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Michael P Jennings

Publications and source records attributed to Michael P Jennings.

At least 19 recordsLinked to original sources
PubMed2007

Evidence for distinctive mechanisms of S-nitrosoglutathione metabolism by AdhC in two closely related species, Neisseria gonorrhoeae and Neisseria meningitidis.

The adhC gene from 11 strains of Neisseria gonorrhoeae was distinguished from its homologue in Neisseria meningitidis by the presence of a premature stop codon caused by a single base insertion. Mutational analysis showed that NADH S-nitrosoglutathione oxidoreductase activity was associated with adhC in Neisseria meningitidis but not in Neisseria gonorrhoeae.

Aldehyde Oxidoreductases↗
PubMed2006

Characterization of the OxyR regulon of Neisseria gonorrhoeae.

OxyR regulates the expression of the majority of H(2)O(2) responses in Gram-negative organisms. In a previous study we reported the OxyR-dependent derepression of catalase expression in the human pathogen Neisseria gonorrhoeae. In the present study we used microarray expression profiling of N. gonorrhoeae wild-type strain 1291 and an oxyR mutant strain to define the OxyR regulon. In addition to katA (encoding catalase), only one other locus displayed a greater than two-fold difference in expression in the wild type : oxyR comparison. This locus encodes an operon of two genes, a putative peroxiredoxin/glutaredoxin (Prx) and a putative glutathione oxidoreductase (Gor). Mutant strains were constructed in which each of these genes was inactivated. A previous biochemical study in Neisseria meningitidis had confirmed function of the glutaredoxin/peroxiredoxin. Assay of the wild-type 1291 cell free extract confirmed Gor activity, which was lost in the gor mutant strain. Phenotypic analysis of the prx mutant strain in H(2)O(2) killing assays revealed increased resistance, presumably due to upregulation of alternative defence mechanisms. The oxyR, prx and gor mutant strains were deficient in biofilm formation, and the oxyR and prx strains had decreased survival in cervical epithelial cells, indicating a key role for the OxyR regulon in these processes.

DNA-Binding Proteins↗
PubMed2006

Rapid and efficient screening of a Representational Difference Analysis library using reverse Southern hybridisation: identification of genetic differences between Haemophilus parasuis isolates.

Representational Difference Analysis (RDA) is an established technique used for isolation of specific genetic differences between or within bacterial species. This method was used to investigate the genetic basis of serovar-specificity and the relationship between serovar and virulence in Haemophilus parasuis. An RDA clone library of 96 isolates was constructed using H. parasuis strains H425(P) (serovar 12) and HS1967 (serovar 4). To screen such a large clone library to determine which clones are strain-specific would typically involved separately labelling each clone for use in Southern hybridisation against genomic DNA from each of the strains. In this study, a novel application of reverse Southern hybridisation was used to screen the RDA library: genomic DNA from each strain was labelled and used to probe the library to identify strain-specific clones. This novel approach represents a significant improvement in methodology that is rapid and efficient.

Animals↗
PubMed2006

Pilin glycosylation in Neisseria meningitidis occurs by a similar pathway to wzy-dependent O-antigen biosynthesis in Escherichia coli.

Pili (type IV fimbriae) of Neisseria meningitidis are glycosylated by the addition of O-linked sugars. Recent work has shown that PglF, a protein with homology to O-antigen 'flippases', is required for the biosynthesis of the pilin-linked glycan and suggests pilin glycosylation occurs in a manner analogous to the wzy-dependent addition of O-antigen to the core-LPS. O-Antigen ligases are crucial in this pathway for the transfer of undecraprenol-linked sugars to the LPS-core in Gram-negative bacteria. An O-antigen ligase homologue, pglL, was identified in N. meningitidis. PglL mutants showed no change in LPS phenotypes but did show loss of pilin glycosylation, confirming PglL is essential for pilin O-linked glycosylation in N. meningitidis.

Escherichia coli↗
PubMed2006

PerR controls Mn-dependent resistance to oxidative stress in Neisseria gonorrhoeae.

In previous studies it has been established that resistance to superoxide by Neisseria gonorrhoeae is dependent on the accumulation of Mn(II) ions involving the ABC transporter, MntABC. A mutant strain lacking the periplasmic binding protein component (MntC) of this transport system is hypersensitive to killing by superoxide anion. In this study the mntC mutant was found to be more sensitive to H2O2 killing than the wild-type. Analysis of regulation of MntC expression revealed that it was de-repressed under low Mn(II) conditions. The N. gonorrhoeae mntABC locus lacks the mntR repressor typically found associated with this locus in other organisms. A search for a candidate regulator of mntABC expression revealed a homologue of PerR, a Mn-dependent peroxide-responsive regulator found in Gram-positive organisms. A perR mutant expressed more MntC protein than wild-type, and expression was independent of Mn(II), consistent with a role for PerR as a repressor of mntABC expression. The PerR regulon of N. gonorrhoeae was defined by microarray analysis and includes ribosomal proteins, TonB-dependent receptors and an alcohol dehydrogenase. Both the mntC and perR mutants had reduced intracellular survival in a human cervical epithelial cell model.

Bacterial Proteins↗
PubMed2006

Defenses against oxidative stress in Neisseria gonorrhoeae: a system tailored for a challenging environment.

Neisseria gonorrhoeae is a host-adapted pathogen that colonizes primarily the human genitourinary tract. This bacterium encounters reactive oxygen and reactive nitrogen species as a consequence of localized inflammatory responses in the urethra of males and endocervix of females and also of the activity of commensal lactobacilli in the vaginal flora. This review describes recent advances in the understanding of defense systems against oxidative stress in N. gonorrhoeae and shows that while some of its defenses have similarities to the paradigm established with Escherichia coli, there are also some key differences. These differences include the presence of a defense system against superoxide based on manganese ions and a glutathione-dependent system for defense against nitric oxide which is under the control of a novel MerR-like transcriptional regulator. An understanding of the defenses against oxidative stress in N. gonorrhoeae and their regulation may provide new insights into the ways in which this bacterium survives challenges from polymorphonuclear leukocytes and urogenital epithelial cells.

Catalase↗
PubMed2005

Differences in macrophage activation by bacterial DNA and CpG-containing oligonucleotides.

Bacterial DNA activates mouse macrophages, B cells, and dendritic cells in a TLR9-dependent manner. Although short ssCpG-containing phosphodiester oligonucleotides (PO-ODN) can mimic the action of bacterial DNA on macrophages, they are much less immunostimulatory than Escherichia coli DNA. In this study we have assessed the structural differences between E. coli DNA and PO-ODN, which may explain the high activity of bacterial DNA on macrophages. DNA length was found to be the most important variable. Double-strandedness was not responsible for the increased activity of long DNA. DNA adenine methyltransferase (Dam) and DNA cytosine methyltransferase (Dcm) methylation of E. coli DNA did not enhance macrophage NO production. The presence of two CpG motifs on one molecule only marginally improved activity at low concentration, suggesting that ligand-mediated TLR9 cross-linking was not involved. The major contribution was from DNA length. Synthetic ODN >44 nt attained the same levels of activity as bacterial DNA. The response of macrophages to CpG DNA requires endocytic uptake. The length dependence of the CpG ODN response was found to correlate with the presence in macrophages of a length-dependent uptake process for DNA. This transport system was absent from B cells and fibroblasts.

Animals↗
PubMed2005

Investigation of ansB and sspA derived promoters for multi- and single-copy antigen expression in attenuated Salmonella enterica var. typhimurium.

Five candidate promoters were examined to determine their utility in directing immunogenic levels of expression of the C fragment from tetanus toxin in attenuated S. enterica used as an oral vaccine in mice. Promoters derived from the genes encoding the stringent starvation protein (sspA) from E. coli and S. enterica, but not ansB derived promoters, expressed immunogenic levels of C fragment from multi-copy plasmids in attenuated S. enterica in vivo and, following oral immunization, induced high titre specific anti-tetanus toxoid serum antibodies. We also demonstrate that not only the choice of promoter, replicon and growth conditions but also how expression constructs are assembled in the chosen plasmid is critical for the successful development of plasmid-based antigen delivery systems using attenuated S. enterica. In addition, the S. enterica sspA promoter is able to elicit anti-tetanus toxoid antibodies in mice when the psspA-tetC expression cassette is integrated in single copy on the S. enterica chromosome.

Adhesins, Bacterial↗
PubMed2005

The phasevarion: a genetic system controlling coordinated, random switching of expression of multiple genes.

Several host-adapted bacterial pathogens contain methyltransferases associated with type III restriction-modification (R-M) systems that are subject to reversible, high-frequency on/off switching of expression (phase variation). To investigate the role of phase-variable expression of R-M systems, we made a mutant strain lacking the methyltransferase (mod) associated with a type III R-M system of Haemophilus influenzae and analyzed its phenotype. By microarray analysis, we identified a number of genes that were either up- or down-regulated in the mod mutant strain. This system reports the coordinated random switching of a set of genes in a bacterial pathogen and may represent a widely used mechanism.

Bacterial Proteins↗
PubMed2005

Copper sensitivity of cueO mutants of Escherichia coli K-12 and the biochemical suppression of this phenotype.

The cueO gene of Escherichia coli encodes a multi-copper oxidase, which contributes to copper tolerance in this bacterium. It was observed that a cueO mutant was highly sensitive to killing by copper ions when cells were grown on defined minimal media. Copper sensitivity was correlated with accumulation of copper in the mutant strain. Growth of the cueO mutant in the presence of copper could be restored by addition of divalent zinc and manganese ions or ferrous iron but not by other first row transition metal ions or magnesium ions. Copper toxicity towards a cueO mutant could also be suppressed by addition of the superoxide quencher 1,2-dihydroxybenzene-3,5-disulfonic acid (tiron), suggesting that a primary cause of copper toxicity is the copper-catalyzed production of superoxide anions in the cytoplasm.

Cell Survival↗
PubMed2005

Recombinant probiotics for treatment and prevention of enterotoxigenic Escherichia coli diarrhea.

BACKGROUND & AIMS: We have developed a therapeutic strategy for gastrointestinal infections that is based on molecular mimicry of host receptors for bacterial toxins on the surface of harmless gut bacteria. The aim of this study was to apply this to the development of a recombinant probiotic for treatment and prevention of diarrheal disease caused by enterotoxigenic Escherichia coli strains that produce heat-labile enterotoxin. METHODS: This was achieved by expressing glycosyltransferase genes from Neisseria meningitidis or Campylobacter jejuni in a harmless Escherichia coli strain (CWG308), resulting in the production of a chimeric lipopolysaccharide capable of binding heat-labile enterotoxin with high avidity. RESULTS: The strongest heat-labile enterotoxin binding was achieved with a construct (CWG308:pLNT) that expresses a mimic of lacto-N-neotetraose, which neutralized > or = 93.8% of the heat-labile enterotoxin activity in culture lysates of diverse enterotoxigenic Escherichia coli strains of both human and porcine origin. When tested with purified heat-labile enterotoxin, it was capable of adsorbing approximately 5% of its own weight of toxin. Weaker toxin neutralization was achieved with a construct that mimicked the ganglioside GM2. Preabsorption with, or coadministration of, CWG308:pLNT also resulted in significant in vivo protection from heat-labile enterotoxin-induced fluid secretion in rabbit ligated ileal loops. CONCLUSIONS: Toxin-binding probiotics such as those described here have considerable potential for prophylaxis and treatment of enterotoxigenic Escherichia coli-induced travelers' diarrhea.

Adrenal Glands↗
PubMed2005

NmlR of Neisseria gonorrhoeae: a novel redox responsive transcription factor from the MerR family.

A MerR-like regulator (NmlR -Neisseria merR-like Regulator) identified in the Neisseria gonorrhoeae genome lacks the conserved cysteines known to bind metal ions in characterized proteins of this family. Phylogenetic analysis indicates that NmlR defines a subfamily of MerR-like transcription factors with a distinctive pattern of conserved cysteines within their primary structure. NmlR regulates itself and three other genes in N. gonorrhoeae encoding a glutathione-dependent dehydrogenase (AdhC), a CPx-type ATPase (CopA) and a thioredoxin reductase (TrxB). An nmlR mutant lacked the ability to survive oxidative stress induced by diamide and cumene hydroperoxide. It also had > 50-fold lower NADH-S-nitrosoglutathione oxidoreductase activity consistent with a role for AdhC in protection against nitric oxide stress. The upstream sequences of the NmlR regulated genes contained typical MerR-like operator/promoter arrangements consisting of a dyad symmetry located between the -35 and -10 elements of the target genes. The NmlR target operator/promoters were cloned into a beta-galactosidase reporter system and promoter activity was repressed by the introduction of NmlR in trans. Promoter activity was activated by NmlR in the presence of diamide. Under metal depleted conditions NmlR did not repress P(AdhC) (or P(CopA)) promoter activity, but this was reversed in the presence of Zn(II), indicating repression was Zn(II)-dependent. Analysis of mutated promoters lacking the dyad symmetry revealed constitutive promoter activity which was independent of NmlR. Gel shift assays further confirmed that NmlR bound to the target promoters possessing the dyad symmetry. Site-directed mutagenesis of the four NmlR cysteine residues revealed that they were essential for activation of gene expression by NmlR.

Amino Acid Sequence↗
PubMed2005

Plasmid-encoded Tet B tetracycline resistance in Haemophilus parasuis.

The complete sequence of two plasmids, pHS-Tet (5.1 kb) and pHS-Rec (9.5 kb), isolated from Haemophilus parasuis strain HS1543 has been obtained. Plasmid pHS-Tet contains four open reading frames including a tet(B) tetracycline resistance gene which unusually did not have an associated tetR repressor gene. From a total of 45 H. parasuis isolates surveyed (15 international reference strains, 15 field isolates selected for their genetic diversity, and 15 recent Australian field isolates), 2 tetracycline-resistant field isolates (HS226 and HS1859) were identified. Analysis of three additional isolates from the same disease outbreak as strain HS1859 revealed a further tetracycline-resistant H. parasuis strain (HS1857, serovar 8) and a tetracycline-resistant Actinobacillus pleuropneumoniae strain (HS1861). An approximately 10.6-kb plasmid was identified in field isolate HS226 and outbreak strains HS1857, HS1859, and HS1861. Southern hybridization analysis of these plasmids showed that the Tet B determinant was present, and restriction digest comparisons suggest that these plasmids are related. This is believed to be the first report of native H. parasuis plasmids and Tet B-mediated tetracycline resistance in this microorganism.

Actinobacillus pleuropneumoniae↗
PubMed2005

Azurin of pathogenic Neisseria spp. is involved in defense against hydrogen peroxide and survival within cervical epithelial cells.

Laz, a lipid-modified azurin of the human pathogens Neisseria gonorrhoeae and Neisseria meningitidis, is involved in defense against oxidative stress and copper toxicity; laz mutant strains are hypersensitive to hydrogen peroxide and copper. The N. gonorrhoeae laz mutant also has decreased survival in an ex vivo primary human ectocervical epithelial assay.

Azurin↗