The genetic and molecular basis of O-antigenic diversity in Burkholderia pseudomallei lipopolysaccharide

Lipopolysaccharide (LPS) is one of the most important virulence and antigenic components of Burkholderia pseudomallei, the causative agent of melioidosis. LPS diversity in B. pseudomallei has been described as typical, atypical or rough, based upon banding patterns on SDS-PAGE. Here, we studied the genetic and molecular basis of these phenotypic differences. Bioinformatics was used to determine the diversity of genes known or predicted to be involved in biosynthesis of the O-antigenic moiety of LPS in B. pseudomallei and its near-relative species. Multiplex-PCR assays were developed to target diversity of the O-antigen biosynthesis gene patterns or LPS genotypes in B. pseudomallei populations. We found that the typical LPS genotype (LPS genotype A) was highly prevalent in strains from Thailand and other countries in Southeast Asia, whereas the atypical LPS genotype (LPS genotype B) was most often detected in Australian strains (~13.8%). In addition, we report a novel LPS ladder pattern, a derivative of the atypical LPS phenotype, associated with an uncommon O-antigen biosynthesis gene cluster that is found in only a small B. pseudomallei sub-population. This new LPS group was designated as genotype B2. We also report natural mutations in the O-antigen biosynthesis genes that potentially cause the rough LPS phenotype. We postulate that the diversity of LPS may correlate with differential immunopathogenicity and virulence among B. pseudomallei strains.     

The mviN homolog in Burkholderia pseudomallei is essential for viability and virulence

The virulence factors of Burkholderia pseudomallei, the causative agent of melioidosis, are not fully understood. We have identified a gene with homology to the Salmonella typhimurium mouse virulence gene, mviN, a member of the mouse virulence factor family. Expression studies with an insertional mutant containing a lux operon demonstrated that the expression of the gene is influenced by free-iron availability in the media and by growth phase. The mutant displayed an increased LD50 value in the hamster infection model and a loss of the ability to invade human lung epithelial cells. The mutant has a slower growth rate than that of the wild type. Both defects were restored to various degrees when complemented in trans with the mviN gene. The mutant contains an insertion at 1229 bp of the 1548 bp gene, resulting in a truncated protein that is presumably responsible for the defects. Deletion mutants of the entire B. pseudomallei mviN gene were obtained only in the presence of the complement vector. This result and the inability of the complemented deletion mutant to lose the plasmid in the absence of antibiotic selection suggest that the gene is essential to B. pseudomallei.     

The wbiA locus is required for the 2-O-acetylation of lipopolysaccharides expressed by Burkholderia pseudomallei and Burkholderia thailandensis

Burkholderia pseudomallei and Burkholderia thailandensis express similar O-antigens (O-PS II) in which their 6-deoxy-alpha-L-talopyranosyl (L-6dTalp) residues are variably substituted with O-acetyl groups at the O-2 or O-4 positions. In previous studies we demonstrated that the protective monoclonal antibody, Pp-PS-W, reacted with O-PS II expressed by wild-type B. pseudomallei strains but not by a B. pseudomallei wbiA null mutant. In the present study we demonstrate that WbiA activity is required for the acetylation of the L-6dTalp residues at the O-2 position and that structural modification of O-PS II molecules at this site is critical for recognition by Pp-PS-W.     

Type III secretion system cluster 3 is required for maximal virulence of Burkholderia pseudomallei in a hamster infection model

Burkholderia pseudomallei, the etiological agent of melioidosis, is an animal pathogen capable of inducing a highly fatal septicemia. B. pseudomallei possesses three type III secretion system (TTSS) clusters, two of which (TTSS1 and TTSS2) are homologous to the TTSS of the plant pathogen Ralstonia solanacearum, and one (TTSS3) is homologous to the Salmonella SPI-1 mammalian pathogenicity island. We have demonstrated that TTSS3 is required for the full virulence of B. pseudomallei in a hamster model of infection. We have also examined the virulence of B. pseudomallei mutants deficient in several putative TTSS3 effector molecules, and found no significant attenuation of B. pseudomallei virulence in the hamster model.     

Facile syntheses of the disaccharide repeating unit of the O-antigenic polysaccharide of Burkholderia pseudomallei strain 304b and its dimer and trimer

A highly efficient strategy for the preparation of a disaccharide-repeating unit of the O-antigenic polysaccharide of Burkholderia pseudomallei strain 304b, and its dimer and trimer, has been developed through a regio- and stereoselective manner using p-methoxylphenyl 2,4,6-tri-O-benzoyl-α-d-glucopyranoside and 3-O-allyloxycarbonyl-2,4-di-O-benzoyl-6-deoxy-α-l-talopyranosyl trichloroacetimidate as the key synthons. The target molecules were equipped with a p-methoxylphenyl handle at the reducing terminus to allow for their further functionalization and attachment to a carrier protein.     

The Burkholderia pseudomallei type III secretion system and BopA are required for evasion of LC3-associated phagocytosis

Burkholderia pseudomallei is the causative agent of melioidosis, a fatal infectious disease endemic in tropical regions worldwide, and especially prevalent in southeast Asia and northern Australia. This intracellular pathogen can escape from phagosomes into the host cytoplasm, where it replicates and infects adjacent cells. We previously demonstrated that, in response to B. pseudomallei infection of macrophage cell line RAW 264.7, a subset of bacteria co-localized with the autophagy marker protein, microtubule-associated protein light chain 3 (LC3), implicating autophagy in host cell defence against infection. Recent reports have suggested that LC3 can be recruited to both phagosomes and autophagosomes, thereby raising questions regarding the identity of the LC3-positive compartments in which invading bacteria reside and the mechanism of the autophagic response to B. pseudomallei infection. Electron microscopy analysis of infected cells demonstrated that the invading bacteria were either free in the cytosol, or sequestered in single-membrane phagosomes rather than double-membrane autophagosomes, suggesting that LC3 is recruited to B. pseudomallei-containing phagosomes. Partial or complete loss of function of type III secretion system cluster 3 (TTSS3) in mutants lacking the BopA (effector) or BipD (translocator) proteins respectively, resulted in delayed or no escape from phagosomes. Consistent with these observations, bopA and bipD mutants both showed a higher level of co-localization with LC3 and the lysosomal marker LAMP1, and impaired survival in RAW264.7 cells, suggesting enhanced killing in phagolysosomes. We conclude that LC3 recruitment to phagosomes stimulates killing of B. pseudomallei trapped in phagosomes. Furthermore, BopA plays an important role in efficient escape of B. pseudomallei from phagosomes.     

Extracellular polysaccharide is required for wild-type virulence of Pseudomonas solanacearum.

Several Pseudomonas solanacearum strains which produced no detectable extracellular polysaccharide (EPS) in planta had been reported to remain highly virulent when tested at high inoculum concentrations (P. Xu, M. Iwata, S. Leong, and L. Sequeira, J. Bacteriol. 172:3946-3951, 1990; P. Xu, S. Leong, and L. Sequeira, J. Bacteriol. 170:617-622, 1988). Two of these mutants, KD700 and KD710, have now been molecularly and genetically mapped to the EPSI gene cluster described by Denny and Baek (Mol. Plant-Microbe Interact. 4:198-206, 1991). When a range of inoculum concentrations was used, these two mutants and all other EPS-defective mutants tested were found to be reduced in virulence to eggplants and tobacco relative to the wild-type strain. Thus, EPS consistently is required for the wild-type level of virulence in P. solanacearum.     

A complete lipopolysaccharide inner core oligosaccharide is required for resistance of Burkholderia cenocepacia to antimicrobial peptides and bacterial survival in vivo

Burkholderia cenocepacia is an important opportunistic pathogen of patients with cystic fibrosis. This bacterium is inherently resistant to a wide range of antimicrobial agents, including high concentrations of antimicrobial peptides. We hypothesized that the lipopolysaccharide (LPS) of B. cenocepacia is important for both virulence and resistance to antimicrobial peptides. We identified hldA and hldD genes in B. cenocepacia strain K56-2. These two genes encode enzymes involved in the modification of heptose sugars prior to their incorporation into the LPS core oligosaccharide. We constructed a mutant, SAL1, which was defective in expression of both hldA and hldD, and by performing complementation studies we confirmed that the functions encoded by both of these B. cenocepacia genes were needed for synthesis of a complete LPS core oligosaccharide. The LPS produced by SAL1 consisted of a short lipid A-core oligosaccharide and was devoid of O antigen. SAL1 was sensitive to the antimicrobial peptides polymyxin B, melittin, and human neutrophil peptide 1. In contrast, another B. cenocepacia mutant strain that produced complete lipid A-core oligosaccharide but lacked polymeric O antigen was not sensitive to polymyxin B or melittin. As determined by the rat agar bead model of lung infection, the SAL1 mutant had a survival defect in vivo since it could not be recovered from the lungs of infected rats 14 days postinfection. Together, these data show that the B. cenocepacia LPS inner core oligosaccharide is needed for in vitro resistance to three structurally unrelated antimicrobial peptides and for in vivo survival in a rat model of chronic lung infection.     

Identification of a bacterial factor required for actin-based motility of Burkholderia pseudomallei

Burkholderia pseudomallei is a Gram-negative facultative intracellular pathogen that enters and escapes from eukaryotic cells using the power of actin polymerization. We have identified a bacterial protein (BimA) that is required for the ability of B. pseudomallei to induce the formation of actin tails. BimA contains proline-rich motifs and WH2-like domains and shares limited homology at the C-terminus with the Yersinia autosecreted adhesin YadA. BimA is located at the pole of the bacterial cell at which actin polymerization occurs and mutation of bimA abolished actin-based motility of the pathogen in J774.2 cells. Transient expression of BimA in HeLa cells resulted in F-actin clustering reminiscent of that seen on WASP overexpression. Antibody-mediated clustering of a CD32 chimera in which the cytoplasmic domain was replaced with BimA resulted in localization of the chimera to the tips of F-actin enriched membrane protrusions. We report that purified truncated BimA protein binds monomeric actin in a concentration-dependent manner in cosedimentation assays and that BimA stimulates actin polymerization in vitro in a manner independent of the cellular Arp2/3 complex.     

The Burkholderia pseudomallei BpeAB-OprB efflux pump: expression and impact on quorum sensing and virulence

BpeAB-OprB is a multidrug efflux pump of the bacterial pathogen Burkholderia pseudomallei and is responsible for conferring antimicrobial resistance to aminoglycosides and macrolides. Expression of bpeAB-oprB is inducible by its substrate erythromycin and upon entry into stationary phase. BpeR, a member of the TetR family, functions as a repressor of the bpeAB-oprB operon. bpeR expression was similarly induced at stationary phase but lagged behind the induction of bpeAB-oprB expression. The induction of bpeAB-oprB expression could be advanced to the early exponential phase by exogenous addition of the B. pseudomallei autoinducers N-octanoyl-homoserine lactone (C8HSL) and N-decanoyl-homoserine lactone (C10HSL), suggesting that the bpeAB-oprB operon may be quorum regulated. On the other hand, acyl-homoserine lactone (acyl-HSL) production was undetectable in the bpeAB-null mutant and strains which overexpress bpeR. The failure of these strains to produce acyl-HSLs seemed to be at the level of synthesis of acyl-HSLs, as growth-phase-dependent expression of the autoinducer synthase BpsI was abolished in the bpeAB-null mutant. bpsI expression remained growth phase dependent in the bpeR mutant which had functional BpeAB-OprB. BpeAB-OprB function is likewise necessary for optimal production of quorum-sensing-controlled virulence factors such as siderophore and phospholipase C and for biofilm formation. Cell invasion and cytotoxicity towards human lung epithelial (A549) and human macrophage (THP-1) cells were also significantly attenuated in both the bpeAB mutant and bpeR-overexpressing strains, thus suggesting the possibility of attenuating B. pseudomallei virulence using inhibitors of the BpeAB-OprB efflux pump.     

A gene cluster required for coordinated biosynthesis of lipopolysaccharide and extracellular polysaccharide also affects virulence of Pseudomonas solanacearum.

Bacterial cell surface components can be important determinants of virulence. At least three gene clusters important for extracellular polysaccharide (EPS) biosynthesis have been previously identified in the plant pathogen Pseudomonas solanacearum. We have found that one of these gene clusters, named ops, is also required for lipopolysaccharide (LPS) biosynthesis. Mutations in any complementation unit of this cluster decreased EPS production, prevented the binding of an LPS-specific phage, and altered the mobility of purified LPS in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, restoration of LPS biosynthesis alone was not sufficient to restore virulence to the wild-type level, suggesting that EPS is important for pathogenesis.     

Experimental and theoretical conformation analysis of the O-antigenic polysaccharide from Pseudomonas cepacia strain 3181. II. The polysaccharide

The spatial structure of Pseudomonas cepacia 3181 polysaccharide in aqueous solution is discussed basing on the data of nuclear Overhauser effect, observed with preirradiation of anomeric protons of all 3 D-rhamnose residues in the repeat unit, and theoretical conformational analysis. It is shown that conformational states of the free disaccharides and corresponding disaccharide units of the polysaccharide are similar. All conformations of the polysaccharide may be described by one shape representing an extended structure with characteristic turns in the D-Rha alpha 1-2-D-Rha beta 1-3-D-Rha units.     

The GATC-binding protein SeqA is required for bile resistance and virulence in Salmonella enterica serovar typhimurium

Disruption of the seqA gene of Salmonella enterica serovar Typhimurium causes defects similar to those described in E. coli: filament formation, aberrant nucleoid segregation, induction of the SOS response, envelope instability, and increased sensitivity to membrane-damaging agents. Differences between SeqA⁻ mutants of E. coli and S. enterica, however, are found. SeqA⁻ mutants of S. enterica form normal colonies and do not exhibit alterations in phage plaquing morphology. Lack of SeqA causes attenuation of S. enterica virulence by the oral route but not by the intraperitoneal route, suggesting a virulence defect in the intestinal stage of infection. However, SeqA⁻ mutants are fully proficient in the invasion of epithelial cells. We hypothesize that attenuation of SeqA⁻ mutants by the oral route may be caused by bile sensitivity, which in turn may be a consequence of envelope instability.     

The structure of the O-antigenic polysaccharide from lipopolysaccharide of Vibrio cholerae strain H11 (non-O1).

After acid degradation of the lipopolysaccharide (LPS) of Vibrio cholerae strain H11 (non-O1), a tetrasaccharide was obtained, the structure of which was determined by quantitative and methylation analyses, periodate oxidation, one- and two-dimensional NMR spectroscopy, and fast-atom-bombardment and four-sector tandem mass spectrometry as beta-D-GalANGro-(1-3)-beta-D-QuiNAc-(1-4)-alpha-D-GalANGr o-(1-4)-NeuAc, in which GalANGro is N-galacturonoyl-2-aminoglycerol and QuiN 2-amino-2,6-dideoxy-glucopyranose. In addition, the trisaccharide beta-D-GalANGro-(1-3)-beta-D-QuiNAc-(1-4)-D-altro-hept ulose and the disaccharide alpha-D-GalANGro-(1-4)-NeuAc were isolated from acid-degraded lipopolysaccharide; the occurrence of sedoheptulose in lipopolysaccharide has not been described before. Based on the result of methylation analysis showing that galacturonic acid was the terminal sugar of the polysaccharide chain, and on the assumption that the tri- and the disaccharide represented the reducing and the non-reducing ends of the polysaccharide, respectively, the chemical structure of the O-specific chain of V. cholerae H11 is proposed as alpha-D-GalANGro-(1-4)-alpha-NeuAc-(2-3)-beta-D-GalANGro-(1- 3)-beta-D-QuiNAc- (1-[4)-alpha-D-GalANGro-(1-4)-alpha-NeuAc-(2-3)-beta-D-GalANGro -(1-3)-beta-D- QuiNAc-(1-]n-(1-4)-D-altro-heptulose. However, other possible structures can not be ruled out since the tri- and the disaccharide could be localised at different positions.     

Structural and serological characterisation of the O-antigenic polysaccharide of the lipopolysaccharide from Acinetobacter baumannii strain 24

Extraction of dry bacteria of Acinetobacter baumannii strain 24 by phenol-water yielded a lipopolysaccharide (LPS) that was studied by serological methods and fatty acid analysis. After immunisation of BALB/c mice with this strain, monoclonal antibody S48-3-13 (IgG(3) isotype) was obtained, which reacted with the LPS in western blot and characterized it as S-form LPS. Degradation of the LPS in aqueous 1% acetic acid followed by GPC gave the O-antigenic polysaccharide, whose structure was determined by compositional analyses and NMR spectroscopy of the polysaccharide and O-deacylated polysaccharide as [carbohydrate structure: see text] where QuiN4N is 2,4-diamino-2,4,6-trideoxyglucose and GalNAcA 2-acetamido-2-deoxygalacturonic acid. The amino group at C-4 of the QuipN4N residues is acetylated in about 2/3 of LPS molecules and (S)-3-hydroxybutyrylated in the rest.     

Macroautophagy is essential for killing of intracellular Burkholderia pseudomallei in human neutrophils

Neutrophils play a key role in the control of Burkholderia pseudomallei, the pathogen that causes melioidosis. Here, we show that survival of intracellular B. pseudomallei was significantly increased in the presence of 3-methyladenine or lysosomal cathepsin inhibitors. The LC3-flux was increased in B. pseudomallei-infected neutrophils. Concordant with this result, confocal microscopy analyses using anti-LC3 antibodies revealed that B. pseudomallei-containing phagosomes partially overlapped with LC3-positive signal at 3 and 6 h postinfection. Electron microscopic analyses of B. pseudomallei-infected neutrophils at 3 h revealed B. pseudomallei-containing phagosomes that occasionally fused with phagophores or autophagosomes. Following infection with a B. pseudomallei mutant lacking the Burkholderia secretion apparatus Bsa Type III secretion system, neither this characteristic structure nor bacterial escape into the cytosol were observed. These findings indicate that human neutrophils are able to recruit autophagic machinery adjacent to B. pseudomallei-containing phagosomes in a Type III secretion system-dependent manner.     

Maturation of Streptococcus pneumoniae lipoproteins by a type II signal peptidase is required for ABC transporter function and full virulence

Cell surface lipoproteins are important for the full virulence of several bacterial pathogens, including Streptococcus pneumoniae. Processing of prolipoproteins seems to be conserved among different bacterial species, and requires type II signal peptidase (Lsp) mediated cleavage of the N-terminal signal peptide to form the mature lipoprotein. Lsp has been suggested as a target for new antibiotic therapies, but at present there are only limited data on the function of Lsp for Gram-positive bacterial pathogens. We have investigated the function and role during disease pathogenesis of the S. pneumoniae Lsp, which, blast searches suggest, is encoded by the gene Sp0928. Expression of Sp0928 protected Escherichia coli against the Lsp antagonist globomycin, and proteomics and immunoblot analysis demonstrated that deletion of Sp0928 prevented processing of S. pneumoniae prolipoproteins to mature lipoproteins. These data strongly suggest that Sp0928 encodes the S. pneumoniae Lsp. However, immunoblots of membrane-associated proteins, immunoelectron microscopy and flow cytometry assays all confirmed that in the absence of Lsp, immature lipoproteins were still attached to the cell surface. Despite preservation of lipoprotein attachment to the cell membrane, loss of S. pneumoniae Lsp resulted in several phenotypes associated with impaired lipoprotein function and reduced S. pneumoniae replication in animal models of infection.     

Hopanoid production is required for low-pH tolerance, antimicrobial resistance, and motility in Burkholderia cenocepacia

Hopanoids are pentacyclic triterpenoids that are thought to be bacterial surrogates for eukaryotic sterols, such as cholesterol, acting to stabilize membranes and to regulate their fluidity and permeability. To date, very few studies have evaluated the role of hopanoids in bacterial physiology. The synthesis of hopanoids depends on the enzyme squalene-hopene cyclase (Shc), which converts the linear squalene into the basic hopene structure. Deletion of the 2 genes encoding Shc enzymes in Burkholderia cenocepacia K56-2, BCAM2831 and BCAS0167, resulted in a strain that was unable to produce hopanoids, as demonstrated by gas chromatography and mass spectrometry. Complementation of the Δshc mutant with only BCAM2831 was sufficient to restore hopanoid production to wild-type levels, while introducing a copy of BCAS0167 alone into the Δshc mutant produced only very small amounts of the hopanoid peak. The Δshc mutant grew as well as the wild type in medium buffered to pH 7 and demonstrated no defect in its ability to survive and replicate within macrophages, despite transmission electron microscopy (TEM) revealing defects in the organization of the cell envelope. The Δshc mutant displayed increased sensitivity to low pH, detergent, and various antibiotics, including polymyxin B and erythromycin. Loss of hopanoid production also resulted in severe defects in both swimming and swarming motility. This suggests that hopanoid production plays an important role in the physiology of B. cenocepacia.     

The structure of the O-specific polysaccharide of the lipopolysaccharide from Burkholderia gladioli pv. agaricicola

A neutral O-specific polysaccharide containing d-mannose, d-rhamnose and d-galactose was obtained by mild acid hydrolysis of the lipopolysaccharide of the plant pathogenic bacterium Burkholderia gladioli pv. agaricicola. By means of compositional analyses and NMR spectroscopy, the chemical repeating unit of the polymer was identified as a linear trisaccharide of the structure shown below, in which the mannose residue was quantitatively acetylated at C-2. [carbohydrate structure: see text]