Jane Newcombe

Generation and characterization of a PhoP homologue mutant of Neisseria meningitidis

Two‐component regulatory systems are important regulators of virulence genes in a number of bacteria. Genes encoding a two‐component regulator system, with homology to the phoP/phoQ system in salmonella, were identified in the meningococcal genome. Allele replacement was used to generate a meningococcal knock‐out mutant of the regulator component of this system, and its phenotype was examined. The mutant displayed many differences in protein profiles compared with wild type, consistent with it being a gene‐regulatory mutation. Many of the growth characteristics of the mutant were similar to those of phoP mutants of salmonella: it was unable to grow at low concentrations of magnesium and was sensitive to defensins and other environmental stresses. Magnesium‐regulated differences in protein expression were abrogated in the mutant, indicating that the meningococcal PhoP/PhoQ system may, as in salmonella, respond to changes in environmental magnesium levels. These results are consistent with the PhoP homologue playing a similar role in the meningococcus to PhoP in salmonella and suggest that it may similarly be involved in the regulation of virulence genes in response to environmental stimuli in the meningococcus. In support of this conclusion, we found the mutant grew was unable to grow in mouse serum and was attenuated in its ability to traverse through a layer of human epithelial cells. Identification of those genes regulated by the meningococcal PhoP may provide a route towards the identification of virulence genes in the meningococcus.

Towards the Immunoproteome of Neisseria meningitidis

Despite the introduction of conjugated polysaccharide vaccines for many of the Neisseria meningitidis serogroups, neisserial infections continue to cause septicaemia and meningitis across the world. This is in part due to the difficulties in developing a, cross-protective vaccine that is effective against all serogroups, including serogroup B meningococci. Although convalescent N. meningitidis patients develop a natural long-lasting cross-protective immunity, the antigens that mediate this response remain unknown. To help define the target of this protective immunity we identified the proteins recognized by IgG in sera from meningococcal patients by a combination of 2D protein gels, western blots and mass spectrometry. Although a number of outer membrane antigens were identified the majority of the antigens were cytoplasmic, with roles in cellular processes and metabolism. When recombinant proteins were expressed and used to raise sera in mice, none of the antigens elicited a positive SBA result, however flow cytometry did demonstrate that some, including the ribosomal protein, RplY were localised to the neisserial cell surface.

Infection with an Avirulent phoP Mutant of Neisseria meningitidis Confers Broad Cross-Reactive Immunity

ABSTRACT Successful vaccines against serogroup A and C meningococcal strains have been developed, but current serogroup B vaccines provide protection against only a limited range of strains. The ideal meningococcal vaccine would provide cross-reactive immunity against the variety of strains that may be encountered in any community, but it is unclear whether the meningococcus possesses immune targets that have the necessary level of cross-reactivity. We have generated a phoP mutant of the meningococcus by allele exchange. PhoP is a component of a two-component regulatory system which in other bacteria is an important regulator of virulence gene expression. Inactivation of the PhoP-PhoQ system in Salmonella leads to avirulence, and phoP mutants have been shown to confer protection against virulent challenge. These mutants have been examined as potential live attenuated vaccines. We here show that a phoP mutant of the meningococcus is avirulent in a mouse model of infection. Moreover, infection of mice with the phoP mutant stimulated a bactericidal immune response that not only killed the infecting strain but also showed cross-reactive bactericidal activity against a range of strains with different serogroup, serotype, and serosubtyping antigens. Sera from the mutant-infected mice contained immunoglobulin G that bound to the surface of a range of meningococcal strains and mediated opsonophagocytosis of meningococci by human phagocytic cells. The meningococcal phoP mutant is thus a candidate live, attenuated vaccine strain and may also be used to identify cross-reactive protective antigens in the meningococcus.

Identification of proteins from tuberculin purified protein derivative (PPD) by LC-MS/MS.

The tuberculin purified protein derivative (PPD) is a widely used diagnostic antigen for tuberculosis, however it is poorly defined. Most mycobacterial proteins are extensively denatured by the procedure employed in its preparation, which explains previous difficulties in identifying constituents from PPD to characterize their behaviour in B- and T-cell reactions. We here described a proteomics-based characterization of PPD from several different sources by LC-MS/MS, which combines the solute separation power of HPLC, with the detection power of a mass spectrometer. The technique is able to identify proteins from complex mixtures of peptide fragments. A total of 171 different proteins were identified among the four PPD samples (two bovine PPD and two avium PPD) from Brazil and UK. The majority of the proteins were cytoplasmic (77.9%) and involved in intermediary metabolism and respiration (24.25%) but there was a preponderance of proteins involved in lipid metabolism. We identified a group of 21 proteins that are present in both bovine PPD but were not detected in avium PPD preparation. In addition, four proteins found in bovine PPD are absent in Mycobacterium bovis BCG vaccine strain. This study provides a better understanding of the tuberculin PPD components leading to the identification of additional antigens useful as reagents for specific diagnosis of tuberculosis.

Naturally occurring insertional inactivation of the porA gene of Neisseria meningitidis by integration of IS1301

Meningococcal meningitis is caused by Neisseria meningitidis. At present, there is an effective vaccine for some of the serogroups of this organism but not for the B strains, which are the most prevalent in the UK, Europe and the USA. The serogroup B polysaccharide is poorly immunogenic, so development of new candidate vaccines has focused on the outer membrane proteins, particularly the porin protein PorA. To develop a non-culture-based subtyping system for the meningococcus, we have used the polymerase chain reaction (PCR) in combination with single-stranded conformational polymorphism analysis (SSCP) to examine the meningococcal porA gene (Newcombe et al., 1997, J Clin Microbiol 35: 1809–1812). The VR1 region of porA was amplified using PCR primers that generated a PCR product of < 300 bp. However, out of the 25 clinical specimens examined, one blood specimen, JP280, generated a 1.1 kb PCR product (Fig. 1). The isolate was obtained from a 12-year-old boy from the Gloucester area in the UK who presented with meningitis, was treated and went on to make an uneventful recovery. The same 1.1 kb PCR product was generated using DNA isolated from a meningococcal strain cultured from the patient’s blood (Fig. 1) and was sequenced. The DNA sequence (EMBL AJ007406, AJ007407) matched the published sequences of the meningococcal porA gene (McGuinness et al., 1990, J Exp Med 171: 1871–1882) but was interrupted by a single copy of the insertion sequence IS1301 (Hammerschmidt et al., 1996, EMBO J 15: 192–198). The IS element had inserted within the VR1 region of porA in an identical position and orientation to that described for a type C strain recently isolated in Quebec, Canada (Arhin et al., 1998, Can J Microbiol 44: 56–63). Strain JP280 was typed by the Meningococcal Reference Laboratory as a C,2a:NT (non-subtypable), it did not react with any of the PorA antibodies, indicating that insertion of IS1301 sequence led to insertional inactivation of PorA expression. We have also examined DNA from 100 microbial isolates of N. meningitidis using PCR-SSCP and found two that were identical to JP280 with regard to both IS1301 insertion into the porA gene and the C,2a:NT typing. The isolates were both from a recent outbreak of meningococcal disease (CDR Weekly, Vol. 7 no. 44, 31 October 1997). The first (M97.252455) was isolated from the cerebrospinal fluid of a patient who had died of meningococcal septicaemia and the second (M97.252456) was isolated from a throat swab obtained from a contact of the first patient. IS1301 is known to be involved in reversible capsulation in Neisseria, by insertion into and excision from the SiaA gene locus (Hammerschmidt et al., 1996, EMBO J 15: 192–198). It is possible that similar excision events may restore expression of PorA in strains like JP280. However, the IS1301 insertion event was also detected in vivo within the blood of one patient (JP280), indicating that the PorA mutant strain was fully virulent and capable of causing both infections and meningitis. PorA is a major antigen of the meningococcus and a target for bactericidal antibodies. For these reasons, a number of vaccines and candidate vaccines have been developed that use PorA as a major immunogen (Ala’Aldeen and Cartwright, 1996, J Infect 33: 153–157). However, meningococcal strains like JP280 with an inactivated porA gene would not be subject to any protection afforded by a PorAbased vaccine. In addition, widespread immunization with a PorA-based vaccine would risk selecting for strains, Molecular Microbiology (1998) 30(2), 453–457

The acute-phase reactant C-reactive protein binds to phosphorylcholine- expressing Neisseria meningitidis and increases uptake by human phagocytes

ABSTRACT Neisseria meningitidis is a global cause of meningitis and septicemia. Immunity to N. meningitidis involves both innate and specific mechanisms with killing by serum bactericidal activity and phagocytic cells. C-reactive protein (CRP) is an acute-phase serum protein that has been shown to help protect the host from several bacterial pathogens, which it recognizes by binding to phosphorylcholine (PC) on their surfaces. Pathogenic Neisseria species can exhibit phase-variable PC modification on type 1 and 2 pili. We have shown that CRP can bind to piliated meningococci in a classical calcium-dependent manner. The binding of CRP to the meningococcus was concentration dependent, of low affinity, and specific for PC. CRP appears to act as an opsonin for N. meningitidis, as CRP-opsonized bacteria showed increased uptake by human macrophages and neutrophils. Further investigation into the downstream effects of CRP-bound N. meningitidis may lead us to a better understanding of meningococcal infection and help direct more effective therapeutic interventions.

Interrogation of global mutagenesis data with a genome scale model of Neisseria meningitidis to assess gene fitness in vitro and in sera

BackgroundNeisseria meningitidis is an important human commensal and pathogen that causes several thousand deaths each year, mostly in young children. How the pathogen replicates and causes disease in the host is largely unknown, particularly the role of metabolism in colonization and disease. Completed genome sequences are available for several strains but our understanding of how these data relate to phenotype remains limited.ResultsTo investigate the metabolism of N. meningitidis we generated and then selected a representative Tn5 library on rich medium, a minimal defined medium and in human serum to identify genes essential for growth under these conditions. To relate these data to a systems-wide understanding of the pathogens biology we constructed a genome-scale metabolic network: Nmb_iTM560. This model was able to distinguish essential and non-essential genes as predicted by the global mutagenesis. These essentiality data, the library and the Nmb_iTM560 model are powerful and widely applicable resources for the study of meningococcal metabolism and physiology. We demonstrate the utility of these resources by predicting and demonstrating metabolic requirements on minimal medium, such as a requirement for phosphoenolpyruvate carboxylase, and by describing the nutritional and biochemical status of N. meningitidis when grown in serum, including a requirement for both the synthesis and transport of amino acids.ConclusionsThis study describes the application of a genome scale transposon library combined with an experimentally validated genome-scale metabolic network of N. meningitidis to identify essential genes and provide novel insight into the pathogens metabolism both in vitro and during infection.

Identification of the immunoproteome of the meningococcus by cell surface immunoprecipitation and MS

Most healthy adults are protected from meningococcal disease by the presence of naturally acquired anti-meningococcal antibodies; however, the identity of the target antigens of this protective immunity remains unclear, particularly for protection against serogroup B disease. To identify the protein targets of natural protective immunity we developed an immunoprecipitation and proteomics approach to define the immunoproteome of the meningococcus. Sera from 10 healthy individuals showing serum bactericidal activity against both a meningococcal C strain (L91543) and the B strain MC58, together with commercially available pooled human sera, were used as probe antisera. Immunoprecipitation was performed with each serum sample and live cells from both meningococcal strains. Immunoprecipitated proteins were identified by MS. Analysis of the immunoproteome from each serum demonstrated both pan-reactive antigens that were recognized by most sera as well as subject-specific antigens. Most antigens were found in both meningococcal strains, but a few were strain-specific. Many of the immunoprecipitated proteins have been characterized previously as surface antigens, including adhesins and proteases, several of which have been recognized as vaccine candidate antigens, e.g. factor H-binding protein, NadA and neisserial heparin-binding antigen. The data demonstrate clearly the presence of meningococcal antibodies in healthy individuals with no history of meningococcal infection and a wide diversity of immune responses. The identification of the immunoreactive proteins of the meningococcus provides a basis for understanding the role of each antigen in the natural immunity associated with carriage and may help to design vaccination strategies.

Phenotypic microarrays suggest Escherichia coli ST131 is not a metabolically distinct lineage of extra-intestinal pathogenic E. coli.

Extraintestinal pathogenic E. coli (ExPEC) are the major aetiological agent of urinary tract infections (UTIs) in humans. The emergence of the CTX-M producing clone E. coli ST131 represents a major challenge to public health worldwide. A recent study on the metabolic potential of E. coli isolates demonstrated an association between the E. coli ST131 clone and enhanced utilisation of a panel of metabolic substrates. The studies presented here investigated the metabolic potential of ST131 and other major ExPEC ST isolates using 120 API test reagents and found that ST131 isolates demonstrated a lower metabolic activity for 5 of 120 biochemical tests in comparison to non-ST131 ExPEC isolates. Furthermore, comparative phenotypic microarray analysis showed a lack of specific metabolic profile for ST131 isolates countering the suggestion that these bacteria are metabolically fitter and therefore more successful human pathogens.

Differences in carbon source utilisation distinguish Campylobacter jejuni from Campylobacter coli

BackgroundCampylobacter jejuni and C. coli are human intestinal pathogens that are the most frequent causes of bacterial foodborne gastroenteritis in humans in the UK. In this study, we aimed to characterise the metabolic diversity of both C. jejuni and C. coli using a diverse panel of clinical strains isolated from the UK, Pakistan and Thailand, thereby representing both the developed and developing world. Our aim was to apply multi genome analysis and Biolog phenotyping to determine differences in carbon source utilisation by C. jejuni and C. coli strains.ResultsWe have identified a core set of carbon sources (utilised by all strains tested) and a set that are differentially utilised for a diverse panel of thirteen C. jejuni and two C. coli strains. This study used multi genome analysis to show that propionic acid is utilised only by C. coli strains tested. A broader PCR screen of 16 C. coli strains and 42 C. jejuni confirmed the absence of the genes needed for propanoate metabolism.ConclusionsFrom our analysis we have identified a phenotypic method and two genotypic methods based on propionic utilisation that might be applicable for distinguishing between C. jejuni and C. coli.