A real-time semi-quantitative RT-PCR assay demonstrates that the pilE sequence dictates the frequency and characteristics of pilin antigenic variation in Neisseria gonorrhoeae

Rohrer, Melissa S.

doi:10.1093/nar/gki650

Cited by 20 publications

(29 citation statements)

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“…The difference in the pilin Av rates could be due to the fact that each strain has a different starting pilE sequence (variant VD300 in MS11 and variant 1-81-S2 in FA1090). It has been demonstrated that the starting pilE sequence plays an important role in directing the pilin Av frequency and rate (34). There may also be other, non-pilE/pilS strain differences that influence the rate of pilin Av that may indicate rate-limiting steps controlling this specialized recombination system.…”

Section: Discussionmentioning

confidence: 99%

“…A disadvantage is that recombination events that do not involve one of the selected pilS copies go undetected. A quantitative PCR-based assay was developed that detected the transfer of the HV sequences of most of the silent copies of strain FA1090, but this assay could only estimate the frequency of pilin Av and was not adaptable to other strains (34). A coconversion assay has also been developed in which a marker is placed downstream of the pilE gene, in the Sma/Cla region, and following certain pilS/pilE recombination events, the marker is lost and replaced with the material downstream of the pilS copy, which is detectable by Southern blotting (15).…”

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confidence: 99%

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

Helm

Seifert

2009

J Bacteriol

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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.Homologous recombination is a widely conserved process used by all organisms to repair DNA damage and to mediate genetic transfer. Usually RecA or a RecA homologue is required for homologous recombination, but the other processing enzymes used to prepare DNA for recombination differ between organisms. Generally, double-stranded DNA (dsDNA) breaks are repaired by the RecBCD complex in gram-negative organisms and the AddAB complex in gram-positive organisms, although exceptions exist in which AddAB has been found in some proteobacteria (1,4,29,33,56). Single-stranded DNA (ssDNA) gaps are typically repaired by the RecF pathway, which is ubiquitous among the Bacteria and is part of a universal step in recombinational repair (31). While the RecBCD and RecF pathways have been extensively studied in Escherichia coli, they are less well understood in the humanspecific pathogen Neisseria gonorrhoeae. N. gonorrhoeae is a gram-negative bacterium that has been isolated only from infected humans and is believed to exist naturally only within the human population. Like E. coli, N. gonorrhoeae has a RecA homologue and possesses most of the genes that define the RecBCD and RecF pathways of E. coli (27).In E. coli, the RecBCD exonuclease begins to degrade DNA at the site of the break, and upon reaching a particular sequence, a chi site, the RecD component is inactivated, leaving RecBC to act as a helicase, processively unwinding the DNA, yielding ssDNA, which is the substrate for RecA (reviewed in refere...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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

Helm

Seifert

2009

J Bacteriol

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“…Since the starting pilE sequence can influence the frequency of pilin Av (34) and there is some variability in measuring PϪ colonies that arise from Pϩ progenitors, we constructed the previously reported RecQ HRDC mutant strains ( Fig. 2A) but made sure that they all had the same pilE sequence, and these assays were per- formed in a recA6 genetic background, where recA is under the control of IPTG, so that the pilE sequence was stable until RecA induction (31,35).…”

Section: Pilin Antigenic Variation and The N Gonorrhoeae Recq Helicasementioning

confidence: 99%

Neisseria gonorrhoeae RecQ Helicase HRDC Domains Are Essential for Efficient Binding and Unwinding of the pilE Guanine Quartet Structure Required for Pilin Antigenic Variation

et al. 2013

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The strict human pathogen Neisseria gonorrhoeae utilizes homologous recombination to antigenically vary the pilus, thus evading the host immune response. High-frequency gene conversion reactions between many silent pilin loci and the expressed pilin locus (pilE) allow for numerous pilus variants per strain to be produced from a single strain. For pilin antigenic variation (Av) to occur, a guanine quartet (G4) structure must form upstream of pilE. The RecQ helicase is one of several recombination or repair enzymes required for efficient levels of pilin Av, and RecQ family members have been shown to bind to and unwind G4 structures. Additionally, the vast majority of RecQ helicase family members encode one "helicase and RNase D C-terminal" (HRDC) domain, whereas the N. gonorrhoeae RecQ helicase gene encodes three HRDC domains, which are critical for pilin Av. Here, we confirm that deletion of RecQ HRDC domains 2 and 3 causes a decrease in the frequency of pilin Av comparable to that obtained with a functional knockout. We demonstrate that the N. gonorrhoeae RecQ helicase can bind and unwind the pilE G4 structure. Deletion of the RecQ HRDC domains 2 and 3 resulted in a decrease in G4 structure binding and unwinding. These data suggest that the decrease in pilin Av observed in the RecQ HRDC domain 2 and 3 deletion mutant is a result of the enzyme's inability to efficiently bind and unwind the pilE G4 structure.

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“…Hippocampal RNA was extracted using TRIzol reagent [3] . RNA was reverse transcribed into cDNA using a Reverse Transcription System Kit according to manufacturer's instructions (Promega Corporation, Promega).…”

Section: Rt-pcrmentioning

confidence: 99%

Expression of the apoptosis-related proteins caspase-3 and NF-κB in the hippocampus of Tg2576 mice

Niu

Zhang

et al. 2010

Neurosci. Bull.

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Objective To investigate the relations between neuroapoptosis and the onset and development of Alzheimer's disease (AD), especially the role of NF-κB in the regulation of neuroapoptosis. Methods expressions in the CA3 region of the hippocampus in APPswe Tg2576 transgenic mice were studied from postnatal day 0-180, using Nissl staining, immunohistochemistry and RT-PCR methods. Results Both neuronal apoptosis and NF-κB activity decreased gradually with the increase of age in wild type and Tg2576 mice. However, the number of caspase-3-positive or NF-κB-positive pyramidal cells in Tg2576 mice was greater than that in age-matched wild type mice, with significant differences after postnatal day 14 (P < 0.01 or P < 0.05). Linear regression analyses of caspase-3 and NF-κB expression demonstrated a correlation between neuroapoptosis and activity of NF-κB. Conclusion The process of neuroapoptosis is consistent with the onset and development of AD. Furthermore, the observed correlation between neuroapoptosis and NF-κB activity suggests a role of NF-κB in hippocampal neuroapoptosis.

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A real-time semi-quantitative RT-PCR assay demonstrates that the pilE sequence dictates the frequency and characteristics of pilin antigenic variation in Neisseria gonorrhoeae

Cited by 20 publications

References 31 publications

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

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

Neisseria gonorrhoeae RecQ Helicase HRDC Domains Are Essential for Efficient Binding and Unwinding of the pilE Guanine Quartet Structure Required for Pilin Antigenic Variation

Expression of the apoptosis-related proteins caspase-3 and NF-κB in the hippocampus of Tg2576 mice

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