Population structure in the Neisseria, and the biological significance of fuzzy species.

Phenotypic and genetic variation in bacteria can take bewilderingly complex forms even within a single genus. One of the most intriguing examples of this is the genus Neisseria, which comprises both pathogens and commensals colonizing a variety of body sites and host species, and causing a range of disease. Complex relatedness among both named species and previously identified lineages of Neisseria makes it challenging to study their evolution. Using the largest publicly available collection of bacterial sequence data in combination with a population genetic analysis and experiment, we probe the contribution of inter-species recombination to neisserial population structure, and specifically whether it is more common in some strains than others. We identify hybrid groups of strains containing sequences typical of more than one species. These groups of strains, typical of a fuzzy species, appear to have experienced elevated rates of inter-species recombination estimated by population genetic analysis and further supported by transformation experiments. In particular, strains of the pathogen Neisseria meningitidis in the fuzzy species boundary appear to follow a different lifestyle, which may have considerable biological implications concerning distribution of novel resistance elements and meningococcal vaccine development. Despite the strong evidence for negligible geographical barriers to gene flow within the population, exchange of genetic material still shows directionality among named species in a non-uniform manner.

Regulation of pga operon expression and biofilm formation in Actinobacillus pleuropneumoniae by sigmaE and H-NS.

Clinical isolates of the porcine pathogen Actinobacillus pleuropneumoniae often form adherent colonies on agar plates due to expression of an operon, pgaABCD, encoding a poly-beta-1,6-N-acetyl-D-glucosamine (PGA) extracellular matrix. The adherent colony phenotype, which correlates with the ability to form biofilms on the surfaces of polystyrene plates, is lost following serial passage in broth culture, and repeated passage of the nonadherent variants on solid media does not result in reversion to the adherent colony phenotype. In order to investigate the regulation of PGA expression and biofilm formation in A. pleuropneumoniae, we screened a bank of transposon mutants of the nonadherent serovar 1 strain S4074(T) and identified mutations in two genes, rseA and hns, which resulted in the formation of the adherent colony phenotype. In other bacteria, including the Enterobacteriaceae, H-NS acts as a global gene regulator, and RseA is a negative regulator of the extracytoplasmic stress response sigma factor sigma(E). Transcription profiling of A. pleuropneumoniae rseA and hns mutants revealed that both sigma(E) and H-NS independently regulate expression of the pga operon. Transcription of the pga operon is initiated from a sigma(E) promoter site in the absence of H-NS, and upregulation of sigma(E) is sufficient to displace H-NS, allowing transcription to proceed. In A. pleuropneumoniae, H-NS does not act as a global gene regulator but rather specifically regulates biofilm formation via repression of the pga operon. Positive regulation of the pga operon by sigma(E) indicates that biofilm formation is part of the extracytoplasmic stress response in A. pleuropneumoniae.

Neisseria meningitidis GNA2132, a heparin-binding protein that induces protective immunity in humans.

GNA2132 is a Neisseria meningitidis antigen of unknown function, discovered by reverse vaccinology, which has been shown to induce bactericidal antibodies in animal models. Here we show that this antigen induces protective immunity in humans and it is recognized by sera of patients after meningococcal disease. The protein binds heparin in vitro through an Arg-rich region and this property correlates with increased survival of the unencapsulated bacterium in human serum. Furthermore, two proteases, the meningococcal NalP and human lactoferrin, cleave the protein upstream and downstream from the Arg-rich region, respectively. We conclude that GNA2132 is an important protective antigen of N. meningitidis and we propose to rename it, Neisserial Heparin Binding Antigen (NHBA).

Meningococcal biofilm growth on an abiotic surface - a model for epithelial colonization?

Neisseria meningitidis colonizes the human nasopharynx asymptomatically, often for prolonged periods, but occasionally invades from this site to cause life-threatening infection. In the nasopharynx aggregated organisms are closely attached to the epithelial surface, in a state in which the expression of components of the bacterial envelope differs significantly from that found in organisms multiplying exponentially in liquid phase culture or in the blood. We and others have hypothesized that here they are in the biofilm state, and to explore this we have investigated biofilm formation by the serogroup B strain MC58 on an abiotic surface, in a sorbarod system. Transcriptional changes were analysed, focusing on alteration in gene expression relevant to polysaccharide capsulation, lipooligosaccharide and outer-membrane protein synthesis - all phenotypes of importance in epithelial colonization. We report downregulation of genes controlling capsulation and the production of core oligosaccharide, and upregulation of genes encoding a range of outer-membrane components, reflecting phenotypic changes that have been established to occur in the colonizing state. A limited comparison with organisms recovered from an extended period of co-cultivation with epithelial cells suggests that this model system may better mirror natural colonization than do short-term meningococcal/epithelial cell co-cultivation systems. Modelling prolonged meningococcal colonization with a sorbarod system offers insight into gene expression during this important, but experimentally relatively inaccessible, phase of human infection.

Presence of copper- and zinc-containing superoxide dismutase in commensal Haemophilus haemolyticus isolates can be used as a marker to discriminate them from nontypeable H. influenzae isolates.

Respiratory isolates of Haemophilus haemolyticus are regularly misclassified as nontypeable (NT) Haemophilus influenzae due to an aberrant hemolytic reaction on blood agar, with implications for treatment. The presence of sodC or its cognate protein, copper-zinc superoxide dismutase, can distinguish respiratory isolates of H. haemolyticus from NT H. influenzae with 100% accuracy.

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.

A novel neisserial shuttle plasmid: a useful new tool for meningococcal research.

We report the identification and nucleotide sequence analysis of a cryptic plasmid pMIDG2830 from the Gram-negative bacterium Neisseria flavescens. The largest open reading frame encodes a protein similar to the replication protein, RepA, found in pAB49 from Acinetobacter baumannii and pNI10 from Pseudomonas. Modified by the incorporation of a kanamycin resistance cassette, the plasmid can be stably maintained in Escherichia coli and Neisseria meningitidis, and can be used as a shuttle plasmid in meningococcal research.

Rapid characterization of outer-membrane proteins in Neisseria lactamica by SELDI-TOF-MS (surface-enhanced laser desorption ionization-time-of-flight MS) for use in a meningococcal vaccine.

Immunological and epidemiological evidence suggests that the development of natural immunity to meningococcal disease results from colonization of the nasopharynx by commensal Neisseria species, particularly with Neisseria lactamica. We have reported previously that immunization with N. lactamica outer-membrane vesicles containing the major OMPs (outer-membrane proteins) protected mice against lethal challenge with meningococci of diverse serogroups and serotypes and has the potential to form the basis of a vaccine against meningococcal diseases [Oliver, Reddin, Bracegirdle et al. (2002) Infect. Immun. 70, 3621-3626]. In the present study, we have shown that biomass production and the profile of outer-membrane vesicle proteins may be affected by fermentation conditions and, in particular, media composition. Ciphergen SELDI-TOF Protein Chips were used as a rapid and sensitive new method in comparison with conventional SDS/PAGE. SELDI-TOF-MS (surface-enhanced laser-desorption ionization-time-of-flight MS) reproducibly identified three major OMPs (NspA, RmpM and PorB) and detected the changes in the protein profile when the growth medium was altered. The findings of this work indicate that SELDI-TOF-MS is a useful tool for the rapid optimization of OMP production in industrial fermentation processes and can be adapted as a Process Analytical Technology.

Expression of heterologous antigens in commensal Neisseria spp.: preservation of conformational epitopes with vaccine potential.

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.

Cloning, mutation and distribution of a putative lipopolysaccharide biosynthesis locus in Campylobacter jejuni.

A region encoding ORFs with homology to known lipopolysaccharide (LPS) biosynthesis genes was isolated from two strains of Campylobacter jejuni. One of the strains produces LPS, but the second strain is reported to produce only lipooligosaccharide (LOS) and therefore lacks the O-chain. The two strains shared six predicted ORFs, but an additional ORF, orfE, of unknown function was identified in the LOS-producing strain. Mutation of the shared wbeE (rfbE) homologue (orfF) or deletion of five of the seven genes reduced core reactivity with specific antiserum without affecting O-chain production. Mutation of either the capD homologue (orfG) or the unique orfE had no detectable effect on LOS or LPS production. The presence or absence of orfE in 36 isolates of C. jejuni did not correlate with LOS/LPS phenotype or serotype. However, after insertion of orfE into a LPS-producing orfE-negative strain the O-chain ladder was no longer detectable on Western blots. We were not able to disrupt the wbaP (rfbP) homologue (orfC) in C jejuni.