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

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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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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Transcriptomic Data Sets To Determine Gene Expression Changes Mediated by the Presence of PBT2 in Growth Medium of Multidrug-Resistant Neisseria gonorrhoeae WHO Z

Microbiology Resource Announcements ◽

10.1128/mra.00283-20 ◽

2020 ◽

Vol 9 (21) ◽

Author(s):

Freda E.-C. Jen ◽

Ibrahim M. El-Deeb ◽

John M. Atack ◽

Mark von Itzstein ◽

Michael P. Jennings

Keyword(s):

Neisseria Gonorrhoeae ◽

Transcriptome Sequencing ◽

Multidrug Resistant ◽

Data Sets ◽

Sequencing Data ◽

Transmitted Infection ◽

High Coverage ◽

Content Type ◽

Sexually Transmitted ◽

Transcriptomic Data

ABSTRACT Neisseria gonorrhoeae causes the sexually transmitted infection gonorrhea. High-coverage (∼3,300-fold) transcriptome sequencing data have been collected from multidrug-resistant N. gonorrhoeae strain WHO Z grown in the presence and absence of PBT2.

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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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Challenges and Controversies Concerning Neisseria gonorrhoeae-Neutrophil Interactions in Pathogenesis

mBio ◽

10.1128/mbio.00721-21 ◽

2021 ◽

Author(s):

Alison K. Criss ◽

Caroline A. Genco ◽

Scott D. Gray-Owen ◽

Ann E. Jerse ◽

H Steven Seifert

Keyword(s):

Neisseria Gonorrhoeae ◽

Sexually Transmitted Infection ◽

Infection Rates ◽

Transmitted Infection ◽

Content Type ◽

Sexually Transmitted

The bacterium Neisseria gonorrhoeae (Ngo) is the main cause of the sexually transmitted infection gonorrhea. The global incidence of 87 million new Ngo infections each year, rising infection rates, and the emergence of Ngo strains that are resistant to all clinically recommended antibiotics have raised the specter of untreatable infections (M.

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Men and Women Have Similar Neisseria gonorrhoeae Bacterial Loads: a Comparison of Three Anatomical Sites

Journal of Clinical Microbiology ◽

10.1128/jcm.01171-20 ◽

2020 ◽

Vol 58 (11) ◽

Author(s):

Brian M. J. W. van der Veer ◽

Christian J. P. A. Hoebe ◽

Nicole H. T. M. Dukers-Muijrers ◽

Lieke B. van Alphen ◽

Petra F. G. Wolffs

Keyword(s):

Neisseria Gonorrhoeae ◽

Bacterial Load ◽

Large Population ◽

Transmitted Infection ◽

Men And Women ◽

Content Type ◽

Transmission Potential ◽

Sexually Transmitted ◽

Standard Curve ◽

Sex With Men

ABSTRACT Neisseria gonorrhoeae is a common bacterial sexually transmitted infection (STI). Currently, there are limited data on the bacterial load in both men and women and on both genital and extragenital sites. Therefore, we quantified N. gonorrhoeae bacterial loads in a large population of women, heterosexual men, and men who have sex with men (MSM) at three different anatomical sites. N. gonorrhoeae-positive samples (n = 1265) of STI clinic consultations (n = 944) were tested for N. gonorrhoeae with the Roche Cobas 4800 system, and quantification cycle (Cq) values were used as an inversely proportional measure for N. gonorrhoeae bacterial load after interpolation from a standard curve. Bacterial loads were compared between sample materials and sexes using t tests. The following mean N. gonorrhoeae loads were observed: urine, 4.5 ± 1.0 log10 CFU/ml; vaginal swabs, 4.3 ± 1.1 log10 CFU/ml; anorectal swabs (women), 4.0 ± 1.2 log10 CFU/ml; anorectal swabs (men), 4.5 ± 1.3 log10 CFU/ml; oropharyngeal swabs (women), 2.8 ± 0.9 log10 CFU/ml; and oropharyngeal swabs (men), 3.2 ± 1.0 log10 CFU/ml. Oropharyngeal swabs had a significantly lower N. gonorrhoeae load (P < 0.001) than genital and anorectal samples. Loads did not differ between men and women. This is the first study that determined N. gonorrhoeae load in both women and men at three anatomical sites. The substantial N. gonorrhoeae load at all sample sites suggest that all sites may have transmission potential. However, the oropharyngeal site presents the lowest bacterial load. Men and women have a similar N. gonorrhoeae loads on separate anatomical sites, arguing for similar transmission potential and similar clinical relevance.

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MetQ of Neisseria gonorrhoeae Is a Surface-Expressed Antigen That Elicits Bactericidal and Functional Blocking Antibodies

Infection and Immunity ◽

10.1128/iai.00898-16 ◽

2016 ◽

Vol 85 (2) ◽

Cited By ~ 26

Author(s):

Evgeny A. Semchenko ◽

Christopher J. Day ◽

Kate L. Seib

Keyword(s):

Epithelial Cells ◽

Neisseria Gonorrhoeae ◽

Transmitted Infection ◽

Content Type ◽

Sexually Transmitted ◽

Bacterial Surface ◽

Functional Blocking ◽

Blocking Antibodies ◽

Candidate Antigen

ABSTRACT Neisseria gonorrhoeae, the causative agent of the sexually transmitted infection (STI) gonorrhea, is a growing public health threat for which a vaccine is urgently needed. We characterized the functional role of the gonococcal MetQ protein, which is the methionine binding component of an ABC transporter system, and assessed its potential as a candidate antigen for inclusion in a gonococcal vaccine. MetQ has been found to be highly conserved in all strains investigated to date, it is localized on the bacterial surface, and it binds l-methionine with a high affinity. MetQ is also involved in gonococcal adherence to cervical epithelial cells. Mutants lacking MetQ have impaired survival in human monocytes, macrophages, and serum. Furthermore, antibodies raised against MetQ are bactericidal and are able to block gonococcal adherence to epithelial cells. These data suggest that MetQ elicits both bactericidal and functional blocking antibodies and is a valid candidate antigen for additional investigation and possible inclusion in a vaccine for prevention of gonorrhea.

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The Iron-Repressed, AraC-Like Regulator MpeR Activates Expression offetAin Neisseria gonorrhoeae

Infection and Immunity ◽

10.1128/iai.05806-11 ◽

2011 ◽

Vol 79 (12) ◽

pp. 4764-4776 ◽

Cited By ~ 23

Author(s):

Aimee Hollander ◽

Alexandra Dubon Mercante ◽

William M. Shafer ◽

Cynthia Nau Cornelissen

Keyword(s):

Neisseria Gonorrhoeae ◽

Iron Transport ◽

Reproductive Tract ◽

Treatment Options ◽

Effective Vaccine ◽

Transmitted Infection ◽

Content Type ◽

Sexually Transmitted ◽

Treatment And Prevention ◽

Resistance Rates

ABSTRACTNeisseria gonorrhoeaeis an obligate human pathogen that causes the common sexually transmitted infection gonorrhea. Gonococcal infections cause significant morbidity, particularly among women, as the organism ascends to the upper reproductive tract, resulting in pelvic inflammatory disease, ectopic pregnancy, and infertility. In the last few years, antibiotic resistance rates have risen dramatically, leading to severe restriction of treatment options for gonococcal disease. Gonococcal infections do not elicit protective immunity, nor is there an effective vaccine to prevent the disease. Thus, further understanding of the expression, function, and regulation of surface antigens could lead to better treatment and prevention modalities in the future. In the current study, we determined that an iron-repressed regulator, MpeR, interacted specifically with the DNA sequence upstream offetAand activated FetA expression. Interestingly, MpeR was previously shown to regulate the expression of gonococcal antimicrobial efflux systems. We confirmed that the outer membrane transporter FetA allows gonococcal strain FA1090 to utilize the xenosiderophore ferric enterobactin as an iron source. However, we further demonstrated that FetA has an extended range of substrates that encompasses other catecholate xenosiderophores, including ferric salmochelin and the dimers and trimers of dihydroxybenzoylserine. We demonstrated thatfetAis part of an iron-repressed, MpeR-activated operon which putatively encodes other iron transport proteins. This is the first study to describe a regulatory linkage between antimicrobial efflux and iron transport inN. gonorrhoeae. The regulatory nidus that links these systems, MpeR, is expressed exclusively by pathogenic neisseriae and is therefore expected to be an important virulence factor.

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Seminal Plasma Promotes Neisseria gonorrhoeae Aggregation and Biofilm Formation

Journal of Bacteriology ◽

10.1128/jb.00165-16 ◽

2016 ◽

Vol 198 (16) ◽

pp. 2228-2235 ◽

Cited By ~ 10

Author(s):

Mark T. Anderson ◽

Luke Byerly ◽

Michael A. Apicella ◽

H. Steven Seifert

Keyword(s):

Neisseria Gonorrhoeae ◽

Seminal Plasma ◽

Biofilm Formation ◽

Seminal Fluid ◽

Sexual Transmission ◽

Type Iv Pilus ◽

Transmitted Infection ◽

Content Type ◽

Bacterial Physiology ◽

Type Iv

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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Neisseria gonorrhoeae Modulates Cell Death in Human Endocervical Epithelial Cells through Export of Exosome-Associated cIAP2

Infection and Immunity ◽

10.1128/iai.00732-15 ◽

2015 ◽

Vol 83 (9) ◽

pp. 3410-3417 ◽

Cited By ~ 8

Author(s):

Kathleen Nudel ◽

Paola Massari ◽

Caroline A. Genco

Keyword(s):

Cell Death ◽

Epithelial Cells ◽

Neisseria Gonorrhoeae ◽

Cell Types ◽

Transmitted Infection ◽

Content Type ◽

Sexually Transmitted ◽

Cell Death Pathways ◽

Host Cell Death ◽

Cervical Epithelial Cells

Several bacterial pathogens persist and survive in the host by modulating host cell death pathways. We previously demonstrated thatNeisseria gonorrhoeae, a Gram-negative pathogen responsible for the sexually transmitted infection gonorrhea, protects against exogenous induction of apoptosis in human cervical epithelial cells. However, induction of cell death byN. gonorrhoeaehas also been reported in other cell types. The mechanisms by whichN. gonorrhoeaemodulates cell death are not clear, although a role for the inhibitor of apoptosis-2 (cIAP2) has been proposed. In this study, we confirmed thatN. gonorrhoeaeinduces production of cIAP2 in human cervical epithelial cells. High levels of intracellular cIAP2 were detected early afterN. gonorrhoeaestimulation, which was followed by a marked decrease at 24 h. At this time point, we observed increased levels of extracellular cIAP2 associated with exosomes and an overall increase in production of exosomes. Inhibition of cIAP2 inN. gonorrhoeae-stimulated epithelial cells resulted in increased cell death and interleukin-1β (IL-1β) production. Collectively these results indicate thatN. gonorrhoeaestimulation of human endocervical epithelial cells induces the release of cIAP2, an essential regulator of cell death and immune signaling.

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Global Network Analysis of Neisseria gonorrhoeae Identifies Coordination between Pathways, Processes, and Regulators Expressed during Human Infection

mSystems ◽

10.1128/msystems.00729-19 ◽

2020 ◽

Vol 5 (1) ◽

Author(s):

Ryan McClure ◽

Ashwini Sunkavalli ◽

Phillip M. Balzano ◽

Paola Massari ◽

Christine Cho ◽

...

Keyword(s):

Network Analysis ◽

Neisseria Gonorrhoeae ◽

Treatment Strategies ◽

High Morbidity ◽

Gene Coexpression Network ◽

Transmitted Infection ◽

Content Type ◽

Sexually Transmitted ◽

Gene Coexpression ◽

New Treatment

ABSTRACT Neisseria gonorrhoeae is a Gram-negative diplococcus that is responsible for the sexually transmitted infection gonorrhea, a high-morbidity disease in the United States and worldwide. Over the past several years, N. gonorrhoeae strains resistant to antibiotics used to treat this infection have begun to emerge across the globe. Thus, new treatment strategies are needed to combat this organism. Here, we utilized N. gonorrhoeae transcriptomic data sets, including those obtained from natural infection of the human genital tract, to infer the first global gene coexpression network of this pathogen. Interrogation of this network revealed genes central to the network that are likely critical for gonococcal growth, metabolism, and virulence, including genes encoding hypothetical proteins expressed during mucosal infection. In addition, network analysis revealed overlap in the response of N. gonorrhoeae to incubation with neutrophils and exposure to hydrogen peroxide stress in vitro. Network analysis also identified new targets of the gonococcal global regulatory protein Fur, while examination of the network neighborhood of genes allowed us to assign additional putative categories to several proteins. Collectively, the characterization of the first gene coexpression network for N. gonorrhoeae described here has revealed new regulatory pathways and new categories for proteins and has shown how processes important to gonococcal infection in both men and women are linked. This information fills a critical gap in our understanding of virulence strategies of this obligate human pathogen and will aid in the development of new treatment strategies for gonorrhea. IMPORTANCE Neisseria gonorrhoeae is the causative agent of the sexually transmitted infection (STI) gonorrhea, a disease with high morbidity worldwide with an estimated 87 million cases annually. Current therapeutic and pharmacologic approaches to treat gonorrhea have been compromised by increased antibiotic resistance worldwide, including to the most recent FDA-approved antibiotic. New treatment strategies are urgently needed to combat this organism. In this study, we used network analysis to interrogate and define the coordination of pathways and processes in N. gonorrhoeae. An analysis of the gonococcal network was also used to assign categories to genes and to expand our understanding of regulatory strategies. Network analysis provides important insights into pathogenic mechanisms of this organism that will guide the design of new strategies for disease treatment.

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