Structural Alterations in a Component of Cytochrome c Oxidase and Molecular Evolution of Pathogenic Neisseria in Humans

Three closely related bacterial species within the genus Neisseria are of importance to human disease and health. Neisseria meningitidis is a major cause of meningitis, while Neisseria gonorrhoeae is the agent of the sexually transmitted disease gonorrhea and Neisseria lactamica is a common, harmless commensal of children. Comparative genomics have yet to yield clear insights into which factors dictate the unique host-parasite relationships exhibited by each since, as a group, they display remarkable conservation at the levels of nucleotide sequence, gene content and synteny. Here, we discovered two rare alterations in the gene encoding the CcoP protein component of cytochrome cbb3 oxidase that are phylogenetically informative. One is a single nucleotide polymorphism resulting in CcoP truncation that acts as a molecular signature for the species N. meningitidis. We go on to show that the ancestral ccoP gene arose by a unique gene duplication and fusion event and is specifically and completely distributed within species of the genus Neisseria. Surprisingly, we found that strains engineered to express either of the two CcoP forms conditionally differed in their capacity to support nitrite-dependent, microaerobic growth mediated by NirK, a nitrite reductase. Thus, we propose that changes in CcoP domain architecture and ensuing alterations in function are key traits in successive, adaptive radiations within these metapopulations. These findings provide a dramatic example of how rare changes in core metabolic proteins can be connected to significant macroevolutionary shifts. They also show how evolutionary change at the molecular level can be linked to metabolic innovation and its reversal as well as demonstrating how genotype can be used to infer alterations of the fitness landscape within a single host

The Meningococcal Vaccine Candidate Neisserial Surface Protein A (NspA) Binds to Factor H and Enhances Meningococcal Resistance to Complement

Complement forms an important arm of innate immunity against invasive meningococcal infections. Binding of the alternative complement pathway inhibitor factor H (fH) to fH-binding protein (fHbp) is one mechanism meningococci employ to limit complement activation on the bacterial surface. fHbp is a leading vaccine candidate against group B Neisseria meningitidis. Novel mechanisms that meningococci employ to bind fH could undermine the efficacy of fHbp-based vaccines. We observed that fHbp deletion mutants of some meningococcal strains showed residual fH binding suggesting the presence of a second receptor for fH. Ligand overlay immunoblotting using membrane fractions from one such strain showed that fH bound to a ∼17 kD protein, identified by MALDI-TOF analysis as Neisserial surface protein A (NspA), a meningococcal vaccine candidate whose function has not been defined. Deleting nspA, in the background of fHbp deletion mutants, abrogated fH binding and mAbs against NspA blocked fH binding, confirming NspA as a fH binding molecule on intact bacteria. NspA expression levels vary among strains and expression correlated with the level of fH binding; over-expressing NspA enhanced fH binding to bacteria. Progressive truncation of the heptose (Hep) I chain of lipooligosaccharide (LOS), or sialylation of lacto-N-neotetraose LOS both increased fH binding to NspA-expressing meningococci, while expression of capsule reduced fH binding to the strains tested. Similar to fHbp, binding of NspA to fH was human-specific and occurred through fH domains 6–7. Consistent with its ability to bind fH, deleting NspA increased C3 deposition and resulted in increased complement-dependent killing. Collectively, these data identify a key complement evasion mechanism with important implications for ongoing efforts to develop meningococcal vaccines that employ fHbp as one of its components

Transcriptome Analysis of Neisseria meningitidis in Human Whole Blood and Mutagenesis Studies Identify Virulence Factors Involved in Blood Survival

During infection Neisseria meningitidis (Nm) encounters multiple environments within the host, which makes rapid adaptation a crucial factor for meningococcal survival. Despite the importance of invasion into the bloodstream in the meningococcal disease process, little is known about how Nm adapts to permit survival and growth in blood. To address this, we performed a time-course transcriptome analysis using an ex vivo model of human whole blood infection. We observed that Nm alters the expression of ≈30% of ORFs of the genome and major dynamic changes were observed in the expression of transcriptional regulators, transport and binding proteins, energy metabolism, and surface-exposed virulence factors. In particular, we found that the gene encoding the regulator Fur, as well as all genes encoding iron uptake systems, were significantly up-regulated. Analysis of regulated genes encoding for surface-exposed proteins involved in Nm pathogenesis allowed us to better understand mechanisms used to circumvent host defenses. During blood infection, Nm activates genes encoding for the factor H binding proteins, fHbp and NspA, genes encoding for detoxifying enzymes such as SodC, Kat and AniA, as well as several less characterized surface-exposed proteins that might have a role in blood survival. Through mutagenesis studies of a subset of up-regulated genes we were able to identify new proteins important for survival in human blood and also to identify additional roles of previously known virulence factors in aiding survival in blood. Nm mutant strains lacking the genes encoding the hypothetical protein NMB1483 and the surface-exposed proteins NalP, Mip and NspA, the Fur regulator, the transferrin binding protein TbpB, and the L-lactate permease LctP were sensitive to killing by human blood. This increased knowledge of how Nm responds to adaptation in blood could also be helpful to develop diagnostic and therapeutic strategies to control the devastating disease cause by this microorganism

Recent progress in the prevention of serogroup B meningococcal disease

The widespread use of meningococcal polysaccharide conjugate vaccines has highlighted the challenge of providing protection against serogroup B disease. Over a period of four decades vaccine development has focused on sub-capsular, protein antigens, first with outer membrane vesicle (OMV) vaccines against epidemic outbreaks, and more recently on new multicomponent vaccines designed to offer better cross-protection against the antigenically diverse strains responsible for endemic disease. Because of the low incidence of meningococcal disease, the protective efficacy of these vaccines has not been determined in clinical studies and their licensure has been based on serological data; however, the serological assays used to predict protective coverage have limitations. As a result, evidence of the effectiveness of these vaccines against different strains and the contribution of specific antigens to protection can only be provided by epidemiological analyses following their implementation in sufficiently large populations. The recent inclusion of the 4CMenB vaccine, Bexsero®, in the infant immunization program in the UK has provided preliminary evidence that the vaccine is effective. On-going surveillance will provide valuable data on its longer-term impact and antigenic coverage. Further development of protein-based vaccines against meningococcal disease is anticipated to improve antigenic coverage and adjust to changes in circulating strains. At the same time, alternative immunization strategies may be explored to improve overall vaccine effectiveness by, for example, protecting the youngest infants or providing herd protection

Recent progress in the prevention of serogroup B meningococcal disease

The widespread use of meningococcal polysaccharide conjugate vaccines has highlighted the challenge of providing protection against serogroup B disease. Over a period of four decades vaccine development has focused on sub-capsular, protein antigens, first with outer membrane vesicle (OMV) vaccines against epidemic outbreaks, and more recently on new multicomponent vaccines designed to offer better cross-protection against the antigenically diverse strains responsible for endemic disease. Because of the low incidence of meningococcal disease, the protective efficacy of these vaccines has not been determined in clinical studies and their licensure has been based on serological data; however, the serological assays used to predict protective coverage have limitations. As a result, evidence of the effectiveness of these vaccines against different strains and the contribution of specific antigens to protection can only be provided by epidemiological analyses following their implementation in sufficiently large populations. The recent inclusion of the 4CMenB vaccine, Bexsero®, in the infant immunization program in the UK has provided preliminary evidence that the vaccine is effective. On-going surveillance will provide valuable data on its longer-term impact and antigenic coverage. Further development of protein-based vaccines against meningococcal disease is anticipated to improve antigenic coverage and adjust to changes in circulating strains. At the same time, alternative immunization strategies may be explored to improve overall vaccine effectiveness by, for example, protecting the youngest infants or providing herd protection

Role of sigma H in expression of the fumarase gene (citG) in vegetative cells of Bacillus subtilis 168.

The fumarase gene (citG) of Bacillus subtilis is transcribed from two promoter regions, citGp1 and citGp2 (P1 and P2); the P2 promoter is used by the E sigma H form of RNA polymerase. In order to study the role of P1 and P2 in citG expression, the promoter region and various deletion derivatives that effectively separate P1 and P2 were fused to the Escherichia coli beta-galactosidase gene (lacZ) and introduced into the chromosome in single copy at the amyE locus. P1 functioned to provide a relatively low and stable basal level of fumarase activity throughout growth. In contrast, P2 activity was found to vary over at least a 50-fold range and was responsible for regulating fumarase activity during growth and sporulation in a rich medium and in response to changes in carbon source. To further investigate the role of sigma H in fumarase regulation, citGp2-lacZ fusions were introduced into a strain in which the expression of the chromosomal spoOH gene was under the control of the isopropylthiogalactopyranoside-inducible spac promoter. Induction of pspac did not lead to P2 induction, suggesting that citG expression is not regulated at the level of spoOH transcription

The effect of iron availability on transcription of the Neisseria meningitidis fHbp gene varies among clonal complexes.

Factor H binding protein (fHbp) is a major antigenic component of novel vaccines designed to protect against meningococcal disease. Prediction of the potential coverage of these vaccines is difficult, as fHbp is antigenically variable and levels of expression differ among isolates. Transcriptional regulation of the fHbp gene is poorly understood, although evidence suggests that oxygen availability is involved. In this study iron accessibility was found to affect fHbp transcription. However, regulation differed among meningococcal clonal complexes (ccs). For the majority of isolates, increased iron concentrations upregulated transcription. This effect was enhanced by the presence of a 181 bp insertion element upstream of fHbp, associated with isolates belonging to cc4 and cc5. Conversely, meningococci belonging to cc32 showed iron-repressed control of fHbp, as regulation was dominated by cotranscription with the iron-repressed upstream gene cbbA. These results highlight the complexity of fHbp regulation and demonstrate that control of transcription can vary among genetic lineages