Phase Variation of NadA in Invasive Neisseria meningitidis Isolates Impacts on Coverage Estimates for 4C-MenB, a MenB Vaccine

ABSTRACT A recombinant NadA protein is one of the four major protective antigens of 4C-MenB (Bexsero), a vaccine developed for serogroup B Neisseria meningitidis (MenB). The meningococcal antigen typing system (MATS) is utilized as a high-throughput assay for assessing the invasive MenB strain coverage of 4C-MenB. Where present, the nadA gene is subject to phase-variable changes in transcription due to a 5′TAAA repeat tract located in a regulatory region. The promoter-containing intergenic region (IGR) sequences and 5′TAAA repeat numbers were determined for 906 invasive meningococcal disease isolates possessing the nadA gene. Exclusion of the 5′TAAA repeats reduced the number of IGR alleles from 82 to 23. Repeat numbers were associated with low and high levels of NadA expression by Western blotting and enzyme-linked immunosorbent assay (ELISA). Low-expression repeat numbers were present in 83% of 179 MenB isolates with NadA-2/3 or NadA-1 peptide variants and 68% of 480 MenW ST-11 complex isolates with NadA-2/3 peptide variants. For isolates with vaccine-compatible NadA variants, 93% of MATS-negative isolates were associated with low-expression repeat numbers, whereas 63% of isolates with MATS relative potency (RP) scores above the 95% confidence interval for the positive bactericidal threshold had high-expression repeat numbers. Analysis of 5′TAAA repeat numbers has potential as a rapid, high-throughput method for assessing strain coverage for the NadA component of 4C-MenB. A key application will be assessing coverage in meningococcal disease cases where confirmation is by PCR only and MATS cannot be applied.

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Genotypic and Phenotypic Characterization of theO-Linked Protein Glycosylation System Reveals High Glycan Diversity in Paired Meningococcal Carriage Isolates

Journal of Bacteriology ◽

10.1128/jb.00794-17 ◽

2018 ◽

Vol 200 (16) ◽

Cited By ~ 8

Author(s):

Bente Børud ◽

Guro K. Bårnes ◽

Ola Brønstad Brynildsrud ◽

Elisabeth Fritzsønn ◽

Dominique A. Caugant

Keyword(s):

Neisseria Meningitidis ◽

Phase Variation ◽

Protein Glycosylation ◽

Phase Variable ◽

Upper Respiratory Tract ◽

Content Type ◽

Variable Expression ◽

Evolutionary Forces ◽

Strain Collection

ABSTRACTSpecies within the genusNeisseriadisplay significant glycan diversity associated with theO-linked protein glycosylation (pgl) systems due to phase variation and polymorphic genes and gene content. The aim of this study was to examine in detail thepglgenotype and glycosylation phenotype in meningococcal isolates and the changes occurring during short-term asymptomatic carriage. Paired meningococcal isolates derived from 50 asymptomatic meningococcal carriers, taken about 2 months apart, were analyzed with whole-genome sequencing. TheO-linked protein glycosylation genes were characterized in detail using the Genome Comparator tool at the https://pubmlst.org/ database. Immunoblotting with glycan-specific antibodies (Abs) was used to investigate the protein glycosylation phenotype. All majorpgllocus polymorphisms identified inNeisseria meningitidisto date were present in our isolate collection, with the variable presence ofpglGandpglH, both in combination with eitherpglBorpglB2. We identified significant changes and diversity in thepglgenotype and/or glycan phenotype in 96% of the paired isolates. There was also a high degree of glycan microheterogeneity, in which different variants of glycan structures were found at a given glycoprotein. The main mechanism responsible for the observed differences was phase-variable expression of the involved glycosyltransferases and theO-acetyltransferase. To our knowledge, this is the first characterization of thepglgenotype and glycosylation phenotype in a larger strain collection. This report thus provides important insight into glycan diversity inN. meningitidisand into the phase variability changes that influence the expressed glycoform repertoire during meningococcal carriage.IMPORTANCEBacterial meningitis is a serious global health problem, and one of the major causative organisms isNeisseria meningitidis, which is also a common commensal in the upper respiratory tract of healthy humans. In bacteria, numerous loci involved in biosynthesis of surface-exposed antigenic structures that are involved in the interaction between bacteria and host are frequently subjected to homologous recombination and phase variation. These mechanisms are well described inNeisseria, and phase variation provides the ability to change these structures reversibly in response to the environment. Protein glycosylation systems are becoming widely identified in bacteria, and yet little is known about the mechanisms and evolutionary forces influencing glycan composition during carriage and disease.

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Influence of the combination and phase variation status of the haemoglobin receptors HmbR and HpuAB on meningococcal virulence

Microbiology ◽

10.1099/mic.0.046946-0 ◽

2011 ◽

Vol 157 (5) ◽

pp. 1446-1456 ◽

Cited By ~ 36

Author(s):

Isfahan Tauseef ◽

Odile B. Harrison ◽

Karl G. Wooldridge ◽

Ian M. Feavers ◽

Keith R. Neal ◽

...

Keyword(s):

Neisseria Meningitidis ◽

Meningococcal Disease ◽

Systemic Disease ◽

Phase Variation ◽

Phase Variable ◽

Insertion Element ◽

Systemic Spread ◽

High Level ◽

Complete Deletion

Neisseria meningitidis can utilize haem, haemoglobin and haemoglobin–haptoglobin complexes as sources of iron via two TonB-dependent phase variable haemoglobin receptors, HmbR and HpuAB. HmbR is over-represented in disease isolates, suggesting a link between haemoglobin acquisition and meningococcal disease. This study compared the distribution of HpuAB and phase variation (PV) status of both receptors in disease and carriage isolates. Meningococcal disease (n = 214) and carriage (n = 305) isolates representative of multiple clonal complexes (CCs) were investigated for the distribution, polyG tract lengths and ON/OFF status of both haemoglobin receptors, and for the deletion mechanism for HpuAB. Strains with both receptors or only hmbR were present at similar frequencies among meningococcal disease isolates as compared with carriage isolates. However, >90 % of isolates from the three CCs CC5, CC8 and CC11 with the highest disease to carriage ratios contained both receptors. Strains with an hpuAB-only phenotype were under-represented among disease isolates, suggesting selection against this receptor during systemic disease, possibly due to the receptor having a high level of immunogenicity or being inefficient in acquisition of iron during systemic spread. Absence of hpuAB resulted from either complete deletion or replacement by an insertion element. In an examination of PV status, one or both receptors were found in an ON state in 91 % of disease and 71 % of carriage isolates. We suggest that expression of a haemoglobin receptor, either HmbR or HpuAB, is of major importance for systemic spread of meningococci, and that the presence of both receptors contributes to virulence in some strains.

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Analysis of Invasive NontypeableHaemophilus influenzaeIsolates Reveals Selection for the Expression State of Particular Phase-Variable Lipooligosaccharide Biosynthetic Genes

Infection and Immunity ◽

10.1128/iai.00093-19 ◽

2019 ◽

Vol 87 (5) ◽

Cited By ~ 6

Author(s):

Zachary N. Phillips ◽

Charles Brizuela ◽

Amy V. Jennison ◽

Megan Staples ◽

Keith Grimwood ◽

...

Keyword(s):

Gene Expression ◽

Phase Variation ◽

Phase Variable ◽

Chronic Infections ◽

Biosynthetic Genes ◽

Content Type ◽

Invasive Infections ◽

Acetyl Group ◽

Overt Disease ◽

Expression Status

ABSTRACTNontypeableHaemophilus influenzae(NTHi) is a major human pathogen, responsible for several acute and chronic infections of the respiratory tract. The incidence of invasive infections caused by NTHi is increasing worldwide. NTHi is able to colonize the nasopharynx asymptomatically, and the exact change(s) responsible for transition from benign carriage to overt disease is not understood. We have previously reported that phase variation (the rapid and reversible ON-OFF switching of gene expression) of particular lipooligosaccharide (LOS) glycosyltransferases occurs during transition from colonizing the nasopharynx to invading the middle ear. Variation in the structure of the LOS is dependent on the ON/OFF expression status of each of the glycosyltransferases responsible for LOS biosynthesis. In this study, we surveyed a collection of invasive NTHi isolates for ON/OFF expression status of seven phase-variable LOS glycosyltransferases. We report that the expression state of the LOS biosynthetic genesoafAON andlic2AOFF shows a correlation with invasive NTHi isolates. We hypothesize that these gene expression changes contribute to the invasive potential of NTHi. OafA expression, which is responsible for the addition of anO-acetyl group onto the LOS, has been shown to impart a phenotype of increased serum resistance and may serve as a marker for invasive NTHi.

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Insights on Population Structure and Within-Host Genetic Changes among Meningococcal Carriage Isolates from U.S. Universities

mSphere ◽

10.1128/msphere.00197-20 ◽

2020 ◽

Vol 5 (2) ◽

Author(s):

Sandeep J. Joseph ◽

Nadav Topaz ◽

How-Yi Chang ◽

Melissa J. Whaley ◽

Jeni T. Vuong ◽

...

Keyword(s):

General Population ◽

Neisseria Meningitidis ◽

Meningococcal Disease ◽

Mass Vaccination ◽

Invasive Meningococcal Disease ◽

Whole Genome ◽

Genetic Changes ◽

Content Type ◽

Host Genetic ◽

Vaccination Campaigns

ABSTRACT In 2015 and 2016, meningococcal carriage evaluations were conducted at two universities in the United States following mass vaccination campaigns in response to Neisseria meningitidis serogroup B (NmB) disease outbreaks. A simultaneous carriage evaluation was also conducted at a university near one of the outbreaks, where no NmB cases were reported and no mass vaccination occurred. A total of ten cross-sectional carriage evaluation rounds were conducted, resulting in 1,514 meningococcal carriage isolates collected from 7,001 unique participants; 1,587 individuals were swabbed at multiple time points (repeat participants). All isolates underwent whole-genome sequencing. The most frequently observed clonal complexes (CC) were CC198 (27.3%), followed by CC1157 (17.4%), CC41/44 (9.8%), CC35 (7.4%), and CC32 (5.6%). Phylogenetic analysis identified carriage isolates that were highly similar to the NmB outbreak strains; comparative genomics between these outbreak and carriage isolates revealed genetic changes in virulence genes. Among repeat participants, 348 individuals carried meningococcal bacteria during at least one carriage evaluation round; 50.3% retained N. meningitidis carriage of a strain with the same sequence type (ST) and CC across rounds, 44.3% only carried N. meningitidis in one round, and 5.4% acquired a new N. meningitidis strain between rounds. Recombination, point mutations, deletions, and simple sequence repeats were the most frequent genetic mechanisms found in isolates collected from hosts carrying a strain of the same ST and CC across rounds. Our findings provide insight on the dynamics of meningococcal carriage among a population that is at higher risk for invasive meningococcal disease than the general population. IMPORTANCE U.S. university students are at a higher risk of invasive meningococcal disease than the general population. The responsible pathogen, Neisseria meningitidis, can be carried asymptomatically in the oropharynx; the dynamics of meningococcal carriage and the genetic features that distinguish carriage versus disease states are not completely understood. Through our analyses, we aimed to provide data to address these topics. We whole-genome sequenced 1,514 meningococcal carriage isolates from individuals at three U.S. universities, two of which underwent mass vaccination campaigns following recent meningococcal outbreaks. We describe the within-host genetic changes among individuals carrying a strain with the same molecular type over time, the primary strains being carried in this population, and the genetic differences between closely related outbreak and carriage strains. Our results provide detailed information on the dynamics of meningococcal carriage and the genetic differences in carriage and outbreak strains, which can inform future efforts to reduce the incidence of invasive meningococcal disease.

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Genome Sequence of a Neisseria meningitidis Capsule Null Locus Strain from the Clonal Complex of Sequence Type 198

Journal of Bacteriology ◽

10.1128/jb.01099-12 ◽

2012 ◽

Vol 194 (18) ◽

pp. 5144-5145 ◽

Cited By ~ 7

Author(s):

Sabine Schork ◽

Andreas Schlüter ◽

Jochen Blom ◽

Susanne Schneiker-Bekel ◽

Alfred Pühler ◽

...

Keyword(s):

Neisseria Meningitidis ◽

Genome Sequence ◽

Meningococcal Disease ◽

Clonal Complex ◽

Sequence Type ◽

Invasive Meningococcal Disease ◽

Content Type ◽

Polysaccharide Capsules

ABSTRACTNeisseria meningitidisis a commensal and accidental pathogen exclusively of humans. Although the production of polysaccharide capsules is considered to be essential for meningococcal virulence, there have been reports of constitutively unencapsulated strains causing invasive meningococcal disease (IMD). Here we report the genome sequence of a capsule null locus (cnl) strain of sequence type 198 (ST-198), which is found in half of the reported cases of IMD caused bycnlmeningococcal strains.

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Phasevarions Mediate Epigenetic Regulation of Antimicrobial Susceptibility in Neisseria meningitidis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00004-14 ◽

2014 ◽

Vol 58 (7) ◽

pp. 4219-4221 ◽

Cited By ~ 22

Author(s):

Freda E.-C. Jen ◽

Kate L. Seib ◽

Michael P. Jennings

Keyword(s):

Gene Expression ◽

Neisseria Meningitidis ◽

Epigenetic Regulation ◽

Antimicrobial Susceptibility ◽

Bacterial Pathogens ◽

Phase Variation ◽

Dna Methyltransferases ◽

Global Methylation ◽

Content Type ◽

Multiple Genes

ABSTRACTPhase variation is a common feature of host-adapted bacterial pathogens such asNeisseria meningitidis. Recently, we reported that this rapid on/off switching of gene expression occurs in DNA methyltransferases, altering expression in multiple genes via changes in global methylation. In the current study, we compared MIC values of strains with ModA11, ModA12, and ModD1 phasevarions, revealing MIC differences due to ModA11 and ModA12 switching, with a ModA11_OFF strain showing 4-fold reduced susceptibilities to ceftazidime and ciprofloxacin.

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Disease-Associated Neisseria meningitidis Isolates Inhibit Wound Repair in Respiratory Epithelial Cells in a Type IV Pilus-Independent Manner

Infection and Immunity ◽

10.1128/iai.02001-14 ◽

2014 ◽

Vol 82 (12) ◽

pp. 5023-5034 ◽

Cited By ~ 2

Author(s):

Xiaoyun Ren ◽

Joanna K. MacKichan

Keyword(s):

Cell Migration ◽

Neisseria Meningitidis ◽

Meningococcal Disease ◽

Wound Repair ◽

Disease Onset ◽

Independent Manner ◽

Content Type ◽

Type Iv ◽

Respiratory Epithelial

ABSTRACTNeisseria meningitidisis the causative agent of meningococcal disease. Onset of meningococcal disease can be extremely rapid and can kill within a matter of hours. However, although a much-feared pathogen,Neisseria meningitidisis frequently found in the nasopharyngeal mucosae of healthy carriers. The bacterial factors that distinguish disease- from carriage-associated meningococci are incompletely understood. Evidence suggesting that disruptions to the nasopharynx may increase the risk of acquiring meningococcal disease led us to evaluate the ability of disease- and carriage-associated meningococcal isolates to inhibit cell migration, using anin vitroassay for wound repair. We found that disease-associated isolates in our collection inhibited wound closure, while carriage-associated isolates were more variable, with many isolates not inhibiting wound repair at all. For isolates selected for further study, we found that actin morphology, such as presence of lamellipodia, correlated with cell migration. We demonstrated that multiple meningococcal virulence factors, including the type IV pili, are dispensable for inhibition of wound repair. Inhibition of wound repair was also shown to be an active process, i.e., requiring live bacteria undergoing active protein synthesis.

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Dissecting meningococcal disease and carriage traits using high throughput phenotypic testing

Access Microbiology ◽

10.1099/acmi.ac2020.po0210 ◽

2020 ◽

Vol 2 (7A) ◽

Author(s):

Megan De Ste Croix ◽

Dave Neelam ◽

Neil Oldfield ◽

Jay Lucidarme ◽

David Turner ◽

...

Keyword(s):

High Throughput ◽

Meningococcal Disease ◽

Association Studies ◽

Phase Variation ◽

Genome Wide Association Studies ◽

Current Policy ◽

Genome Sequences ◽

Phenotypic Differences ◽

Genome Wide ◽

The Uk

Despite on-going vaccination programmes, Neisseria meningitidis causes over 700 cases of invasive meningococcal disease (IMD) in the UK each year. In 2017-18, the MenW and MenY capsular groups caused 38% of all IMD cases. Current policy is to generate genome sequences of all meningococcal disease isolates. Using this resource, we aim to understand how genetic variation contributes to phenotypic differences between carriage and disease isolates. We are adapting a variety of assays, designed to mimic carriage and disease behaviours, for high throughput phenotypic testing of multiple meningococcal isolates from carriage and cases of IMD. We have selected 335 MenW cc11 and MenY cc23 isolates and are currently testing subsets of isolates in cell culture (CaLu3), growth and biofilm assays. Phenotypic differences will be utilised as input data for Genome Wide Association Studies that aim to identify the specific genomic variants, or combinations of variants, determining observed differences. Genomic data will include whole genome sequences and repeat-mediated phase variation states. Our preliminary data has detected variation in the ability of cc11 and cc23 isolates to disrupt monolayers of CaLu3 cells, indicating that minor genetic differences in phylogentically similar organisms may be physiologically important for both carriage and disease. We will also discuss progress in establishing successful, high-throughput assays for testing multiple isolates.

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The Major Phase-Variable Outer Membrane Protein ofEscherichia coli Structurally Resembles the Immunoglobulin A1 Protease Class of Exported Protein and Is Regulated by a Novel Mechanism Involving Dam and OxyR

Journal of Bacteriology ◽

10.1128/jb.181.7.2132-2141.1999 ◽

1999 ◽

Vol 181 (7) ◽

pp. 2132-2141 ◽

Cited By ~ 120

Author(s):

Ian R. Henderson ◽

Peter Owen

Keyword(s):

Membrane Protein ◽

Outer Membrane ◽

Dna Sequences ◽

Outer Membrane Protein ◽

Signal Sequence ◽

Regulatory Region ◽

Specific Antigen ◽

Phase Variation ◽

Phase Variable ◽

Phase Switching

ABSTRACT Here we report the characterization of an Escherichia coli gene (agn43) which encodes the principal phase-variable outer membrane protein termed antigen 43 (Ag43). Theagn43 gene encodes a precursor protein of 107 kDa containing a 52-amino-acid signal sequence. Posttranslational processing generates an α43 subunit (predictedM r of 49,789) and a C-terminal domain (β43) with features typical of a bacterial integral outer membrane protein (predicted M r of 51,642). Secondary structure analysis predicts that β43 exists as an 18-stranded β barrel and that Ag43 shows structural organization closely resembling that of immunoglobulin A1 protease type of exoprotein produced by pathogenic Neisseria andHaemophilus spp. The correct processing of the polyprotein to α43 and β43 in OmpT, OmpP, and DegP protease-deficient E. coli strains points to an autocatalytic cleavage mechanism, a hypothesis supported by the occurrence of an aspartyl protease active site within α43. Ag43, a species-specific antigen, possesses two RGD motifs of the type implicated in binding to human integrins. The mechanism of reversible phase variation was studied by immunochemical analysis of a panel of well-defined regulatory mutants and by analysis of DNA sequences upstream of agn43. Evidence strongly suggests that phase variation is regulated by both deoxyadenosine methylase (Dam) and by OxyR. Thus, oxyR mutants are locked on for Ag43 expression, whereas dam mutants are locked off for Ag43 expression. We propose a novel mechanism for the regulation of phase switching in which OxyR competes with Dam for unmethylated GATC sites in the regulatory region of the agn43 gene.

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Stabilizing Genetically Unstable Simple Sequence Repeats in the Campylobacter jejuni Genome by Multiplex Genome Editing: a Reliable Approach for Delineating Multiple Phase-Variable Genes

mBio ◽

10.1128/mbio.01401-21 ◽

2021 ◽

Author(s):

Shouji Yamamoto ◽

Sunao Iyoda ◽

Makoto Ohnishi

Keyword(s):

Campylobacter Jejuni ◽

Simple Sequence Repeat ◽

Phase Variation ◽

Developed Countries ◽

Phase Variable ◽

Phenotypic Stability ◽

Content Type ◽

Multiple Phase ◽

Simple Sequence ◽

Variable Genes

Campylobacter jejuni is the leading bacterial cause of foodborne gastroenteritis in developed countries and occasionally progresses to the autoimmune disease Guillain-Barré syndrome. A relatively large number of hypermutable simple sequence repeat (SSR) tracts in the C. jejuni genome markedly decreases its phenotypic stability through reversible changes in the ON or OFF expression states of the genes in which they reside, a phenomenon called phase variation.

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