Seminal Plasma Promotes Neisseria gonorrhoeae Aggregation and Biofilm Formation

ABSTRACTNeisseria gonorrhoeaecauses the human-specific disease gonorrhea and is transmitted from person to person primarily via sexual contact. During transmission,N. gonorrhoeaeis often exposed to seminal fluid and must adapt to this change in environment. Previous work demonstrated that seminal fluid facilitatesN. gonorrhoeaemotility and alters epithelial cell interactions. In this study, exposure to seminal fluid was found to decrease surface adherence of gonococci in a manner that was independent of Opa adhesin proteins or type IV pilus retraction. Semen was also shown to cause dispersal of bacteria that had previously established surface adherence. Although surface adherence decreased, interbacterial interactions were increased by seminal plasma both in long-term static culture and on a cell-to-cell basis over shorter time periods. The result of increased bacterium-bacterium interactions resulted in the formation of microcolonies, an important step in theN. gonorrhoeaeinfectious process. Seminal fluid also facilitated increased bacterial aggregation in the form of shear-resistant three-dimensional biofilms. These results emphasize the importance of the gonococcal response to the influx of seminal fluid within the genital niche. Further characterization of theN. gonorrhoeaeresponse to semen will advance our understanding of the mechanisms behind the establishment of infection in naive hosts and the process of transmission.IMPORTANCEN. gonorrhoeaeis the causative agent of the globally prevalent sexually transmitted infection gonorrhea. An understudied aspect of this human-adapted pathogen is the change in bacterial physiology that occurs during sexual transmission.N. gonorrhoeaeencounters semen when transmitted from host to host, and it is known that, whenN. gonorrhoeaeis exposed to seminal fluid, alterations in bacterial motility and type IV pilus arrangement occur. This work extends our previous observations on this modulation of gonococcal physiology by seminal fluid and demonstrates that seminal plasma decreases surface adherence, promotes interbacterial interactions, and enhances biofilm formation.

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Seminal Plasma Initiates a Neisseria gonorrhoeae Transmission State

mBio ◽

10.1128/mbio.01004-13 ◽

2014 ◽

Vol 5 (2) ◽

Cited By ~ 9

Author(s):

Mark T. Anderson ◽

Lena Dewenter ◽

Berenike Maier ◽

H. Steven Seifert

Keyword(s):

Neisseria Gonorrhoeae ◽

Seminal Plasma ◽

Seminal Fluid ◽

Sexual Transmission ◽

Type Iv Pili ◽

Twitching Motility ◽

Type Iv Pilus ◽

Content Type ◽

Host Environment ◽

Type Iv

ABSTRACTNiche-restricted pathogens are evolutionarily linked with the specific biological fluids that are encountered during infection.Neisseria gonorrhoeaecauses the genital infection gonorrhea and is exposed to seminal fluid during sexual transmission. Treatment ofN. gonorrhoeaewith seminal plasma or purified semen proteins lactoferrin, serum albumin, and prostate-specific antigen each facilitated type IV pilus-mediated twitching motility of the bacterium. Motility in the presence of seminal plasma was characterized by high velocity and low directional persistence. In addition, infection of epithelial cells withN. gonorrhoeaein the presence of seminal plasma resulted in enhanced microcolony formation. Close association of multiple pili in the form of bundles was also disrupted after seminal plasma treatment leading to an increase in the number of single pilus filaments on the bacterial surface. Thus, exposure ofN. gonorrhoeaeto seminal plasma is proposed to alter bacterial motility and aggregation characteristics to influence the processes of transmission and colonization.IMPORTANCEThere are greater than 100 million estimated new cases of gonorrhea annually worldwide. Research characterizing the mechanisms of pathogenesis and transmission ofNeisseria gonorrhoeaeis important for developing new prevention strategies, since antibiotic resistance of the organism is becoming increasingly prevalent. Our work identifies seminal plasma as a mediator ofN. gonorrhoeaetwitching motility and microcolony formation through functional modification of the type IV pilus. These findings provide insight into motility dynamics and epithelial cell colonization under conditions that are relevant to sexual transmission. Type IV pili are common virulence factors with diverse functions among bacterial pathogens, and this work identifies interactions between type IV pili and the host environment. Finally, this work illustrates the importance of the host environment and niche-specific fluids on microbial pathogenesis.

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Discovery of a New Neisseria gonorrhoeae Type IV Pilus Assembly Factor, TfpC

mBio ◽

10.1128/mbio.02528-20 ◽

2020 ◽

Vol 11 (5) ◽

Author(s):

Linda I. Hu ◽

Shaohui Yin ◽

Egon A. Ozer ◽

Lee Sewell ◽

Saima Rehman ◽

...

Keyword(s):

Cell Surface ◽

Neisseria Gonorrhoeae ◽

Bacterial Cell ◽

Bacterial Species ◽

Type Iv Pili ◽

Type Iv Pilus ◽

Transmitted Infection ◽

Content Type ◽

Sexually Transmitted ◽

Type Iv

ABSTRACT Neisseria gonorrhoeae relies on type IV pili (T4p) to promote colonization of their human host and to cause the sexually transmitted infection gonorrhea. This organelle cycles through a process of extension and retraction back into the bacterial cell. Through a genetic screen, we identified the NGO0783 locus of N. gonorrhoeae strain FA1090 as containing a gene encoding a protein required to stabilize the type IV pilus in its extended, nonretracted conformation. We have named the gene tfpC and the protein TfpC. Deletion of tfpC produces a nonpiliated colony morphology, and immuno-transmission electron microscopy confirms that the pili are lost in the ΔtfpC mutant, although there is some pilin detected near the bacterial cell surface. A copy of the tfpC gene expressed from a lac promoter restores pilus expression and related phenotypes. A ΔtfpC mutant shows reduced levels of pilin protein, but complementation with a tfpC gene restored pilin to normal levels. Bioinformatic searches show that there are orthologues in numerous bacterial species, but not all type IV pilin-expressing bacteria contain orthologous genes. Coevolution and nuclear magnetic resonance (NMR) analysis indicates that TfpC contains an N-terminal transmembrane helix, a substantial extended/unstructured region, and a highly charged C-terminal coiled-coil domain. IMPORTANCE Most bacterial species express one or more extracellular organelles called pili/fimbriae that are required for many properties of each bacterial cell. The Neisseria gonorrhoeae type IV pilus is a major virulence and colonization factor for the sexually transmitted infection gonorrhea. We have discovered a new protein of Neisseria gonorrhoeae called TfpC that is required to maintain type IV pili on the bacterial cell surface. There are similar proteins found in other members of the Neisseria genus and many other bacterial species important for human health.

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Discovery of a new Neisseria gonorrhoeae Type IV pilus assembly factor, TfpC

10.1101/2020.09.26.314724 ◽

2020 ◽

Author(s):

Linda I. Hu ◽

Shaohui Yin ◽

Egon A. Ozer ◽

Lee Sewell ◽

Saima Rehman ◽

...

Keyword(s):

Cell Surface ◽

Neisseria Gonorrhoeae ◽

Sexually Transmitted Infection ◽

Bacterial Cell ◽

Bacterial Species ◽

Type Iv Pili ◽

Type Iv Pilus ◽

Transmitted Infection ◽

Sexually Transmitted ◽

Type Iv

AbstractNeisseria gonorrhoeae rely on Type IV pili (T4p) to promote colonization of their human host and to cause the sexually transmitted infection, gonorrhea. This organelle cycles through a process of extension and retraction back into the bacterial cell. Through a genetic screen, we identified the NGO0783 locus of N. gonorrhoeae strain FA1090 as containing a gene encoding a protein required to stabilize the Type IV pilus in its extended, non-retracted conformation. We have named the gene tfpC and the protein TfpC. Deletion of tfpC produces a nonpiliated colony morphology and immuno-transmission electron microscopy confirms that the pili are lost in the ΔtfpC mutant, although there is some pilin detected near the bacterial cell surface. A copy of the tfpC gene expressed from a lac promoter restores pilus expression and related phenotypes. A ΔtfpC mutant shows reduced levels of pilin protein, but complementation with a tfpC gene restored pilin to normal levels. Bioinformatic searches show there are orthologues in numerous bacteria species but not all Type IV pilin expressing bacteria contain orthologous genes. Co-evolution and NMR analysis indicates that TfpC contains an N-terminal transmembrane helix, a substantial extended/unstructured region and a highly charge C-terminal coiled-coil domain.ImportanceMost bacterial species express one or more extracellular organelles called pili/fimbriae that are required for many properties of each bacterial cell. The Neisseria gonorrhoeae Type IV pilus is a major virulence and colonization factor for the sexually transmitted infection, gonorrhea. We have discovered a new protein of Neisseria gonorrhoeae called TfpC that is required to maintain the Type IV pili on the bacterial cell surface. There are similar proteins found in the other members of the Neisseria genus and many other bacterial species important for human health.

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

mBio ◽

10.1128/mbio.00399-13 ◽

2013 ◽

Vol 4 (4) ◽

Cited By ~ 28

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.

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Mutation of the Conserved Calcium-Binding Motif in Neisseria gonorrhoeae PilC1 Impacts Adhesion but Not Piliation

Infection and Immunity ◽

10.1128/iai.00493-13 ◽

2013 ◽

Vol 81 (11) ◽

pp. 4280-4289 ◽

Cited By ~ 16

Author(s):

Yuan Cheng ◽

Michael D. L. Johnson ◽

Christine Burillo-Kirch ◽

Jeffrey C. Mocny ◽

James E. Anderson ◽

...

Keyword(s):

Neisseria Gonorrhoeae ◽

Calcium Binding ◽

Critical Role ◽

Full Length ◽

Binding Motif ◽

Type Iv Pilus ◽

Kingella Kingae ◽

Content Type ◽

Terminal Domain ◽

Type Iv

ABSTRACTNeisseria gonorrhoeaePilC1 is a member of the PilC family of type IV pilus-associated adhesins found inNeisseriaspecies and other type IV pilus-producing genera. Previously, a calcium-binding domain was described in the C-terminal domains of PilY1 ofPseudomonas aeruginosaand in PilC1 and PilC2 ofKingella kingae. Genetic analysis ofN. gonorrhoeaerevealed a similar calcium-binding motif in PilC1. To evaluate the potential significance of this calcium-binding region inN. gonorrhoeae, we produced recombinant full-length PilC1 and a PilC1 C-terminal domain fragment. We show that, while alterations of the calcium-binding motif disrupted the ability of PilC1 to bind calcium, they did not grossly affect the secondary structure of the protein. Furthermore, we demonstrate that both full-length wild-type PilC1 and full-length calcium-binding-deficient PilC1 inhibited gonococcal adherence to cultured human cervical epithelial cells, unlike the truncated PilC1 C-terminal domain. Similar to PilC1 inK. kingae, but in contrast to the calcium-binding mutant ofP. aeruginosaPilY1, an equivalent mutation inN. gonorrhoeaePilC1 produced normal amounts of pili. However, theN. gonorrhoeaePilC1 calcium-binding mutant still had partial defects in gonococcal adhesion to ME180 cells and genetic transformation, which are both essential virulence factors in this human pathogen. Thus, we conclude that calcium binding to PilC1 plays a critical role in pilus function inN. gonorrhoeae.

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Streptococcus sanguinisNoncodingcia-Dependent Small RNAs Negatively Regulate Expression of Type IV Pilus Retraction ATPase PilT and Biofilm Formation

Infection and Immunity ◽

10.1128/iai.00894-17 ◽

2017 ◽

Vol 86 (3) ◽

Cited By ~ 1

Author(s):

Chiaki Ota ◽

Hirobumi Morisaki ◽

Masanobu Nakata ◽

Takafumi Arimoto ◽

Haruka Fukamachi ◽

...

Keyword(s):

Biofilm Formation ◽

Small Rnas ◽

Regulatory System ◽

Luciferase Reporter ◽

Nucleotide Exchange ◽

Type Iv Pilus ◽

Mobility Shift ◽

Putative Gene ◽

Content Type ◽

Type Iv

ABSTRACTSmall noncoding RNAs (sRNAs) have been identified as important regulators of gene expression in various cellular processes.cia-dependent small RNAs (csRNAs), a group of sRNAs that are controlled by the two-component regulatory system CiaRH, are widely conserved in streptococci, but their targets have been identified only inStreptococcus pneumoniae.Streptococcus sanguinis, a pioneer colonizer of teeth and one of the most predominant bacteria in the early oral biofilm, has been shown to have six csRNAs. Using computational target prediction and the luciferase reporter assay, we identifiedpilT, a constituent of the type IV pilus operon, as a negative regulatory target for one of the csRNAs, namely, csRNA1-1, inS. sanguinis. RNA-RNA electrophoretic mobility shift assay using a nucleotide exchange mutant of csRNA1-1 revealed that csRNA1-1 binds directly topilTmRNA. In addition, csRNA1-1 and csRNA1-2, a putative gene duplication product of csRNA1-1 that is tandemly located in theS. sanguinisgenome, negatively regulatedS. sanguinisbiofilm formation. These results suggest the involvement of csRNAs in the colonization step ofS. sanguinis.

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Analyzing Neisseria gonorrhoeae Pilin Antigenic Variation Using 454 Sequencing Technology

Journal of Bacteriology ◽

10.1128/jb.00330-16 ◽

2016 ◽

Vol 198 (18) ◽

pp. 2470-2482 ◽

Cited By ~ 11

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.

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

mSphere ◽

10.1128/msphere.00562-19 ◽

2019 ◽

Vol 4 (5) ◽

Cited By ~ 1

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.

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Type IV Pilus Assembly Proficiency and Dynamics Influence Pilin Subunit Phospho-Form Macro- and Microheterogeneity in Neisseria gonorrhoeae

PLoS ONE ◽

10.1371/journal.pone.0096419 ◽

2014 ◽

Vol 9 (5) ◽

pp. e96419 ◽

Cited By ~ 1

Author(s):

Åshild Vik ◽

Jan Haug Anonsen ◽

Finn Erik Aas ◽

Finn Terje Hegge ◽

Norbert Roos ◽

...

Keyword(s):

Neisseria Gonorrhoeae ◽

Type Iv Pilus ◽

Type Iv ◽

Pilin Subunit ◽

Pilus Assembly

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Identification and initial characterization of a new pair of sibling sRNAs of Neisseria gonorrhoeae involved in type IV pilus biogenesis

Microbiology ◽

10.1099/mic.0.001080 ◽

2021 ◽

Vol 167 (9) ◽

Author(s):

Marie Zachary ◽

Susanne Bauer ◽

Maximilian Klepsch ◽

Katharina Wagler ◽

Bruno Hüttel ◽

...

Keyword(s):

Target Genes ◽

Target Prediction ◽

Type Iv Pilus ◽

Content Type ◽

Type Iv ◽

Gene Expression Control ◽

Initial Characterization ◽

Pilus Biogenesis ◽

Regulatory Rnas

Non-coding regulatory RNAs mediate post-transcriptional gene expression control by a variety of mechanisms relying mostly on base-pairing interactions with a target mRNA. Though a plethora of putative non-coding regulatory RNAs have been identified by global transcriptome analysis, knowledge about riboregulation in the pathogenic Neisseriae is still limited. Here we report the initial characterization of a pair of sRNAs of N. gonorrhoeae , TfpR1 and TfpR2, which exhibit a similar secondary structure and identical single-stranded seed regions, and therefore might be considered as sibling sRNAs. By combination of in silico target prediction and sRNA pulse expression followed by differential RNA sequencing we identified target genes of TfpR1 which are involved in type IV pilus biogenesis and DNA damage repair. We provide evidence that members of the TfpR1 regulon can also be targeted by the sibling TfpR2.

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