PacBio amplicon sequencing method to measure pilin antigenic variation frequencies of Neisseria gonorrhoeae

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.

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Ecf, an Alternative Sigma Factor from Neisseria gonorrhoeae, Controls Expression of msrAB, Which Encodes Methionine Sulfoxide Reductase

Journal of Bacteriology ◽

10.1128/jb.188.10.3463-3469.2006 ◽

2006 ◽

Vol 188 (10) ◽

pp. 3463-3469 ◽

Cited By ~ 23

Author(s):

Ishara C. Gunesekere ◽

Charlene M. Kahler ◽

Catherine S. Ryan ◽

Lori A. S. Snyder ◽

Nigel J. Saunders ◽

...

Keyword(s):

Neisseria Gonorrhoeae ◽

Sigma Factor ◽

Reductase Activity ◽

Methionine Sulfoxide ◽

Methionine Sulfoxide Reductase ◽

Transcriptional Unit ◽

Alternative Sigma Factor ◽

Gene Encoding ◽

Strain Fa1090

ABSTRACT A DNA microarray was used to identify genes transcribed in Neisseria gonorrhoeae using Ecf, an alternative sigma factor. No differences between the transcriptional profiles of strain FA1090 and a mutant where ecf had been inactivated could be detected when both were grown in vitro. We therefore constructed a gonococcal strain in which Ecf can be overexpressed. Some differentially expressed genes are clustered with ecf on the genome and appear to form a single transcriptional unit. Expression of the gene encoding MsrAB, which possesses methionine sulfoxide reductase activity, was also dependent on Ecf, suggesting that the regulon responds to oxidative damage. Western blotting confirmed that the increased level of MsrAB protein is dependent on the presence of Ecf.

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Pilin Antigenic Variation Occurs Independently of the RecBCD Pathway in Neisseria gonorrhoeae

Journal of Bacteriology ◽

10.1128/jb.00535-09 ◽

2009 ◽

Vol 191 (18) ◽

pp. 5613-5621 ◽

Cited By ~ 20

Author(s):

R. Allen Helm ◽

H. Steven Seifert

Keyword(s):

Dna Damage ◽

Neisseria Gonorrhoeae ◽

Antigenic Variation ◽

Risk Groups ◽

Gene Encoding ◽

Indirect Methods ◽

Strand Breaks ◽

Type Iv ◽

Recombination Pathway

ABSTRACT Type IV pilus expression has been strongly implicated in the virulence of Neisseria gonorrhoeae, the causative agent of gonorrhea. In Neisseria, these pili undergo frequent antigenic variation (Av), which is presumed to allow reinfection of high-risk groups. Pilin Av is the result of RecA-mediated recombination events between the gene encoding the major pilin subunit (pilE) and multiple silent pilin locus (pilS) copies, utilizing a RecF-like recombination pathway. The role of RecBCD in pilin Av has been controversial. Previous studies measuring pilin Av in recB and recD mutants in two independent strains of N. gonorrhoeae (MS11 and FA1090) by indirect methods yielded conflicting results. In addition, these two laboratory strains have been suggested to express very different DNA repair capabilities. We show that the FA1090 and MS11 parental strains have similar abilities to repair DNA damage via UV-induced DNA damage, nalidixic acid-induced double-strand breaks, and methyl methanesulfonate-induced alkylation and that RecB and RecD are involved in the repair of these lesions. To test the role of the RecBCD pathway in pilin Av, the rate and frequency of pilin Av were directly measured by sequencing the pilE locus in randomly selected piliated progeny of both MS11 and FA1090 in recB and recD mutants. Our results definitively show that recB and recD mutants undergo pilin Av at rates similar to those of the parents in both strain backgrounds, demonstrating that efficient pilin Av is neither enhanced nor inhibited by the RecBCD complex.

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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.

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Evaluation of the immunogenic property of NT H. influenzae protein D with Neisseria meningitidis OMV in BALB/c

The Journal of Infection in Developing Countries ◽

10.3855/jidc.7513 ◽

2016 ◽

Vol 10 (12) ◽

pp. 1345-1351 ◽

Cited By ~ 3

Author(s):

Ava Behrouzi ◽

Saeid Bouzari ◽

Seyed Davar Siadat ◽

Mana Oloomi ◽

Mehdi Davari ◽

...

Keyword(s):

Antigenic Variation ◽

Membrane Vesicle ◽

Negative Control Group ◽

Negative Control ◽

Enzyme Linked Immunosorbent Assay ◽

Control Group ◽

Gene Encoding ◽

Immunogenic Property ◽

Vaccine Candidates ◽

Subcutaneous Immunization

Introduction: Identifying ideal non typeable Haemophilus influenzae (NTHi) vaccine candidates has not been easy due to extensive sequence and antigenic variation among gene products interacting with the immune system. Protein D (PD) is a highly conserved 42 kDa surface lipoprotein available in all H. influenzae, including NTHi. Methodology: In this study, the gene encoding PD was cloned from H. influenzae and expressed in Escheriachia coli TOPO10 cell in pBAD vector. Arabinose was used to express recombinant protein. In order to purify the protein, Ni-NTA agarose was used to perform affinity chromatography. Purified PD and PD mixed with outer membrane vesicle (OMV) and alum adjuvant were used for subcutaneous immunization in BALB/c mice. After vaccination, IgG responses to PD-OMV, PD-alum, and PD alone were determined by enzyme-linked immunosorbent assay (ELISA). Results: The recombinant PD containing His6 residues showed a molecular weight of 42 kDa. Anti-PD IgG was detected after first immunization in all groups of mice compared to the negative control group, and it increased after first vaccination, but results showed that the addition of OMV to PD led to a remarkable increase in IgG responses. Conclusions: Our results suggest an important role for OMV as an adjuvant and show how it could potentially be used when conjugated to H. influenzae PD or other safe subunit vaccine candidates.

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Molecular mechanisms of antigenic variation in Neisseria gonorrhoeae

Molecular and Cell Biology of Sexually Transmitted Diseases ◽

10.1007/978-94-011-2384-6_1 ◽

1992 ◽

pp. 1-22 ◽

Cited By ~ 1

Author(s):

H. Steven Seifert

Keyword(s):

Neisseria Gonorrhoeae ◽

Molecular Mechanisms ◽

Antigenic Variation

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Protocols for 16S rDNA Array Analyses of Microbial Communities by Sequence-Specific Labeling of DNA Probes

The Scientific World JOURNAL ◽

10.1100/tsw.2003.44 ◽

2003 ◽

Vol 3 ◽

pp. 578-584 ◽

Cited By ~ 9

Author(s):

Knut Rudi ◽

Janneke Treimo ◽

Hilde Nissen ◽

Gerd Vegarud

Keyword(s):

Microbial Communities ◽

Pcr Amplification ◽

Dna Probes ◽

Dna Array ◽

Gene Encoding ◽

Variable Regions ◽

Conserved Regions ◽

Rdna Array ◽

Array Hybridization ◽

Array Analyses

Analyses of complex microbial communities are becoming increasingly important. Bottlenecks in these analyses, however, are the tools to actually describe the biodiversity. Novel protocols for DNA array-based analyses of microbial communities are presented. In these protocols, the specificity obtained by sequence-specific labeling of DNA probes is combined with the possibility of detecting several different probes simultaneously by DNA array hybridization. The gene encoding 16S ribosomal RNA was chosen as the target in these analyses. This gene contains both universally conserved regions and regions with relatively high variability. The universally conserved regions are used for PCR amplification primers, while the variable regions are used for the specific probes. Protocols are presented for DNA purification, probe construction, probe labeling, and DNA array hybridizations.

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Reaction of O2with a diiron protein generates a mixed-valent Fe2+/Fe3+center and peroxide

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1809913116 ◽

2019 ◽

Vol 116 (6) ◽

pp. 2058-2067 ◽

Cited By ~ 10

Author(s):

Justin M. Bradley ◽

Dimitri A. Svistunenko ◽

Jacob Pullin ◽

Natalie Hill ◽

Rhona K. Stuart ◽

...

Keyword(s):

Electron Transfer ◽

High Resolution ◽

Long Range ◽

Copper Stress ◽

Common Mechanism ◽

Gene Encoding ◽

X Ray ◽

Helical Bundle ◽

Ferroxidase Center ◽

Diiron Site

The gene encoding the cyanobacterial ferritinSynFtn is up-regulated in response to copper stress. Here, we show that, whileSynFtn does not interact directly with copper, it is highly unusual in several ways. First, its catalytic diiron ferroxidase center is unlike those of all other characterized prokaryotic ferritins and instead resembles an animal H-chain ferritin center. Second, as demonstrated by kinetic, spectroscopic, and high-resolution X-ray crystallographic data, reaction of O2with the di-Fe2+center results in a direct, one-electron oxidation to a mixed-valent Fe2+/Fe3+form. Iron–O2chemistry of this type is currently unknown among the growing family of proteins that bind a diiron site within a four α-helical bundle in general and ferritins in particular. The mixed-valent form, which slowly oxidized to the more usual di-Fe3+form, is an intermediate that is continually generated during mineralization. Peroxide, rather than superoxide, is shown to be the product of O2reduction, implying that ferroxidase centers function in pairs via long-range electron transfer through the protein resulting in reduction of O2bound at only one of the centers. We show that electron transfer is mediated by the transient formation of a radical on Tyr40, which lies ∼4 Å from the diiron center. As well as demonstrating an expansion of the iron–O2chemistry known to occur in nature, these data are also highly relevant to the question of whether all ferritins mineralize iron via a common mechanism, providing unequivocal proof that they do not.

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Rapid Engineering of Foot-and-Mouth Disease Vaccine and Challenge Viruses

Journal of Virology ◽

10.1128/jvi.00155-17 ◽

2017 ◽

Vol 91 (16) ◽

Cited By ~ 11

Author(s):

Seo-Yong Lee ◽

Yeo-Joo Lee ◽

Rae-Hyung Kim ◽

Jeong-Nam Park ◽

Min-Eun Park ◽

...

Keyword(s):

Vaccine Strain ◽

Antigenic Variation ◽

Foot And Mouth Disease ◽

Vaccine Virus ◽

Mouth Disease ◽

Economic Losses ◽

Gene Encoding ◽

Virus Challenge ◽

Mouth Disease Virus ◽

Foot And Mouth

ABSTRACT There are seven antigenically distinct serotypes of foot-and-mouth disease virus (FMDV), each of which has intratypic variants. In the present study, we have developed methods to efficiently generate promising vaccines against seven serotypes or subtypes. The capsid-encoding gene (P1) of the vaccine strain O1/Manisa/Turkey/69 was replaced with the amplified or synthetic genes from the O, A, Asia1, C, SAT1, SAT2, and SAT3 serotypes. Viruses of the seven serotype were rescued successfully. Each chimeric FMDV with a replacement of P1 showed serotype-specific antigenicity and varied in terms of pathogenesis in pigs and mice. Vaccination of pigs with an experimental trivalent vaccine containing the inactivated recombinants based on the main serotypes O, A, and Asia1 effectively protected them from virus challenge. This technology could be a potential strategy for a customized vaccine with challenge tools to protect against epizootic disease caused by specific serotypes or subtypes of FMDV. IMPORTANCE Foot-and-mouth disease (FMD) virus (FMDV) causes significant economic losses. For vaccine preparation, the selection of vaccine strains was complicated by high antigenic variation. In the present study, we suggested an effective strategy to rapidly prepare and evaluate mass-produced customized vaccines against epidemic strains. The P1 gene encoding the structural proteins of the well-known vaccine virus was replaced by the synthetic or amplified genes of viruses of seven representative serotypes. These chimeric viruses generally replicated readily in cell culture and had a particle size similar to that of the original vaccine strain. Their antigenicity mirrored that of the original serotype from which their P1 gene was derived. Animal infection experiments revealed that the recombinants varied in terms of pathogenicity. This strategy will be a useful tool for rapidly generating customized FMD vaccines or challenge viruses for all serotypes, especially for FMD-free countries, which have prohibited the import of FMDVs.

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Altering the Neisseria gonorrhoeae pilE Guanine Quadruplex Loop Bases Affects Pilin Antigenic Variation

Biochemistry ◽

10.1021/acs.biochem.9b01038 ◽

2020 ◽

Vol 59 (10) ◽

pp. 1104-1112

Author(s):

Lauren L. Prister ◽

Shaohui Yin ◽

Laty A. Cahoon ◽

H Steven Seifert

Keyword(s):

Neisseria Gonorrhoeae ◽

Antigenic Variation ◽

Guanine Quadruplex

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