Ferric quinate (QPLEX) inhibits the interaction of major outer membrane protein (MOMP) with the Lewis b (Leb) antigen and limits Campylobacter colonization in broilers.

Campylobacter jejuni colonizes hosts by interacting with Blood Group Antigens (BgAgs) on the surface of gastrointestinal epithelia. Genetic variations in BgAg expression affects host susceptibility to C. jejuni. Here, we show that the essential major outer membrane protein (MOMP) of C. jejuni NCTC11168 binds to the Lewis b (Leb) antigen on the gastrointestinal epithelia of host tissues and this interaction can be competitively inhibited by ferric quinate (QPLEX), a ferric chelate structurally similar to bacterial siderophores. We provide evidence that QPLEX competitively inhibits the MOMP-Leb interaction. Furthermore, we demonstrate that QPLEX can be used as a feed additive in broiler farming to significantly reduce C. jejuni colonization. Our results indicate that QPLEX can be a viable alternative to the preventative use of antibiotics in broiler farming to combat C. jejuni infections.

Variable disruption of epithelial monolayers by Neisseria meningitidis carriage isolates of the hypervirulent MenW cc11 and MenY cc23 lineages.

Colonization of mucosal tissues by Neisseria meningitidis requires adhesion mediated by the type IV pilus and multiple outer-membrane proteins. Penetration of the mucosa and invasion of epithelial cells are thought to contribute to host persistence and invasive disease. Using Calu-3 cell monolayers grown at an air-liquid interface, we examined adhesion, invasion and monolayer disruption by carriage isolates of two clonal complexes of N. meningitidis. Carriage isolates of both the serogroup Y cc23 and the hypervirulent serogroup W cc11 lineages exhibited high levels of cellular adhesion, and a variable disruption phenotype across independent isolates. Inactivation of the gene encoding the main pilus sub-unit in multiple cc11 isolates abrogated both adhesive capacity and ability to disrupt epithelial monolayers. Contrastingly, inactivation of the phase-variable opa or nadA genes reduced adhesion and invasion, but not disruption of monolayer integrity. Adherence of tissue-disruptive meningococci correlated with loss of staining for the tight junction protein, occludin. Intriguingly, in a pilus-negative strain background, we observed compensatory ON switching of opa genes, which facilitated continued adhesion. We conclude that disruption of epithelial monolayers occurs in multiple meningococcal lineages but can vary during carriage and is intimately linked to pilus-mediated adhesion.

Ferric quinate (QPLEX) interacts with the major outer membrane protein (MOMP) of Campylobacter jejuni and enters through the porin channel into the periplasmic space.

Ferric chelates like ferric tyrosinate (TYPLEX) and the closely related ferric quinate (QPLEX) are structural mimics of bacterial siderophores. TYPLEX has been trialled as a feed additive in farming of commercial broilers, reducing Campylobacter loads by 2-3 log10 and leading to faster growth and better feed consumption. These ferric chelates offer a good alternative feed additive to antibiotics helping to reduce the indiscriminate use of preventative antibiotics in broiler farming to control Campylobacter infections. In this study, we show that QPLEX binds to the Major Outer Membrane Protein (MOMP) of C. jejuni NCTC11168. MOMP is an essential and abundant outer membrane porin on the surface of the bacteria, acting as an adhesin to help establish infection by mediating attachment of C. jejuni onto the gut epithelium of broilers and establish infection. Using carbene footprinting, we map the MOMP-QPLEX interaction and show by complementary in silico docking that QPLEX enters the porin channel through interactions at the extracellular face, translocates down the channel through a dipole transverse electric field towards the opposite end and is released into the periplasm at the intracellular face of MOMP. Our studies suggest a potential mechanism for the non-antibiotic anti-Campylobacter activity of these ferric chelates.

Rapid Transmission of a Hyper-Virulent Meningococcal Clone Due to High Effective Contact Numbers and Super Spreaders.

Rapid transmission, a critical contributory factor in outbreaks of invasive meningococcal disease, requires naïve populations of sufficient size and intermingling. We examined genomic variability and transmission dynamics in a student population subject to an 11-fold increase in carriage of a hypervirulent Neisseria meningitidis serogroup W ST-11 clone. Phylogenetic clusters, mutation and recombination rates were derived by bioinformatic analyses of whole-genome sequencing data. Transmission dynamics were determined by combining observed carriage rates, cluster sizes and distributions with simple SIS models. Between 9 and 15 genetically-distinct clusters were detected and associated with seven residential halls. Clusters had low mutation accumulation rates and infrequent recombination events. Modeling indicated that effective contacts decreased from 10 to 2 per day between the start and mid-point of the university term. Transmission rates fluctuated between 1 and 4% while the R(t) for carriage decreased from an initial rate of 47 to 1. Decreases in transmission values correlated with a rise in vaccine-induced immunity. Observed carriage dynamics could be mimicked by populations containing 20% of super spreaders with 2.3-fold higher effective contact rates. We conclude that spread of this hypervirulent ST-11 meningococcal clone depends on the levels of effective contacts and immunity rather than genomic variability. Additionally, we propose that super-spreaders enhance meningococcal transmission and that a 70% MenACWY immunization level is sufficient to retard, but not fully prevent, meningococcal spread in close-contact populations.

A role for fibroblast growth factor receptor 1 in the pathogenesis of Neisseria meningitidis.

Neisseria meningitidis (the meningococcus) remains an important cause of human disease, including meningitis and sepsis. Adaptation to the host environment includes many interactions with specific cell surface receptors, resulting in intracellular signalling and cytoskeletal rearrangements that contribute to pathogenesis. Here, we assessed the interactions between meningococci and Fibroblast Growth Factor Receptor 1-IIIc (FGFR1-IIIc): a receptor specific to endothelial cells of the microvasculature, including that of the blood-brain barrier. We show that the meningococcus recruits FGFR1-IIIc onto the surface of human blood microvascular endothelial cells (HBMECs). Furthermore, we demonstrate that expression of FGFR1-IIIc is required for optimal invasion of HBMECs by meningococci. We show that the ability of N. meningitidis to interact with the ligand-binding domain of FGFR1-IIIc is shared with the other pathogenic Neisseria species, N. gonorrhoeae, but not with commensal bacteria including non-pathogenic Neisseria species.

Localized Hypermutation is the Major Driver of Meningococcal Genetic Variability during Persistent Asymptomatic Carriage.

Host persistence of bacteria is facilitated by mutational and recombinatorial processes that counteract loss of genetic variation during transmission and selection from evolving host responses. Genetic variation was investigated during persistent asymptomatic carriage of Neisseria meningitidis Interrogation of whole-genome sequences for paired isolates from 25 carriers showed that de novo mutations were infrequent, while horizontal gene transfer occurred in 16% of carriers. Examination of multiple isolates per time point enabled separation of sporadic and transient allelic variation from directional variation. A comprehensive comparative analysis of directional allelic variation with hypermutation of simple sequence repeats and hyperrecombination of class 1 type IV pilus genes detected an average of seven events per carrier and 2:1 bias for changes due to localized hypermutation. Directional genetic variation was focused on the outer membrane with 69% of events occurring in genes encoding enzymatic modifiers of surface structures or outer membrane proteins. Multiple carriers exhibited directional and opposed switching of allelic variants of the surface-located Opa proteins that enables continuous expression of these adhesins alongside antigenic variation. A trend for switching from PilC1 to PilC2 expression was detected, indicating selection for specific alterations in the activities of the type IV pilus, whereas phase variation of restriction modification (RM) systems, as well as associated phasevarions, was infrequent. We conclude that asymptomatic meningococcal carriage on mucosal surfaces is facilitated by frequent localized hypermutation and horizontal gene transfer affecting genes encoding surface modifiers such that optimization of adhesive functions occurs alongside escape of immune responses by antigenic variation.IMPORTANCE Many bacterial pathogens coexist with host organisms, rarely causing disease while adapting to host responses. Neisseria meningitidis, a major cause of meningitis and septicemia, is a frequent persistent colonizer of asymptomatic teenagers/young adults. To assess how genetic variation contributes to host persistence, whole-genome sequencing and hypermutable sequence analyses were performed on multiple isolates obtained from students naturally colonized with meningococci. High frequencies of gene transfer were observed, occurring in 16% of carriers and affecting 51% of all nonhypermutable variable genes. Comparative analyses showed that hypermutable sequences were the major mechanism of variation, causing 2-fold more changes in gene function than other mechanisms. Genetic variation was focused on genes affecting the outer membrane, with directional changes in proteins responsible for bacterial adhesion to host surfaces. This comprehensive examination of genetic plasticity in individual hosts provides a significant new platform for rationale design of approaches to prevent the spread of this pathogen.

Corrigendum: Variant Signal Peptides of Vaccine Antigen, FHbp, Impair Processing Affecting Surface Localization and Antibody-Mediated Killing in Most Meningococcal Isolates.

[This corrects the article DOI: 10.3389/fmicb.2019.02847.].

The moonlighting peroxiredoxin-glutaredoxin in Neisseria meningitidis binds plasminogen via a C-terminal lysine residue and contributes to survival in a whole blood model.

Neisseria meningitidis is a human-restricted bacterium that can invade the bloodstream and cross the blood-brain barrier resulting in life-threatening sepsis and meningitis. Meningococci express a cytoplasmic peroxiredoxin-glutaredoxin (Prx5-Grx) hybrid protein that has also been identified on the bacterial surface. Here, recombinant Prx5-Grx was confirmed as a plasminogen (Plg)-binding protein, in an interaction which could be inhibited by the lysine analogue ε-aminocapronic acid. rPrx5-Grx derivatives bearing a substituted C-terminal lysine residue (rPrx5-GrxK244A), but not the active site cysteine residue (rPrx5-GrxC185A) or the sub-terminal rPrx5-GrxK230A lysine residue, exhibited significantly reduced Plg-binding. The absence of Prx5-Grx did not significantly reduce the ability of whole meningococcal cells to bind Plg, but under hydrogen peroxide-mediated oxidative stress, the N. meningitidis Δpxn5-grx mutant survived significantly better than the wild-type or complemented strains. Significantly, using human whole blood as a model of meningococcal bacteremia, it was found that the N. meningitidis Δpxn5-grx mutant had a survival defect compared with the parental or complemented strain, confirming an important role for Prx5-Grx in meningococcal pathogenesis.

Potentiation of Phase Variation in Multiple Outer-Membrane Proteins During Spread of the Hyperinvasive Neisseria meningitidis Serogroup W ST-11 Lineage.

BACKGROUND: Since 2009, increases in the incidence of invasive meningococcal disease have occurred in the United Kingdom due to a sublineage of the Neisseria meningitidis serogroup W ST-11 clonal complex (hereafter, the "original UK strain"). In 2013, a descendent substrain (hereafter, the "2013 strain") became the dominant disease-causing variant. Multiple outer-membrane proteins of meningococci are subject to phase-variable switches in expression due to hypermutable simple-sequence repeats. We investigated whether alterations in phase-variable genes may have influenced the relative prevalence of the original UK and 2013 substrains, using multiple disease and carriage isolates. METHODS: Repeat numbers were determined by either bioinformatics analysis of whole-genome sequencing data or polymerase chain reaction amplification and sizing of fragments from genomic DNA extracts. Immunoblotting and sequence-translation analysis was performed to identify expression states. RESULTS: Significant increases in repeat numbers were detected between the original UK and 2013 strains in genes encoding PorA, NadA, and 2 Opa variants. Invasive and carriage isolates exhibited similar repeat numbers, but the absence of pilC gene expression was frequently associated with disease. CONCLUSIONS: Elevated repeat numbers in outer-membrane protein genes of the 2013 strain are indicative of higher phase-variation rates, suggesting that rapid expansion of this strain was due to a heightened ability to evade host immune responses during transmission and asymptomatic carriage.

Variant Signal Peptides of Vaccine Antigen, FHbp, Impair Processing Affecting Surface Localization and Antibody-Mediated Killing in Most Meningococcal Isolates.

Meningococcal lipoprotein, Factor H binding protein (FHbp), is the sole antigen of the Trumenba vaccine (Pfizer) and one of four antigens of the Bexsero vaccine (GSK) targeting Neisseria meningitidis serogroup B isolates. Lipidation of FHbp is assumed to occur for all isolates. We show in the majority of a collection of United Kingdom isolates (1742/1895) non-synonymous single nucleotide polymorphisms (SNPs) in the signal peptide (SP) of FHbp. A single SNP, common to all, alters a polar amino acid that abolishes processing: lipidation and SP cleavage. Whilst some of the FHbp precursor is retained in the cytoplasm due to reduced binding to SecA, remarkably some is translocated and further surface-localized by Slam. Thus we show Slam is not lipoprotein-specific. In a panel of isolates tested, the overall reduced surface localization of the precursor FHbp, compared to isolates with an intact SP, corresponded with decreased susceptibility to antibody-mediated killing. Our findings shed new light on the canonical pathway for lipoprotein processing and translocation of important relevance for lipoprotein-based vaccines in development and in particular for Trumenba.

Uptake of Neisserial autotransporter lipoprotein (NalP) promotes an increase in human brain microvascular endothelial cell metabolic activity.

Neisseria meningitidis is normally a human nasopharyngeal commensal but is also capable of causing life-threatening sepsis and meningitis. N. meningitidis secretes several virulence-associated proteins including Neisserial autotransporter lipoprotein (NalP), an immunogenic, type Va autotransporter harboring an S8-family serine endopeptidase domain. NalP has been previously characterized as a cell-surface maturation protease which processes other virulence-associated meningococcal surface proteins, and as a factor contributing to the survival of meningococci in human serum due to its ability to cleave complement factor C3. Here, recombinant NalP (rNalP) fragments were purified and used to investigate the interaction of NalP with host cells. Flow cytometry and confocal microscopy demonstrated binding and uptake of rNalP into different human cell types. High-resolution microscopy confirmed that internalized rNalP predominantly localized to the perinuclear region of cells. Abolition of rNalP protease activity using site-directed mutagenesis did not influence uptake or sub-cellular localization, but inactive rNalP (rNalPS426A) was unable to induce an increase in human brain microvascular endothelial cell metabolic activity provoked by proteolytically-active rNalP. Our data suggests a more complex and multifaceted role for NalP in meningococcal pathogenesis than was previously understood which includes novel intra-host cell functions.

Limited Impact of Adolescent Meningococcal ACWY Vaccination on Neisseria meningitidis Serogroup W Carriage in University Students.

Background: In the United Kingdom, rising levels of disease due to Neisseria meningitidis serogroup W clonal complex (cc) sequence type (ST) 11 (MenW:cc11) strains led to introduction of meningococcal conjugate vaccine (MenACWY) for teenagers. We investigated the impact of immunization on carriage of meningococci targeted by the vaccine, using whole-genome sequencing of isolates recovered from a cohort of vaccinated university students. Methods: Strain designation data were extracted from whole-genome sequencing data. Genomes from carried and invasive MenW:cc11 strains were compared using a gene-by-gene approach. Serogrouping identified isolates expressing capsule antigens targeted by the vaccine. Results: Isolates with a W: P1.5,2: F1-1: ST-11 (cc11) designation and belonging to the emerging 2013-strain of the South American-United Kingdom MenW:cc11 sublineage were responsible for an increase in carried group W strains. A multifocal expansion was evident, with close transmission networks extending beyond individual dormitories. Carried group Y isolates were predominantly from cc23 but showed significant heterogeneity, and individual strain designations were only sporadically recovered. No shifts toward acapsulate phenotypes were detected in targeted meningococcal populations. Conclusions: In a setting with high levels of MenACWY use, expansion of capsule-expressing isolates from the 2013-strain of MenW:cc11 but not MenY:cc23 isolates is indicative of differential susceptibilities to vaccine-induced immunity.

Rise in Group W Meningococcal Carriage in University Students, United Kingdom.

MenACWY conjugate vaccination was recently introduced in the United Kingdom for adolescents and young adults to reduce disease from infection by Neisseria meningitidis group W. We conducted a cross-sectional meningococcal carriage study in first-year UK university students. Despite 71% MenACWY vaccine coverage, carriage of group W increased substantially.

Fructose-1,6-bisphosphate aldolase of Neisseria meningitidis binds human plasminogen via its C-terminal lysine residue.

Neisseria meningitidis is a leading cause of fatal sepsis and meningitis worldwide. As for commensal species of human neisseriae, N. meningitidis inhabits the human nasopharynx and asymptomatic colonization is ubiquitous. Only rarely does the organism invade and survive in the bloodstream leading to disease. Moonlighting proteins perform two or more autonomous, often dissimilar, functions using a single polypeptide chain. They have been increasingly reported on the surface of both prokaryotic and eukaryotic organisms and shown to interact with a variety of host ligands. In some organisms moonlighting proteins perform virulence-related functions, and they may play a role in the pathogenesis of N. meningitidis. Fructose-1,6-bisphosphate aldolase (FBA) was previously shown to be surface-exposed in meningococci and involved in adhesion to host cells. In this study, FBA was shown to be present on the surface of both pathogenic and commensal neisseriae, and surface localization and anchoring was demonstrated to be independent of aldolase activity. Importantly, meningococcal FBA was found to bind to human glu-plasminogen in a dose-dependent manner. Site-directed mutagenesis demonstrated that the C-terminal lysine residue of FBA was required for this interaction, whereas subterminal lysine residues were not involved.

Genomic Analysis of Serogroup Y Neisseria meningitidis Isolates Reveals Extensive Similarities Between Carriage-Associated and Disease-Associated Organisms.

BACKGROUND: Neisseria meningitidis is a frequent colonizer of the human nasopharynx, with asymptomatic carriage providing the reservoir for invasive, disease-causing strains. Serogroup Y (MenY) strains are a major cause of meningococcal disease. High-resolution genetic analyses of carriage and disease isolates can establish epidemiological relationships and identify potential virulence factors. METHODS: Whole-genome sequence data were obtained for 99 MenY carriage isolates recovered in the United Kingdom during 1997-2010. Sequences were compared to those of 73 MenY invasive isolates recovered during 2010-2011, using a gene-by-gene approach. RESULTS: Comparisons across 1605 core genes resolved 91% of isolates into one of 8 clusters containing closely related disease and carriage isolates. Six clusters contained carried meningococci isolated during 1997-2001, suggesting temporal stability. One cluster of isolates, predominately sharing the designation Y: P1.5-1,10-1: F4-1: ST-1655 (cc23), was resolved into one subcluster with 86% carriage isolates and a second with 90% invasive isolates. These subclusters were defined by specific allelic differences in 5 core genes encoding glycerate kinase (glxK), valine-pyruvate transaminase (avtA), superoxide dismutase (sodB), and 2 hypothetical proteins. CONCLUSIONS: High-resolution genetic analyses detected long-term temporal stability and temporally overlapping carriage and disease populations for MenY clones but also evidence of a disease-associated clone.

Optimization of Molecular Approaches to Genogroup Neisseria meningitidis Carriage Isolates and Implications for Monitoring the Impact of New Serogroup B Vaccines.

The reservoir for Neisseria meningitidis (Nm) is the human oropharynx. Implementation of Nm serogroup C (NmC) glycoconjugate vaccines directly reduced NmC carriage. Prophylactic vaccines are now available to prevent disease caused by the five major Nm disease causing serogroups (ABCWY). Nm serogroup B (NmB) vaccines are composed of antigens that are conserved across Nm serogroups and therefore have the potential to impact all Nm carriage. To assess the effect of these vaccines on carriage, standardized approaches to identify and group Nm are required. Real-time PCR (rt-PCR) capsule grouping assays that were internally controlled to confirm Nm species were developed for eight serogroups associated with carriage (A, B, C, E, W, X, Y and Z). The grouping scheme was validated using diverse bacterial species associated with carriage and then used to evaluate a collection of diverse Nm carriage isolates (n=234). A scheme that also included porA and ctrA probes was able to speciate the isolates, while ctrA also provided insights on the integrity of the polysaccharide loci. Isolates were typed for the Nm vaccine antigen factor H binding protein (fHbp), and were found to represent the known diversity of this antigen. The porA rt-PCR yielded positive results with all 234 of the Nm carriage isolates. Genogrouping assays classified 76.5% (179/234) of these isolates to a group, categorized 53 as nongenogroupable (NGG) and two as mixed results. Thirty seven NGG isolates evidenced a disrupted capsular polysaccharide operon judged by a ctrA negative result. Only 28.6% (67/234) of the isolates were serogrouped by slide agglutination (SASG), highlighting the reduced capability of carriage strains to express capsular polysaccharide. These rt-PCR assays provide a comprehensive means to identify and genogroup N. meningitidis in carriage studies used to guide vaccination strategies and to assess the impact of novel fHbp containing vaccines on meningococcal carriage.

Nuclear trafficking, histone cleavage and induction of apoptosis by the meningococcal App and MspA autotransporters.

Neisseria meningitidis, a major cause of bacterial meningitis and septicaemia, secretes multiple virulence factors, including the adhesion and penetration protein (App) and meningococcal serine protease A (MspA). Both are conserved, immunogenic, type Va autotransporters harbouring S6-family serine endopeptidase domains. Previous work suggested that both could mediate adherence to human cells, but their precise contribution to meningococcal pathogenesis was unclear. Here, we confirm that App and MspA are in vivo virulence factors since human CD46-expressing transgenic mice infected with meningococcal mutants lacking App, MspA or both had improved survival rates compared with mice infected with wild type. Confocal imaging showed that App and MspA were internalized by human cells and trafficked to the nucleus. Cross-linking and enzyme-linked immuno assay (ELISA) confirmed that mannose receptor (MR), transferrin receptor 1 (TfR1) and histones interact with MspA and App. Dendritic cell (DC) uptake could be blocked using mannan and transferrin, the specific physiological ligands for MR and TfR1, whereas in vitro clipping assays confirmed the ability of both proteins to proteolytically cleave the core histone H3. Finally, we show that App and MspA induce a dose-dependent increase in DC death via caspase-dependent apoptosis. Our data provide novel insights into the roles of App and MspA in meningococcal infection.

Fructose-1,6-bisphosphate aldolase (FBA)-a conserved glycolytic enzyme with virulence functions in bacteria: 'ill met by moonlight'.

Moonlighting proteins constitute an intriguing class of multifunctional proteins. Metabolic enzymes and chaperones, which are often highly conserved proteins in bacteria, archaea and eukaryotic organisms, are among the most commonly recognized examples of moonlighting proteins. Fructose-1,6-bisphosphate aldolase (FBA) is an enzyme involved in the Embden-Meyerhof-Parnas (EMP) glycolytic pathway and in gluconeogenesis. Increasingly, it is also recognized that FBA has additional functions beyond its housekeeping role in central metabolism. In the present review, we summarize the current knowledge of the moonlighting functions of FBA in bacteria.

Deciphering the complex three-way interaction between the non-integrin laminin receptor, galectin-3 and Neisseria meningitidis.

The non-integrin laminin receptor (LAMR1/RPSA) and galectin-3 (Gal-3) are multi-functional host molecules with roles in diverse pathological processes, particularly of infectious or oncogenic origins. Using bimolecular fluorescence complementation and confocal imaging, we demonstrate that the two proteins homo- and heterodimerize, and that each isotype forms a distinct cell surface population. We present evidence that the 37 kDa form of LAMR1 (37LRP) is the precursor of the previously described 67 kDa laminin receptor (67LR), whereas the heterodimer represents an entity that is distinct from this molecule. Site-directed mutagenesis confirmed that the single cysteine (C(173)) of Gal-3 or lysine (K(166)) of LAMR1 are critical for heterodimerization. Recombinant Gal-3, expressed in normally Gal-3-deficient N2a cells, dimerized with endogenous LAMR1 and led to a significantly increased number of internalized bacteria (Neisseria meningitidis), confirming the role of Gal-3 in bacterial invasion. Contact-dependent cross-linking determined that, in common with LAMR1, Gal-3 binds the meningococcal secretin PilQ, in addition to the major pilin PilE. This study adds significant new mechanistic insights into the bacterial-host cell interaction by clarifying the nature, role and bacterial ligands of LAMR1 and Gal-3 isotypes during colonization.

Phase variation mediates reductions in expression of surface proteins during persistent meningococcal carriage.

Asymptomatic and persistent colonization of the upper respiratory tract by Neisseria meningitidis occurs despite elicitation of adaptive immune responses against surface antigens. A putative mechanism for facilitating host persistence of this bacterial commensal and pathogen is alterations in expression of surface antigens by simple sequence repeat (SSR)-mediated phase variation. We investigated how often phase variation occurs during persistent carriage by analyzing the SSRs of eight loci in multiple isolates from 21 carriers representative of 1 to 6 months carriage. Alterations in repeat number were detected by a GeneScan analysis and occurred at 0.06 mutations/gene/month of carriage. The expression states were determined by Western blotting and two genes, fetA and nadA, exhibited trends toward low expression states. A critical finding from our unique examination of combinatorial expression states, "phasotypes," was for significant reductions in expression of multiple phase-variable surface proteins during persistent carriage of some strains. The immune responses in these carriers were examined by measuring variant-specific PorA IgG antibodies, capsular group Y IgG antibodies and serum bactericidal activity in concomitant serum samples. Persistent carriage was associated with high levels of specific IgG antibodies and serum bactericidal activity while recent strain acquisition correlated with a significant induction of antibodies. We conclude that phase-variable genes are driven into lower expression states during long-term persistent meningococcal carriage, in part due to continuous exposure to antibody-mediated selection, suggesting localized hypermutation has evolved to facilitate host persistence.