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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Cj1136 is required for lipooligosaccharide biosynthesis, hyperinvasion, and chick colonization by Campylobacter jejuni.

Campylobacter jejuni is a major cause of bacterial food-borne enteritis worldwide, and invasion into intestinal epithelial cells is an important virulence mechanism. Recently we reported the identification of hyperinvasive C. jejuni strains and created a number of transposon mutants of one of these strains, some of which exhibited reduced invasion into INT-407 and Caco-2 cells. In one such mutant the transposon had inserted into a homologue of cj1136, which encodes a putative galactosyltransferase according to the annotation of the C. jejuni NCTC11168 genome. In the current study, we investigated the role of cj1136 in C. jejuni virulence, lipooligosaccharide (LOS) biosynthesis, and host colonization by targeted mutagenesis and complementation of the mutation. The cj1136 mutant showed a significant reduction in invasion into human intestinal epithelial cells compared to the wild-type strain 01/51. Invasion levels were partially restored on complementing the mutation. The inactivation of cj1136 resulted in the production of truncated LOS, while biosynthesis of a full-length LOS molecule was restored in the complemented strain. The cj1136 mutant showed an increase in sensitivity to the bile salts sodium taurocholate and sodium deoxycholate and significantly increased sensitivity to polymyxin B compared to the parental strain. Importantly, the ability of the mutant to colonize 1-day-old chicks was also significantly impaired. This study confirms that a putative galactosyltransferase encoded by cj1136 is involved in LOS biosynthesis and is important for C. jejuni virulence, as disruption of this gene and the resultant truncation of LOS affect both colonization in vivo and invasiveness in vitro.

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.

The moonlighting protein fructose-1, 6-bisphosphate aldolase of Neisseria meningitidis: surface localization and role in host cell adhesion.

Fructose-1, 6-bisphosphate aldolases (FBA) are cytoplasmic glycolytic enzymes, which despite lacking identifiable secretion signals, have also been found localized to the surface of several bacteria where they bind host molecules and exhibit non-glycolytic functions. Neisseria meningitidis is an obligate human nasopharyngeal commensal, which has the capacity to cause life-threatening meningitis and septicemia. Recombinant native N. meningitidis FBA was purified and used in a coupled enzymic assay confirming that it has fructose bisphosphate aldolase activity. Cell fractionation experiments showed that meningococcal FBA is localized both to the cytoplasm and the outer membrane. Flow cytometry demonstrated that outer membrane-localized FBA was surface-accessible to FBA-specific antibodies. Mutational analysis and functional complementation was used to identify additional functions of FBA. An FBA-deficient mutant was not affected in its ability to grow in vitro, but showed a significant reduction in adhesion to human brain microvascular endothelial and HEp-2 cells compared to its isogenic parent and its complemented derivative. In summary, FBA is a highly conserved, surface exposed protein that is required for optimal adhesion of meningococci to human cells.

Persistence, replacement, and rapid clonal expansion of meningococcal carriage isolates in a 2008 university student cohort.

A study of meningococcal carriage dynamics was performed with a cohort of 190 first-year students recruited from six residential halls at Nottingham University, United Kingdom. Pharyngeal swabs were obtained on four occasions between November 2008 and May 2009. Direct plating and culture on selective media were succeeded by identification and characterization of meningococci using PCR-based methodologies. Three serogroup Y clones and one serogroup 29E clone were highly prevalent in particular residential halls in November 2008, which is indicative of rapid clonal expansion since the start of the academic year. Persistent carriage of the same meningococcal strain for at least 5 to 6 months was observed in 45% of carriers, with infrequent evidence of antigenic variation in PorA. Sequential carriage of heterologous meningococcal strains occurred in 36% of carriers and involved strains with different capsules and antigenic variants of PorA and FetA in 83% of the cases. These clonal replacement strains also exhibited frequent differences in the presence and antigenic structures of two other surface proteins, NadA and HmbR. This study highlights the low level of antigenic variation associated with persistent carriage but, conversely, the importance of alterations in the repertoire of antigenic variants for sequential carriage of meningococcal strains. Rapid clonal expansion of potentially pathogenic strains in residential halls has implications for the implementation of public health interventions in university populations.

The role of glyceraldehyde 3-phosphate dehydrogenase (GapA-1) in Neisseria meningitidis adherence to human cells.

BACKGROUND: Glyceraldehyde 3-phosphate dehydrogenases (GAPDHs) are cytoplasmic glycolytic enzymes, which although lacking identifiable secretion signals, have also been found localized to the surface of several bacteria (and some eukaryotic organisms); where in some cases they have been shown to contribute to the colonization and invasion of host tissues. Neisseria meningitidis is an obligate human nasopharyngeal commensal which can cause life-threatening infections including septicaemia and meningitis. N. meningitidis has two genes, gapA-1 and gapA-2, encoding GAPDH enzymes. GapA-1 has previously been shown to be up-regulated on bacterial contact with host epithelial cells and is accessible to antibodies on the surface of capsule-permeabilized meningococcal cells. The aims of this study were: 1) to determine whether GapA-1 was expressed across different strains of N. meningitidis; 2) to determine whether GapA-1 surface accessibility to antibodies was dependent on the presence of capsule; 3) to determine whether GapA-1 can influence the interaction of meningococci and host cells, particularly in the key stages of adhesion and invasion. RESULTS: In this study, expression of GapA-1 was shown to be well conserved across diverse isolates of Neisseria species. Flow cytometry confirmed that GapA-1 could be detected on the cell surface, but only in a siaD-knockout (capsule-deficient) background, suggesting that GapA-1 is inaccessible to antibody in in vitro-grown encapsulated meningococci. The role of GapA-1 in meningococcal pathogenesis was addressed by mutational analysis and functional complementation. Loss of GapA-1 did not affect the growth of the bacterium in vitro. However, a GapA-1 deficient mutant showed a significant reduction in adhesion to human epithelial and endothelial cells compared to the wild-type and complemented mutant. A similar reduction in adhesion levels was also apparent between a siaD-deficient meningococcal strain and an isogenic siaD gapA-1 double mutant. CONCLUSIONS: Our data demonstrates that meningococcal GapA-1 is a constitutively-expressed, highly-conserved surface-exposed protein which is antibody-accessible only in the absence of capsule. Mutation of GapA-1 does not affect the in vitro growth rate of N. meningitidis, but significantly affects the ability of the organism to adhere to human epithelial and endothelial cells in a capsule-independent process suggesting a role in the pathogenesis of meningococcal infection.

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.].

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.

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.