Bacterial rhomboid proteases mediate quality control of orphan membrane proteins.

Although multiprotein membrane complexes play crucial roles in bacterial physiology and virulence, the mechanisms governing their quality control remain incompletely understood. In particular, it is not known how unincorporated, orphan components of protein complexes are recognised and eliminated from membranes. Rhomboids, the most widespread and largest superfamily of intramembrane proteases, are known to play key roles in eukaryotes. In contrast, the function of prokaryotic rhomboids has remained enigmatic. Here, we show that the Shigella sonnei rhomboid proteases GlpG and the newly identified Rhom7 are involved in membrane protein quality control by specifically targeting components of respiratory complexes, with the metastable transmembrane domains (TMDs) of rhomboid substrates protected when they are incorporated into a functional complex. Initial cleavage by GlpG or Rhom7 allows subsequent degradation of the orphan substrate. Given the occurrence of this strategy in an evolutionary ancient organism and the presence of rhomboids in all domains of life, it is likely that this form of quality control also mediates critical events in eukaryotes and protects cells from the damaging effects of orphan proteins.

Genome-wide association studies reveal the role of polymorphisms affecting factor H binding protein expression in host invasion by Neisseria meningitidis.

Many invasive bacterial diseases are caused by organisms that are ordinarily harmless components of the human microbiome. Effective interventions against these microbes require an understanding of the processes whereby symbiotic or commensal relationships transition into pathology. Here, we describe bacterial genome-wide association studies (GWAS) of Neisseria meningitidis, a common commensal of the human respiratory tract that is nevertheless a leading cause of meningitis and sepsis. An initial GWAS discovered bacterial genetic variants, including single nucleotide polymorphisms (SNPs), associated with invasive meningococcal disease (IMD) versus carriage in several loci across the meningococcal genome, encoding antigens and other extracellular components, confirming the polygenic nature of the invasive phenotype. In particular, there was a significant peak of association around the fHbp locus, encoding factor H binding protein (fHbp), which promotes bacterial immune evasion of human complement by recruiting complement factor H (CFH) to the meningococcal surface. The association around fHbp with IMD was confirmed by a validation GWAS, and we found that the SNPs identified in the validation affected the 5' region of fHbp mRNA, altering secondary RNA structures, thereby increasing fHbp expression and enhancing bacterial escape from complement-mediated killing. This finding is consistent with the known link between complement deficiencies and CFH variation with human susceptibility to IMD. These observations demonstrate the importance of human and bacterial genetic variation across the fHbp:CFH interface in determining IMD susceptibility, the transition from carriage to disease.

Commensal Neisseria cinerea impairs Neisseria meningitidis microcolony development and reduces pathogen colonisation of epithelial cells.

It is increasingly being recognised that the interplay between commensal and pathogenic bacteria can dictate the outcome of infection. Consequently, there is a need to understand how commensals interact with their human host and influence pathogen behaviour at epithelial surfaces. Neisseria meningitidis, a leading cause of sepsis and meningitis, exclusively colonises the human nasopharynx and shares this niche with several other Neisseria species, including the commensal Neisseria cinerea. Here, we demonstrate that during adhesion to human epithelial cells N. cinerea co-localises with molecules that are also recruited by the meningococcus, and show that, similar to N. meningitidis, N. cinerea forms dynamic microcolonies on the cell surface in a Type four pilus (Tfp) dependent manner. Finally, we demonstrate that N. cinerea colocalises with N. meningitidis on the epithelial cell surface, limits the size and motility of meningococcal microcolonies, and impairs the effective colonisation of epithelial cells by the pathogen. Our data establish that commensal Neisseria can mimic and affect the behaviour of a pathogen on epithelial cell surfaces.

Sugar coating: bacterial protein glycosylation and host-microbe interactions.

Bacterial surfaces are rich in glycoconjugates such as capsules, lipopolysaccharides, and peptidoglycans. The discovery of prokaryotic protein glycosylation systems has revealed that many bacteria also have the capacity to synthesise a diverse array of protein glycans, in some cases using novel strategies that differ from those of eukaryotes. Despite advances in our understanding of glycan biosynthesis and the proteins that are targets of glycosylation in bacteria, the roles of these modifications are relatively less well explored. We present an overview of bacterial protein glycosylation systems in bacteria that are relevant to human health, and discuss current evidence which indicates that glycosylation of proteins may impact upon fundamental processes such as bacterial motility, adhesion, and the modulation of immune responses.

Contribution of σ70 and σN Factors to Expression of Class II pilE in Neisseria meningitidis.

Neisseria meningitidis expresses multicomponent organelles called type four pili (Tfp), which are key virulence factors required for attachment to human cells during carriage and disease. Pilin (PilE) is the main component of Tfp, and N. meningitidis isolates either have a class I pilE locus and express pilins that undergo antigenic variation or have a class II pilE locus and express invariant pilins. The transcriptional regulation of class I pilE has been studied in both N. meningitidis and Neisseria gonorrhoeae, while the control of expression of class II pilE has been elucidated in the nonpathogenic species Neisseria elongata However, the factors that govern the regulation of the class II pilE gene in N. meningitidis are not known. In this work, we have bioinformatically and experimentally identified the class II pilE promoter. We confirmed the presence of conserved σ70 and σN-dependent promoters upstream of pilE in a collection of meningococcal genomes and demonstrated that class II pilE expression initiates from the σ70 family-dependent promoter. By deletion or overexpression of sigma factors, we showed that σN, σH, and σE do not affect class II pilin expression. These findings are consistent with a role of the housekeeping σD in expression of this important component of Tfp. Taken together, our data indicate that the σ-dependent network responsible for the expression of class II pilE has been selected to maintain pilE expression, consistent with the essential roles of Tfp in colonization and pathogenesis.IMPORTANCE The type four pilus (Tfp) of Neisseria meningitidis contributes to fundamental processes such as adhesion, transformation, and disease pathology. Meningococci express one of two distinct classes of Tfp (class I or class II), which can be distinguished antigenically or by the major subunit (pilE) locus and its genetic context. The factors that govern transcription of the class II pilE gene are not known, even though it is present in isolates that cause epidemic disease. Here we show that the transcription of class II pilE is maintained throughout growth and under different stress conditions and is driven by a σ70-dependent promoter. This is distinct from Tfp regulation in nonpathogenic Neisseria spp. and may confer an advantage during host-cell interaction and infection.

Neisseria cinerea isolates can adhere to human epithelial cells by type IV pilus-independent mechanisms.

In pathogenic Neisseria species the type IV pili (Tfp) are of primary importance in host-pathogen interactions. Tfp mediate initial bacterial attachment to cell surfaces and formation of microcolonies via pilus-pilus interactions. Based on genome analysis, many non-pathogenic Neisseria species are predicted to express Tfp, but aside from studies on Neisseria elongata, relatively little is known about the formation and function of pili in these organisms. Here, we have analysed pilin expression and the role of Tfp in Neisseria cinerea. This non-pathogenic species shares a close taxonomic relationship to the pathogen Neisseria meningitidis and also colonizes the human oropharyngeal cavity. Through analysis of non-pathogenic Neisseria genomes we identified two genes with homology to pilE, which encodes the major pilin of N. meningitidis. We show which of the two genes is required for Tfp expression in N. cinerea and that Tfp in this species are required for DNA competence, similar to other Neisseria. However, in contrast to the meningococcus, deletion of the pilin gene did not impact the association of N. cinerea to human epithelial cells, demonstrating that N. cinerea isolates can adhere to human epithelial cells by Tfp-independent mechanisms.

Characterization of a novel antisense RNA in the major pilin locus of Neisseria meningitidis influencing antigenic variation.

UNLABELLED: Expression of type four pili (Tfp) is essential for virulence in Neisseria meningitidis. Pili mediate adhesion, bacterial aggregation, and DNA uptake. In N. meningitidis, the major pilin subunit is encoded by the pilE gene. In some strains, PilE is subject to phase and antigenic variation, which can alter Tfp properties and together offer a possible mechanism of immune escape. Pilin expression and antigenic variation can be modulated in response to environmental cues; however, the precise mechanisms of such regulation remain unclear. We identified a promoter in the pilE locus, 3' of the pilE coding sequence, on the antisense (AS) strand which is conserved in meningococci. We show that this promoter directs transcription of an AS RNA that is expressed during specific growth phases and in response to salt stress. Furthermore, we demonstrate that the transcript encompasses sequences complementary to the entire pilE coding sequence and 5' untranslated region. AS RNAs can regulate the gene on the sense strand by altering transcript stability or translation. However, by using Northern blotting, quantitative reverse transcription-PCR (RT-PCR), and Western blotting, we found no significant AS RNA-dependent changes in pilE transcript or protein level. Instead, our data indicate that the AS RNA influences pilin antigenic variation. This work provides further insights into the complex regulation of pilin expression and variation in pathogenic Neisseria. IMPORTANCE: Pathogenic Neisseria spp. express type four pili (Tfp) which are important for adhesion, aggregation and transformation. Some strains of N. meningitidis are able to vary the sequence of the major subunit (PilE) of the Tfp. The mechanisms underlying this variation are not fully defined, but the process requires several noncoding elements that are found adjacent to the pilE gene. In this work, we identified a cis-encoded RNA antisense to pilE in N. meningitidis. By using Northern blotting and RT-PCR analysis, we found that the RNA is expressed in stationary phase or following salt stress. Our work also indicates that this RNA does not significantly affect pilE or pilin expression levels but instead appears to modulate pilin variation.

Non-pathogenic Neisseria: members of an abundant, multi-habitat, diverse genus.

The genus Neisseria contains the important pathogens Neisseria meningitidis and Neisseria gonorrhoeae. These Gram-negative coccoid bacteria are generally thought to be restricted to humans and inhabit mucosal surfaces in the upper respiratory and genito-urinary tracts. While the meningococcus and gonococcus have been widely studied, far less attention has been paid to other Neisseria species. Here we review current knowledge of the distribution of commensal Neisseria in humans and other hosts. Analysis of the microbiome has revealed that Neisseria is an abundant member of the oropharyngeal flora, and we review its potential impact on health and disease. Neisseria also exhibit remarkable diversity, exhibiting both coccoid and rod-shaped morphologies, as well as environmental strains which are capable of degrading complex organic molecules.

Distinct binding and immunogenic properties of the gonococcal homologue of meningococcal factor h binding protein.

Neisseria meningitidis is a leading cause of sepsis and meningitis. The bacterium recruits factor H (fH), a negative regulator of the complement system, to its surface via fH binding protein (fHbp), providing a mechanism to avoid complement-mediated killing. fHbp is an important antigen that elicits protective immunity against the meningococcus and has been divided into three different variant groups, V1, V2 and V3, or families A and B. However, immunisation with fHbp V1 does not result in cross-protection against V2 and V3 and vice versa. Furthermore, high affinity binding of fH could impair immune responses against fHbp. Here, we investigate a homologue of fHbp in Neisseria gonorrhoeae, designated as Gonococcal homologue of fHbp (Ghfp) which we show is a promising vaccine candidate for N. meningitidis. We demonstrate that Gfhp is not expressed on the surface of the gonococcus and, despite its high level of identity with fHbp, does not bind fH. Substitution of only two amino acids in Ghfp is sufficient to confer fH binding, while the corresponding residues in V3 fHbp are essential for high affinity fH binding. Furthermore, immune responses against Ghfp recognise V1, V2 and V3 fHbps expressed by a range of clinical isolates, and have serum bactericidal activity against N. meningitidis expressing fHbps from all variant groups.

Sequence, distribution and chromosomal context of class I and class II pilin genes of Neisseria meningitidis identified in whole genome sequences.

BACKGROUND: Neisseria meningitidis expresses type four pili (Tfp) which are important for colonisation and virulence. Tfp have been considered as one of the most variable structures on the bacterial surface due to high frequency gene conversion, resulting in amino acid sequence variation of the major pilin subunit (PilE). Meningococci express either a class I or a class II pilE gene and recent work has indicated that class II pilins do not undergo antigenic variation, as class II pilE genes encode conserved pilin subunits. The purpose of this work was to use whole genome sequences to further investigate the frequency and variability of the class II pilE genes in meningococcal isolate collections. RESULTS: We analysed over 600 publically available whole genome sequences of N. meningitidis isolates to determine the sequence and genomic organization of pilE. We confirmed that meningococcal strains belonging to a limited number of clonal complexes (ccs, namely cc1, cc5, cc8, cc11 and cc174) harbour a class II pilE gene which is conserved in terms of sequence and chromosomal context. We also identified pilS cassettes in all isolates with class II pilE, however, our analysis indicates that these do not serve as donor sequences for pilE/pilS recombination. Furthermore, our work reveals that the class II pilE locus lacks the DNA sequence motifs that enable (G4) or enhance (Sma/Cla repeat) pilin antigenic variation. Finally, through analysis of pilin genes in commensal Neisseria species we found that meningococcal class II pilE genes are closely related to pilE from Neisseria lactamica and Neisseria polysaccharea, suggesting horizontal transfer among these species. CONCLUSIONS: Class II pilins can be defined by their amino acid sequence and genomic context and are present in meningococcal isolates which have persisted and spread globally. The absence of G4 and Sma/Cla sequences adjacent to the class II pilE genes is consistent with the lack of pilin subunit variation in these isolates, although horizontal transfer may generate class II pilin diversity. This study supports the suggestion that high frequency antigenic variation of pilin is not universal in pathogenic Neisseria.

Design and evaluation of meningococcal vaccines through structure-based modification of host and pathogen molecules.

Neisseria meningitis remains a leading cause of sepsis and meningitis, and vaccines are required to prevent infections by this important human pathogen. Factor H binding protein (fHbp) is a key antigen that elicits protective immunity against the meningococcus and recruits the host complement regulator, fH. As the high affinity interaction between fHbp and fH could impair immune responses, we sought to identify non-functional fHbps that could act as effective immunogens. This was achieved by alanine substitution of fHbps from all three variant groups (V1, V2 and V3 fHbp) of the protein; while some residues affected fH binding in each variant group, the distribution of key amino underlying the interaction with fH differed between the V1, V2 and V3 proteins. The atomic structure of V3 fHbp in complex with fH and of the C-terminal barrel of V2 fHbp provide explanations to the differences in the precise nature of their interactions with fH, and the instability of the V2 protein. To develop transgenic models to assess the efficacy of non-functional fHbps, we determined the structural basis of the low level of interaction between fHbp and murine fH; in addition to changes in amino acids in the fHbp binding site, murine fH has a distinct conformation compared with the human protein that would sterically inhibit binding to fHbp. Non-functional V1 fHbps were further characterised by binding and structural studies, and shown in non-transgenic and transgenic mice (expressing chimeric fH that binds fHbp and precisely regulates complement system) to retain their immunogenicity. Our findings provide a catalogue of non-functional fHbps from all variant groups that can be included in new generation meningococcal vaccines, and establish proof-in-principle for clinical studies to compare their efficacy with wild-type fHbps.

Galectin-3 binds Neisseria meningitidis and increases interaction with phagocytic cells.

Galectin-3 is expressed and secreted by immune cells and has been implicated in multiple aspects of the inflammatory response. It is a glycan binding protein which can exert its functions within cells or exogenously by binding cell surface ligands, acting as a molecular bridge or activating signalling pathways. In addition, this lectin has been shown to bind to microorganisms. In this study we investigated the interaction between galectin-3 and Neisseria meningitidis, an important extracellular human pathogen, which is a leading cause of septicaemia and meningitis. Immunohistochemical analysis indicated that galectin-3 is expressed during meningococcal disease and colocalizes with bacterial colonies in infected tissues from patients. We show that galectin-3 binds to N. meningitidis and we demonstrate that this interaction requiresfull-length, intact lipopolysaccharide molecules. We found that neither exogenous nor endogenous galectin-3 contributes to phagocytosis of N. meningitidis; instead exogenous galectin-3 increases adhesion to monocytes and macrophages but not epithelial cells. Finally we used galectin-3 deficient (Gal-3(-/-) ) mice to evaluate the contribution of galectin-3 to meningococcal bacteraemia. We found that Gal-3(-/-) mice had significantly lower levels of bacteraemia compared with wild-type mice after challenge with live bacteria, indicating that galectin-3 confers an advantage to N. meningitidis during systemic infection.

Glutamate utilization promotes meningococcal survival in vivo through avoidance of the neutrophil oxidative burst.

Polymorphonuclear neutrophil leucocytes (PMNs) are a critical part of innate immune defence against bacterial pathogens, and only a limited subset of microbes can escape killing by these phagocytic cells. Here we show that Neisseria meningitidis, a leading cause of septicaemia and meningitis, can avoid killing by PMNs and this is dependent on the ability of the bacterium to acquire L-glutamate through its GltT uptake system. We demonstrate that the uptake of available L-glutamate promotes N. meningitidis evasion of PMN reactive oxygen species produced by the oxidative burst. In the meningococcus, L-glutamate is converted to glutathione, a key molecule for maintaining intracellular redox potential, which protects the bacterium from reactive oxygen species such as hydrogen peroxide. We show that this mechanism contributes to the ability of N. meningitidis to cause bacteraemia, a critical step in the disease process during infections caused by this important human pathogen.

Et tu, Neisseria? Conflicts of Interest Between Neisseria Species.

Neisseria meningitidis and Neisseria gonorrhoeae are two obligate human pathogens that have evolved to be uniquely adapted to their host. The meningococcus is frequently carried asymptomatically in the nasopharynx, while gonococcal infection of the urogenital tract usually elicits a marked local inflammatory response. Other members of the Neisseria genus are abundant in the upper airway where they could engage in co-operative or competitive interactions with both these pathogens. Here, we briefly outline the potential sites of contact between Neisseria spp. in the body, with emphasis on the upper airway, and describe the growing yet circumstantial evidence for antagonism from carriage studies and human volunteer challenge models with Neisseria lactamica. Recent laboratory studies have characterized antagonistic mechanisms that enable competition between Neisseria species. Several of these mechanisms, including Multiple Adhesin family (Mafs), Two Partner Secretion Systems, and Type VI secretion system, involve direct contact between bacteria; the genetic organisation of these systems, and the domain structure of their effector molecules have striking similarities. Additionally, DNA from one species of Neisseria can be toxic to another species, following uptake. More research is needed to define the full repertoire of antagonistic mechanisms in Neisseria spp., their distribution in strains, their range of activity, and contribution to survival in vivo. Understanding the targets of effectors could reveal how antagonistic relationships between close relatives shape subsequent interactions between pathogens and their hosts.

Available carbon source influences the resistance of Neisseria meningitidis against complement.

Neisseria meningitidis is an important cause of septicaemia and meningitis. To cause disease, the bacterium must acquire essential nutrients for replication in the systemic circulation, while avoiding exclusion by host innate immunity. Here we show that the utilization of carbon sources by N. meningitidis determines its ability to withstand complement-mediated lysis, through the intimate relationship between metabolism and virulence in the bacterium. The gene encoding the lactate permease, lctP, was identified and disrupted. The lctP mutant had a reduced growth rate in cerebrospinal fluid compared with the wild type, and was attenuated during bloodstream infection through loss of resistance against complement-mediated killing. The link between lactate and complement was demonstrated by the restoration of virulence of the lctP mutant in complement (C3(-/-))-deficient animals. The underlying mechanism for attenuation is mediated through the sialic acid biosynthesis pathway, which is directly connected to central carbon metabolism. The findings highlight the intimate relationship between bacterial physiology and resistance to innate immune killing in the meningococcus.

Type VI secretion system killing by commensal Neisseria is influenced by expression of type four pili.

Type VI Secretion Systems (T6SSs) are widespread in bacteria and can dictate the development and organisation of polymicrobial ecosystems by mediating contact dependent killing. In Neisseria species, including Neisseria cinerea a commensal of the human respiratory tract, interbacterial contacts are mediated by Type four pili (Tfp) which promote formation of aggregates and govern the spatial dynamics of growing Neisseria microcolonies. Here, we show that N. cinerea expresses a plasmid-encoded T6SS that is active and can limit growth of related pathogens. We explored the impact of Tfp on N. cinerea T6SS-dependent killing within a colony and show that pilus expression by a prey strain enhances susceptibility to T6SS compared to a non-piliated prey, by preventing segregation from a T6SS-wielding attacker. Our findings have important implications for understanding how spatial constraints during contact-dependent antagonism can shape the evolution of microbial communities.

Transcellular passage of Neisseria meningitidis across a polarized respiratory epithelium.

Neisseria meningitidis is a major cause of sepsis and meningitis but is also a common commensal, present in the nasopharynx of between 8 and 20% of healthy individuals. During carriage, the bacterium is found on the surface of the nasopharyngeal epithelium and in deeper tissues, while to develop disease the meningococcus must spread across the respiratory epithelium and enter the systemic circulation. Therefore, investigating the pathways by which N. meningitidis crosses the epithelial barrier is relevant for understanding carriage and disease but has been hindered by the lack of appropriate models. Here, we have established a physiologically relevant model of the upper respiratory epithelial cell barrier to investigate the mechanisms responsible for traversal of N. meningitidis. Calu-3 human respiratory epithelial cells were grown on permeable cell culture membranes to form polarized monolayers of cells joined by tight junctions. We show that the meningococcus crosses the epithelial cell barrier by a transcellular route; traversal of the layer did not disrupt its integrity, and bacteria were detected within the cells of the monolayer. We demonstrate that successful traversal of the epithelial cell barrier by N. meningitidis requires expression of its type 4 pili (Tfp) and capsule and is dependent on the host cell microtubule network. The Calu-3 model should be suitable for dissecting the pathogenesis of infections caused by other respiratory pathogens, as well as the meningococcus.

Identification of meningococcal genes necessary for colonization of human upper airway tissue.

Neisseria meningitidis is an exclusively human pathogen that has evolved primarily to colonize the nasopharynx rather than to cause systemic disease. Colonization is the most frequent outcome following meningococcal infection and a prerequisite for invasive disease. The mechanism of colonization involves attachment of the organism to epithelial cells via bacterial type IV pili (Tfp), but subsequent events during colonization remain largely unknown. We analyzed 576 N. meningitidis mutants for their capacity to colonize human nasopharyngeal tissue in an organ culture model to identify bacterial genes required for colonization. Eight colonization-defective mutants were isolated. Two mutants were unable to express Tfp and were defective for adhesion to epithelial cells, which is likely to be the basis of their attenuation in nasopharyngeal tissue. Three other mutants are predicted to have lost previously uncharacterized surface molecules, while the remaining mutants have transposon insertions in genes of unknown function. We have identified novel meningococcal colonization factors, and this should provide insights into the survival of this important pathogen in its natural habitat.

Interactions between Neisseria meningitidis and the complement system.

Meningococcal infection remains a worldwide health problem, and understanding the mechanisms by which Neisseria meningitidis evades host innate and acquired immunity is crucial. The complement system is vital for protecting individuals against N. meningitidis. However, this pathogen has evolved several mechanisms to avoid killing by human complement. Bacterial structures such as polysaccharide capsule and those which mimic or bind host molecules function to prevent complement-mediated lysis and phagocytosis. This review provides an update on the recent findings on the diverse mechanisms by which N. meningitidis avoids complement-mediated killing, and how polymorphisms in genes encoding human complement proteins affect susceptibility to this important human pathogen.

Competition between antagonistic complement factors for a single protein on N. meningitidis rules disease susceptibility.

Genome-wide association studies have found variation within the complement factor H gene family links to host susceptibility to meningococcal disease caused by infection with Neisseria meningitidis (Davila et al., 2010). Mechanistic insights have been challenging since variation within this locus is complex and biological roles of the factor H-related proteins, unlike factor H, are incompletely understood. N. meningitidis subverts immune responses by hijacking a host-immune regulator, complement factor H (CFH), to the bacterial surface (Schneider et al., 2006; Madico et al., 2007; Schneider et al., 2009). We demonstrate that complement factor-H related 3 (CFHR3) promotes immune activation by acting as an antagonist of CFH. Conserved sequences between CFH and CFHR3 mean that the bacterium cannot sufficiently distinguish between these two serum proteins to allow it to hijack the regulator alone. The level of protection from complement attack achieved by circulating N. meningitidis therefore depends on the relative levels of CFH and CFHR3 in serum. These data may explain the association between genetic variation in both CFH and CFHR3 and susceptibility to meningococcal disease.