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1.  Lipid A’s Structure Mediates Neisseria gonorrhoeae Fitness during Experimental Infection of Mice and Men 
mBio  2013;4(6):e00892-13.
ABSTRACT
Phosphoethanolamine (PEA) on Neisseria gonorrhoeae lipid A influences gonococcal inflammatory signaling and susceptibility to innate host defenses in in vitro models. Here, we evaluated the role of PEA-decorated gonococcal lipid A in competitive infections in female mice and in male volunteers. We inoculated mice and men with mixtures of wild-type N. gonorrhoeae and an isogenic mutant that lacks the PEA transferase, LptA. LptA production conferred a marked survival advantage for wild-type gonococci in the murine female genital tract and in the human male urethra. Our studies translate results from test tube to animal model and into the human host and demonstrate the utility of the mouse model for studies of virulence factors of the human-specific pathogen N. gonorrhoeae that interact with non-host-restricted elements of innate immunity. These results validate the use of gonococcal LptA as a potential target for development of novel immunoprophylactic strategies or antimicrobial treatments.
IMPORTANCE Gonorrhea is one of the most common bacterial sexually transmitted infections, and increasing antibiotic resistance threatens the use of currently available antimicrobial therapies. In this work, encompassing in vitro studies and in vivo studies of animal and human models of experimental genital tract infection, we document the importance of lipid A’s structure, mediated by a single bacterial enzyme, LptA, in enhancing the fitness of Neisseria gonorrhoeae. The results of these studies suggest that novel agents targeting LptA may offer urgently needed prevention or treatment strategies for gonorrhea.
IMPORTANCE
Gonorrhea is one of the most common bacterial sexually transmitted infections, and increasing antibiotic resistance threatens the use of currently available antimicrobial therapies. In this work, encompassing in vitro studies and in vivo studies of animal and human models of experimental genital tract infection, we document the importance of lipid A’s structure, mediated by a single bacterial enzyme, LptA, in enhancing the fitness of Neisseria gonorrhoeae. The results of these studies suggest that novel agents targeting LptA may offer urgently needed prevention or treatment strategies for gonorrhea.
doi:10.1128/mBio.00892-13
PMCID: PMC3870242  PMID: 24255126
2.  Identification of the Mutation Responsible for the Temperature-Sensitive Lipopolysaccharide O-Antigen Defect in the Pseudomonas aeruginosa Cystic Fibrosis Isolate 2192 
Journal of Bacteriology  2013;195(7):1504-1514.
Pseudomonas aeruginosa in the lungs of cystic fibrosis (CF) patients is characterized by a series of genotypic and phenotypic changes that reflect the transition from acute to chronic infection. These include the overproduction of the exopolysaccharide alginate and the loss of complete lipopolysaccharide (LPS). LPS is a major component of the Gram-negative outer membrane and is composed of lipid A, core oligosaccharide, and O antigen. In this report, we show that the LPS defect of the P. aeruginosa chronic infection isolate 2192 is temperature sensitive. When grown at 25°C, 2192 expresses serotype O1 LPS with a moderate chain length and in reduced amounts relative to those of a wild-type serotype O1 laboratory strain (stO1). In contrast, 2192 expresses no LPS O antigen when grown at 37°C. This is the first time that a temperature-sensitive defect in O-antigen production has been reported. Using complementation analyses with a constructed wbpM deletion mutant of stO1, we demonstrate that the temperature-sensitive O-antigen production defect in 2192 is due to a mutation in wbpM, which encodes a UDP-4,6-GlcNAc dehydratase involved in O-antigen synthesis. The mutation, a deletion of a single amino acid (V636) from the extreme C terminus of WbpM, renders the protein less stable than its wild-type counterpart. This residue of WbpM, which is critical for stability and function, is located outside of the recognized domains of the protein and may provide insight into the structure-function relationship of this enzyme, which is found in all 20 serotypes of P. aeruginosa. We also identify a promoter of wbpM, map a transcriptional start site of wbpM, and show that mucoidy plays a role in the loss of expression of high-molecular-weight LPS in this CF isolate.
doi:10.1128/JB.01999-12
PMCID: PMC3624535  PMID: 23354750
3.  Localization and structural analysis of a conserved pyruvylated epitope in Bacillus anthracis secondary cell wall polysaccharides and characterization of the galactose-deficient wall polysaccharide from avirulent B. anthracis CDC 684 
Glycobiology  2012;22(8):1103-1117.
Bacillus anthracis CDC 684 is a naturally occurring, avirulent variant and close relative of the highly pathogenic B. anthracis Vollum. Bacillus anthracis CDC 684 contains both virulence plasmids, pXO1 and pXO2, yet is non-pathogenic in animal models, prompting closer scrutiny of the molecular basis of attenuation. We structurally characterized the secondary cell wall polysaccharide (SCWP) of B. anthracis CDC 684 (Ba684) using chemical and NMR spectroscopy analysis. The SCWP consists of a HexNAc trisaccharide backbone having identical structure as that of B. anthracis Pasteur, Sterne and Ames, →4)-β-d-ManpNAc-(1 → 4)-β-d-GlcpNAc-(1 → 6)-α-d-GlcpNAc-(1→. Remarkably, although the backbone is fully polymerized, the SCWP is the devoid of all galactosyl side residues, a feature which normally comprises 50% of the glycosyl residues on the highly galactosylated SCWPs from pathogenic strains. This observation highlights the role of defective wall assembly in virulence and indicates that polymerization occurs independently of galactose side residue attachment. Of particular interest, the polymerized Ba684 backbone retains the substoichiometric pyruvate acetal, O-acetate and amino group modifications found on SCWPs from normal B. anthracis strains, and immunofluorescence analysis confirms that SCWP expression coincides with the ability to bind the surface layer homology (SLH) domain containing S-layer protein extractable antigen-1. Pyruvate was previously demonstrated as part of a conserved epitope, mediating SLH-domain protein attachment to the underlying peptidoglycan layer. We find that a single repeating unit, located at the distal (non-reducing) end of the Ba684 SCWP, is structurally modified and that this modification is present in identical manner in the SCWPs of normal B. anthracis strains. These polysaccharides terminate in the sequence: (S)-4,6-O-(1-carboxyethylidene)-β-d-ManpNAc-(1 → 4)-[3-O-acetyl]-β-d-GlcpNAc-(1 → 6)-α-d-GlcpNH2-(1→.
doi:10.1093/glycob/cws080
PMCID: PMC3382348  PMID: 22556058
Bacillus anthracis; cell wall; polysaccharide; pyruvylation; structure
4.  Characterization of the lipopolysaccharide from a wbjE mutant of the serogroup O11 Pseudomonas aeruginosa strain, PA103 
Carbohydrate research  2007;343(2):238-248.
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,
a pentasaccharide,
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.
doi:10.1016/j.carres.2007.11.003
PMCID: PMC2243183  PMID: 18039536
Pseudomonas aeruginosa; Lipopolysaccharide; Inner core; wbjE; Biosynthesis
5.  Secondary cell wall polysaccharides from Bacillus cereus strains G9241, 03BB87 and 03BB102 causing fatal pneumonia share similar glycosyl structures with the polysaccharides from Bacillus anthracis 
Glycobiology  2011;21(7):934-948.
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.
doi:10.1093/glycob/cwr026
PMCID: PMC3110489  PMID: 21421577
Bacillus anthracis; Bacillus cereus; cell wall; polysaccharide; structure
6.  Mutant Lipooligosaccharide-Based Conjugate Vaccine Demonstrates a Broad-Spectrum Effectiveness against Moraxella catarrhalis 
Vaccine  2011;29(25):4210-4217.
There is no licensed vaccine available against Moraxella catarrhalis, an exclusive human pathogen responsible for otitis media in children and respiratory infections in adults. We previously developed conjugate vaccine candidates based on lipooligosaccharides (LOSs) of M. catarrhalis serotypes A, B, and C, each of which was shown to cover a portion of the clinical strains. To generate conserved LOS antigens and eliminate a potential autoimmune response to a similar epitope between M. catarrhalis LOS moiety Galα1-4Galβ1-4Glc and human Pk antigen, two LOS mutants from strain O35E were constructed. Mutant O35Elgt5 or O35EgalE revealed a deletion of one or two terminal galactose residues of wild type O35E LOS. Each LOS molecule was purified, characterized, detoxified, and coupled to tetanus toxoid (TT) to form conjugates, namely dLOS-TT. Three subcutaneous immunizations using dLOS-TT from O35Elgt5 or O35EgalE elicited significant increases (a 729- or 1263-fold above the preimmune serum levels) of serum immunoglobulin (Ig)G against O35E LOS in rabbits with an adjuvant or without an adjuvant (an 140- or 140-fold above the preimmune serum levels). Rabbit antisera demonstrated elevated complement-mediated bactericidal activities against the wild type strain O35E. The rabbit sera elicited by O35Elgt5 dLOS-TT were further examined and showed cross bactericidal activity against all additional 19 M. catarrhalis strains and clinical isolates studied. Moreover, the rabbit sera displayed cross-reactivity not only among three serotype strains but also clinical isolates in a whole-cell enzyme-linked immunosorbent assay (ELISA), which was further confirmed under transmission electron microscopy. In conclusion, O35Elgt5 dLOS-TT may act as a vaccine against most M. catarrhalis strains and therefore can be used for further in vivo efficacy studies.
doi:10.1016/j.vaccine.2011.03.102
PMCID: PMC3109615  PMID: 21501641
Moraxella catarrhalis; lipooligosaccharide mutant; conjugate vaccine; cross-reactivity; conserved antigen
7.  An acpXL Mutant of Rhizobium leguminosarum bv. phaseoli Lacks 27-Hydroxyoctacosanoic Acid in Its Lipid A and Is Developmentally Delayed during Symbiotic Infection of the Determinate Nodulating Host Plant Phaseolus vulgaris ▿  
Journal of Bacteriology  2011;193(18):4766-4778.
Rhizobium leguminosarum is a Gram-negative bacterium that forms nitrogen-fixing symbioses with compatible leguminous plants via intracellular invasion and establishes a persistent infection within host membrane-derived subcellular compartments. Notably, an unusual very-long-chain fatty acid (VLCFA) is found in the lipid A of R. leguminosarum as well as in the lipid A of the medically relevant pathogens Brucella abortus, Brucella melitensis, Bartonella henselae, and Legionella pneumophila, which are also able to persist within intracellular host-derived membranes. These bacterial symbionts and pathogens each contain a homologous gene region necessary for the synthesis and transfer of the VLCFA to the lipid A. Within this region lies a gene that encodes the specialized acyl carrier protein AcpXL, on which the VLCFA is built. This study describes the biochemical and infection phenotypes of an acpXL mutant which lacks the VLCFA. The mutation was created in R. leguminosarum bv. phaseoli strain 8002, which forms symbiosis with Phaseolus vulgaris, a determinate nodulating legume. Structural analysis using gas chromatography and mass spectrometry revealed that the mutant lipid A lacked the VLCFA. Compared to the parent strain, the mutant was more sensitive to the detergents deoxycholate and dodecyl sulfate and the antimicrobial peptide polymyxin B, suggesting a compromise to membrane stability. In addition, the mutant was more sensitive to higher salt concentrations. Passage through the plant restored salt tolerance. Electron microscopic examination showed that the mutant was developmentally delayed during symbiotic infection of the host plant Phaseolus vulgaris and produced abnormal symbiosome structures.
doi:10.1128/JB.00392-11
PMCID: PMC3165650  PMID: 21764936
8.  Structures of the lipopolysaccharides from Rhizobium leguminosarum RBL5523 and its UDP-glucose dehydrogenase mutant (exo5) 
Glycobiology  2010;21(1):55-68.
Rhizobial lipopolysaccharide (LPS) is required to establish an effective symbiosis with its host plant. An exo5 mutant of Rhizobium leguminosarum RBL5523, strain RBL5808, is defective in UDP-glucose (Glc) dehydrogenase that converts UDP-Glc to UDP-glucuronic acid (GlcA). This mutant is unable to synthesize either UDP-GlcA or UDP-galacturonic acid (GalA) and is unable to synthesize extracellular and capsular polysaccharides, lacks GalA in its LPS and is defective in symbiosis (Laus MC, Logman TJ, van Brussel AAN, Carlson RW, Azadi P, Gao MY, Kijne JW. 2004. Involvement of exo5 in production of surface polysaccharides in Rhizobium leguminosarum and its role in nodulation of Vicia sativa subsp. nigra. J Bacteriol. 186:6617–6625). Here, we determined and compared the structures of the RBL5523 parent and RBL5808 mutant LPSs. The parent LPS core oligosaccharide (OS), as with other R. leguminosarum and Rhizobium etli strains, is a Gal1Man1GalA3Kdo3 octasaccharide in, which each of the GalA residues is terminally linked. The core OS from the mutant lacks all three GalA residues. Also, the parent lipid A consists of a fatty acylated GlcNGlcNonate or GlcNGlcN disaccharide that has a GalA residue at the 4′-position, typical of other R. leguminosarum and R. etli lipids A. The mutant lipid A lacks the 4′-GalA residue, and the proximal glycosyl residue was only present as GlcNonate. In spite of these alterations to the lipid A and core OSs, the mutant was still able to synthesize an LPS containing a normal O-chain polysaccharide (OPS), but at reduced levels. The structure of the OPS of the mutant LPS was identical to that of the parent and consists of an O-acetylated →4)-α-d-Glcp-(1→3)-α-d-QuipNAc-(1→ repeating unit.
doi:10.1093/glycob/cwq131
PMCID: PMC2998983  PMID: 20817634
lipopolysaccharide; Rhizobium leguminosarum; structure; symbiosis
9.  Antibody Responses to a Spore Carbohydrate Antigen as a Marker of Nonfatal Inhalation Anthrax in Rhesus Macaques ▿ 
The Bacillus anthracis exosporium protein BclA contains an O-linked antigenic tetrasaccharide whose terminal sugar is known as anthrose (J. M. Daubenspeck et al., J. Biol. Chem. 279:30945–30953, 2004). We hypothesized that serologic responses to anthrose may have diagnostic value in confirming exposure to aerosolized B. anthracis. We evaluated the serologic responses to a synthetic anthrose-containing trisaccharide (ATS) in a group of five rhesus macaques that survived inhalation anthrax following exposure to B. anthracis Ames spores. Two of five animals (RM2 and RM3) were treated with ciprofloxacin starting at 48 hours postexposure and two (RM4 and RM5) at 72 h postexposure; one animal (RM1) was untreated. Infection was confirmed by blood culture and detection of anthrax toxin lethal factor (LF) in plasma. Anti-ATS IgG responses were determined at 14, 21, 28, and 35 days postexposure, with preexposure serum as a control. All animals, irrespective of ciprofloxacin treatment, mounted a specific, measurable anti-ATS IgG response. The earliest detectable responses were on days 14 (RM1, RM2, and RM5), 21 (RM4), and 28 (RM3). Specificity of the anti-ATS responses was demonstrated by competitive-inhibition enzyme immunoassay (CIEIA), in which a 2-fold (wt/wt) excess of carbohydrate in a bovine serum albumin (BSA) conjugate of the oligosaccharide (ATS-BSA) effected >94% inhibition, whereas a structural analog lacking the 3-hydroxy-3-methyl-butyryl moiety at the C-4" of the anthrosyl residue had no inhibition activity. These data suggest that anti-ATS antibody responses may be used to identify aerosol exposure to B. anthracis spores. The anti-ATS antibody responses were detectable during administration of ciprofloxacin.
doi:10.1128/CVI.00475-10
PMCID: PMC3122534  PMID: 21389148
10.  Requirement of NMB0065 for connecting assembly and export of sialic acid capsular polysaccharides in Neisseria meningitidis 
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.
doi:10.1016/j.micinf.2010.02.009
PMCID: PMC2883662  PMID: 20215001
CAPSULE; SIALIC ACID; CAPSULAR POLYSACCHARIDE; NEISSERIA MENINGITIDIS
11.  Francisella Tularensis Blue–Gray Phase Variation Involves Structural Modifications of Lipopolysaccharide O-Antigen, Core and Lipid A and Affects Intramacrophage Survival and Vaccine Efficacy 
Francisella tularensis is a CDC Category A biological agent and a potential bioterrorist threat. There is no licensed vaccine against tularemia in the United States. A long-standing issue with potential Francisella vaccines is strain phase variation to a gray form that lacks protective capability in animal models. Comparisons of the parental strain (LVS) and a gray variant (LVSG) have identified lipopolysaccharide (LPS) alterations as a primary change. The LPS of the F. tularensis variant strain gains reactivity to F. novicida anti-LPS antibodies, suggesting structural alterations to the O-antigen. However, biochemical and structural analysis of the F. tularensis LVSG and LVS LPS demonstrated that LVSG has less O-antigen but no major O-antigen structural alterations. Additionally, LVSG possesses structural differences in both the core and lipid A regions, the latter being decreased galactosamine modification. Recent work has identified two genes important in adding galactosamine (flmF2 and flmK) to the lipid A. Quantitative real-time PCR showed reduced transcripts of both of these genes in the gray variant when compared to LVS. Loss of flmF2 or flmK caused less frequent phase conversion but did not alter intramacrophage survival or colony morphology. The LVSG strain demonstrated an intramacrophage survival defect in human and rat but not mouse macrophages. Consistent with this result, the LVSG variant demonstrated little change in LD50 in the mouse model of infection. Furthermore, the LVSG strain lacks the protective capacity of F. tularensis LVS against virulent Type A challenge. These data suggest that the LPS of the F. tularensis LVSG phase variant is dramatically altered. Understanding the mechanism of blue to gray phase variation may lead to a way to inhibit this variation, thus making future F. tularensis vaccines more stable and efficacious.
doi:10.3389/fmicb.2010.00129
PMCID: PMC3109528  PMID: 21687776
Francisella; LPS; phase variation; tularemia; vaccine
12.  Phosphoglucomutase of Yersinia pestis Is Required for Autoaggregation and Polymyxin B Resistance▿  
Infection and Immunity  2009;78(3):1163-1175.
Yersinia pestis, the causative agent of plague, autoaggregates within a few minutes of cessation of shaking when grown at 28°C. To identify the autoaggregation factor of Y. pestis, we performed mariner-based transposon mutagenesis. Autoaggregation-defective mutants from three different pools were identified, each with a transposon insertion at a different position within the gene encoding phosphoglucomutase (pgmA; y1258). Targeted deletion of pgmA in Y. pestis KIM5 also resulted in loss of autoaggregation. Given the previously defined role for phosphoglucomutase in antimicrobial peptide resistance in other organisms, we tested the KIM5 ΔpgmA mutant for antimicrobial peptide sensitivity. The ΔpgmA mutant displayed >1,000-fold increased sensitivity to polymyxin B compared to the parental Y. pestis strain, KIM5. This sensitivity is not due to changes in lipopolysaccharide (LPS) since the LPSs from both Y. pestis KIM5 and the ΔpgmA mutant are identical based on a comparison of their structures by mass spectrometry (MS), tandem MS, and nuclear magnetic resonance analyses. Furthermore, the ability of polymyxin B to neutralize LPS toxicity was identical for LPS purified from both KIM5 and the ΔpgmA mutant. Our results indicate that increased polymyxin B sensitivity of the ΔpgmA mutant is due to changes in surface structures other than LPS. Experiments with mice via the intravenous and intranasal routes did not demonstrate any virulence defect for the ΔpgmA mutant, nor was flea colonization or blockage affected. Our findings suggest that the activity of PgmA results in modification and/or elaboration of a surface component of Y. pestis responsible for autoaggregation and polymyxin B resistance.
doi:10.1128/IAI.00997-09
PMCID: PMC2825912  PMID: 20028810
13.  Rhizobium leguminosarum biovar viciae 3841, deficient in 27-hydroxyoctacosanoate-modified lipopolysaccharide, is impaired in desiccation tolerance, biofilm formation and motility 
Microbiology  2009;155(Pt 9):3055-3069.
The lipopolysaccharide (LPS) of the Gram-negative legume symbiont Rhizobium leguminosarum biovar viciae 3841 contains several unique modifications, including the addition of a 27-hydroxyoctacosanoic acid (27OHC28 : 0), also termed the very long chain fatty acid (VLCFA), attached at the 2′ position of lipid A. A transposon mutant that lacks expression of two putative 3-oxo-acyl [acyl-carrier protein] synthase II genes, fabF1 and fabF2, from the VLCFA biosynthetic cluster, was isolated and characterized. MS indicated that the lipid A of the mutant lacked the VLCFA modification, and sodium deoxycholate (DOC)-PAGE of the LPS indicated further structural alterations. The mutant was characteristically sensitive to several stresses that would be experienced in the soil environment, such as desiccation and osmotic stresses. An increase in the excretion of neutral surface polysaccharides was observed in the mutant. This mutant was also altered in its attachment to solid surfaces, and was non-motile, with most of the mutant cells lacking flagella. Despite the pleiotropic effects of the mutation, these mutants were still able to nodulate legumes and fix atmospheric nitrogen. This report emphasizes that a structurally intact VLCFA-containing lipid A is critical to cellular traits that are important for survival in the rhizosphere.
doi:10.1099/mic.0.025031-0
PMCID: PMC2850257  PMID: 19460825
14.  Secondary cell wall polysaccharides of Bacillus anthracis are antigens that contain specific epitopes which cross-react with three pathogenic Bacillus cereus strains that caused severe disease, and other epitopes common to all the Bacillus cereus strains tested 
Glycobiology  2009;19(6):665-673.
The immunoreactivities of hydrogen fluoride (HF)-released cell wall polysaccharides (HF-PSs) from selected Bacillus anthracis and Bacillus cereus strains were compared using antisera against live and killed B. anthracis spores. These antisera bound to the HF-PSs from B. anthracis and from three clinical B. cereus isolates (G9241, 03BB87, and 03BB102) obtained from cases of severe or fatal human pneumonia but did not bind to the HF-PSs from the closely related B. cereus ATCC 10987 or from B. cereus type strain ATCC 14579. Antiserum against a keyhole limpet hemocyanin conjugate of the B. anthracis HF-PS (HF-PS-KLH) also bound to HF-PSs and cell walls from B. anthracis and the three clinical B. cereus isolates, and B. anthracis spores. These results indicate that the B. anthracis HF-PS is an antigen in both B. anthracis cell walls and spores, and that it shares cross-reactive, and possibly pathogenicity-related, epitopes with three clinical B. cereus isolates that caused severe disease. The anti-HF-PS-KLH antiserum cross-reacted with the bovine serum albumin (BSA)-conjugates of all B. anthracis and all B. cereus HF-PSs tested, including those from nonclinical B. cereus ATCC 10987 and ATCC 14579 strains. Finally, the serum of vaccinated (anthrax vaccine adsorbed (AVA)) Rhesus macaques that survived inhalation anthrax contained IgG antibodies that bound the B. anthracis HF-PS-KLH conjugate. These data indicate that HF-PSs from the cell walls of the bacilli tested here are (i) antigens that contain (ii) a potentially virulence-associated carbohydrate antigen motif, and (iii) another antigenic determinant that is common to B. cereus strains.
doi:10.1093/glycob/cwp036
PMCID: PMC2682610  PMID: 19270075
antigens; Bacillus anthracis; Bacillus cereus; polysaccharides; specificity
15.  Identification of a novel ABC transporter required for desiccation tolerance, and biofilm formation in Rhizobium leguminosarum bv. viciae 3841 
FEMS microbiology ecology  2009;71(3):327-340.
Rhizobium leguminosarum is a soil bacterium with the ability to form nitrogen-fixing nodules on the roots of leguminous plants. Soil-dwelling, free-living R. leguminosarum often encounters desiccation stress, which impacts its survival within the soil. The mechanisms by which soil bacteria resist the effects of desiccation stress have been described. However, the role of the cell envelope in the desiccation tolerance mechanisms of rhizobia is relatively uncharacterized. Using a transposon mutagenesis approach, a mutant of R. leguminosarum bv. viciae was isolated that was highly sensitive to desiccation. The mutation is located in the ATP-binding protein of an uncharacterized ATP-binding cassette transporter operon (RL2975–RL2977). Exopolysaccharide accumulation was significantly lower in the mutant and the decrease in desiccation tolerance was attributed to the decreased accumulation of exopolysaccharide. In addition to desiccation sensitivity, the mutant was severely impaired in biofilm formation, an important adaptation used by soil bacteria for survival. This work has identified a novel transporter required for physiological traits that are important for the survival of R. leguminosarum in the rhizosphere environment.
doi:10.1111/j.1574-6941.2009.00824.x
PMCID: PMC2868943  PMID: 20030718
ABC transporter; desiccation; biofilm; rhizobium; exopolysaccharide; cell envelope
16.  Rhizobium leguminosarum biovar viciae 3841, deficient in 27-hydroxyoctacosanoate-modified lipopolysaccharide, is impaired in desiccation tolerance, biofilm formation and motility 
Microbiology (Reading, England)  2009;155(Pt 9):3055-3069.
The lipopolysaccharide (LPS) of the Gram-negative legume symbiont Rhizobium leguminosarum biovar viciae 3841 contains several unique modifications, including the addition of a 27-hydroxyoctacosanoic acid (27OHC28 : 0), also termed the very long chain fatty acid (VLCFA), attached at the 2′ position of lipid A. A transposon mutant that lacks expression of two putative 3-oxo-acyl [acyl-carrier protein] synthase II genes, fabF1 and fabF2, from the VLCFA biosynthetic cluster, was isolated and characterized. MS indicated that the lipid A of the mutant lacked the VLCFA modification, and sodium deoxycholate (DOC)-PAGE of the LPS indicated further structural alterations. The mutant was characteristically sensitive to several stresses that would be experienced in the soil environment, such as desiccation and osmotic stresses. An increase in the excretion of neutral surface polysaccharides was observed in the mutant. This mutant was also altered in its attachment to solid surfaces, and was non-motile, with most of the mutant cells lacking flagella. Despite the pleiotropic effects of the mutation, these mutants were still able to nodulate legumes and fix atmospheric nitrogen. This report emphasizes that a structurally intact VLCFA-containing lipid A is critical to cellular traits that are important for survival in the rhizosphere.
doi:10.1099/mic.0.025031-0
PMCID: PMC2850257  PMID: 19460825
17.  Chemical Synthesis and Immunological Properties of Oligosaccharides Derived from the Vegetative Cell Wall of Bacillus anthracis 
Bacillus anthracis vaccine candidate: Sera of rabbits exposed to live and irradiated-killed spores of B. anthracis Sterne 34F2 or immunized with B. anthracis polysaccharide conjugated to KLH elicited antibodies that recognize isolated polysaccharide and two synthetic trisaccharides providing a proof-of-concept step in the development of vegetative and spore-specific reagents for detection and targeting of non-protein structures of B. anthracis.
doi:10.1002/cbic.200800210
PMCID: PMC2832322  PMID: 18563773
Bacillus anthracis; glycoconjugate; oligosaccharide; vaccine; protein conjugation
18.  The structure of the L9 immunotype lipooligosaccharide from Neisseria meningitidis NMA Z2491 
Carbohydrate research  2008;343(17):2971-2979.
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.
doi:10.1016/j.carres.2008.08.026
PMCID: PMC2784147  PMID: 18804756
Neisseria meningitidis; NMA; L9 Immunotype; Lipooligosaccharide; LOS; Structure; Phosphoethanolamine
19.  Identification of Two Late Acyltransferase Genes Responsible for Lipid A Biosynthesis in Moraxella catarrhalis 
The FEBS journal  2008;275(20):5201-5214.
Lipid A is a biological component of the lipooligosaccharide (LOS) of a human pathogen, Moraxella catarrhalis. No other acyltransferases except UDP-GlcNAc acyltransferase responsible for lipid A biosynthesis in M. catarrhalis have been elucidated. By informatics, two late acyltransferase genes lpxX and lpxL responsible for lipid A biosynthesis were identified, and knockout mutants of each gene in M. catarrhalis strain O35E was constructed and named as O35ElpxX and O35ElpxL. Structural analysis of lipid A from the parental strain and derived mutants showed that O35ElpxX lacked two decanoic acids (C10:0) while O35ElpxL lacked one dodecanoic (lauric) acid (C12:0), suggesting that lpxX gene encoded decanoyl transferase and lpxL gene encoded dodecanoyl transferase. Phenotypic analysis revealed that both mutants were similar to the parental strain in their toxicity in vitro. However, the O35ElpxX was sensitive to bactericidal activity of normal human serum and hydrophobic reagents. It had a reduced growth rate in broth and an accelerated bacterial clearance at 3 h (p <0.01) or 6 h (p <0.05) after an aerosol challenge in a murine model of bacterial pulmonary clearance. Meanwhile, the O35ElpxL presented alike patterns as the parental strain except it was slightly sensitive to the hydrophobic reagents. These results indicate that these two genes, particularly lpxX, encoding late acyltransferases responsible for incorporation of the acyloxyacyl linked secondary acyl chains into the lipid A are important for biological activities of M. catarrhalis.
doi:10.1111/j.1742-4658.2008.06651.x
PMCID: PMC2585779  PMID: 18795947
Moraxella catarrhalis; late acyltransferase; lipooligosaccharide; lpxX; lpxL
20.  Cell Wall Carbohydrate Compositions of Strains from the Bacillus cereus Group of Species Correlate with Phylogenetic Relatedness▿  
Journal of Bacteriology  2007;190(1):112-121.
Members of the Bacillus cereus group contain cell wall carbohydrates that vary in their glycosyl compositions. Recent multilocus sequence typing (MLST) refined the relatedness of B. cereus group members by separating them into clades and lineages. Based on MLST, we selected several B. anthracis, B. cereus, and B. thuringiensis strains and compared their cell wall carbohydrates. The cell walls of different B. anthracis strains (clade 1/Anthracis) were composed of glucose (Glc), galactose (Gal), N-acetyl mannosamine (ManNAc), and N-acetylglucosamine (GlcNAc). In contrast, the cell walls from clade 2 strains (B. cereus type strain ATCC 14579 and B. thuringiensis strains) lacked Gal and contained N-acetylgalactosamine (GalNAc). The B. cereus clade 1 strains had cell walls that were similar in composition to B. anthracis in that they all contained Gal. However, the cell walls from some clade 1 strains also contained GalNAc, which was not present in B. anthracis cell walls. Three recently identified clade 1 strains of B. cereus that caused severe pneumonia, i.e., strains 03BB102, 03BB87, and G9241, had cell wall compositions that closely resembled those of the B. anthracis strains. It was also observed that B. anthracis strains cell wall glycosyl compositions differed from one another in a plasmid-dependent manner. When plasmid pXO2 was absent, the ManNAc/Gal ratio decreased, while the Glc/Gal ratio increased. Also, deletion of atxA, a global regulatory gene, from a pXO2− strain resulted in cell walls with an even greater level of Glc.
doi:10.1128/JB.01292-07
PMCID: PMC2223722  PMID: 17981984
21.  Identification of an Orphan Response Regulator Required for the Virulence of Francisella spp. and Transcription of Pathogenicity Island Genes▿  
Infection and Immunity  2007;75(7):3305-3314.
Francisella tularensis is a category A agent of biowarfare/biodefense. Little is known about the regulation of virulence gene expression in Francisella spp. Comparatively few regulatory factors exist in Francisella, including those belonging to two-component systems (TCS). However, orphan members of typical TCS can be identified. To determine if orphan TCS members affect Francisella gene expression, a gene encoding a product with high similarity to the Salmonella PmrA response regulator (FTT1557c/FNU0663.2) was deleted in Francisella novicida (a model organism for F. tularensis). The F. novicida pmrA mutant was defective in survival/growth within human and murine macrophage cell lines and was 100% defective in virulence in mice at a dose of up to 108 CFU. In addition, the mutant strain demonstrated increased susceptibility to antimicrobial peptide killing, but no differences were observed between the lipid A of the mutant and the parental strain, as has been observed with pmrA mutants of other microbes. The F. novicida pmrA mutant was 100% protective as a single-dose vaccine when challenge was with 106 CFU of F. novicida but did not protect against type A Schu S4 wild-type challenge. DNA microarray analysis identified 65 genes regulated by PmrA. The majority of these genes were located in the region surrounding pmrA or within the Francisella pathogenicity island (FPI). These FPI genes are also regulated by MglA, but MglA does not regulate pmrA, nor does PmrA regulate MglA. Thus, the orphan response regulator PmrA is an important factor in controlling virulence in F. novicida, and a pmrA mutant strain is an effective vaccine against homologous challenge.
doi:10.1128/IAI.00351-07
PMCID: PMC1932945  PMID: 17452468
22.  Exo-Oligosaccharides of Rhizobium sp. Strain NGR234 Are Required for Symbiosis with Various Legumes 
Journal of Bacteriology  2006;188(17):6168-6178.
Rhizobia are nitrogen-fixing bacteria that establish endosymbiotic associations with legumes. Nodule formation depends on various bacterial carbohydrates, including lipopolysaccharides, K-antigens, and exopolysaccharides (EPS). An acidic EPS from Rhizobium sp. strain NGR234 consists of glucosyl (Glc), galactosyl (Gal), glucuronosyl (GlcA), and 4,6-pyruvylated galactosyl (PvGal) residues with β-1,3, β-1,4, β-1,6, α-1,3, and α-1,4 glycoside linkages. Here we examined the role of NGR234 genes in the synthesis of EPS. Deletions within the exoF, exoL, exoP, exoQ, and exoY genes suppressed accumulation of EPS in bacterial supernatants, a finding that was confirmed by chemical analyses. The data suggest that the repeating subunits of EPS are assembled by an ExoQ/ExoP/ExoF-dependent mechanism, which is related to the Wzy polymerization system of group 1 capsular polysaccharides in Escherichia coli. Mutation of exoK (NGRΩexoK), which encodes a putative glycanase, resulted in the absence of low-molecular-weight forms of EPS. Analysis of the extracellular carbohydrates revealed that NGRΩexoK is unable to accumulate exo-oligosaccharides (EOSs), which are O-acetylated nonasaccharide subunits of EPS having the formula Gal(Glc)5(GlcA)2PvGal. When used as inoculants, both the exo-deficient mutants and NGRΩexoK were unable to form nitrogen-fixing nodules on some hosts (e.g., Albizia lebbeck and Leucaena leucocephala), but they were able to form nitrogen-fixing nodules on other hosts (e.g., Vigna unguiculata). EOSs of the parent strain were biologically active at very low levels (yield in culture supernatants, ∼50 μg per liter). Thus, NGR234 produces symbiotically active EOSs by enzymatic degradation of EPS, using the extracellular endo-β-1,4-glycanase encoded by exoK (glycoside hydrolase family 16). We propose that the derived EOSs (and not EPS) are bacterial components that play a crucial role in nodule formation in various legumes.
doi:10.1128/JB.00365-06
PMCID: PMC1595362  PMID: 16923883
24.  The Pea Nodule Environment Restores the Ability of a Rhizobium leguminosarum Lipopolysaccharide acpXL Mutant To Add 27-Hydroxyoctacosanoic Acid to Its Lipid A 
Journal of Bacteriology  2006;188(6):2126-2133.
Members of the Rhizobiaceae contain 27-hydroxyoctacosanoic acid (27OHC28:0) in their lipid A. A Rhizobium leguminosarum 3841 acpXL mutant (named here Rlv22) lacking a functional specialized acyl carrier lacked 27OHC28:0 in its lipid A, had altered growth and physiological properties (e.g., it was unable to grow in the presence of an elevated salt concentration [0.5% NaCl]), and formed irregularly shaped bacteroids, and the synchronous division of this mutant and the host plant-derived symbiosome membrane was disrupted. In spite of these defects, the mutant was able to persist within the root nodule cells and eventually form, albeit inefficiently, nitrogen-fixing bacteroids. This result suggested that while it is in a host root nodule, the mutant may have some mechanism by which it adapts to the loss of 27OHC28:0 from its lipid A. In order to further define the function of this fatty acyl residue, it was necessary to examine the lipid A isolated from mutant bacteroids. In this report we show that addition of 27OHC28:0 to the lipid A of Rlv22 lipopolysaccharides is partially restored in Rlv22 acpXL mutant bacteroids. We hypothesize that R. leguminosarum bv. viciae 3841 contains an alternate mechanism (e.g., another acp gene) for the synthesis of 27OHC28:0, which is activated when the bacteria are in the nodule environment, and that it is this alternative mechanism which functionally replaces acpXL and is responsible for the synthesis of 27OHC28:0-containing lipid A in the Rlv22 acpXL bacteroids.
doi:10.1128/JB.188.6.2126-2133.2006
PMCID: PMC1428142  PMID: 16513742
25.  Moraxella catarrhalis Bacterium without Endotoxin, a Potential Vaccine Candidate  
Infection and Immunity  2005;73(11):7569-7577.
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.
doi:10.1128/IAI.73.11.7569-7577.2005
PMCID: PMC1273912  PMID: 16239560

Results 1-25 (37)