scholarly journals Antigenic Variation in Neisseria gonorrhoeae Occurs Independently of RecQ-Mediated Unwinding of the pilE G Quadruplex

2019 ◽  
Vol 202 (3) ◽  
Author(s):  
Andrew F. Voter ◽  
Melanie M. Callaghan ◽  
Ramreddy Tippana ◽  
Sua Myong ◽  
Joseph P. Dillard ◽  
...  
Keyword(s):  
Immune System ◽  
Homologous Recombination ◽  
Neisseria Gonorrhoeae ◽  
Antigenic Variation ◽  
Dna Unwinding ◽  
Duplex Dna ◽  
Recq Helicase ◽  
Content Type ◽  
G4 Dna ◽  
Guanine Quadruplex

ABSTRACT The obligate human pathogen Neisseria gonorrhoeae alters its cell surface antigens to evade the immune system in a process known as antigenic variation (AV). During pilin AV, portions of the expressed pilin gene (pilE) are replaced with segments of silent pilin genes (pilS) through homologous recombination. The pilE-pilS exchange is initiated by formation of a parallel guanine quadruplex (G4) structure near the pilE gene, which recruits the homologous recombination machinery. The RecQ helicase, which has been proposed to aid AV by unwinding the pilE G4 structure, is an important component of this machinery. However, RecQ also promotes homologous recombination through G4-independent duplex DNA unwinding, leaving the relative importance of its G4 unwinding activity unclear. Previous investigations revealed a guanine-specific pocket (GSP) on the surface of RecQ that is required for G4, but not duplex, DNA unwinding. To determine whether RecQ-mediated G4 resolution is required for AV, N. gonorrhoeae strains that encode a RecQ GSP variant that cannot unwind G4 DNA were created. In contrast to the hypothesis that G4 unwinding by RecQ is important for AV, the RecQ GSP variant N. gonorrhoeae strains had normal AV levels. Analysis of a purified RecQ GSP variant confirmed that it retained duplex DNA unwinding activity but had lost its ability to unwind antiparallel G4 DNA. Interestingly, neither the GSP-deficient RecQ variant nor the wild-type RecQ could unwind the parallel pilE G4 nor the prototypical c-myc G4. Based on these results, we conclude that N. gonorrhoeae AV occurs independently of RecQ-mediated pilE G4 resolution. IMPORTANCE The pathogenic bacteria Neisseria gonorrhoeae avoids clearance by the immune system through antigenic variation (AV), the process by which immunogenic surface features of the bacteria are exchanged for novel variants. RecQ helicase is critical in AV and its role has been proposed to stem from its ability to unwind a DNA secondary structure known as a guanine quadruplex (G4) that is central to AV. In this work, we demonstrate that the role of RecQ in AV is independent of its ability to resolve G4s and that RecQ is incapable of unwinding the G4 in question. We propose a new model of RecQ’s role in AV where the G4 might recruit or orient RecQ to facilitate homologous recombination.

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 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.

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.

scholarly journals Guanine Quadruplex DNA Regulates Gamma Radiation Response of Genome Functions in the Radioresistant Bacterium Deinococcus radiodurans

2019 ◽  
Vol 201 (17) ◽  
Author(s):  
Shruti Mishra ◽  
Reema Chaudhary ◽  
Sudhir Singh ◽  
Swathi Kota ◽  
Hari S. Misra
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Deinococcus Radiodurans ◽  
Dna Structure ◽  
Protein Translation ◽  
Content Type ◽  
Cellular Processes ◽  
G4 Dna ◽  
Guanine Quadruplex ◽  
Damage Response

ABSTRACT Guanine quadruplex (G4) DNA/RNA are secondary structures that regulate the various cellular processes in both eukaryotes and bacteria. Deinococcus radiodurans, a Gram-positive bacterium known for its extraordinary radioresistance, shows a genomewide occurrence of putative G4 DNA-forming motifs in its GC-rich genome. N-Methyl mesoporphyrin (NMM), a G4 DNA structure-stabilizing drug, did not affect bacterial growth under normal conditions but inhibited the postirradiation recovery of gamma-irradiated cells. Transcriptome sequencing analysis of cells treated with both radiation and NMM showed repression of gamma radiation-responsive gene expression, which was observed in the absence of NMM. Notably, this effect of NMM on the expression of housekeeping genes involved in other cellular processes was not observed. Stabilization of G4 DNA structures mapped at the upstream of recA and in the encoding region of DR_2199 had negatively affected promoter activity in vivo, DNA synthesis in vitro and protein translation in Escherichia coli host. These results suggested that G4 DNA plays an important role in DNA damage response and in the regulation of expression of the DNA repair proteins required for radioresistance in D. radiodurans. IMPORTANCE Deinococcus radiodurans can recover from extensive DNA damage caused by many genotoxic agents. It lacks LexA/RecA-mediated canonical SOS response. Therefore, the molecular mechanisms underlying the regulation of DNA damage response would be worth investigating in this bacterium. D. radiodurans genome is GC-rich and contains numerous islands of putative guanine quadruplex (G4) DNA structure-forming motifs. Here, we showed that in vivo stabilization of G4 DNA structures can impair DNA damage response processes in D. radiodurans. Essential cellular processes such as transcription, DNA synthesis, and protein translation, which are also an integral part of the double-strand DNA break repair pathway, are affected by the arrest of G4 DNA structure dynamics. Thus, the role of DNA secondary structures in DNA damage response and radioresistance is demonstrated.

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

Biochemistry ◽  
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

scholarly journals Cross-Reactive Immune Responses as Primary Drivers of Malaria Chronicity

2013 ◽  
Vol 82 (1) ◽  
pp. 140-151 ◽  
Author(s):  
Eili Y. Klein ◽  
Andrea L. Graham ◽  
Manuel Llinás ◽  
Simon Levin
Keyword(s):  
Immune System ◽  
Immune Responses ◽  
Malaria Infection ◽  
Antigenic Variation ◽  
Cross Reactivity ◽  
Primary Response ◽  
Small Subset ◽  
Host Immune System ◽  
Primary Role ◽  
Content Type

ABSTRACTThe within-host dynamics of an infection with the malaria parasitePlasmodium falciparumare the result of a complex interplay between the host immune system and parasite. Continual variation of theP. falciparumerythrocyte membrane protein (PfEMP1) antigens displayed on the surface of infected red blood cells enables the parasite to evade the immune system and prolong infection. Despite the importance of antigenic variation in generating the dynamics of infection, our understanding of the mechanisms by which antigenic variation generates long-term chronic infections is still limited. We developed a model to examine the role of cross-reactivity in generating infection dynamics that are comparable to those of experimental infections. The hybrid computational model we developed is attuned to the biology of malaria by mixing discrete replication events, which mimics the synchrony of parasite replication and invasion, with continuous interaction with the immune system. Using simulations, we evaluated the dynamics of a single malaria infection over time. We then examined three major mechanisms by which the dynamics of a malaria infection can be structured: cross-reactivity of the immune response to PfEMP1, differences in parasite clearance rates, and heterogeneity in the rate at which antigens switch. The results of our simulations demonstrate that cross-reactive immune responses play a primary role in generating the dynamics observed in experimentally untreated infections and in lengthening the period of infection. Importantly, we also find that it is the primary response to the initially expressed PfEMP1, or small subset thereof, that structures the cascading cross-immune dynamics and allows for elongation of the infection.

scholarly journals A DOT1B/Ribonuclease H2 Protein Complex Is Involved in R-Loop Processing, Genomic Integrity, and Antigenic Variation in Trypanosoma brucei

mBio ◽  
2021 ◽  
Author(s):  
Nicole Eisenhuth ◽  
Tim Vellmer ◽  
Elisa T. Rauh ◽  
Falk Butter ◽  
Christian J. Janzen
Keyword(s):  
Immune System ◽  
Trypanosoma Brucei ◽  
Protein Complex ◽  
Antigenic Variation ◽  
Surface Coat ◽  
Genomic Integrity ◽  
Trypanosome Infection ◽  
Protein Surface ◽  
Content Type ◽  
Periodic Changes

Trypanosoma brucei is a unicellular parasite that causes devastating diseases like sleeping sickness in humans and the “nagana” disease in cattle in Africa. Fundamental to the establishment and prolongation of a trypanosome infection is the parasite's ability to escape the mammalian host's immune system by antigenic variation, which relies on periodic changes of a protein surface coat.

scholarly journals Fission Yeast RecQ Helicase Rqh1 Is Required for the Maintenance of Circular Chromosomes

2013 ◽  
Vol 33 (6) ◽  
pp. 1175-1187 ◽  
Author(s):  
Tomoko Nanbu ◽  
Katsunori Takahashi ◽  
Johanne M. Murray ◽  
Naoya Hirata ◽  
Shinobu Ukimori ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Fission Yeast ◽  
Synthetic Lethality ◽  
Recq Helicase ◽  
Triple Mutant ◽  
Content Type ◽  
Recq Helicases ◽  
Synthetically Lethal ◽  
Circular Chromosomes ◽  
Multiple Stages

Protection of telomeres protein 1 (Pot1) binds to single-stranded telomere overhangs and protects chromosome ends. RecQ helicases regulate homologous recombination at multiple stages, including resection, strand displacement, and resolution. Fission yeastpot1and RecQ helicaserqh1double mutants are synthetically lethal, but the mechanism is not fully understood. Here, we show that the synthetic lethality ofpot1Δrqh1Δ double mutants is due to inappropriate homologous recombination, as it is suppressed by the deletion ofrad51+. The expression of Rad51 in thepot1Δrqh1Δrad51Δ triple mutant, which has circular chromosomes, is lethal. Reduction of the expression of Rqh1 in apot1disruptant with circular chromosomes caused chromosome missegregation, and this defect was partially suppressed by the deletion ofrad51+. Taken together, our results suggest that Rqh1 is required for the maintenance of circular chromosomes when homologous recombination is active. Crossovers between circular monomeric chromosomes generate dimers that cannot segregate properly inEscherichia coli. We propose that Rqh1 inhibits crossovers between circular monomeric chromosomes to suppress the generation of circular dimers.

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.

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