified and both strands of the PCR product were sequenced to verify allelic exchange.
Construction of a B. pseudomallei boaA boaB double mutant strain
A 0.8-kb PCR product, which corresponds to a region located within the 5'end of the B. pseudomallei DD503 boaB ORF, was amplified with Platinum®Pfx DNA Polymerase (Invitrogen™) using primers P14 (5'-CTC GGG CTC AAT AAC ATG GC-3') and P15 (5'-CGG AAT TC C GGT TCG TGT TGT TGG CT-3'; EcoRI site underlined). This amplicon was digested with EcoR1 and directionally cloned into the EcoRV and EcoR1 sites of the suicide vector pKAS46, yielding the plasmid pKASboaB5'. This construct was digested with ApaLI to remove a 0.8-kb fragment corresponding to the ampicillin-resistance marker of pKAS46 and the resulting plasmid, pKASboaB5'AmpS , was introduced into the B. pseudomallei mutant strain DD503.boaA by conjugation as described above. Conjugants shown to be PmBR zeocinR KanR SmS were screened by PCR using the MasterAmp™ Extra-Long PCR kit (EPICENTRE® Biotechnologies) with primers P13 and P10 to identify the mutant strain DD503.boaA.boaB. These primers amplified PCR products of 5.2-kb in B. pseudomallei DD503 as well as in the single mutant DD503.boaA, and of 11.0-kb in the double mutant strain DD503.boaA.boaB. These results indicated that the boaB gene in DD503.boaA.boaB had been disrupted by integration of the entire pKASboaB5'AmpS plasmid into the genome of B. pseudomallei.
Quantitative reverse-transcriptase PCR (qRT-PCR)
Total RNA was extracted from 108 bacteria with the RNeasy Kit (Qiagen). One μg of total RNA was treated with RQ1 RNAse-Free DNase (Promega) and reverse transcribed with Improm II™ Reverse transcriptase (Promega) using random hexamers (Invitrogen™) under the manufacturer's recommended conditions. PCR quantification of specific cDNA levels was performed using a LightCycler® (Roche Applied Science) rapid fluorescence temperature cycler as reported elsewhere . Briefly, amplification was performed in a 10 μl final volume containing 50 mM Tris (pH 8.3), 3 mM MgCl2, 4.5 μg of bovine serum albumin, 200 μM deoxynucleotide triphosphates, a 1:10,000 dilution of SYBR® Green I (Molecular Probes, Inc.), 1 μM each primer, and 1 unit of Platinum®Taq DNA Polymerase (Invitrogen™). Amplification was performed for 40 cycles, with each run consisting of an initial melting at 95°C for 2 minutes, followed by melting, annealing, extension, and acquiring temperatures specific to each primer set. Serial dilutions of a representative template cDNA were amplified using each primer set to create a standard curve. Particular transcript levels in experimental samples were calculated by comparison to the corresponding standard curve. All calculated values for the boaA and boaB genes are normalized to either the Burkholderia recA or E. coli recA levels. A primer set for Borrelia burgdorferi recA was used as a non-Burkholderia control to further demonstrate primer specificity (control in Fig 4). Negative controls in which the reverse transcriptase enzyme was not added to reaction mixtures were included in all experiments (data not shown). The boa and recA transcripts were amplified from the same sets of samples. The primer sets used were: boaAF (5'-ATC GCG AAC AAT GCG AAC GAT GTC-3') and boaAR (5'-AAG CGA ATA AGC CTG ACC TGC GAT-3'), boaBF (5'-AAT GCC GTA GCC AAC AAC ACG AAC-3') and boaBR (5'-TCG TCG AGT AAA GTT GCG AAC CGT-3'), recABURKF (5'-CAC GAA CTG CCT CGT GAT CTT CAT-3') and recABURKR (5'-AAA TGC CTT CGC CGT ACA GGA TGT-3'), recACOLIF (5'-ATG GCT ATC GAC GAA AAC-3') and recACOLIR (5'-GGT TTT ACC GGA AGA TTC C-3'), and recABbF (5'-GTC GAT CTA TTG TAT TAG ATG AGG CTC TCG-3') and recABbR (5'-GCC AAA GTT CTG CAA CAT TAA CAC CTA AAG-3').
Nucleotide sequence analyses
PCR products and plasmids were sequenced at the University of Michigan Sequencing Core. Chromatograms were assembled using the Sequencher 4.9 software (Gene Codes Corporation). The nucleotide sequences of the B. pseudomallei DD503 boaA (EF423807) and boaB (EF423808) genes were deposited in GenBank under the indicated accession number.
Sequence analyses were performed using Vector NTI (Invitrogen) and the various online tools available through the ExPASy Proteomics Server (http://au.expasy.org/). Signal sequence cleavage sites were determined using the SignalP 3.0 server (http://www.cbs.dtu.dk/services/SignalP/). The B. mallei ATCC23344 boaA gene product (locus tag BMAA0649) was identified by searching the genome of the organism for the presence of a YadA-like C-terminal domain (Pfam database number PF03895) through the NCBI genomic BLAST service using the tblastn and blastp programs (http://www.ncbi.nlm.nih.gov/sutils/genom_table.cgi). The other boaA and boaB gene products described in this study were identified by using the predicted aa sequence of the B. mallei ATCC23344 BoaA protein to search the genomes of the B. mallei as well as B. pseudomallei strains available through the NCBI genomic BLAST service utilizing the tblastn and blastp programs. Structural features of the Boa proteins (e.g. helical regions, β-strands) were identified using the PSIPRED Protein Structure Prediction Server (http://bioinf.cs.ucl.ac.uk/psipred/).
Epithelial cell adherence assays
Quantitative attachment assays were performed as previously described by our laboratory [61, 67]. Monolayers of A549 and HEp2 cells and cultures of NHBE were infected with B. mallei, B. pseudomallei or recombinant E. coli strains at a MOI of 100. Duplicate assays were repeated on at least 3 occasions for each strain, and adherence is expressed as the percentage (± standard error) of bacteria attached to epithelial cells relative to the inoculum. Statistical analyses were performed using the Mann-Whitney test (GraphPad Prism software) and P values < 0.05 are reported as statistically significant.
Biofilm and bactericidal assays
Macrophage survival assays
Plate-grown bacteria were suspended in 5-ml of sterile PBS supplemented with 0.15% gelatin (PBSG) to a density of 109 CFU/ml. These suspensions were used to infect two identical sets of duplicate monolayers of J774A.1 cells (105 cells/well; 24-well tissue culture plate) at an MOI of 10. The inoculated tissue culture plates were centrifuged (5-min, 165 × g) and incubated for 1-hr at 37°C, time after which the medium covering the monolayers was replaced with fresh tissue culture medium containing 50 μg/ml gentamicin. After a 2-hr incubation (i.e. 3-hr post infection), the wells of one tissue culture plate were washed, J774A.1 cells were lysed with a solution containing Saponin, and serial dilutions of the well contents were spread onto agar plates to determine the number of bacteria phagocytosed by the macrophages. The wells of the other tissue culture plate were washed once, fresh medium without antibiotics was added, and the plate was incubated for an additional 5-hr. Following this incubation (i.e. 8-hr post-infection), the wells were processed as described above in order to enumerate bacteria. These experiments were repeated on at least 3 separate occasions. Statistical analyses were performed using the Mann-Whitney test (GraphPad Prism software) and P values < 0.05 are reported as statistically significant.
Epithelial cell invasion and survival assays
These experiments were performed as described above for macrophage survival assays with some modifications. Specifically, epithelial cells were infected with an MOI of 100. The inoculated tissue culture plates were centrifuged and incubated for 3-hr at 37°C, time after which the medium covering the monolayers was replaced with fresh tissue culture medium containing 50 μg/ml gentamicin. After a 2-hr incubation (i.e. 5-hr post infection), the wells of one tissue culture plate were washed and processed to enumerate intracellular bacteria as described above. The wells of the other tissue culture plate were washed once, fresh medium without antibiotics was added to wells, and the plate was incubated for an additional 3-hr. Following this incubation (i.e. 8-hr post-infection), the wells were processed as described above. These experiments were repeated on at least 3 separate occasions. Statistical analyses were performed using the Mann-Whitney test (GraphPad Prism software) and P values < 0.05 are reported as statistically significant
Protein preparations, western blot, and antibody production
Sarkosyl-insoluble OM proteins were obtained as previously described by Carlone et al. The methods used to prepare whole cell lysates and perform western blot experiments are described elsewhere [61, 62, 67, 104, 105]. To obtain antibodies directed against BoaA, the peptide PEPA (NYLGGLFGFG PQTS MANWG DSSN) was synthesized and conjugated to maleimide-activated keyhole limpet hemocyanin (mcKLH, Thermo Scientific) under the manufacturer's recommended conditions. The sequence of PEPA corresponds to residues 78-100 of B. pseudomallei DD503 BoaA and encompasses aa 79-101 of B. mallei ATCC23344 BoaA (underlined residues in the PEPA sequence being perfectly conserved). The mcKLH-PEPA conjugate was emulsified in Freund's adjuvants and used to immunize female BALB/c mice as previously reported . BoaB-specific antibodies were obtained by immunizing mice with mcKLH conjugated to the synthetic peptide PEPB (GWLLGTTSQTTDPGPLYPGPGAENN), which specifies aa 131-155 of B. pseudomallei DD503 BoaB. These animal studies were performed in compliance with institutional, as well as governmental, rules and regulations.
Immunofluorescence labeling of E. coli and microscopy
Plate-grown bacteria were suspended in 5-ml of sterile PBSG to a density of 108 CFU/ml. Portions of these suspensions were spotted onto glass slides and dried using a warming plate. The slides were fixed with PBSG supplemented with 4% paraformaldehyde for 30-min at room temperature, washed with PBS supplemented with 0.05% Tween 20 (PBST), and blocked overnight at 4°C using PBST supplemented with 10% goat serum (SIGMA-ALDRICH®). Next, bacteria were probed for 1-hr at room temperature with murine α-BoaA or α-BoaB antibodies diluted (1:200) in PBST supplemented with 10% goat serum. After this incubation, the slides were washed with PBST to remove unbound antibodies and incubated for 30-min at room temperature with a goat α-mouse antibody labeled with Alexa Fluor® 546 (Molecular Probes, Inc) and diluted (1:400) in PBST supplemented with 10% goat serum. Following this incubation, the slides were washed with PBST to remove unbound antibody and bacterial cells were stained using the nucleic acid dye DAPI (Molecular Probes, Inc). Slides were mounted with SlowFade® reagent (Invitrogen™) and examined by microscopy using a Zeiss LSM 510 Meta confocal system.
open reading frame
oligomeric coiled-coil adhesin
colony forming units
quantitative reverse-transcriptase PCR
This study was supported by a grant from NIH/NIAID (AI062775) and startup funds from the University of Georgia College of Veterinary Medicine to ERL. The authors would like to thank Lauren Snipes and Frank Michel at the University of Georgia for their technical assistance.
- Cheng AC, Currie BJ: Melioidosis: epidemiology, pathophysiology, and management. Clin Microbiol Rev. 2005, 18 (2): 383-416. 10.1128/CMR.18.2.383-416.2005.PubMed CentralPubMedGoogle Scholar
- Wiersinga WJ, van der Poll T, White NJ, Day NP, Peacock SJ: Melioidosis: insights into the pathogenicity of Burkholderia pseudomallei. Nat Rev Microbiol. 2006, 4 (4): 272-282. 10.1038/nrmicro1385.PubMedGoogle Scholar
- Currie BJ, Fisher DA, Anstey NM, Jacups SP: Melioidosis: acute and chronic disease, relapse and re-activation. Trans R Soc Trop Med Hyg. 2000, 94 (3): 301-304. 10.1016/S0035-9203(00)90333-X.PubMedGoogle Scholar
- Currie BJ, Fisher DA, Howard DM, Burrow JN, Lo D, Selva-Nayagam S, Anstey NM, Huffam SE, Snelling PL, Marks PJ, Stephens DP, Lum GD, Jacups SP, Krause VL: Endemic melioidosis in tropical northern Australia: a 10-year prospective study and review of the literature. Clin Infect Dis. 2000, 31 (4): 981-986. 10.1086/318116.PubMedGoogle Scholar
- Adler NR, Govan B, Cullinane M, Harper M, Adler B, Boyce JD: The molecular and cellular basis of pathogenesis in melioidosis: how does Burkholderia pseudomallei cause disease?. FEMS Microbiol Rev. 2009, 33 (6): 1079-1099. 10.1111/j.1574-6976.2009.00189.x.PubMedGoogle Scholar
- Wiersinga WJ, van der Poll T: Immunity to Burkholderia pseudomallei. Curr Opin Infect Dis. 2009, 22 (2): 102-108. 10.1097/QCO.0b013e328322e727.PubMedGoogle Scholar
- Vietri NJ, Deshazer D: Melioidosis. Medical Aspects of Biological Warfare. 2007, U.S Army Medical Department Borden Insitute Textbooks of Biological Warfare, 147-166.Google Scholar
- Bondi SK, Goldberg JB: Strategies toward vaccines against Burkholderia mallei and Burkholderia pseudomallei. Expert Rev Vaccines. 2008, 7 (9): 1357-1365. 10.1586/14760518.104.22.1687.PubMed CentralPubMedGoogle Scholar
- Galyov EE, Brett PJ, Deshazer D: Molecular Insights into Burkholderia pseudomallei and Burkholderia mallei Pathogenesis. Annu Rev Microbiol. 2010, 64: 495-517. 10.1146/annurev.micro.112408.134030.PubMedGoogle Scholar
- DeShazer D, Brett PJ, Woods DE: The type II O-antigenic polysaccharide moiety of Burkholderia pseudomallei lipopolysaccharide is required for serum resistance and virulence. Mol Microbiol. 1998, 30 (5): 1081-1100. 10.1046/j.1365-2958.1998.01139.x.PubMedGoogle Scholar
- Egan AM, Gordon DL: Burkholderia pseudomallei activates complement and is ingested but not killed by polymorphonuclear leukocytes. Infect Immun. 1996, 64 (12): 4952-4959.PubMed CentralPubMedGoogle Scholar
- Reckseidler-Zenteno SL, DeVinney R, Woods DE: The capsular polysaccharide of Burkholderia pseudomallei contributes to survival in serum by reducing complement factor C3b deposition. Infect Immun. 2005, 73 (2): 1106-1115. 10.1128/IAI.73.2.1106-1115.2005.PubMed CentralPubMedGoogle Scholar
- Jones AL, DeShazer D, Woods DE: Identification and characterization of a two-component regulatory system involved in invasion of eukaryotic cells and heavy-metal resistance in Burkholderia pseudomallei. Infect Immun. 1997, 65 (12): 4972-4977.PubMed CentralPubMedGoogle Scholar
- Jones AL, Beveridge TJ, Woods DE: Intracellular survival of Burkholderia pseudomallei. Infect Immun. 1996, 64 (3): 782-790.PubMed CentralPubMedGoogle Scholar
- Burtnick MN, Woods DE: Isolation of polymyxin B-susceptible mutants of Burkholderia pseudomallei and molecular characterization of genetic loci involved in polymyxin B resistance. Antimicrob Agents Chemother. 1999, 43 (11): 2648-2656.PubMed CentralPubMedGoogle Scholar
- Stevens JM, Ulrich RL, Taylor LA, Wood MW, Deshazer D, Stevens MP, Galyov EE: Actin-binding proteins from Burkholderia mallei and Burkholderia thailandensis can functionally compensate for the actin-based motility defect of a Burkholderia pseudomallei bimA mutant. J Bacteriol. 2005, 187 (22): 7857-7862. 10.1128/JB.187.22.7857-7862.2005.PubMed CentralPubMedGoogle Scholar
- Stevens MP, Stevens JM, Jeng RL, Taylor LA, Wood MW, Hawes P, Monaghan P, Welch MD, Galyov EE: Identification of a bacterial factor required for actin-based motility of Burkholderia pseudomallei. Mol Microbiol. 2005, 56 (1): 40-53. 10.1111/j.1365-2958.2004.04528.x.PubMedGoogle Scholar
- Stevens MP, Galyov EE: Exploitation of host cells by Burkholderia pseudomallei. Int J Med Microbiol. 2004, 293 (7-8): 549-555. 10.1078/1438-4221-00292.PubMedGoogle Scholar
- Holden MT, Titball RW, Peacock SJ, Cerdeno-Tarraga AM, Atkins T, Crossman LC, Pitt T, Churcher C, Mungall K, Bentley SD, Sebaihia M, Thomson NR, Bason N, Beacham IR, Brooks K, Brown KA, Brown NF, Challis GL, Cherevach I, Chillingworth T, Cronin A, Crossett B, Davis P, DeShazer D, Feltwell T, Fraser A, Hance Z, Hauser H, Holroyd S, Jagels K, Keith KE, Maddison M, Moule S, Price C, Quail MA, Rabbinowitsch E, Rutherford K, Sanders M, Simmonds M, Songsivilai S, Stevens K, Tumapa S, Vesaratchavest M, Whitehead S, Yeats C, Barrell BG, Oyston PC, Parkhill J: Genomic plasticity of the causative agent of melioidosis, Burkholderia pseudomallei. Proc Natl Acad Sci USA. 2004, 101 (39): 14240-14245. 10.1073/pnas.0403302101.PubMed CentralPubMedGoogle Scholar
- Arun S, Neubauer H, Gurel A, Ayyildiz G, Kuscu B, Yesildere T, Meyer H, Hermanns W: Equine glanders in Turkey. Vet Rec. 1999, 144 (10): 255-258. 10.1136/vr.144.10.255.PubMedGoogle Scholar
- Neubauer H, Meyer H, Finke EJ: Human glanders. Revue Internationale des Services de Sante des Forces Armees. 1997, 70: 258-265.Google Scholar
- Whitlock GC, Estes DM, Torres AG: Glanders: off to the races with Burkholderia mallei. FEMS Microbiol Lett. 2007, 277 (2): 115-122. 10.1111/j.1574-6968.2007.00949.x.PubMedGoogle Scholar
- Srinivasan A, Kraus CN, DeShazer D, Becker PM, Dick JD, Spacek L, Bartlett JG, Byrne WR, Thomas DL: Glanders in a military research microbiologist. N Engl J Med. 2001, 345 (4): 256-258. 10.1056/NEJM200107263450404.PubMedGoogle Scholar
- Gregory BC, Waag DM: Glanders. Medical Aspects of Biological Warfare. 2007, U.S Army Medical Department Borden Insitute Textbooks of Biological Warfare, 121-146.Google Scholar
- Waag DM, Deshazer D: Glanders: New insights into an old disease. Biological Weapons Defense: Infectious Diseases and Counterbioterrorism. Edited by: Lindler LE LF, Korch GW. 2004, Totowa, New Jersey: Humana Press IncGoogle Scholar
- Nierman WC, DeShazer D, Kim HS, Tettelin H, Nelson KE, Feldblyum T, Ulrich RL, Ronning CM, Brinkac LM, Daugherty SC, Davidsen TD, Deboy RT, Dimitrov G, Dodson RJ, Durkin AS, Gwinn ML, Haft DH, Khouri H, Kolonay JF, Madupu R, Mohammoud Y, Nelson WC, Radune D, Romero CM, Sarria S, Selengut J, Shamblin C, Sullivan SA, White O, Yu Y, Zafar N, Zhou L, Fraser CM: Structural flexibility in the Burkholderia mallei genome. Proc Natl Acad Sci USA. 2004, 101 (39): 14246-14251. 10.1073/pnas.0403306101.PubMed CentralPubMedGoogle Scholar
- Kumar A, Chua KL, Schweizer HP: Method for regulated expression of single-copy efflux pump genes in a surrogate Pseudomonas aeruginosa strain: identification of the BpeEF-OprC chloramphenicol and trimethoprim efflux pump of Burkholderia pseudomallei 1026b. Antimicrob Agents Chemother. 2006, 50 (10): 3460-3463. 10.1128/AAC.00440-06.PubMed CentralPubMedGoogle Scholar
- Harland DN, Dassa E, Titball RW, Brown KA, Atkins HS: ATP-binding cassette systems in Burkholderia pseudomallei and Burkholderia mallei. BMC Genomics. 2007, 8: 83-10.1186/1471-2164-8-83.PubMed CentralPubMedGoogle Scholar
- Tribuddharat C, Moore RA, Baker P, Woods DE: Burkholderia pseudomallei class a beta-lactamase mutations that confer selective resistance against ceftazidime or clavulanic acid inhibition. Antimicrob Agents Chemother. 2003, 47 (7): 2082-2087. 10.1128/AAC.47.7.2082-2087.2003.PubMed CentralPubMedGoogle Scholar
- Dance DA, Wuthiekanun V, Chaowagul W, White NJ: The antimicrobial susceptibility of Pseudomonas pseudomallei. Emergence of resistance in vitro and during treatment. J Antimicrob Chemother. 1989, 24 (3): 295-309. 10.1093/jac/24.3.295.PubMedGoogle Scholar
- Jenney AW, Lum G, Fisher DA, Currie BJ: Antibiotic susceptibility of Burkholderia pseudomallei from tropical northern Australia and implications for therapy of melioidosis. Int J Antimicrob Agents. 2001, 17 (2): 109-113. 10.1016/S0924-8579(00)00334-4.PubMedGoogle Scholar
- Thibault FM, Hernandez E, Vidal DR, Girardet M, Cavallo JD: Antibiotic susceptibility of 65 isolates of Burkholderia pseudomallei and Burkholderia mallei to 35 antimicrobial agents. J Antimicrob Chemother. 2004, 54 (6): 1134-1138. 10.1093/jac/dkh471.PubMedGoogle Scholar
- Wuthiekanun V, Cheng AC, Chierakul W, Amornchai P, Limmathurotsakul D, Chaowagul W, Simpson AJ, Short JM, Wongsuvan G, Maharjan B, White NJ, Peacock SJ: Trimethoprim/sulfamethoxazole resistance in clinical isolates of Burkholderia pseudomallei. J Antimicrob Chemother. 2005, 55 (6): 1029-1031. 10.1093/jac/dki151.PubMedGoogle Scholar
- Ho PL, Cheung TK, Yam WC, Yuen KY: Characterization of a laboratory-generated variant of BPS beta-lactamase from Burkholderia pseudomallei that hydrolyses ceftazidime. J Antimicrob Chemother. 2002, 50 (5): 723-726. 10.1093/jac/dkf208.PubMedGoogle Scholar
- Cheung TK, Ho PL, Woo PC, Yuen KY, Chau PY: Cloning and expression of class A beta-lactamase gene blaA(BPS) in Burkholderia pseudomallei. Antimicrob Agents Chemother. 2002, 46 (4): 1132-1135. 10.1128/AAC.46.4.1132-1135.2002.PubMed CentralPubMedGoogle Scholar
- Niumsup P, Wuthiekanun V: Cloning of the class D beta-lactamase gene from Burkholderia pseudomallei and studies on its expression in ceftazidime-susceptible and -resistant strains. J Antimicrob Chemother. 2002, 50 (4): 445-455. 10.1093/jac/dkf165.PubMedGoogle Scholar
- Wuthiekanun V, Peacock SJ: Management of melioidosis. Expert Rev Anti Infect Ther. 2006, 4 (3): 445-455. 10.1586/1478722.214.171.1245.PubMedGoogle Scholar
- Peacock SJ, Schweizer HP, Dance DA, Smith TL, Gee JE, Wuthiekanun V, DeShazer D, Steinmetz I, Tan P, Currie BJ: Management of accidental laboratory exposure to Burkholderia pseudomallei and B. mallei. Emerg Infect Dis. 2008, 14 (7): e2-10.3201/eid1407.071501.PubMed CentralPubMedGoogle Scholar
- Cheng AC, McBryde ES, Wuthiekanun V, Chierakul W, Amornchai P, Day NP, White NJ, Peacock SJ: Dosing regimens of cotrimoxazole (trimethoprim-sulfamethoxazole) for melioidosis. Antimicrob Agents Chemother. 2009, 53 (10): 4193-4199. 10.1128/AAC.01301-08.PubMed CentralPubMedGoogle Scholar
- Burtnick MN, Brett PJ, Woods DE: Molecular and physical characterization of Burkholderia mallei O antigens. J Bacteriol. 2002, 184 (3): 849-852. 10.1128/JB.184.3.849-852.2002.PubMed CentralPubMedGoogle Scholar
- Ribot WJ, Ulrich RL: The animal pathogen-like type III secretion system is required for the intracellular survival of Burkholderia mallei within J774.2 macrophages. Infect Immun. 2006, 74 (7): 4349-4353. 10.1128/IAI.01939-05.PubMed CentralPubMedGoogle Scholar
- Kenny DJ, Russell P, Rogers D, Eley SM, Titball RW: In vitro susceptibilities of Burkholderia mallei in comparison to those of other pathogenic Burkholderia spp. Antimicrob Agents Chemother. 1999, 43 (11): 2773-2775.PubMed CentralPubMedGoogle Scholar
- Schell MA, Ulrich RL, Ribot WJ, Brueggemann EE, Hines HB, Chen D, Lipscomb L, Kim HS, Mrazek J, Nierman WC, Deshazer D: Type VI secretion is a major virulence determinant in Burkholderia mallei. Mol Microbiol. 2007, 64 (6): 1466-1485. 10.1111/j.1365-2958.2007.05734.x.PubMedGoogle Scholar
- Shalom G, Shaw JG, Thomas MS: In vivo expression technology identifies a type VI secretion system locus in Burkholderia pseudomallei that is induced upon invasion of macrophages. Microbiology. 2007, 153 (Pt 8): 2689-2699. 10.1099/mic.0.2007/006585-0.PubMedGoogle Scholar
- Warawa J, Woods DE: Type III secretion system cluster 3 is required for maximal virulence of Burkholderia pseudomallei in a hamster infection model. FEMS Microbiol Lett. 2005, 242 (1): 101-108. 10.1016/j.femsle.2004.10.045.PubMedGoogle Scholar
- Ulrich RL, DeShazer D: Type III secretion: a virulence factor delivery system essential for the pathogenicity of Burkholderia mallei. Infect Immun. 2004, 72 (2): 1150-1154. 10.1128/IAI.72.2.1150-1154.2004.PubMed CentralPubMedGoogle Scholar
- Stevens MP, Haque A, Atkins T, Hill J, Wood MW, Easton A, Nelson M, Underwood-Fowler C, Titball RW, Bancroft GJ, Galyov EE: Attenuated virulence and protective efficacy of a Burkholderia pseudomallei bsa type III secretion mutant in murine models of melioidosis. Microbiology. 2004, 150 (Pt 8): 2669-2676. 10.1099/mic.0.27146-0.PubMedGoogle Scholar
- Stevens MP, Wood MW, Taylor LA, Monaghan P, Hawes P, Jones PW, Wallis TS, Galyov EE: An Inv/Mxi-Spa-like type III protein secretion system in Burkholderia pseudomallei modulates intracellular behaviour of the pathogen. Mol Microbiol. 2002, 46 (3): 649-659. 10.1046/j.1365-2958.2002.03190.x.PubMedGoogle Scholar
- Burtnick MN, DeShazer D, Nair V, Gherardini FC, Brett PJ: Burkholderia mallei cluster 1 type VI secretion mutants exhibit growth and actin polymerization defects in RAW 264.7 murine macrophages. Infect Immun. 78 (1): 88-99. 10.1128/IAI.00985-09.
- St Geme JW: Bacterial adhesins: determinants of microbial colonization and pathogenicity. Adv Pediatr. 1997, 44: 43-72.PubMedGoogle Scholar
- Boyle EC, Finlay BB: Bacterial pathogenesis: exploiting cellular adherence. Curr Opin Cell Biol. 2003, 15 (5): 633-639. 10.1016/S0955-0674(03)00099-1.PubMedGoogle Scholar
- Samrakandi MM, Ridenour DA, Yan L, Cirillo JD: Entry into host cells by Legionella. Front Biosci. 2002, 7: d1-11. 10.2741/samrakan.PubMedGoogle Scholar
- Inglis TJ, Robertson T, Woods DE, Dutton N, Chang BJ: Flagellum-mediated adhesion by Burkholderia pseudomallei precedes invasion of Acanthamoeba astronyxis. Infect Immun. 2003, 71 (4): 2280-2282. 10.1128/IAI.71.4.2280-2282.2003.PubMed CentralPubMedGoogle Scholar
- Boddey JA, Flegg CP, Day CJ, Beacham IR, Peak IR: Temperature-regulated microcolony formation by Burkholderia pseudomallei requires pilA and enhances association with cultured human cells. Infect Immun. 2006, 74 (9): 5374-5381. 10.1128/IAI.00569-06.PubMed CentralPubMedGoogle Scholar
- Hoiczyk E, Roggenkamp A, Reichenbecher M, Lupas A, Heesemann J: Structure and sequence analysis of Yersinia YadA and Moraxella UspAs reveal a novel class of adhesins. Embo J. 2000, 19 (22): 5989-5999. 10.1093/emboj/19.22.5989.PubMed CentralPubMedGoogle Scholar
- Roggenkamp A, Ackermann N, Jacobi CA, Truelzsch K, Hoffmann H, Heesemann J: Molecular analysis of transport and oligomerization of the Yersinia enterocolitica adhesin YadA. J Bacteriol. 2003, 185 (13): 3735-3744. 10.1128/JB.185.13.3735-3744.2003.PubMed CentralPubMedGoogle Scholar
- Nummelin H, Merckel MC, Leo JC, Lankinen H, Skurnik M, Goldman A: The Yersinia adhesin YadA collagen-binding domain structure is a novel left-handed parallel beta-roll. Embo J. 2004, 23 (4): 701-711. 10.1038/sj.emboj.7600100.PubMed CentralPubMedGoogle Scholar
- Yeo HJ, Cotter SE, Laarmann S, Juehne T, St Geme JW, Waksman G: Structural basis for host recognition by the Haemophilus influenzae Hia autotransporter. Embo J. 2004, 23 (6): 1245-1256. 10.1038/sj.emboj.7600142.PubMed CentralPubMedGoogle Scholar
- Laarmann S, Cutter D, Juehne T, Barenkamp SJ, St Geme JW: The Haemophilus influenzae Hia autotransporter harbours two adhesive pockets that reside in the passenger domain and recognize the same host cell receptor. Mol Microbiol. 2002, 46 (3): 731-743. 10.1046/j.1365-2958.2002.03189.x.PubMedGoogle Scholar
- Surana NK, Cutter D, Barenkamp SJ, St Geme JW: The Haemophilus influenzae Hia autotransporter contains an unusually short trimeric translocator domain. J Biol Chem. 2004, 279 (15): 14679-14685. 10.1074/jbc.M311496200.PubMedGoogle Scholar
- Bullard B, Lipski SL, Lafontaine ER: Hag directly mediates the adherence of Moraxella catarrhalis to human middle ear cells. Infect Immun. 2005, 73 (8): 5127-5136. 10.1128/IAI.73.8.5127-5136.2005.PubMed CentralPubMedGoogle Scholar
- Bullard B, Lipski S, Lafontaine ER: Regions important for the adhesin activity of Moraxella catarrhalis Hag. BMC Microbiol. 2007, 7: 65-10.1186/1471-2180-7-65.PubMed CentralPubMedGoogle Scholar
- Henderson IR, Navarro-Garcia F, Desvaux M, Fernandez RC, Ala'Aldeen D: Type V protein secretion pathway: the autotransporter story. Microbiol Mol Biol Rev. 2004, 68 (4): 692-744. 10.1128/MMBR.68.4.692-744.2004.PubMed CentralPubMedGoogle Scholar
- Linke D, Riess T, Autenrieth IB, Lupas A, Kempf VA: Trimeric autotransporter adhesins: variable structure, common function. Trends Microbiol. 2006, 14 (6): 264-270. 10.1016/j.tim.2006.04.005.PubMedGoogle Scholar
- Cotter SE, Surana NK, St Geme JW: Trimeric autotransporters: a distinct subfamily of autotransporter proteins. Trends Microbiol. 2005, 13 (5): 199-205. 10.1016/j.tim.2005.03.004.PubMedGoogle Scholar
- Balder R, Hassel J, Lipski S, Lafontaine ER: Moraxella catarrhalis strain O35E expresses two filamentous hemagglutinin-like proteins that mediate adherence to human epithelial cells. Infect Immun. 2007, 75 (6): 2765-2775. 10.1128/IAI.00079-07.PubMed CentralPubMedGoogle Scholar
- Balder R, Krunkosky TM, Nguyen CQ, Feezel L, Lafontaine ER: Hag mediates adherence of Moraxella catarrhalis to ciliated human airway cells. Infect Immun. 2009, 77 (10): 4597-4608. 10.1128/IAI.00212-09.PubMed CentralPubMedGoogle Scholar
- Krunkosky TM, Fischer BM, Martin LD, Jones N, Akley NJ, Adler KB: Effects of TNF-alpha on expression of ICAM-1 in human airway epithelial cells in vitro. Signaling pathways controlling surface and gene expression. Am J Respir Cell Mol Biol. 2000, 22 (6): 685-692.PubMedGoogle Scholar
- Krunkosky TM, Jordan JL, Chambers E, Krause DC: Mycoplasma pneumoniae host-pathogen studies in an air-liquid culture of differentiated human airway epithelial cells. Microb Pathog. 2007, 42 (2-3): 98-103. 10.1016/j.micpath.2006.11.003.PubMedGoogle Scholar
- Capecchi B, Adu-Bobie J, Di Marcello F, Ciucchi L, Masignani V, Taddei A, Rappuoli R, Pizza M, Arico B: Neisseria meningitidis NadA is a new invasin which promotes bacterial adhesion to and penetration into human epithelial cells. Mol Microbiol. 2005, 55 (3): 687-698. 10.1111/j.1365-2958.2004.04423.x.PubMedGoogle Scholar
- Valle J, Mabbett AN, Ulett GC, Toledo-Arana A, Wecker K, Totsika M, Schembri MA, Ghigo JM, Beloin C: UpaG, a new member of the trimeric autotransporter family of adhesins in uropathogenic Escherichia coli. J Bacteriol. 2008, 190 (12): 4147-4167. 10.1128/JB.00122-08.PubMed CentralPubMedGoogle Scholar
- Fexby S, Bjarnsholt T, Jensen PO, Roos V, Hoiby N, Givskov M, Klemm P: Biological Trojan horse: Antigen 43 provides specific bacterial uptake and survival in human neutrophils. Infect Immun. 2007, 75 (1): 30-34. 10.1128/IAI.01117-06.PubMed CentralPubMedGoogle Scholar
- Attia AS, Lafontaine ER, Latimer JL, Aebi C, Syrogiannopoulos GA, Hansen EJ: The UspA2 protein of Moraxella catarrhalis is directly involved in the expression of serum resistance. Infect Immun. 2005, 73 (4): 2400-2410. 10.1128/IAI.73.4.2400-2410.2005.PubMed CentralPubMedGoogle Scholar
- Pearson MM, Hansen EJ: Identification of Gene Products Involved in Biofilm Production by Moraxella catarrhalis ETSU-9 In Vitro. Infect Immun. 2007, 75 (9): 4316-4325. 10.1128/IAI.01347-06.PubMed CentralPubMedGoogle Scholar
- Wang W, Pearson MM, Attia AS, Blick RJ, Hansen EJ: A UspA2H-negative variant of Moraxella catarrhalis strain O46E has a deletion in a homopolymeric nucleotide repeat common to uspA2H genes. Infect Immun. 2007, 75 (4): 2035-2045. 10.1128/IAI.00609-06.PubMed CentralPubMedGoogle Scholar
- Farn JL, Strugnell RA, Hoyne PA, Michalski WP, Tennent JM: Molecular characterization of a secreted enzyme with phospholipase B activity from Moraxella bovis. J Bacteriol. 2001, 183 (22): 6717-6720. 10.1128/JB.183.22.6717-6720.2001.PubMed CentralPubMedGoogle Scholar
- Timpe JM, Holm MM, Vanlerberg SL, Basrur V, Lafontaine ER: Identification of a Moraxella catarrhalis outer membrane protein exhibiting both adhesin and lipolytic activities. Infect Immun. 2003, 71 (8): 4341-4350. 10.1128/IAI.71.8.4341-4350.2003.PubMed CentralPubMedGoogle Scholar
- Maroncle NM, Sivick KE, Brady R, Stokes FE, Mobley HL: Protease activity, secretion, cell entry, cytotoxicity, and cellular targets of secreted autotransporter toxin of uropathogenic Escherichia coli. Infect Immun. 2006, 74 (11): 6124-6134. 10.1128/IAI.01086-06.PubMed CentralPubMedGoogle Scholar
- Lafontaine ER, Cope LD, Aebi C, Latimer JL, McCracken GH, Hansen EJ: The UspA1 protein and a second type of UspA2 protein mediate adherence of Moraxella catarrhalis to human epithelial cells in vitro. J Bacteriol. 2000, 182 (5): 1364-1373. 10.1128/JB.182.5.1364-1373.2000.PubMed CentralPubMedGoogle Scholar
- Sherlock O, Schembri MA, Reisner A, Klemm P: Novel roles for the AIDA adhesin from diarrheagenic Escherichia coli: cell aggregation and biofilm formation. J Bacteriol. 2004, 186 (23): 8058-8065. 10.1128/JB.186.23.8058-8065.2004.PubMed CentralPubMedGoogle Scholar
- Tiyawisutsri R, Holden MT, Tumapa S, Rengpipat S, Clarke SR, Foster SJ, Nierman WC, Day NP, Peacock SJ: Burkholderia Hep_Hap autotransporter (BuHA) proteins elicit a strong antibody response during experimental glanders but not human melioidosis. BMC Microbiol. 2007, 7: 19-10.1186/1471-2180-7-19.PubMed CentralPubMedGoogle Scholar
- Schell MA, Lipscomb L, DeShazer D: Comparative genomics and an insect model rapidly identify novel virulence genes of Burkholderia mallei. J Bacteriol. 2008, 190 (7): 2306-2313. 10.1128/JB.01735-07.PubMed CentralPubMedGoogle Scholar
- Kespichayawattana W, Intachote P, Utaisincharoen P, Sirisinha S: Virulent Burkholderia pseudomallei is more efficient than avirulent Burkholderia thailandensis in invasion of and adherence to cultured human epithelial cells. Microb Pathog. 2004, 36 (5): 287-292. 10.1016/j.micpath.2004.01.001.PubMedGoogle Scholar
- Deshazer D: Virulence of clinical and environmental isolates of Burkholderia oklahomensis and Burkholderia thailandensis in hamsters and mice. FEMS Microbiol Lett. 2007, 277 (1): 64-69. 10.1111/j.1574-6968.2007.00946.x.PubMedGoogle Scholar
- Brett PJ, Deshazer D, Woods DE: Characterization of Burkholderia pseudomallei and Burkholderia pseudomallei-like strains. Epidemiol Infect. 1997, 118 (2): 137-148. 10.1017/S095026889600739X.PubMed CentralPubMedGoogle Scholar
- Smith MD, Angus BJ, Wuthiekanun V, White NJ: Arabinose assimilation defines a nonvirulent biotype of Burkholderia pseudomallei. Infect Immun. 1997, 65 (10): 4319-4321.PubMed CentralPubMedGoogle Scholar
- Ulett GC, Currie BJ, Clair TW, Mayo M, Ketheesan N, Labrooy J, Gal D, Norton R, Smith CA, Barnes J, Warner J, Hirst RG: Burkholderia pseudomallei virulence: definition, stability and association with clonality. Microbes Infect. 2001, 3 (8): 621-631. 10.1016/S1286-4579(01)01417-4.PubMedGoogle Scholar
- DeShazer D, Waag DM, Fritz DL, Woods DE: Identification of a Burkholderia mallei polysaccharide gene cluster by subtractive hybridization and demonstration that the encoded capsule is an essential virulence determinant. Microb Pathog. 2001, 30 (5): 253-269. 10.1006/mpat.2000.0430.PubMedGoogle Scholar
- Kim HS, Schell MA, Yu Y, Ulrich RL, Sarria SH, Nierman WC, DeShazer D: Bacterial genome adaptation to niches: divergence of the potential virulence genes in three Burkholderia species of different survival strategies. BMC Genomics. 2005, 6: 174-10.1186/1471-2164-6-174.PubMed CentralPubMedGoogle Scholar
- Druar C, Yu F, Barnes JL, Okinaka RT, Chantratita N, Beg S, Stratilo CW, Olive AJ, Soltes G, Russell ML, Limmathurotsakul D, Norton RE, Ni SX, Picking WD, Jackson PJ, Stewart DI, Tsvetnitsky V, Picking WL, Cherwonogrodzky JW, Ketheesan N, Peacock SJ, Wiersma EJ: Evaluating Burkholderia pseudomallei Bip proteins as vaccines and Bip antibodies as detection agents. FEMS Immunol Med Microbiol. 2008, 52 (1): 78-87. 10.1111/j.1574-695X.2007.00345.x.PubMedGoogle Scholar
- Chantratita N, Wuthiekanun V, Boonbumrung K, Tiyawisutsri R, Vesaratchavest M, Limmathurotsakul D, Chierakul W, Wongratanacheewin S, Pukritiyakamee S, White NJ, Day NP, Peacock SJ: Biological relevance of colony morphology and phenotypic switching by Burkholderia pseudomallei. J Bacteriol. 2007, 189 (3): 807-817. 10.1128/JB.01258-06.PubMed CentralPubMedGoogle Scholar
- Felgner PL, Kayala MA, Vigil A, Burk C, Nakajima-Sasaki R, Pablo J, Molina DM, Hirst S, Chew JS, Wang D, Tan G, Duffield M, Yang R, Neel J, Chantratita N, Bancroft G, Lertmemongkolchai G, Davies DH, Baldi P, Peacock S, Titball RW: A Burkholderia pseudomallei protein microarray reveals serodiagnostic and cross-reactive antigens. Proc Natl Acad Sci USA. 2009, 106 (32): 13499-13504. 10.1073/pnas.0812080106.PubMed CentralPubMedGoogle Scholar
- Arjcharoen S, Wikraiphat C, Pudla M, Limposuwan K, Woods DE, Sirisinha S, Utaisincharoen P: Fate of a Burkholderia pseudomallei lipopolysaccharide mutant in the mouse macrophage cell line RAW 264.7: possible role for the O-antigenic polysaccharide moiety of lipopolysaccharide in internalization and intracellular survival. Infect Immun. 2007, 75 (9): 4298-4304. 10.1128/IAI.00285-07.PubMed CentralPubMedGoogle Scholar
- Tangsudjai S, Pudla M, Limposuwan K, Woods DE, Sirisinha S, Utaisincharoen P: Involvement of the MyD88-independent pathway in controlling the intracellular fate of Burkholderia pseudomallei infection in the mouse macrophage cell line RAW 264.7. Microbiol Immunol. 54 (5): 282-290.
- Felek S, Krukonis ES: The Yersinia pestis Ail protein mediates binding and Yop delivery to host cells required for plague virulence. Infect Immun. 2009, 77 (2): 825-836. 10.1128/IAI.00913-08.PubMed CentralPubMedGoogle Scholar
- Lipski SL, Holm MM, Lafontaine ER: Identification of a Moraxella catarrhalis gene that confers adherence to various human epithelial cell lines in vitro. FEMS Microbiol Lett. 2007, 267 (2): 207-213. 10.1111/j.1574-6968.2006.00549.x.PubMedGoogle Scholar
- Lipski SL, Akimana C, Timpe JM, Wooten RM, Lafontaine ER: The Moraxella catarrhalis Autotransporter McaP Is a Conserved Surface Protein That Mediates Adherence to Human Epithelial Cells through Its N-Terminal Passenger Domain. Infect Immun. 2007, 75 (1): 314-324. 10.1128/IAI.01330-06.PubMed CentralPubMedGoogle Scholar
- Sambrook J, Russell DW: Molecular Cloning: A Laboratory Manual (Third Edition). 2001, Cold Spring Harbor Laboratory Press, ThirdGoogle Scholar
- Burtnick M, Bolton A, Brett P, Watanabe D, Woods D: Identification of the acid phosphatase (acpA) gene homologues in pathogenic and non-pathogenic Burkholderia spp. facilitates TnphoA mutagenesis. Microbiology. 2001, 147 (Pt 1): 111-120.PubMedGoogle Scholar
- Lazarus JJ, Meadows MJ, Lintner RE, Wooten RM: IL-10 deficiency promotes increased Borrelia burgdorferi clearance predominantly through enhanced innate immune responses. J Immunol. 2006, 177 (10): 7076-7085.PubMedGoogle Scholar
- Aebi C, Lafontaine ER, Cope LD, Latimer JL, Lumbley SL, McCracken GH, Hansen EJ: Phenotypic effect of isogenic uspA1 and uspA2 mutations on Moraxella catarrhalis 035E. Infect Immun. 1998, 66 (7): 3113-3119.PubMed CentralPubMedGoogle Scholar
- Holm MM, Vanlerberg SL, Foley IM, Sledjeski DD, Lafontaine ER: The Moraxella catarrhalis porin-like outer membrane protein CD is an adhesin for human lung cells. Infect Immun. 2004, 72 (4): 1906-1913. 10.1128/IAI.72.4.1906-1913.2004.PubMed CentralPubMedGoogle Scholar
- Carlone GM, Thomas ML, Rumschlag HS, Sottnek FO: Rapid microprocedure for isolating detergent-insoluble outer membrane proteins from Haemophilus species. J Clin Microbiol. 1986, 24 (3): 330-332.PubMed CentralPubMedGoogle Scholar
- Cope LD, Lafontaine ER, Slaughter CA, Hasemann CA, Aebi C, Henderson FW, McCracken GH, Hansen EJ: Characterization of the Moraxella catarrhalis uspA1 and uspA2 genes and their encoded products. J Bacteriol. 1999, 181 (13): 4026-4034.PubMed CentralPubMedGoogle Scholar
- Patrick CC, Kimura A, Jackson MA, Hermanstorfer L, Hood A, McCracken GH, Hansen EJ: Antigenic characterization of the oligosaccharide portion of the lipooligosaccharide of nontypable Haemophilus influenzae. Infect Immun. 1987, 55 (12): 2902-2911.PubMed CentralPubMedGoogle Scholar
- Lafontaine ER, Wagner NJ, Hansen EJ: Expression of the Moraxella catarrhalis UspA1 protein undergoes phase variation and is regulated at the transcriptional level. J Bacteriol. 2001, 183 (5): 1540-1551. 10.1128/JB.183.5.1540-1551.2001.PubMed CentralPubMedGoogle Scholar
- Moore RA, DeShazer D, Reckseidler S, Weissman A, Woods DE: Efflux-mediated aminoglycoside and macrolide resistance in Burkholderia pseudomallei. Antimicrob Agents Chemother. 1999, 43 (3): 465-470.PubMed CentralPubMedGoogle Scholar
- Simon R, Priefer U, Puhler A: A broad host range mobilisation system for in vivo genetic engineering: transposon mutagenesis in gram-negative bacteria. Bio/Technology. 1983, 1: 784-791. 10.1038/nbt1183-784.Google Scholar
- Skorupski K, Taylor RK: Positive selection vectors for allelic exchange. Gene. 1996, 169 (1): 47-52. 10.1016/0378-1119(95)00793-8.PubMedGoogle Scholar
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