Secondary cell wall polysaccharides (SCWPs) are important structural components of the Bacillus cell wall and contribute to the array of antigens presented by these organisms in both spore and vegetative forms. We previously found that antisera raised to Bacillus anthracis spore preparations cross-reacted with SCWPs isolated from several strains of pathogenic B. cereus, but did not react with other phylogenetically related but nonpathogenic Bacilli, suggesting that the SCWP from B. anthracis and pathogenic B. cereus strains share specific structural features. In this study, SCWPs from three strains of B. cereus causing severe or fatal pneumonia (G9241, 03BB87 and 03BB102) were isolated and subjected to structural analysis and their structures were compared to SCWPs from B. anthracis. Complete structural analysis was performed for the B. cereus G9241 SCWP using NMR spectroscopy, mass spectrometry and derivatization methods. The analyses show that SCWPs from B. cereus G9241 has a glycosyl backbone identical to that of B. anthracis SCWP, consisting of multiple trisaccharide repeats of: →6)-α-d-GlcpNAc-(1 → 4)-β-d-ManpNAc-(1 → 4)-β-d-GlcpNAc-(1→. Both the B. anthracis and pathogenic B. cereus SCWPs are highly substituted at all GlcNAc residues with α- and β-Gal residues, however, only the SCWPs from B. cereus G9241 and 03BB87 carry an additional α-Gal substitution at O-3 of ManNAc residues, a feature lacking in the B. anthracis SCWPs. Both the B. anthracis and B. cereus SCWPs are pyruvylated, with an approximate molecular mass of ≈12,000 Da. The implications of these findings regarding pathogenicity and cell wall structure are discussed.
Bacillus anthracis; Bacillus cereus; cell wall; polysaccharide; structure
Nonulosonic acids (NulOs) encompass a large group of structurally diverse nine-carbon backbone α-keto sugars widely distributed among the three domains of life. Mammals express a specialized version of NulOs called sialic acids, which are displayed in prominent terminal positions of cell surface and secreted glycoconjugates. Within bacteria, the ability to synthesize NulOs has been demonstrated in a number of human pathogens and is phylogenetically widespread. Here we examine the distribution, diversity, evolution, and function of NulO biosynthesis pathways in members of the family Vibrionaceae. Among 27 species of Vibrionaceae examined at the genomic level, 12 species contained nab gene clusters. We document examples of duplication, divergence, horizontal transfer, and recombination of nab gene clusters in different Vibrionaceae lineages. Biochemical analyses, including mass spectrometry, confirmed that many species do, in fact, produce di-N-acetylated NulOs. A library of clinical and environmental isolates of Vibrio vulnificus served as a model for further investigation of nab allele genotypes and levels of NulO expression. The data show that lineage I isolates produce about 20-fold higher levels of NulOs than lineage II isolates. Moreover, nab gene alleles found in a subset of V. vulnificus clinical isolates express 40-fold higher levels of NulOs than nab alleles associated with environmental isolates. Taken together, the data implicate the family Vibrionaceae as a “hot spot” of NulO evolution and suggest that these molecules may have diverse roles in environmental persistence and/or animal virulence.
Capsule expression in Neisseria meningitidis is encoded by the cps locus comprised of genes required for biosynthesis and surface translocation. Located adjacent to the gene encoding the polysialyltransferase in serogroups expressing sialic acid-containing capsule, NMB0065 is likely a member of the cps locus, but it is not found in serogroups A or X that express non-sialic acid capsules. To further understand its role in CPS expression, NMB0065 mutants were created in the serogroups B, C and Y strains. The mutants were as sensitive as unencapsulated strains to killing by normal human serum, despite producing near wild-type levels of CPS. Absence of surface expression of capsule was suggested by increased surface hydrophobicity and confirmed by immunogold electron microscopy, which revealed the presence of large vacuoles containing CPS within the cell. GC-MS and NMR analyses of purified capsule from the mutant revealed no apparent changes in polymer structures and lipid anchors. Mutants of NMB0065 homologues in other sialic acid CPS expressing meningococcal serogroups had similar phenotypes. Thus, NMB0065 (CtrG) is not involved in biosynthesis or lipidation of sialic acid-containing capsule but encodes a protein required for proper coupling of the assembly complex to the membrane transport complex allowing surface expression of CPS.
CAPSULE; SIALIC ACID; CAPSULAR POLYSACCHARIDE; NEISSERIA MENINGITIDIS
The strict human pathogen Neisseria gonorrhoeae has caused the sexually transmitted infection termed gonorrhea for thousands of years. Over the millennia, the gonococcus has likely evolved mechanisms to evade host defense systems that operate on the genital mucosal surfaces in both males and females. Past research has shown that the presence or modification of certain cell envelope structures can significantly impact levels of gonococcal susceptibility to host-derived antimicrobial compounds that bathe genital mucosal surfaces and participate in innate host defense against invading pathogens. In order to facilitate the identification of gonococcal genes that are important in determining levels of bacterial susceptibility to mediators of innate host defense, we used the Himar I mariner in vitro mutagenesis system to construct a transposon insertion library in strain F62. As proof of principle that this strategy would be suitable for this purpose, we screened the library for mutants expressing decreased susceptibility to the bacteriolytic action of normal human serum (NHS). We found that a transposon insertion in the lgtD gene, which encodes an N-acetylgalactosamine transferase involved in the extension of the α-chain of lipooligosaccharide (LOS), could confer decreased susceptibility of strain F62 to complement-mediated killing by NHS. By complementation and chemical analyses, we demonstrated both linkage of the transposon insertion to the NHS-resistance phenotype and chemical changes in LOS structure that resulted from loss of LgtD production. Further truncation of the LOS α-chain or loss of phosphoethanolamine (PEA) from the lipid A region of LOS also impacted levels of NHS-resistance. PEA decoration of lipid A also increased gonococcal resistance to the model cationic antimicrobial polymyxin B. Taken together, we conclude that the Himar I mariner in vitro mutagenesis procedure can facilitate studies on structures involved in gonococcal pathogenesis.
Neisseria gonorrhoeae; mutagenesis; complement; antimicrobial; peptide; resistance; lipooligosaccharide
Capsular polysaccharides are important factors in bacterial pathogenesis and have been the target of a number of successful vaccines. Francisella tularensis has been considered to express a capsular antigen but none has been isolated or characterized. We have developed a monoclonal antibody, 11B7, which recognizes the capsular polysaccharide of F. tularensis migrating on Western blot as a diffuse band between 100 kDa and 250 kDa. The capsule stains poorly on SDS-PAGE with silver stain but can be visualized using ProQ Emerald glycoprotein stain. The capsule appears to be highly conserved among strains of F. tularensis as antibody 11B7 bound to the capsule of 14 of 14 F. tularensis type A and B strains on Western blot. The capsular material can be isolated essentially free of LPS, is phenol and proteinase K resistant, ethanol precipitable and does not dissociate in sodium dodecyl sulfate. Immunoelectron microscopy with colloidal gold demonstrates 11B7 circumferentially staining the surface of F. tularensis which is typical of a polysaccharide capsule. Mass spectrometry, compositional analysis and NMR indicate that the capsule is composed of a polymer of the tetrasaccharide repeat, 4)-α-D-GalNAcAN-(1->4)-α-D-GalNAcAN-(1->3)-β-D-QuiNAc-(1->2)-β-D-Qui4NFm-(1-, which is identical to the previously described F. tularensis O-antigen subunit. This indicates that the F. tularensis capsule can be classified as an O-antigen capsular polysaccharide. Our studies indicate that F. tularensis O-antigen glycosyltransferase mutants do not make a capsule. An F. tularensis acyltransferase and an O-antigen polymerase mutant had no evidence of an O-antigen but expressed a capsular antigen. Passive immunization of BALB/c mice with 75 µg of 11B7 protected against a 150 fold lethal challenge of F. tularensis LVS. Active immunization of BALB/c mice with 10 µg of capsule showed a similar level of protection. These studies demonstrate that F. tularensis produces an O-antigen capsule that may be the basis of a future vaccine.
The lipooligosaccharide (LOS) from the N. meningitidis prototype serogroup A strain NMA Z2491, an L9 immunotype LOS, was isolated and structurally characterized using glycosyl composition and linkage determination, mass spectrometry and both 1- and 2-D nuclear resonance spectroscopy. The results show that the L9 LOS has an identical structure to that of an L4 LOS structure with the exception that it does not contain a sialic acid residue linked to position 3 of the lactoneotetraose terminal galactosyl residue. Further, two oligosaccharides are present in the Z2491 LOS preparation, OS1 and OS2. They differ from one another only in that OS2 contains an added glycine moiety, presumably at O-7 on the inner core Hep II residue. The structures of these oligosaccharides are as follows:
Where R = H or Gly.
Neisseria meningitidis; NMA; L9 Immunotype; Lipooligosaccharide; LOS; Structure; Phosphoethanolamine
The capacity of Neisseria gonorrhoeae to cause disseminated gonococcal infection requires that such strains resist the bactericidal action of normal human serum. The bactericidal action of normal human serum against N. gonorrhoeae is mediated by the classical complement pathway through an antibody-dependent mechanism. The mechanism(s) by which certain strains of gonococci resist normal human serum is not fully understood, but alterations in lipooligosaccharide structure can affect such resistance. During an investigation of the biological significance of phosphoethanolamine extensions from lipooligosaccharide, we found that phosphoethanolamine substitutions from the heptose II group of the lipooligosaccharide β-chain did not impact levels of gonococcal (strain FA19) resistance to normal human serum or polymyxin B. However, loss of phosphoethanolamine substitution from the lipid A component of lipooligosaccharide, due to insertional inactivation of lptA, resulted in increased gonococcal susceptibility to polymyxin B, as reported previously for Neisseria meningitidis. In contrast to previous reports with N. meningitidis, loss of phosphoethanolamine attached to lipid A rendered strain FA19 susceptible to complement killing. Serum killing of the lptA mutant occurred through the classical complement pathway. Both serum and polymyxin B resistance as well as phosphoethanolamine decoration of lipid A were restored in the lptA-null mutant by complementation with wild-type lptA. Our results support a role for lipid A phosphoethanolamine substitutions in resistance of this strict human pathogen to innate host defenses.
The lipooligosaccharide (LOS) of Neisseria meningitidis can be decorated with phosphoethanolamine (PEA) at the 4′ position of lipid A and at the O-3 and O-6 positions of the inner core of the heptose II residue. The biological role of PEA modification in N. meningitidis remains unclear. During the course of our studies to elucidate the pathogenicity of the ST-2032 (invasive) meningococcal clonal group, disruption of lptA, the gene that encodes the PEA transferase for 4′ lipid A, led to a approximately 10-fold decrease in N. meningitidis adhesion to four kinds of human endothelial and epithelial cell lines at an multiplicity of infection of 5,000. Complementation of the lptA gene in a ΔlptA mutant restored wild-type adherence. By matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analysis, PEA was lost from the lipid A of the ΔlptA mutant compared to that of the wild-type strain. The effect of LptA on meningococcal adhesion was independent of other adhesins such as pili, Opc, Opa, and PilC but was inhibited by the presence of capsule. These results indicate that modification of LOS with PEA by LptA enhances meningococcal adhesion to human endothelial and epithelial cells in unencapsulated N. meningitidis.
The lipopolyaccharide (LPS) of a wbjE mutant of Pseudomonas aeruginosa PA103, a serogroup O11 strain consists of both high and low molecular weight (HMW and LMW) LPSs. The HMW LPS consisted exclusively of rhamnan A-band LPS however no B-band LPS was detected in the wbjE mutant. Interestingly, the LMW LPS from the wbjE mutant showed that it contained a variety of oligosaccharides, each with two or three phosphate groups present as mono- or pyrophosphates.
These oligosaccharides consisted of the complete core octasaccharide,
and a tetrasaccharide,
The GalN residue was present as an N-acetylated residue in all of these oligosaccharides except the tetrasaccharide in which it is present as an N-alanylated residue. None of these oligosaccharides contained either a D- or L-FucpNAc residue. These results are discussed with regard to the role of WbjE in the biosynthesis of P. aeruginosa PA103 B-band LPS.
Pseudomonas aeruginosa; Lipopolysaccharide; Inner core; wbjE; Biosynthesis
Lipooligosaccharide (LOS) is a major surface component of Moraxella catarrhalis and a possible virulence factor in the pathogenesis of human infections caused by this organism. The presence of LOS on the bacterium is an obstacle to the development of vaccines derived from whole cells or outer membrane components of the bacterium. An lpxA gene encoding UDP-N-acetylglucosamine acyltransferase responsible for the first step of lipid A biosynthesis was identified by the construction and characterization of an isogenic M. catarrhalis lpxA mutant in strain O35E. The resulting mutant was viable despite the complete loss of LOS. The mutant strain showed significantly decreased toxicity by the Limulus amebocyte lysate assay, reduced resistance to normal human serum, reduced adherence to human epithelial cells, and enhanced clearance in lungs and nasopharynx in a mouse aerosol challenge model. Importantly, the mutant elicited high levels of antibodies with bactericidal activity and provided protection against a challenge with the wild-type strain. These data suggest that the null LOS mutant is attenuated and may be a potential vaccine candidate against M. catarrhalis.
Lipooligosaccharide (LOS), a major outer membrane component of Moraxella catarrhalis, is a possible virulence factor in the pathogenesis of human infections caused by the organism. However, information about the roles of the oligosaccharide chain from LOS in bacterial infection remains limited. Here, a kdtA gene encoding 3-deoxy-d-manno-2-octulosonic acid (Kdo) transferase, which is responsible for adding Kdo residues to the lipid A portion of the LOS, was identified by transposon mutagenesis and construction of an isogenic kdtA mutant in strain O35E. The resulting O35EkdtA mutant produced only lipid A without any core oligosaccharide, and it was viable. Physicochemical and biological analysis revealed that the mutant was susceptible to hydrophobic reagents and a hydrophilic glycopeptide and was sensitive to bactericidal activity of normal human serum. Importantly, the mutant showed decreased toxicity by the Limulus amebocyte lysate assay, reduced adherence to human epithelial cells, and enhanced clearance in lungs and nasopharynx in a mouse aerosol challenge model. These data suggest that the oligosaccharide moiety of the LOS is important for the biological activity of the LOS and the virulence capability of the bacteria in vitro and in vivo. This study may bring new insights into novel vaccines or therapeutic interventions against M. catarrhalis infections.
Burkholderia cenocepacia is an opportunistic bacterium that infects patients with cystic fibrosis. B. cenocepacia strains J2315, K56-2, C5424, and BC7 belong to the ET12 epidemic clone, which is transmissible among patients. We have previously shown that transposon mutants with insertions within the O antigen cluster of strain K56-2 are attenuated for survival in a rat model of lung infection. From the genomic DNA sequence of the O antigen-deficient strain J2315, we have identified an O antigen lipopolysaccharide (LPS) biosynthesis gene cluster that has an IS402 interrupting a predicted glycosyltransferase gene. A comparison with the other clonal isolates revealed that only strain K56-2, which produced O antigen and displayed serum resistance, lacked the insertion element inserted within the putative glycosyltransferase gene. We cloned the uninterrupted gene and additional flanking sequences from K56-2 and conjugated this plasmid into strains J2315, C5424, and BC7. All the exconjugants recovered the ability to form LPS O antigen. We also determined that the structure of the strain K56-2 O antigen repeat, which was absent from the LPS of strain J2315, consisted of a trisaccharide unit made of rhamnose and two N-acetylgalactosamine residues. The complexity of the gene organization of the K56-2 O antigen cluster was also investigated by reverse transcription-PCR, revealing several transcriptional units, one of which also contains genes involved in lipid A-core oligosaccharide biosynthesis.