Biomedical subjects

Paul G Hitchen

Publications and source records attributed to Paul G Hitchen.

15 recordsLinked to original sources

PubMed2006

The immunologically distinct O antigens from Francisella tularensis subspecies tularensis and Francisella novicida are both virulence determinants and protective antigens.

We have determined the sequence of the gene cluster encoding the O antigen in Francisella novicida and compared it to the previously reported O-antigen cluster in Francisella tularensis subsp. tularensis. Immunization with purified lipopolysaccharide (LPS) from F. tularensis subsp. tularensis or F. novicida protected against challenge with Francisella tularensis subsp. holarctica and F. novicida, respectively. The LPS from F. tularensis subsp. tularensis did not confer protection against challenge with F. novicida, and the LPS from F. novicida did not confer protection against challenge with F. tularensis subsp. holarctica. Allelic replacement mutants of F. tularensis subsp. tularensis or F. novicida which failed to produce O antigen were attenuated, but exposure to these mutants did not induce a protective immune response. The O antigen of F. tularensis subsp. tularensis appeared to be important for intracellular survival whereas the O antigen of F. novicida appeared to be critical for serum resistance and less important for intracellular survival.

Amino Acid Sequence↗

PubMed2006

Neisseria gonorrhoeae type IV pili undergo multisite, hierarchical modifications with phosphoethanolamine and phosphocholine requiring an enzyme structurally related to lipopolysaccharide phosphoethanolamine transferases.

The zwitterionic phospho-forms phosphoethanolamine and phosphocholine are recognized as influential and important substituents of pathogen cell surfaces. PilE, the major pilin subunit protein of the type IV pilus (Tfp) colonization factor of Neisseria gonorrhoeae undergoes unique, post-translational modifications with these moieties. These phospho-form modifications have been shown to be O-linked alternately to a specific, conserved serine residue of PilE. However, the enzymes and precursors involved in their addition are unknown, and the full spectrum of PilE post-translational modifications has yet to be defined. Here, an intact protein-based mass spectrometric approach was integrated with bioinformatics and reverse genetics to address these matters. Specifically we show that a protein limited in its distribution to pathogenic Neisseria species and structurally related to enzymes implicated in phosphoethanolamine modification of lipopolysaccharide is necessary for PilE covalent modification with phosphoethanolamine and phosphocholine. These findings strongly suggest that protein phospho-form modification is mechanistically similar to processes underlying analogous modifications of prokaryotic saccharolipid glycans. We also show that PilE undergoes multisite and hierarchical phospho-form modifications and that the stoichiometries of site occupancy can be influenced by PilE primary structure and the abundance of the pilin-like protein PilV. Together, these findings have important implications for the structure and antigenicity of PilE.

Amino Acid Sequence↗

PubMed2006

Bacterial glycoproteomics.

Glycosylated proteins are ubiquitous components of eukaryote cellular surfaces, where the glycan moieties are implicated in a wide range of cell-cell recognition events. Once thought to be restricted to eukaryotes, glycosylation is now being increasingly reported in prokaryotes. Many of these discoveries have grown from advances in analytical technologies and genome sequencing. This review highlights the capabilities of high-sensitivity mass spectrometry for carbohydrate structure determination of bacterial glycoproteins and the emergence of glycoproteomic strategies that have evolved from proteomics and genomics for the functional analysis of bacterial glycosylation.

Bacteria↗

PubMed2005

LosA, a key glycosyltransferase involved in the biosynthesis of a novel family of glycosylated acyltrehalose lipooligosaccharides from Mycobacterium marinum.

Members of the genus Mycobacterium are characterized by cell envelopes rich in unusual free lipids, interacting with a covalently anchored mycolyl-arabinogalactan matrix. Previous studies have shown that Mycobacterium marinum produces large amounts of a diacylglycosylphenolphthiocerol, "phenolic" glycolipid. When cultivated on liquid Sauton medium, traces of a polar lipooligosaccharide (LOS) glycolipid antigen were also previously indicated. In this study, it was found that growth of the type strain of M. marinum on solid Sauton or Middlebrook 7H10 agar gave substantial, but different, amounts of a family of four major trehalose-based LOSs. The core pentasaccharide LOS-I was a rhamnosyl diglucosyl-acylated trehalose. The heptasaccharide, LOS-II, was derived from LOS-I by adding xylose accompanied by a novel sugar (X); repeated addition of this sugar unit X gave the octasaccharide LOS-III. LOS-IV has a decasaccharide component with two additional unusual sugar units, YZ. In a recent study (Alexander, D. C., Jones, J. R., Tan, T., Chen, J. M., and Liu, J. (2004) J. Biol. Chem. 279, 18824-18833), chromatographically similar glycolipids were assigned to the family of phosphatidylinositol mannosides (PIMs) and a "PimF" (Rv1500) glycosyltransferase implicated in the conversion of a supposed "PIM5" to a "PIM7." The present study indicates that these putative PIMs are in fact members of the phosphorus-free LOS family of glycolipids and that the protein product of Rv1500, which we have now termed LosA, is a glycosyltransferase involved in transferring sugars to LOS-III to form LOS-IV of M. marinum.

Base Sequence↗

PubMed2005

Grey variants of the live vaccine strain of Francisella tularensis lack lipopolysaccharide O-antigen, show reduced ability to survive in macrophages and do not induce protective immunity in mice.

Francisella tularensis live vaccine strain (LVS) produces two colony types when grown on solid media, often referred to as blue variants (BV) and grey variants (GV). Whereas blue variant bacteria possessed a lipopolysaccharide O-side chain, grey variant bacteria lacked O-side chains. Grey variant bacteria appeared in stationary phase bacterial cultures and could be identified using a novel FACS-based assay. Compared to blue variant bacteria, grey variants showed a reduced ability to infect and survive in macrophages. The immunisation of mice with blue variant bacteria, but not grey variant bacteria, induced protective immunity towards fully virulent F. tularensis.

Animals↗

PubMed2005

Deletion of Cg-emb in corynebacterianeae leads to a novel truncated cell wall arabinogalactan, whereas inactivation of Cg-ubiA results in an arabinan-deficient mutant with a cell wall galactan core.

The cell wall of Mycobacterium tuberculosis has a complex ultrastructure that consists of mycolic acids connected to peptidoglycan via arabinogalactan (AG) and abbreviated as the mAGP complex. The mAGP complex is crucial for the survival and pathogenicity of M. tuberculosis and is the target of several anti-tubercular agents. Apart from sharing a similar mAGP and the availability of the complete genome sequence, Corynebacterium glutamicum has proven useful in the study of orthologous M. tuberculosis genes essential for viability. Here we examined the effects of particular genes involved in AG polymerization by gene deletion in C. glutamicum. The anti-tuberculosis drug ethambutol is thought to target a set of arabinofuranosyltransferases (Emb) that are involved in arabinan polymerization. Deletion of emb in C. glutamicum results in a slow growing mutant with profound morphological changes. Chemical analysis revealed a dramatic reduction of arabinose resulting in a novel truncated AG structure possessing only terminal arabinofuranoside (t-Araf) residues with a corresponding loss of cell wall bound mycolic acids. Treatment of wild-type C. glutamicum with ethambutol and subsequent cell wall analyses resulted in an identical phenotype comparable to the C. glutamicum emb deletion mutant. Additionally, disruption of ubiA in C. glutamicum, the first enzyme involved in the biosynthesis of the sugar donor decaprenol phosphoarabinose (DPA), resulted in a complete loss of cell wall arabinan. Herein, we establish for the first time, (i) that in contrast to M. tuberculosis embA and embB mutants, deletion of C. glutamicum emb leads to a highly truncated AG possessing t-Araf residues, (ii) the exact site of attachment of arabinan chains in AG, and (iii) DPA is the only Araf sugar donor in AG biosynthesis suggesting the presence of a novel enzyme responsible for "priming" the galactan domain for further elaboration by Emb, resulting in the final maturation of the native AG polysaccharide.

Arabinose↗

PubMed2005

Identification of a capsular polysaccharide from Moraxella bovis.

The bacterium Moraxella bovis is the causative agent of an economically important disease of cattle: Infectious Bovine Keratoconjunctivitis (IBK), otherwise known as pinkeye. Little is known regarding the structure of the carbohydrates produced by M. bovis. The structure of a capsular polysaccharide from M. bovis (strain Mb25) has been determined using NMR and MS analysis. From these data it is concluded that the polysaccharide is composed of the unmodified chondroitin disaccharide repeat unit.

Bacterial Capsules↗

PubMed2005

Engineering N-linked protein glycosylation with diverse O antigen lipopolysaccharide structures in Escherichia coli.

Campylobacter jejuni has a general N-linked protein glycosylation system that can be functionally transferred to Escherichia coli. In this study, we engineered E. coli cells in a way that two different pathways, protein N-glycosylation and lipopolysaccharide (LPS) biosynthesis, converge at the step in which PglB, the key enzyme of the C. jejuni N-glycosylation system, transfers O polysaccharide from a lipid carrier (undecaprenyl pyrophosphate) to an acceptor protein. PglB was the only protein of the bacterial N-glycosylation machinery both necessary and sufficient for the transfer. The relaxed specificity of the PglB oligosaccharyltransferase toward the glycan structure was exploited to create novel N-glycan structures containing two distinct E. coli or Pseudomonas aeruginosa O antigens. PglB-mediated transfer of polysaccharides might be valuable for in vivo production of O polysaccharides-protein conjugates for use as antibacterial vaccines.

Bacterial Vaccines↗

PubMed2005

Altered expression profile of mycobacterial surface glycopeptidolipids following treatment with the antifungal azole inhibitors econazole and clotrimazole.

The azole antifungal drugs econazole and clotrimazole are known cytochrome P450 enzyme inhibitors. This study shows that these drugs are potent inhibitors of mycobacterial growth and are more effective against Mycobacterium smegmatis than isoniazid and ethionamide, two established anti-mycobacterial drugs. Several non-tuberculous mycobacteria, including the pathogenic members of the Mycobacterium avium-intracellulare complex (MAC) and the fast-growing saprophytic organism M. smegmatis, produce an array of serovar-specific (ss) and non-serovar-specific (ns) glycopeptidolipids (GPLs). GPL biosynthesis has been investigated for several years but has still not been fully elucidated. The authors demonstrate here that econazole and clotrimazole inhibit GPL biosynthesis in M. smegmatis. In particular, clotrimazole inhibits all four types of nsGPLs found in M. smegmatis, suggesting an early and common target within their biosynthetic pathway. Altogether, the data suggest that an azole-specific target, most likely a cytochrome P450, may be involved in the hydroxylation of the N-acyl chain in GPL biosynthesis. Azole antifungal drugs and potential derivatives could represent an interesting new range of anti-mycobacterial drugs, especially against opportunistic human pathogens including MAC, M. scrofulaceum, M. peregrinum, M. chelonae and M. abscessus.

Antitubercular Agents↗

PubMed2005

Functional analysis of the Campylobacter jejuni N-linked protein glycosylation pathway.

We describe in this report the characterization of the recently discovered N-linked glycosylation locus of the human bacterial pathogen Campylobacter jejuni, the first such system found in a species from the domain Bacteria. We exploited the ability of this locus to function in Escherichia coli to demonstrate through mutational and structural analyses that variant glycan structures can be transferred onto protein indicating the relaxed specificity of the putative oligosaccharyltransferase PglB. Structural data derived from these variant glycans allowed us to infer the role of five individual glycosyltransferases in the biosynthesis of the N-linked heptasaccharide. Furthermore, we show that C. jejuni- and E. coli-derived pathways can interact in the biosynthesis of N-linked glycoproteins. In particular, the E. coli encoded WecA protein, a UDP-GlcNAc: undecaprenylphosphate GlcNAc-1-phosphate transferase involved in glycolipid biosynthesis, provides for an alternative N-linked heptasaccharide biosynthetic pathway bypassing the requirement for the C. jejuni-derived glycosyltransferase PglC. This is the first experimental evidence that biosynthesis of the N-linked glycan occurs on a lipid-linked precursor prior to transfer onto protein. These findings provide a framework for understanding the process of N-linked protein glycosylation in Bacteria and for devising strategies to exploit this system for glycoengineering.

Bacterial Proteins↗

PubMed2004

Unique modifications with phosphocholine and phosphoethanolamine define alternate antigenic forms of Neisseria gonorrhoeae type IV pili.

Several major bacterial pathogens and related commensal species colonizing the human mucosa express phosphocholine (PC) at their cell surfaces. PC appears to impact host-microbe biology by serving as a ligand for both C-reactive protein and the receptor for platelet-activating factor. Type IV pili of Neisseria gonorrhoeae (Ng) and Neisseria meningitidis, filamentous protein structures critical to the colonization of their human hosts, are known to react variably with monoclonal antibodies recognizing a PC epitope. However, the structural basis for this reactivity has remained elusive. To address this matter, we exploited the finding that the PilE pilin subunit in Ng mutants lacking the PilV protein acquired the PC epitope independent of changes in pilin primary structure. Specifically, we show by using mass spectrometry that PilE derived from the pilV background is composed of a mixture of subunits bearing O-linked forms of either phosphoethanolamine (PE) or PC at the same residue, whereas the wild-type background carries only PE at that same site. Therefore, PilV can influence pilin structure and antigenicity by modulating the incorporation of these alternative modifications. The disaccharide covalently linked to Ng pilin was also characterized because it is present on the same peptides bearing the PE and PC modifications and, contrary to previous reports, was found to be linked by means of 2,4-diacetamido-2,4,6-trideoxyhexose. Taken together, these findings provide new insights into Ng type IV pilus structure and antigenicity and resolve long-standing issues regarding the nature of both the PC epitope and the pilin glycan.

Amino Acid Sequence↗

PubMed2003

Characterization of the O antigen gene cluster and structural analysis of the O antigen of Francisella tularensis subsp. tularensis.

A gene cluster encoding enzymes involved in LPS O antigen biosynthesis was identified from the partial genome sequence of Francisella tularensis subsp. tularensis Schu S4. All of the genes within the cluster were assigned putative functions based on sequence similarity with genes from O antigen biosynthetic clusters from other bacteria. Ten pairs of overlapping primers were designed to amplify the O antigen biosynthetic cluster by PCR from nine strains of F. tularensis. Although the gene cluster was present in all strains, there was a size difference in one of the PCR products between subsp. tularensis strains and subsp. holarctica strains. LPS was purified from F. tularensis subsp. tularensis Schu S4 and the O antigen was shown by mass spectrometry to have a structure similar to that of F. tularensis subsp. holarctica strain 15. When LPS from F. tularensis subsp. tularensis Schu S4 was used to immunize mice that were then challenged with F. tularensis subsp. tularensis Schu S4, an extended time to death was observed.

Animals↗

PubMed2002

N-linked glycosylation in Campylobacter jejuni and its functional transfer into E. coli.

N-linked protein glycosylation is the most abundant posttranslation modification of secretory proteins in eukaryotes. A wide range of functions are attributed to glycan structures covalently linked to asparagine residues within the asparagine-X-serine/threonine consensus sequence (Asn-Xaa-Ser/Thr). We found an N-linked glycosylation system in the bacterium Campylobacter jejuni and demonstrate that a functional N-linked glycosylation pathway could be transferred into Escherichia coli. Although the bacterial N-glycan differs structurally from its eukaryotic counterparts, the cloning of a universal N-linked glycosylation cassette in E. coli opens up the possibility of engineering permutations of recombinant glycan structures for research and industrial applications.

Bacterial Proteins↗

PubMed2002

Characterization of a putative alpha-mannosyltransferase involved in phosphatidylinositol trimannoside biosynthesis in Mycobacterium tuberculosis.

Phosphatidyl-myo-inositol mannosides (PIMs), lipomannan (LM) and lipoarabinomannan (LAM) are an important class of bacterial factors termed modulins that are found in tuberculosis and leprosy. Although their structures are well established, little is known with respect to the molecular aspects of the biosynthetic machinery involved in the synthesis of these glycolipids. On the basis of sequence similarity to other glycosyltransferases and our previous studies defining an alpha-mannosyltransferase from Mycobacterium tuberculosis, named PimB [Schaeffer, Khoo, Besra, Chatterjee, Brennan, Belisle and Inamine (1999) J. Biol. Chem. 274, 31625-31631], which catalysed the formation of triacyl (Ac(3))-PIM(2) (i.e. the dimannoside), we have identified a related gene from M. tuberculosis CDC1551, now designated pimC. The use of a cell-free assay containing GDP-[(14)C]mannose, amphomycin and membranes from Myobacterium smegmatis overexpressing PimC led to the synthesis of a new alkali-labile PIM product. Fast-atom-bombardment MS established the identity of the new enzymically synthesized product as Ac(3)PIM(3) (i.e. the trimannoside). The results indicate that pimC encodes an alpha-mannosyltransferase involved in Ac(3)PIM(3) biosynthesis. However, inactivation of pimC in Myobacterium bovis Bacille Calmette-Guérin (BCG) did not affect the production of higher PIMs, LM and LAM when compared with wild-type M. bovis BCG, suggesting the existence of redundant gene(s) or an alternate pathway that may compensate for this PimC deficiency. Further analyses, which compared the distribution of pimC in a panel of M. tuberculosis strains, revealed that pimC was present in only 22% of the clinical isolates examined.

Amino Acid Sequence↗

PubMed2002

Structural characterization of lipo-oligosaccharide (LOS) from Yersinia pestis: regulation of LOS structure by the PhoPQ system.

The two-component regulatory system PhoPQ has been shown to regulate the expression of virulence factors in a number of bacterial species. For one such virulence factor, lipopolysaccharide (LPS), the PhoPQ system has been shown to regulate structural modifications in Salmonella enterica var Typhimurium. In Yersinia pestis, which expresses lipo-oligosaccharide (LOS), a PhoPQ regulatory system has been identified and an isogenic mutant constructed. To investigate potential modifications to LOS from Y. pestis, which to date has not been fully characterized, purified LOS from wild-type plague and the phoP defective mutant were analysed by mass spectrometry. Here we report the structural characterization of LOS from Y. pestis and the direct comparison of LOS from a phoP mutant. Structural modifications to lipid A, the host signalling portion of LOS, were not detected but analysis of the core revealed the expression of two distinct molecular species in wild-type LOS, differing in terminal galactose or heptose. The phoP mutant was restricted to the expression of a single molecular species, containing terminal heptose. The minimum inhibitory concentration of cationic antimicrobial peptides for the two strains was determined and compared with the wild-type: the phoP mutant was highly sensitive to polymyxin. Thus, LOS modification is under the control of the PhoPQ regulatory system and the ability to alter LOS structure may be required for survival of Y. pestis within the mammalian and/or flea host.

Bacterial Proteins↗