scholarly journals Mismatch Correction Modulates Mutation Frequency and Pilus Phase and Antigenic Variation in Neisseria gonorrhoeae

2009 ◽  
Vol 192 (1) ◽  
pp. 316-325 ◽  
Author(s):  
Alison K. Criss ◽  
Kevin M. Bonney ◽  
Rhoda A. Chang ◽  
Paul M. Duffin ◽  
Brian E. LeCuyer ◽  
...  
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Antigenic Variation ◽  
Phase Variation ◽  
Single Nucleotide ◽  
Mismatch Correction ◽  
Nucleotide Mismatch ◽  
Specific Pathogen ◽  
Dna Mismatches ◽  
Nucleotide Insertions ◽  
Human Specific

ABSTRACT The mismatch correction (MMC) system repairs DNA mismatches and single nucleotide insertions or deletions postreplication. To test the functions of MMC in the obligate human pathogen Neisseria gonorrhoeae, homologues of the core MMC genes mutS and mutL were inactivated in strain FA1090. No mutH homologue was found in the FA1090 genome, suggesting that gonococcal MMC is not methyl directed. MMC mutants were compared to a mutant in uvrD, the helicase that functions with MMC in Escherichia coli. Inactivation of MMC or uvrD increased spontaneous resistance to rifampin and nalidixic acid, and MMC/uvrD double mutants exhibited higher mutation frequencies than any single mutant. Loss of MMC marginally enhanced the transformation efficiency of DNA carrying a single nucleotide mismatch but not that of DNA with a 1-kb insertion. Unlike the exquisite UV sensitivity of the uvrD mutant, inactivating MMC did not affect survival after UV irradiation. MMC and uvrD mutants exhibited increased PilC-dependent pilus phase variation. mutS-deficient gonococci underwent an increased frequency of pilin antigenic variation, whereas uvrD had no effect. Recombination tracts in the mutS pilin variants were longer than in parental gonococci but utilized the same donor pilS loci. These results show that gonococcal MMC repairs mismatches and small insertion/deletions in DNA and also affects the recombination events underlying pilin antigenic variation. The differential effects of MMC and uvrD in gonococci unexpectedly reveal that MMC can function independently of uvrD in this human-specific pathogen.

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

2015 ◽  
Vol 197 (10) ◽  
pp. 1828-1838 ◽  
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.

Effects of recA Mutations on Pilus Antigenic Variation and Phase Transitions in Neisseria gonorrhoeae

Genetics ◽  
1987 ◽  
Vol 117 (3) ◽  
pp. 391-398
Author(s):  
Michael Koomey ◽  
Emil C Gotschlich ◽  
Ken Robbins ◽  
Sven Bergström ◽  
John Swanson
Keyword(s):  
Homologous Recombination ◽  
Neisseria Gonorrhoeae ◽  
Genetic Basis ◽  
Antigenic Variation ◽  
Phase Variation ◽  
Intragenic Recombination ◽  
Gene Copies ◽  
Reduced Rate ◽  
Pilin Gene ◽  
Isogenic Strains

ABSTRACT Intragenic recombination between the single complete pilin gene (expression locus) and multiple, distinct, partial pilin gene copies (silent, storage loci) is thought to account for the generation of pilus antigenic diversity and piliation phase (on-off) changes exhibited by Neisseria gonorrhoeae. The mechanisms operating in the genomic rearrangements associated with these forms of pilus variation were investigated through the study of isogenic strains of gonococci bearing either wild-type or altered recA alleles. Examination of the rates of pilus phase variation and the genetic basis for changes in piliation status displayed by these strains show that recA mediated homologous recombination is required for these high frequency events and confirm that the nonpiliated state results from mutations in the expressed pilin gene. In a strain that is deficient in recA mediated homologous recombination, pilus phase variation occurs at a 100-1000-fold reduced rate and results predominantly from one class of spontaneous frameshift mutations within the pilin structural gene.

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

mBio ◽  
2013 ◽  
Vol 4 (4) ◽  
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.

scholarly journals Analyzing Neisseria gonorrhoeae Pilin Antigenic Variation Using 454 Sequencing Technology

2016 ◽  
Vol 198 (18) ◽  
pp. 2470-2482 ◽  
Author(s):  
Ella Rotman ◽  
David M. Webber ◽  
H. Steven Seifert
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Biological Diversity ◽  
Antigenic Variation ◽  
Immune Surveillance ◽  
454 Sequencing ◽  
Phase Variation ◽  
Transmitted Infection ◽  
Major Barrier ◽  
Content Type ◽  
Type Iv

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.

scholarly journals Antisense Oligonucleotide Screening to Optimize the Rescue of the Splicing Defect Caused by the Recurrent Deep-Intronic ABCA4 Variant c.4539+2001G>A in Stargardt Disease

Genes ◽  
2019 ◽  
Vol 10 (6) ◽  
pp. 452 ◽  
Author(s):  
Alejandro Garanto ◽  
Lonneke Duijkers ◽  
Tomasz Z. Tomkiewicz ◽  
Rob W. J. Collin
Keyword(s):  
Pluripotent Stem Cells ◽  
Antisense Oligonucleotides ◽  
Autosomal Recessive ◽  
Stargardt Disease ◽  
Single Nucleotide ◽  
Significant Correction ◽  
Nucleotide Mismatch ◽  
Single Nucleotide Mismatch ◽  
Splicing Defects ◽  
Induced Pluripotent

Deep-sequencing of the ABCA4 locus has revealed that ~10% of autosomal recessive Stargardt disease (STGD1) cases are caused by deep-intronic mutations. One of the most recurrent deep-intronic variants in the Belgian and Dutch STGD1 population is the c.4539+2001G>A mutation. This variant introduces a 345-nt pseudoexon to the ABCA4 mRNA transcript in a retina-specific manner. Antisense oligonucleotides (AONs) are short sequences of RNA that can modulate splicing. In this work, we designed 26 different AONs to perform a thorough screening to identify the most effective AONs to correct splicing defects associated with c.4539+2001G>A. All AONs were tested in patient-derived induced pluripotent stem cells (iPSCs) that were differentiated to photoreceptor precursor cells (PPCs). AON efficacy was assessed through RNA analysis and was based on correction efficacy, and AONs were grouped and their properties assessed. We (a) identified nine AONs with significant correction efficacies (>50%), (b) confirmed that a single nucleotide mismatch was sufficient to significantly decrease AON efficacy, and (c) found potential correlations between efficacy and some of the parameters analyzed. Overall, our results show that AON-based splicing modulation holds great potential for treating Stargardt disease caused by splicing defects in ABCA4.

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

mSphere ◽  
2019 ◽  
Vol 4 (5) ◽  
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.

Identifying DNA mismatches at single-nucleotide resolution by probing individual surface potentials of DNA-capped nanoparticles

Nanoscale ◽  
2018 ◽  
Vol 10 (2) ◽  
pp. 538-547 ◽  
Author(s):  
Hyungbeen Lee ◽  
Sang Won Lee ◽  
Gyudo Lee ◽  
Wonseok Lee ◽  
Kihwan Nam ◽  
...  
Keyword(s):  
Kelvin Probe Force Microscopy ◽  
Powerful Method ◽  
Kelvin Probe ◽  
Single Nucleotide ◽  
Force Microscopy ◽  
Surface Potentials ◽  
Dna Mismatches ◽  
Nucleotide Resolution ◽  
Single Nucleotide Resolution

Here, we demonstrate a powerful method to discriminate DNA mismatches at single-nucleotide resolution from 0 to 5 mismatches (χ0 to χ5) using Kelvin probe force microscopy (KPFM).

scholarly journals Paired Siglec receptors generate opposite inflammatory responses to a human‐specific pathogen

2017 ◽  
Vol 36 (6) ◽  
pp. 751-760 ◽  
Author(s):  
Flavio Schwarz ◽  
Corinna S Landig ◽  
Shoib Siddiqui ◽  
Ismael Secundino ◽  
Joshua Olson ◽  
...  
Keyword(s):  
Inflammatory Responses ◽  
Specific Pathogen ◽  
Human Specific

scholarly journals A Double-Strand Break Does Not PromoteNeisseria gonorrhoeaePilin Antigenic Variation

2019 ◽  
Vol 201 (13) ◽  
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.

scholarly journals 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 ◽  
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.

Sign in / Sign up

Export Citation Format

Share Document