Analyzing Neisseria gonorrhoeae Pilin Antigenic Variation Using 454 Sequencing Technology

ABSTRACTMany pathogens use homologous recombination to vary surface antigens in order to avoid immune surveillance.Neisseria gonorrhoeae, the bacterium responsible for the sexually transmitted infection gonorrhea, achieves this in part by changing the sequence of the major subunit of the type IV pilus in a process termed pilin antigenic variation (Av). TheN. gonorrhoeaechromosome contains one expression locus (pilE) and many promoterless, partial-coding silent copies (pilS) that act as reservoirs for variant pilin information. Pilin Av occurs by high-frequency gene conversion reactions, which transferpilSsequences into thepilElocus. We have developed a 454 sequencing-based assay to analyze the frequency and characteristics of pilin Av that allows a more robust analysis of pilin Av than previous assays. We used this assay to analyze mutations and conditions previously shown to affect pilin Av, confirming many but not all of the previously reported phenotypes. We show that mutations or conditions that cause growth defects can result in Av phenotypes when analyzed by phase variation-based assays. Adapting the 454 sequencing to analyze pilin Av demonstrates the utility of this technology to analyze any diversity generation system that uses recombination to develop biological diversity.IMPORTANCEMeasuring and analyzing complex recombination-based systems constitute a major barrier to understanding the mechanisms used to generate diversity. We have analyzed the contributions of many gonococcal mutations or conditions to the process of pilin antigenic variation.

Download Full-text

PacBio Amplicon Sequencing Method To Measure Pilin Antigenic Variation Frequencies of Neisseria gonorrhoeae

mSphere ◽

10.1128/msphere.00562-19 ◽

2019 ◽

Vol 4 (5) ◽

Cited By ~ 1

Author(s):

Egon A. Ozer ◽

Lauren L. Prister ◽

Shaohui Yin ◽

Billy H. Ward ◽

Stanimir Ivanov ◽

...

Keyword(s):

Neisseria Gonorrhoeae ◽

Antigenic Variation ◽

Amplicon Sequencing ◽

Common Mechanism ◽

Macrophage Infection ◽

Gene Encoding ◽

Transmitted Infection ◽

Variable Regions ◽

Content Type ◽

Strain Fa1090

ABSTRACT Gene diversification is a common mechanism pathogens use to alter surface structures to aid in immune avoidance. Neisseria gonorrhoeae uses a gene conversion-based diversification system to alter the primary sequence of the gene encoding the major subunit of the pilus, pilE. Antigenic variation occurs when one of the nonexpressed 19 silent copies donates part of its DNA sequence to pilE. We have developed a method using Pacific Biosciences (PacBio) amplicon sequencing and custom software to determine pilin antigenic variation frequencies. The program analyzes 37 variable regions across the strain FA1090 1-81-S2 pilE gene and can be modified to determine sequence variation from other starting pilE sequences or other diversity generation systems. Using this method, we measured pilin antigenic variation frequencies for various derivatives of strain FA1090 and showed we can also analyze pilin antigenic variation frequencies during macrophage infection. IMPORTANCE Diversity generation systems are used by many unicellular organism to provide subpopulations of cell with different properties that are available when needed. We have developed a method using the PacBio DNA sequencing technology and a custom computer program to analyze the pilin antigenic variation system of the organism that is the sole cause of the sexually transmitted infection, gonorrhea.

Download Full-text

Discovery of a New Neisseria gonorrhoeae Type IV Pilus Assembly Factor, TfpC

mBio ◽

10.1128/mbio.02528-20 ◽

2020 ◽

Vol 11 (5) ◽

Author(s):

Linda I. Hu ◽

Shaohui Yin ◽

Egon A. Ozer ◽

Lee Sewell ◽

Saima Rehman ◽

...

Keyword(s):

Cell Surface ◽

Neisseria Gonorrhoeae ◽

Bacterial Cell ◽

Bacterial Species ◽

Type Iv Pili ◽

Type Iv Pilus ◽

Transmitted Infection ◽

Content Type ◽

Sexually Transmitted ◽

Type Iv

ABSTRACT Neisseria gonorrhoeae relies on type IV pili (T4p) to promote colonization of their human host and to cause the sexually transmitted infection gonorrhea. This organelle cycles through a process of extension and retraction back into the bacterial cell. Through a genetic screen, we identified the NGO0783 locus of N. gonorrhoeae strain FA1090 as containing a gene encoding a protein required to stabilize the type IV pilus in its extended, nonretracted conformation. We have named the gene tfpC and the protein TfpC. Deletion of tfpC produces a nonpiliated colony morphology, and immuno-transmission electron microscopy confirms that the pili are lost in the ΔtfpC mutant, although there is some pilin detected near the bacterial cell surface. A copy of the tfpC gene expressed from a lac promoter restores pilus expression and related phenotypes. A ΔtfpC mutant shows reduced levels of pilin protein, but complementation with a tfpC gene restored pilin to normal levels. Bioinformatic searches show that there are orthologues in numerous bacterial species, but not all type IV pilin-expressing bacteria contain orthologous genes. Coevolution and nuclear magnetic resonance (NMR) analysis indicates that TfpC contains an N-terminal transmembrane helix, a substantial extended/unstructured region, and a highly charged C-terminal coiled-coil domain. IMPORTANCE Most bacterial species express one or more extracellular organelles called pili/fimbriae that are required for many properties of each bacterial cell. The Neisseria gonorrhoeae type IV pilus is a major virulence and colonization factor for the sexually transmitted infection gonorrhea. We have discovered a new protein of Neisseria gonorrhoeae called TfpC that is required to maintain type IV pili on the bacterial cell surface. There are similar proteins found in other members of the Neisseria genus and many other bacterial species important for human health.

Download Full-text

Neisseria gonorrhoeae MutS Affects Pilin Antigenic Variation through Mismatch Correction and Not bypilEGuanine Quartet Binding

Journal of Bacteriology ◽

10.1128/jb.02594-14 ◽

2015 ◽

Vol 197 (10) ◽

pp. 1828-1838 ◽

Cited By ~ 15

Author(s):

Ella Rotman ◽

H. Steven Seifert

Keyword(s):

Homologous Recombination ◽

Neisseria Gonorrhoeae ◽

Antigenic Variation ◽

Synthetic Lethality ◽

Phase Variation ◽

Surface Proteins ◽

Loss Of Function ◽

Content Type ◽

Mismatch Correction ◽

Guanine Quartet

ABSTRACTMany pathogens use homologous recombination to vary surface antigens to avoid immune surveillance.Neisseria gonorrhoeaeachieves this in part by changing the properties of its surface pili in a process called pilin antigenic variation (AV). Pilin AV occurs by high-frequency gene conversion reactions that transfer silentpilSsequences into the expressedpilElocus and requires the formation of an upstream guanine quartet (G4) DNA structure to initiate this process. The MutS and MutL proteins of the mismatch correction (MMC) system act to correct mismatches after replication and prevent homeologous (i.e., partially homologous) recombination, but MutS orthologs can also bind to G4 structures. A previous study showed that mutation of MutS resulted in a 3-fold increase in pilin AV, which could be due to the loss of MutS antirecombination properties or loss of G4 binding. We tested two site-directed separation-of-function MutS mutants that are both predicted to bind to G4s but are not able to perform MMC. Pilus phase variation assays and DNA sequence analysis ofpilEvariants produced in these mutants showed that all threemutSmutants and amutLmutant had similar increased frequencies of pilin AV. Moreover, themutSmutants all showed similar increased levels of pilin AV-dependent synthetic lethality. These results show that antirecombination by MMC is the reason for the effect that MutS has on pilin AV and is not due topilEG4 binding by MutS.IMPORTANCENeisseria gonorrhoeaecontinually changes its outer surface proteins to avoid recognition by the immune system.N. gonorrhoeaealters the antigenicity of the pilus by directed recombination between partially homologous pilin copies in a process that requires a guanine quartet (G4) structure. The MutS protein of the mismatch correction (MMC) system prevents recombination between partially homologous sequences and can also bind to G4s. We confirmed that loss of MMC increases the frequency of pilin antigenic variation and that two MutS mutants that are predicted to separate the two different functions of MutS inhibit pilin variation similarly to a complete-loss-of-function mutant, suggesting that interaction of MutS with the G4 structure is not a major factor in this process.

Download Full-text

Neisseria gonorrhoeae Metalloprotease NGO1686 Is Required for Full Piliation, and Piliation Is Required for Resistance to H2O2- and Neutrophil-Mediated Killing

mBio ◽

10.1128/mbio.00399-13 ◽

2013 ◽

Vol 4 (4) ◽

Cited By ~ 28

Author(s):

Elizabeth A. Stohl ◽

Erin M. Dale ◽

Alison K. Criss ◽

H. Steven Seifert

Keyword(s):

Neisseria Gonorrhoeae ◽

Virulence Factor ◽

Transmitted Infection ◽

Content Type ◽

Sexually Transmitted ◽

Type Iv ◽

Specific Pathogen ◽

Associated Functions ◽

Host Tissues ◽

Human Specific

ABSTRACTThe sexually transmitted infection gonorrhea is caused exclusively by the human-specific pathogenNeisseria gonorrhoeae. Type IV pili are an essential virulence factor uniformly expressed on clinical gonococcal isolates and are required for several aspects of gonococcal pathogenesis, including adherence to host tissues, autoagglutination, twitching motility, and the uptake of DNA during transformation. Symptomatic gonococcal infection is characterized by the influx of neutrophils or polymorphonuclear leukocytes (PMNs) to the site of infection. PMNs are a key component of gonococcal pathogenesis, mediating the innate immune response through the use of oxidative and nonoxidative killing mechanisms. The M23B family zinc metallopeptidase NGO1686 is required for gonococci to survive oxidative killing by H2O2- and PMN-mediated killing through unknown mechanisms, but the only known target of NGO1686 is peptidoglycan. We report that the effect of NGO1686 on survival after exposure to H2O2and PMNs is mediated through its role in elaborating pili and that nonpiliated mutants ofN. gonorrhoeaeare less resistant to killing by H2O2, LL-37, and PMNs than the corresponding piliated strains. These findings add to the various virulence-associated functions attributable to gonococcal pili and may explain the selection basis for piliation in clinical isolates ofN. gonorrhoeae.IMPORTANCESuccessful infectious agents need to overcome host defense systems to establish infection. We show that theNeisseriapilus, a major virulence factor of this organism, which causes gonorrhea, helps protect the bacterium from two major killing mechanisms used by the host to combat infections. We also show that to express the pilus, an enzyme needs to partially degrade the cell wall of the bacterium.

Download Full-text

Seminal Plasma Promotes Neisseria gonorrhoeae Aggregation and Biofilm Formation

Journal of Bacteriology ◽

10.1128/jb.00165-16 ◽

2016 ◽

Vol 198 (16) ◽

pp. 2228-2235 ◽

Cited By ~ 10

Author(s):

Mark T. Anderson ◽

Luke Byerly ◽

Michael A. Apicella ◽

H. Steven Seifert

Keyword(s):

Neisseria Gonorrhoeae ◽

Seminal Plasma ◽

Biofilm Formation ◽

Seminal Fluid ◽

Sexual Transmission ◽

Type Iv Pilus ◽

Transmitted Infection ◽

Content Type ◽

Bacterial Physiology ◽

Type Iv

ABSTRACTNeisseria gonorrhoeaecauses the human-specific disease gonorrhea and is transmitted from person to person primarily via sexual contact. During transmission,N. gonorrhoeaeis often exposed to seminal fluid and must adapt to this change in environment. Previous work demonstrated that seminal fluid facilitatesN. gonorrhoeaemotility and alters epithelial cell interactions. In this study, exposure to seminal fluid was found to decrease surface adherence of gonococci in a manner that was independent of Opa adhesin proteins or type IV pilus retraction. Semen was also shown to cause dispersal of bacteria that had previously established surface adherence. Although surface adherence decreased, interbacterial interactions were increased by seminal plasma both in long-term static culture and on a cell-to-cell basis over shorter time periods. The result of increased bacterium-bacterium interactions resulted in the formation of microcolonies, an important step in theN. gonorrhoeaeinfectious process. Seminal fluid also facilitated increased bacterial aggregation in the form of shear-resistant three-dimensional biofilms. These results emphasize the importance of the gonococcal response to the influx of seminal fluid within the genital niche. Further characterization of theN. gonorrhoeaeresponse to semen will advance our understanding of the mechanisms behind the establishment of infection in naive hosts and the process of transmission.IMPORTANCEN. gonorrhoeaeis the causative agent of the globally prevalent sexually transmitted infection gonorrhea. An understudied aspect of this human-adapted pathogen is the change in bacterial physiology that occurs during sexual transmission.N. gonorrhoeaeencounters semen when transmitted from host to host, and it is known that, whenN. gonorrhoeaeis exposed to seminal fluid, alterations in bacterial motility and type IV pilus arrangement occur. This work extends our previous observations on this modulation of gonococcal physiology by seminal fluid and demonstrates that seminal plasma decreases surface adherence, promotes interbacterial interactions, and enhances biofilm formation.

Download Full-text

Analysis of Pilin Antigenic Variation inNeisseria meningitidisby Next-Generation Sequencing

Journal of Bacteriology ◽

10.1128/jb.00465-18 ◽

2018 ◽

Vol 200 (22) ◽

Cited By ~ 5

Author(s):

Jing Xu ◽

H Steven Seifert

Keyword(s):

Next Generation Sequencing ◽

Neisseria Gonorrhoeae ◽

Neisseria Meningitidis ◽

Antigenic Variation ◽

Immune Surveillance ◽

Human Pathogen ◽

Next Generation ◽

Content Type ◽

Generation Sequencing ◽

Host Immune Surveillance

ABSTRACTMany pathogenic microbes evade host immune surveillance by varying the surface antigens, a process termed antigenic variation. While the process of pilin antigenic variation has been extensively studied in the human pathogenNeisseria gonorrhoeae(gonococcus [Gc]), relatively few studies of pilin antigenic variation have been conducted withNeisseria meningitidis(meningococcus [Mc]). Mc is usually a commensal organism that colonizes the human nasopharynx, but when it translocates to the bloodstream or meninges, it results in the severe and often deadly meningococcal disease. The type IV pili of Mc isolates play a critical role in host surface adherence, and its major pilin component (PilE) can undergo antigenic variation. In this study, Roche 454 pyrosequencing was used to examine the pilin antigenic variation of Mc strain 8013, as well as 8013recA,recX,recQ,rep, andrecJmutants, Gc orthologues which have been shown to play a role in pilin antigenic variation. This study confirms that the McrecA,rep, andrecJgenes are essential for pilin antigenic variation. While the McrecQandrecXgene products contribute to normal frequencies of antigenic variation, the loss of these factors does not alter the types of pilin variants produced. Overall, this study shows that the mechanisms of pilin antigenic variation are conserved between Gc and Mc.IMPORTANCEAntigenic variation is a strategy used by many pathogens to escape host immune surveillance and establish persistent infections. This study successfully applies next-generation sequencing to study pilin antigenic variation in the human pathogenNeisseria meningitidis. This assay provides an affordable and efficient solution for quantifying antigenic variation frequency in mutant strains and for defining the recombination products of the process. We determined that there is a nonuniformity of silent donor copies used during meningococcus antigenic variation, and by the analysis of selected mutants deficient for specific recombination pathways, we show for the first time that the processes are conserved betweenN. meningitidisandNeisseria gonorrhoeae.

Download Full-text

A Double-Strand Break Does Not PromoteNeisseria gonorrhoeaePilin Antigenic Variation

Journal of Bacteriology ◽

10.1128/jb.00256-19 ◽

2019 ◽

Vol 201 (13) ◽

Cited By ~ 1

Author(s):

Lauren L. Prister ◽

Jing Xu ◽

H Steven Seifert

Keyword(s):

Neisseria Gonorrhoeae ◽

Antigenic Variation ◽

Dna Structure ◽

Strand Break ◽

Double Strand Break ◽

Content Type ◽

G4 Dna ◽

Recombination System ◽

Guanine Quadruplex ◽

Pilin Gene

ABSTRACTThe major subunit of the type IV pilus (T4p) ofNeisseria gonorrhoeaeundergoes antigenic variation (AV) dependent on a guanine quadruplex (G4) DNA structure located upstream of the pilin gene. Since the presence of G4 DNA induces genome instability in both eukaryotic and prokaryotic chromosomes, we tested whether a double-strand break (DSB) at the site of thepilEG4 sequence could substitute for G4-directed pilin AV. The G4 motif was replaced by an I-SceI cut site, and the cut site was also introduced to locations near the origin of replication and the terminus. Expression of the I-SceI endonuclease from an irrelevant chromosomal site confirmed that the endonuclease functions to induce double-strand breaks at all three locations. No antigenic variants were detected when the G4 was replaced with the I-SceI cut site, but there was a growth defect from having a DSB in the chromosome, and suppressor mutations that were mainly deletions of the cut site and/or the entirepilEgene accumulated. Thus, thepilEG4 does not act to promote pilin AV by generating a DSB but requires either a different type of break, a nick, or more complex interactions with other factors to stimulate this programmed recombination system.IMPORTANCENeisseria gonorrhoeae, the causative agent of gonorrhea, possesses a DNA recombination system to change one of its surface-exposed antigens. This recombination system, known as antigenic variation, uses an alternate DNA structure to initiate variation. The guanine quadruplex DNA structure is known to cause nicks or breaks in DNA; however, much remains unknown about how this structure functions in cells. We show that inducing a break by different means does not allow antigenic variation, indicating that the DNA structure may have a more complicated role.

Download Full-text

The Lst Sialyltransferase of Neisseria gonorrhoeae Can Transfer Keto-Deoxyoctanoate as the Terminal Sugar of Lipooligosaccharide: a Glyco-Achilles Heel That Provides a New Strategy for Vaccines to Prevent Gonorrhea

mBio ◽

10.1128/mbio.03666-20 ◽

2021 ◽

Vol 12 (2) ◽

Author(s):

Freda E.-C. Jen ◽

Margaret R. Ketterer ◽

Evgeny A. Semchenko ◽

Christopher J. Day ◽

Kate L. Seib ◽

...

Keyword(s):

Monoclonal Antibody ◽

Neisseria Gonorrhoeae ◽

Phase Variation ◽

Multidrug Resistant ◽

Content Type ◽

Acetylneuraminic Acid ◽

New Strategy ◽

Opsonophagocytic Killing ◽

Further Development

ABSTRACT The lipooligosaccharide (LOS) of Neisseria gonorrhoeae plays key roles in pathogenesis and is composed of multiple possible glycoforms. These glycoforms are generated by the process of phase variation and by differences in the glycosyltransferase gene content of particular strains. LOS glycoforms of N. gonorrhoeae can be terminated with an N-acetylneuraminic acid (Neu5Ac), which imparts resistance to the bactericidal activity of serum. However, N. gonorrhoeae cannot synthesize the CMP-Neu5Ac required for LOS biosynthesis and must acquire it from the host. In contrast, Neisseria meningitidis can synthesize endogenous CMP-Neu5Ac, the donor molecule for Neu5Ac, which is a component of some meningococcal capsule structures. Both species have an almost identical LOS sialyltransferase, Lst, that transfers Neu5Ac from CMP-Neu5Ac to the terminus of LOS. Lst is homologous to the LsgB sialyltransferase of nontypeable Haemophilus influenzae (NTHi). Studies in NTHi have demonstrated that LsgB can transfer keto-deoxyoctanoate (KDO) from CMP-KDO to the terminus of LOS in place of Neu5Ac. Here, we show that Lst can also transfer KDO to LOS in place of Neu5Ac in both N. gonorrhoeae and N. meningitidis. Consistent with access to the pool of CMP-KDO in the cytoplasm, we present data indicating that Lst is localized in the cytoplasm. Lst has previously been reported to be localized on the outer membrane. We also demonstrate that KDO is expressed as a terminal LOS structure in vivo in samples from infected women and further show that the anti-KDO monoclonal antibody 6E4 can mediate opsonophagocytic killing of N. gonorrhoeae. Taken together, these studies indicate that KDO expressed on gonococcal LOS represents a new antigen for the development of vaccines against gonorrhea. IMPORTANCE The emergence of multidrug-resistant N. gonorrhoeae strains that are resistant to available antimicrobials is a current health emergency, and no vaccine is available to prevent gonococcal infection. Lipooligosaccharide (LOS) is one of the major virulence factors of N. gonorrhoeae. The sialic acid N-acetylneuraminic acid (Neu5Ac) is present as the terminal glycan on LOS in N. gonorrhoeae. In this study, we made an unexpected discovery that KDO can be incorporated as the terminal glycan on LOS of N. gonorrhoeae by the alpha-2,3-sialyltransferase Lst. We showed that N. gonorrhoeae express KDO on LOS in vivo and that the KDO-specific monoclonal antibody 6E4 can direct opsonophagocytic killing of N. gonorrhoeae. These data support further development of KDO-LOS structures as vaccine antigens for the prevention of infection by N. gonorrhoeae.

Download Full-text

Cryo-EM Structures of a Gonococcal Multidrug Efflux Pump Illuminate a Mechanism of Drug Recognition and Resistance

mBio ◽

10.1128/mbio.00996-20 ◽

2020 ◽

Vol 11 (3) ◽

Cited By ~ 5

Author(s):

Meinan Lyu ◽

Mitchell A. Moseng ◽

Jennifer L. Reimche ◽

Concerta L. Holley ◽

Vijaya Dhulipala ◽

...

Keyword(s):

Electron Microscopy ◽

Drug Resistance ◽

Neisseria Gonorrhoeae ◽

Antimicrobial Agents ◽

Efflux Pump ◽

Multidrug Efflux ◽

Multidrug Efflux Pump ◽

Transmitted Infection ◽

Content Type ◽

Cryo Electron Microscopy

ABSTRACT Neisseria gonorrhoeae is an obligate human pathogen and causative agent of the sexually transmitted infection (STI) gonorrhea. The most predominant and clinically important multidrug efflux system in N. gonorrhoeae is the multiple transferrable resistance (Mtr) pump, which mediates resistance to a number of different classes of structurally diverse antimicrobial agents, including clinically used antibiotics (e.g., β-lactams and macrolides), dyes, detergents and host-derived antimicrobials (e.g., cationic antimicrobial peptides and bile salts). Recently, it has been found that gonococci bearing mosaic-like sequences within the mtrD gene can result in amino acid changes that increase the MtrD multidrug efflux pump activity, probably by influencing antimicrobial recognition and/or extrusion to elevate the level of antibiotic resistance. Here, we report drug-bound solution structures of the MtrD multidrug efflux pump carrying a mosaic-like sequence using single-particle cryo-electron microscopy, with the antibiotics bound deeply inside the periplasmic domain of the pump. Through this structural approach coupled with genetic studies, we identify critical amino acids that are important for drug resistance and propose a mechanism for proton translocation. IMPORTANCE Neisseria gonorrhoeae has become a highly antimicrobial-resistant Gram-negative pathogen. Multidrug efflux is a major mechanism that N. gonorrhoeae uses to counteract the action of multiple classes of antibiotics. It appears that gonococci bearing mosaic-like sequences within the gene mtrD, encoding the most predominant and clinically important transporter of any gonococcal multidrug efflux pump, significantly elevate drug resistance and enhance transport function. Here, we report cryo-electron microscopy (EM) structures of N. gonorrhoeae MtrD carrying a mosaic-like sequence that allow us to understand the mechanism of drug recognition. Our work will ultimately inform structure-guided drug design for inhibiting these critical multidrug efflux pumps.

Download Full-text

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

mBio ◽

10.1128/mbio.03068-19 ◽

2020 ◽

Vol 11 (2) ◽

Cited By ~ 2

Author(s):

Luke R. Green ◽

Ali A. Al-Rubaiawi ◽

Mohammad A. R. M. Al-Maeni ◽

Odile B. Harrison ◽

Matthew Blades ◽

...

Keyword(s):

Genetic Variation ◽

Gene Transfer ◽

Outer Membrane ◽

Antigenic Variation ◽

Allelic Variation ◽

Host Responses ◽

Content Type ◽

Type Iv ◽

Asymptomatic Carriage ◽

Genes Encoding

ABSTRACT 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.

Download Full-text