Paul R. Langford

Actinobacillus pleuropneumoniae : pathobiology and pathogenesis of infection

Actinobacillus pleuropneumoniae causes porcine pleuropneumonia, a highly contagious disease for which there is no effective vaccine. This review considers how adhesins, iron-acquisition factors, capsule and lipopolysaccharide, RTX cytotoxins and other potential future vaccine components contribute to colonisation, to avoidance of host clearance mechanisms and to damage of host tissues.

Diagnosis of Childhood Tuberculosis and Host RNA Expression in Africa

BACKGROUND Improved diagnostic tests for tuberculosis in children are needed. We hypothesized that transcriptional signatures of host blood could be used to distinguish tuberculosis from other diseases in African children who either were or were not infected with the human immunodeficiency virus (HIV). METHODS The study population comprised prospective cohorts of children who were undergoing evaluation for suspected tuberculosis in South Africa (655 children), Malawi (701 children), and Kenya (1599 children). Patients were assigned to groups according to whether the diagnosis was culture-confirmed tuberculosis, culture-negative tuberculosis, diseases other than tuberculosis, or latent tuberculosis infection. Diagnostic signatures distinguishing tuberculosis from other diseases and from latent tuberculosis infection were identified from genomewide analysis of RNA expression in host blood. RESULTS We identified a 51-transcript signature distinguishing tuberculosis from other diseases in the South African and Malawian children (the discovery cohort). In the Kenyan children (the validation cohort), a risk score based on the signature for tuberculosis and for diseases other than tuberculosis showed a sensitivity of 82.9% (95% confidence interval [CI], 68.6 to 94.3) and a specificity of 83.6% (95% CI, 74.6 to 92.7) for the diagnosis of culture-confirmed tuberculosis. Among patients with cultures negative for Mycobacterium tuberculosis who were treated for tuberculosis (those with highly probable, probable, or possible cases of tuberculosis), the estimated sensitivity was 62.5 to 82.3%, 42.1 to 80.8%, and 35.3 to 79.6%, respectively, for different estimates of actual tuberculosis in the groups. In comparison, the sensitivity of the Xpert MTB/RIF assay for molecular detection of M. tuberculosis DNA in cases of culture-confirmed tuberculosis was 54.3% (95% CI, 37.1 to 68.6), and the sensitivity in highly probable, probable, or possible cases was an estimated 25.0 to 35.7%, 5.3 to 13.3%, and 0%, respectively; the specificity of the assay was 100%. CONCLUSIONS RNA expression signatures provided data that helped distinguish tuberculosis from other diseases in African children with and those without HIV infection. (Funded by the European Union Action for Diseases of Poverty Program and others).

Bacterial copper- and zinc-cofactored superoxide dismutase contributes to the pathogenesis of systemic salmonellosis.

Copper/zinc‐cofactored superoxide dismutase ([Cu,Zn]‐SOD) has been found in the periplasm of many bacterial species but its biological function is unknown. Here we report the cloning and characterization of sodC, encoding [Cu,Zn]‐SOD, from Salmonella typhimurium. The predicted protein sequence shows only 58% identity to Escherichia coli SodC, and from this its chromosomal location and its immediate proximity to a phage gene, sodC, in Salmonella is speculated to have been acquired by bacteriophage‐mediated horizontal transfer from an unknown donor. A sodC mutant of S. typhimurium was unimpaired on aerobic growth in rich medium but showed enhanced sensitivity in vitro to the microbicidal action of superoxide. S. typhimurium, S. choleraesuis and S. dublin sodC mutants showed reduced lethality in a mouse model of oral infection and persisted in significantly lower numbers in livers and spleens after intraperitoneal infection, suggesting that [Cu,Zn]‐SOD plays a role in pathogenicity, protecting Salmonella against oxygen radical‐mediated host defences. There was, however, no observable difference compared with wild type in the interaction of sodC mutants with porcine pleural, mouse peritoneal or J774 macrophages in vitro, perhaps reflecting the hierarchical capacity of different macrophage lines to kill Salmonella, the most efficient overwhelming the proposed protective effect of periplasmic SOD.

Bacterial [Cu,Zn]-superoxide dismutase : phylogenetically distinct from the eukaryotic enzyme, and not so rare after all !

Copper- and zinc-containing superoxide dismutases ([Cu,Zn]-SODs) are generally considered almost exclusively eukaryotic enzymes, protecting the cytosol and extracellular compartments of higher organisms from damage by oxygen free-radicals. The recent description of a few examples of bacterial forms of the enzyme, located in the periplasm of different Gram-negative micro-organisms, prompted a re-evaluation of this general perception. A PCR-based approach has been developed and used successfully to identify bacterial genes encoding [Cu,Zn]-SOD in a wide range of important human and animal pathogens-members of the Haemophilus, Actinobacillus and Pasteurella (HAP) group, and Neisseria meningitidis. Comparison of [Cu,Zn]-SOD peptide sequences found in Haemophilus ducreyi, Actinobacillus pleuropneumoniae, Actinobacillus actinomycetemcomitans, Pasteurella multocida, and N. meningitidis with previously described bacterial proteins and examples of eukaryotic [Cu,Zn]-SOD has shown that the bacterial proteins constitute a distinct family apparently widely separated in evolutionary terms from the eukaryotic examples. The widespread occurrence of [Cu,Zn]-SOD in the periplasm of bacterial pathogens, appropriately located to dismute exogenously derived superoxide radical anions, suggests that this enzyme may play a role in the interactive biology of organisms with their hosts and so contribute to their capacity to cause disease.

Dysbiosis Anticipating Necrotizing Enterocolitis in Very Premature Infants

Using 16S rRNA gene sequencing and targeted culture, we compared microbiota in fecal samples from infants with necrotizing enterocolitis (NEC) and controls. Two significant signatures were associated with NEC: 1 with dominant Clostridium perfringens and 1 with dominant Enterobacteriaceae.

Factor H, a regulator of complement activity, is a major determinant of meningococcal disease susceptibility in UK Caucasian patients

Defence against Neisseria meningitidis involves complement-mediated bactericidal activity. Factor H (fH) down-regulates complement activation. A putatively functional single-nucleotide-polymorphism (SNP) exists within a presumed nuclear-factor-kappa-B responsive element (NF-kB) in the fH gene (C-496T). Genetic and functional investigations were carried out to determine whether C-496T has a role in meningococcal disease (MD) susceptibility. Genetic susceptibility was investigated in 2 independent studies, a case-control and family-based transmission-disequilibrium-test (TDT), using 2 separate cohorts of UK Caucasian patients. MD susceptibility was both genetically associated with the C/C homozygous genotype (OR = 2.0, 95% CI 1.3 – 3.2, p = 0.001) and linked to the C allele (p = 0.04), the association being most significant in serogroup C infected patients (OR = 2.9, 95% CI 1.6 – 5.5, p = 0.0002). FH serum concentrations were also associated with C-496T genotype, with highest fH concentrations in C/C homozygous individuals (p = 0.01). Functional studies showed NF-kappa-B binding to the C-496T-containing region and that pre-incubation of fH with meningococci reduced bactericidal activity and increased meningococci B and C survival in blood. This study shows that C-496T is both associated and linked with MD and that individuals possessing the fH C-496T C/C genotype are more likely to have increased serum fH protein levels, have reduced bactericidal activity against meningococci and be at an increased risk of contracting MD.

Identification of Actinobacillus pleuropneumoniae Genes Important for Survival during Infection in Its Natural Host

ABSTRACT Actinobacillus pleuropneumoniae is a strict respiratory tract pathogen of swine and is the causative agent of porcine pleuropneumonia. We have used signature-tagged mutagenesis (STM) to identify genes required for survival of the organism within the pig. A total of 2,064 signature-tagged Tn10 transposon mutants were assembled into pools of 48 each, and used to inoculate pigs by the endotracheal route. Out of 105 mutants that were consistently attenuated in vivo, only 11 mutants showed a >2-fold reduction in growth in vitro compared to the wild type, whereas 8 of 14 mutants tested showed significant levels of attenuation in pig as evidenced from competitive index experiments. Inverse PCR was used to generate DNA sequence of the chromosomal domains flanking each transposon insertion. Only one sibling pair of mutants was identified, but three apparent transposon insertion hot spots were found—an anticipated consequence of the use of a Tn10-based system. Transposon insertions were found within 55 different loci, and similarity (BLAST) searching identified possible analogues or homologues for all but four of these. Matches included proteins putatively involved in metabolism and transport of various nutrients or unknown substances, in stress responses, in gene regulation, and in the production of cell surface components. Ten of the sequences have homology with genes involved in lipopolysaccharide and capsule production. The results highlight the importance of genes involved in energy metabolism, nutrient uptake and stress responses for the survival of A. pleuropneumoniae in its natural host: the pig.

The Complete Genome Sequence of Actinobacillus pleuropneumoniae L20 (Serotype 5b)

There are 16 capsule-based serotypes of Actinobacillus pleuropneumoniae, all of which are capable of causing disease in pigs. Here we report the finished and annotated genome sequence of the reference serotype 5b strain L20. This strain has a rough appearance and readily forms biofilms, as is typical for most field isolates.

Biomarker discovery in infectious diseases using SELDI

Surface enhanced laser desorption ionization-time of flight is a mass spectrometric-based method that requires a minimal amount of sample for analysis and can be used for high-throughput screening. It has been used to discover serum or tissue protein signatures and biomarkers for infectious diseases in the fields of virology (hepatitis B and C viruses, severe acute respiratory syndrome, HIV-1, human T-cell leukemia virus-1 and BK virus), parasitology (trypanosomiasis) and bacteriology (intra-amniotic inflammation, tuberculosis and bacterial endocarditis). The protein signatures, or biomarkers, can be used to diagnose infection, predict disease states and to inform on disease processes. Careful attention to experimental design, sample handling and storage, and the use of appropriate internal controls is crucial to success.

Expression of Heterologous Antigens in Commensal Neisseria spp.: Preservation of Conformational Epitopes with Vaccine Potential

ABSTRACT Commensal neisseriae share with Neisseria meningitidis (meningococcus) a tendency towards overproduction of the bacterial outer envelope, leading to the formation and release during growth of outer membrane vesicles (OMVs). OMVs from both meningococci and commensal neisseriae have shown promise as vaccines to protect against meningococcal disease. We report here the successful expression at high levels of heterologous proteins in commensal neisseriae and the display, in its native conformation, of one meningococcal outer membrane protein vaccine candidate, NspA, in OMVs prepared from such a recombinant Neisseria flavescens strain. These NspA-containing OMVs conferred protection against otherwise lethal intraperitoneal challenge of mice with N. meningitidis serogroup B, and sera raised against them mediated opsonophagocytosis of meningococcal strains expressing this antigen. This development promises to facilitate the design of novel vaccines containing membrane protein antigens that are otherwise difficult to present in native conformation that provide cross-protective efficacy in the prevention of meningococcal disease.