Computational prediction of crucial genes involved in gonorrhea infection and neoplastic cell transformation: A multiomics approach.

Neisseria gonorrheae, the causative agent of genitourinary infections, has been associated with asymptomatic or recurrent infections and has the potential to form biofilms and induce inflammation and cell transformation. Herein, we aimed to use computational analysis to predict novel associations between chronic inflammation caused by gonorrhea infection and neoplastic transformation. Prioritization and gene enrichment strategies based on virulence and resistance genes utilizing essential genes from the DEG and PANTHER databases, respectively, were performed. Using the STRING database, protein‒protein interaction networks were constructed with 55 nodes of bacterial proteins and 72 nodes of proteins involved in the host immune response. MCODE and cytoHubba were used to identify 12 bacterial hub proteins (murA, murB, murC, murD, murE, purN, purL, thyA, uvrB, kdsB, lpxC, and ftsH) and 19 human hub proteins, of which TNF, STAT3 and AKT1 had high significance. The PPI networks are based on the connectivity degree (K), betweenness centrality (BC), and closeness centrality (CC) values. Hub genes are vital for cell survival and growth, and their significance as potential drug targets is discussed. This computational study provides a comprehensive understanding of inflammation and carcinogenesis pathways that are activated during gonorrhea infection.

Neisseria gonorrhoeae subverts formin-dependent actin polymerization to colonize human macrophages.

Dynamic reorganization of the actin cytoskeleton dictates plasma membrane morphogenesis and is frequently subverted by bacterial pathogens for entry and colonization of host cells. The human-adapted bacterial pathogen Neisseria gonorrhoeae can colonize and replicate when cultured with human macrophages, however the basic understanding of how this process occurs is incomplete. N. gonorrhoeae is the etiological agent of the sexually transmitted disease gonorrhea and tissue resident macrophages are present in the urogenital mucosa, which is colonized by the bacteria. We uncovered that when gonococci colonize macrophages, they can establish an intracellular or a cell surface-associated niche that support bacterial replication independently. Unlike other intracellular bacterial pathogens, which enter host cells as single bacterium, establish an intracellular niche and then replicate, gonococci invade human macrophages as a colony. Individual diplococci are rapidly phagocytosed by macrophages and transported to lysosomes for degradation. However, we found that surface-associated gonococcal colonies of various sizes can invade macrophages by triggering actin skeleton rearrangement resulting in plasma membrane invaginations that slowly engulf the colony. The resulting intracellular membrane-bound organelle supports robust bacterial replication. The gonococci-occupied vacuoles evaded fusion with the endosomal compartment and were enveloped by a network of actin filaments. We demonstrate that gonococcal colonies invade macrophages via a process mechanistically distinct from phagocytosis that is regulated by the actin nucleating factor FMNL3 and is independent of the Arp2/3 complex. Our work provides insights into the gonococci life-cycle in association with human macrophages and defines key host determinants for macrophage colonization.

Septic Arthritis: Diagnosis and Treatment.

Septic arthritis must be considered and promptly diagnosed in any patient presenting with acute atraumatic joint pain, swelling, and fever. Risk factors for septic arthritis include age older than 80 years, diabetes mellitus, rheumatoid arthritis, recent joint surgery, hip or knee prosthesis, skin infection, and immunosuppressive medication use. A delay in diagnosis and treatment can result in permanent morbidity and mortality. Physical examination findings and serum markers, including erythrocyte sedimentation rate and C-reactive protein, are helpful in the diagnosis but are nonspecific. Synovial fluid studies are required to confirm the diagnosis. History and Gram stain aid in determining initial antibiotic selection. Staphylococcus aureus is the most common pathogen isolated in septic arthritis; however, other bacteria, viruses, fungi, and mycobacterium can cause the disease. After synovial fluid has been obtained, empiric antibiotic therapy should be initiated if there is clinical concern for septic arthritis. Oral antibiotics can be given in most cases because they are not inferior to intravenous therapy. Total duration of therapy ranges from two to six weeks; however, certain infections require longer courses. Consideration for microorganisms such as Neisseria gonorrhoeae, Borrelia burgdorferi, and fungal infections should be based on history findings and laboratory results.

Neisseria gonorrhoeae: un patógeno díscolo: conceptos microbiológicos, resistencia a antimicrobianos y su vigilancia epidemiológica en Chile / Neisseria gonorrhoeae: a wayward pathogen: microbiological concepts, antimicrobial resistance and its epidemiological surveillance in Chile

Resumen Neisseria gonorrhoeae es un diplococo gramnegativo, no móvil, esporulado, aerobio o anaerobio facultativo, catalasa y oxidasa positivas. Las infecciones de transmisión sexual causadas por este microorganismo son un problema de salud pública definido como tal desde el siglo XIX, representando una gran amenaza para la salud humana debido a la su alta prevalencia y multirresistencia a antimicrobianos. En las últimas décadas han aumentado los reportes de cepas resistentes a penicilina, fluoroquinolonas, sulfonamidas, tetraciclina, macrólidos, y más recientemente a cefalosporinas y azitromicina. Tal panorama ha generado preocupación a nivel mundial, debido al aumento de casos de gonorrea asociados a cepas multirresistentes. En Chile se desarrolló desde el 2010 hasta el 2018 el Programa de Vigilancia de N. gonorrhoeae a nivel nacional con el objeto de caracterizar esta infección en las regiones y registrar la resistencia a los antimicrobianos. Esta revisión presenta un análisis sistemático bibliográfico, actualizado, de los principales aspectos de este microorganismo, su respuesta a antimicrobianos, y entrega pautas de diagnóstico y tratamiento, a la espera de avanzar en la comprensión del mecanismo molecular y las interacciones metabólicas e inmunológicas que determinan la infección, con miras a diseñar una vacuna efectiva.

Abstract Neisseria gonorrhoeae is a nonmotile, sporulated, aerobic or facultative anaerobic gram-negative diplococcus, catalase and oxidase positive. Sexually transmitted infections caused by this microorganism were established as public health problem since the 19th century, representing a great threat to human health due to its high prevalence and multi-resistance to antimicrobials. In recent decades, reports of strains resistant to penicillin, fluoroquinolones, sulfonamides, tetracycline, macrolides, and more recently to cephalosporins and azithromycin have increased. Such a panorama has generated concern worldwide, due to the increase in cases of gonorrhea associated with multi-resistant strains. In Chile, from 2010 to 2018, the National Surveillance Program for N. gonorrhoeae was developed in order to characterize this infection in the regions and record antimicrobial resistance. This review presents an updated, systematic bibliographic analysis of the main aspects of this microorganism, its response to antimicrobials, and provides diagnostic and treatment guidelines, while waiting to advance in the understanding of the molecular mechanism and the metabolic and immunological interactions that determine infection, with a view to designing an effective vaccine.

Incidence and risk factors of C. trachomatis and N. gonorrhoeae among young women from the Western Cape, South Africa: The EVRI study.

OBJECTIVE: Young women in South Africa are highly affected by sexually transmitted infections (STI), like C. trachomatis (CT) and N. gonorrhoeae (NG). We aimed to estimate the incidence of CT and NG, and its determinants, among young women from the Western Cape, South Africa, participating in an HPV vaccine trial (the EVRI study). METHODS: HIV-negative women aged 16-24 years were enrolled between October 2012 and July 2013. At enrolment and month 6 participants were screened for CT and NG (Anyplex CT/NG real-time detection method). A questionnaire on demographic and sexual history characteristics was completed at enrolment and month 7. Treatment for CT and/or NG was offered to infected participants. Incidence rates (IR) of CT and NG were estimated. Determinants of incident CT and NG infections were assessed using Poisson regression. RESULTS: 365 women were tested for CT and/or NG at least twice. Prevalence of CT and NG at baseline was 33.7% and 10.4%, respectively. Prevalence of co-infection with CT and NG was 7.1%. During 113.3 person-years (py), 48 incident CT infections were diagnosed (IR = 42.4 per 100 py, 95% confidence interval (CI) 31.9-56.2). Twenty-nine incident NG were diagnosed during 139.3 py (IR = 20.8 per 100 py, 95%CI 14.5-29.9). Prevalent CT infection at baseline was associated with incident CT (adjusted incidence rate ratio (aIRR) 5.8, 95%CI 3.0-11.23. More than three lifetime sex partners increased the risk for incident NG (3-4 partners aIRR = 7.3, 95%CI 2.1-26.0; ≥5 partners aIRR = 4.3, 95%CI 1.1-17.5). CONCLUSIONS: The IR of bacterial STIs among young women in the Western Cape is very high. Besides being previously infected and a higher lifetime number of sex partners, no other risk factors were found for CT and NG, suggesting that the majority of these women were at risk. This indicates the need for intensified prevention of STIs as well as screening and treatment programs to increase sexual health in this region.

Etiologies of genital inflammation and ulceration in symptomatic Rwandan men and women responding to radio promotions of free screening and treatment services.

INTRODUCTION: The longstanding inadequacies of syndromic management for genital ulceration and inflammation are well-described. The Rwanda National Guidelines for sexually transmitted infection (STI) syndromic management are not yet informed by the local prevalence and correlates of STI etiologies, a component World Health Organization guidelines stress as critical to optimize locally relevant algorithms. METHODS: Radio announcements and pharmacists recruited symptomatic patients to seek free STI services in Kigali. Clients who sought services were asked to refer sexual partners and symptomatic friends. Demographic, behavioral risk factor, medical history, and symptom data were collected. Genital exams were performed by trained research nurses and physicians. We conducted phlebotomy for rapid HIV and rapid plasma reagin (RPR) serologies and vaginal pool swab for microscopy of wet preparation to diagnose Trichomonas vaginalis (TV), bacterial vaginosis (BV), and vaginal Candida albicans (VCA). GeneXpert testing for Neisseria gonorrhoeae (NG) and Chlamydia trachomatis (CT) were conducted. Here we assess factors associated with diagnosis of NG and CT in men and women. We also explore factors associated with TV, BV and VCA in women. Finally, we describe genital ulcer and RPR results by HIV status, gender, and circumcision in men. RESULTS: Among 974 men (with 1013 visits), 20% were positive for CT and 74% were positive for NG. Among 569 women (with 579 visits), 17% were positive for CT and 27% were positive for NG. In multivariate analyses, factors associated with CT in men included younger age, responding to radio advertisements, <17 days since suspected exposure, and not having dysuria. Factors associated with NG in men included not having higher education or full-time employment, <17 days since suspected exposure, not reporting a genital ulcer, and having urethral discharge on physical exam. Factors associated with CT in women included younger age and < = 10 days with symptoms. Factors associated with NG in women included younger age, lower education and lack of full-time employment, sometimes using condoms vs. never, using hormonal vs. non-hormonal contraception, not having genital ulcer or itching, having symptoms < = 10 days, HIV+ status, having BV, endocervical discharge noted on speculum exam, and negative vaginal wet mount for VCA. In multivariate analyses, only reporting >1 partner was associated with BV; being single and RPR+ was associated with TV; and having < = 1 partner in the last month, being pregnant, genital itching, discharge, and being HIV and RPR negative were associated with VCA. Genital ulcers and positive RPR were associated with being HIV+ and lack of circumcision among men. HIV+ women were more likely to be RPR+. In HIV+ men and women, ulcers were more likely to be herpetic rather than syphilitic compared with their HIV- counterparts. CONCLUSIONS: Syndromic management guidelines in Rwanda can be improved with consideration of the prevalence of confirmed infections from this study of symptomatic men and women representative of those who would seek care at government health centers. Inclusion of demographic and risk factor measures shown to be predictive of STI and non-STI dysbioses may also increase diagnostic accuracy.

Neisseria gonorrhoeae Multivalent Maxibody with a Broad Spectrum of Strain Specificity and Sensitivity for Gonorrhea Diagnosis.

Gonorrhea is one of the most common, but still hidden and insidious, sexually transmitted diseases caused by Neisseria gonorrhoeae (gonococci). However, the diagnosis and treatment of gonorrhea are hampered by antigenic variability among gonococci, the lack of acquired immunity, and antimicrobial resistance. Further, strains resistant to cephalosporins, including ceftriaxone, the last line of defense, represent a growing threat, which prompted us to develop gonococci-specific diagnostic antibodies with broad-spectrum binding to gonococci strains to generate gonorrhea-detecting reagents. This study reports the identification of gonococci antibodies via bio-panning on gonococci cells using scFv-phage libraries. Reformatting the lead scFv-phage Clones 1 and 4 to a multivalent scFv1-Fc-scFv4 maxibody increased the sensitivity by up to 20-fold compared to the single scFv-Fc (maxibody) alone. Moreover, the multivalent maxibody showed broader cross-reactivity with clinical isolates and the ceftriaxone antibiotic-resistant World Health Organization (WHO) reference strain L. In contrast, the selected antibodies in the scFv-phage, maxibody, and multivalent maxibody did not bind to N. sicca, N. meningitides, and N. lactamica, suggesting the clinical and pharmaceutical diagnostic value of these selected antibodies for gonorrheal infections. The present study illustrates the advantages and potential application of multivalent maxibodies to develop rapid and sensitive diagnostic reagents for infectious diseases and cancer.

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.

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.

Protocolo Brasileiro para Infecções Sexualmente Transmissíveis 2020: doença inflamatória pélvica / Brazilian Protocol for Sexually Transmitted Infections 2020: pelvic inflammatory disease / Protocolo Brasileño para Infecciones de Transmisión Sexual 2020: enfermedad inflamatoria pélvica

O tema doença inflamatória pélvica está contemplado no Protocolo Clínico e Diretrizes Terapêuticas para Atenção Integral às Pessoas com Infecções Sexualmente Transmissíveis, publicado pelo Ministério da Saúde do Brasil em 2020. A doença inflamatória pélvica é a infecção aguda do trato genital superior feminino decorrente da ascensão canalicular de microrganismos cervicovaginais endógenos e, principalmente, os de transmissão sexual. Entre os agentes etiológicos envolvidos, destacam-se Chlamydia trachomatis e Neisseria gonorrhoeae. As sequelas mais importantes são dor pélvica crônica, infertilidade e gravidez ectópica. O diagnóstico clínico apresenta-se como a abordagem prática mais importante. O tratamento com antibióticos deve ser iniciado imediatamente diante da suspeição clínica. Descrevem-se orientações para gestores e profissionais de saúde sobre testes diagnósticos, tratamento preconizado, seguimento, aconselhamento, notificação, manejo de parcerias sexuais e de populações especiais. Com a maior disponibilidade da técnica de biologia molecular no Brasil, recomenda-se o rastreio de C. trachomatis e N. gonorrhoeae como estratégia preventiva da doença.

Pelvic Inflammatory Disease is a topic included in the Clinical Protocol and Therapeutic Guidelines for Comprehensive Care for People with Sexually Transmitted Infections, published by the Brazilian Ministry of Health in 2020. Pelvic inflammatory disease is an acute infection of the upper female genital tract due to canalicular spread of endogenous cervicovaginal microorganisms, in particular sexually transmitted organisms. Standing out among the etiological agents involved are Chlamydia trachomatis and Neisseria gonorrhoeae. The most important sequels are chronic pelvic pain, infertility and ectopic pregnancy. Clinical diagnosis is the most important practical approach. Antibiotic treatment should start immediately upon clinical suspicion. The article contains guidance for health service managers and health professionals on diagnostic tests, treatment, follow-up, counseling, notification, handling of sexual partnerships and special populations. In view of increased availability of the molecular biology technique in Brazil, C. trachomatis and N. gonorrhoeae screening is recommended as a disease prevention strategy.

El tema de la enfermedad inflamatoria pélvica está incluido en el Protocolo Clínico y Directrices Terapéuticas para la Atención Integral para Personas con Infecciones de Transmisión Sexual, publicado por el Ministerio de Salud de Brasil en 2020. La enfermedad inflamatoria pélvica es una infección aguda del tracto genital superior femenino resultante del ascenso canalicular de microorganismos cervicovaginales endógenos y, principalmente, los de transmisión sexual. Entre los agentes etiológicos involucrados, se destacan Chlamydia trachomatis y Neisseria gonorrhoeae. Las secuelas más importantes son: dolor pélvico crónico, infertilidad y embarazo ectópico. El diagnóstico clínico es el enfoque práctico más importante. El tratamiento con antibiótico debe iniciarse inmediatamente ante la sospecha clínica. Se describen pautas para gestores y profesionales de la salud sobre pruebas de diagnóstico, tratamiento, seguimiento, asesoramiento, notificación, manejo de parejas sexuales y poblaciones especiales. Con la mayor disponibilidad de la técnica de biología molecular, se recomienda el cribado de C. trachomatis y N. gonorrhoeae como estrategia preventiva para la enfermedad.

Synthesis and Biological Evaluation of 1,3-Dideazapurine-Like 7-Amino-5-Hydroxymethyl-Benzimidazole Ribonucleoside Analogues as Aminoacyl-tRNA Synthetase Inhibitors.

Aminoacyl-tRNA synthetases (aaRSs) have become viable targets for the development of antimicrobial agents due to their crucial role in protein translation. A series of six amino acids were coupled to the purine-like 7-amino-5-hydroxymethylbenzimidazole nucleoside analogue following an optimized synthetic pathway. These compounds were designed as aaRS inhibitors and can be considered as 1,3-dideazaadenine analogues carrying a 2-hydroxymethyl substituent. Despite our intentions to obtain N1-glycosylated 4-aminobenzimidazole congeners, resembling the natural purine nucleosides glycosylated at the N9-position, we obtained the N3-glycosylated benzimidazole derivatives as the major products, resembling the respective purine N7-glycosylated nucleosides. A series of X-ray crystal structures of class I and II aaRSs in complex with newly synthesized compounds revealed interesting interactions of these "base-flipped" analogues with their targets. While the exocyclic amine of the flipped base mimics the reciprocal interaction of the N3-purine atom of aminoacyl-sulfamoyl adenosine (aaSA) congeners, the hydroxymethyl substituent of the flipped base apparently loses part of the standard interactions of the adenine N1 and the N6-amine as seen with aaSA analogues. Upon the evaluation of the inhibitory potency of the newly obtained analogues, nanomolar inhibitory activities were noted for the leucine and isoleucine analogues targeting class I aaRS enzymes, while rather weak inhibitory activity against the corresponding class II aaRSs was observed. This class bias could be further explained by detailed structural analysis.

Neisseria gonorrhoeae infects the heterogeneous epithelia of the human cervix using distinct mechanisms.

Sexually transmitted infections are a critical public health issue. However, the mechanisms underlying sexually transmitted infections in women and the link between the infection mechanism and the wide range of clinical outcomes remain elusive due to a lack of research models mimicking human infection in vivo. We established a human cervical tissue explant model to mimic local Neisseria gonorrhoeae (GC) infections. We found that GC preferentially colonize the ectocervix by activating integrin-ß1, which inhibits epithelial shedding. GC selectively penetrate into the squamocolumnar junction (TZ) and endocervical epithelia by inducing ß-catenin phosphorylation, which leads to E-cadherin junction disassembly. Epithelial cells in various cervical regions differentially express carcinoembryonic antigen-related cell adhesion molecules (CEACAMs), the host receptor for GC opacity-associated proteins (OpaCEA). Relatively high levels were detected on the luminal membrane of ecto/endocervical epithelial cells but very low levels intracellularly in TZ epithelial cells. CEACAM-OpaCEA interaction increased ecto/endocervical colonization and reduced endocervical penetration by increasing integrin-ß1 activation and inhibiting ß-catenin phosphorylation respectively, through CEACAM downstream signaling. Thus, the intrinsic properties of cervical epithelial cells and phase-variation of bacterial surface molecules both play a role in controlling GC infection mechanisms and infectivity, preferential colonization or penetration, potentially leading to asymptomatic or symptomatic infection.

Molecular Detection of Pathogens Causing Sexually Transmissible Infections in Patients with Prostate Cancer and Hyperplasia by Quantitative TaqMan Real-Time PCR Assay.

BACKGROUND: Prostate cancer is considered the most prevalent cancer among men. Recent studies suggest that sex-ually transmissible infections (STIs) may be related to prostate carcinogenesis. Therefore, the aim of this study was to investigate whether STI pathogens (Atopobium vaginae (ATO), Neisseria gonorrhoeae (NG), Chlamydia tra-chomatis (CT), Treponema pallidum (TP), Ureaplasma urealyticum (UU), Gardnerella vaginalis (GV), Herpes Sim-plex Virus (HSV), Cytomegalovirus (CMV), Human herpesvirus (HHV), Human papillomavirus (HPV), and Tricho-monas vaginalis (TV)) presence in prostate tissues are associated with the risk of prostate cancer. METHODS: Paraffin-embedded prostate tissues obtained from patients with hyperplasia and prostate cancer were extracted. Determination of infectious microorganisms of interest was done by quantitative TaqMan real-time PCR assay. RESULTS: STI DNA was detected in 53/243 (21.8%) of the prostate tissues samples (ATO 3.7%, UU 2.88%, GV 2.46%, HSV-2 2.05%, CT 2.05%, CMV 1.64%, NG 1.64%, TP 1.64%, HHV-8 1.23%, HPV 1.23%, and TV 1.23%.) The statistical analysis revealed significant correlation between prevalence of Gardnerella vaginalis (GV) and Herpes Simplex Virus (HSV-2) between hyperplasia and cancerous groups (p = 0.02), respectively. CONCLUSIONS: No statistically significant difference was observed in the prevalence of most candidate infectious or-ganisms between hyperplasia and cancerous groups except for GV and HSV-2. It appears that inflammation in the prostate gland is more associated with prostate hyperplasia than prostate cancer. According to the role of in-fectious microorganisms in induction of chronic inflammation, we cannot exclude the importance of these patho-gens in progression of cancer. More studies are required to explore the associations of cancer with different infec-tious organisms.

Glycointeractome of Neisseria gonorrhoeae: Identification of Host Glycans Targeted by the Gonococcus To Facilitate Adherence to Cervical and Urethral Epithelial Cells.

Neisseria gonorrhoeae is a significant threat to global health for which a vaccine and novel treatment options are urgently needed. Glycans expressed by human cells are commonly targeted by pathogens to facilitate interactions with the host, and thus characterization of these interactions can aid identification of bacterial receptors that can be exploited as vaccine and/or drug targets. Using glycan array analysis, we identified 247 specific interactions between N. gonorrhoeae and glycans representative of those found on human cells. Interactions included those with mannosylated, fucosylated, and sialylated glycans, glycosaminoglycans (GAGs), and glycans terminating with galactose (Gal), N-acetylgalactosamine (GalNAc), and N-acetylglucosamine (GlcNAc). By investigating the kinetics of interactions with selected glycans, we demonstrate that whole-cell N. gonorrhoeae has a high affinity for mannosylated glycans (dissociation constant [KD ], 0.14 to 0.59 µM), which are expressed on the surface of cervical and urethral epithelial cells. Using chromatography coupled with mass spectrometric (MS) analysis, we identified potential mannose-binding proteins in N. gonorrhoeae Pretreatment of cells with mannose-specific lectin (concanavalin A) or free mannose competitor (α-methyl-d-mannopyranoside) substantially reduced gonococcal adherence to epithelial cells. This suggests that N. gonorrhoeae targets mannosyl glycans to facilitate adherence to host cells and that mannosides or similar compounds have the potential to be used as a novel treatment option for N. gonorrhoeaeIMPORTANCE Multidrug-resistant strains of Neisseria gonorrhoeae are emerging worldwide, and novel treatment and prevention strategies are needed. Glycans are ubiquitously expressed by all human cells and can be specifically targeted by pathogens to facilitate association with host cells. Here we identify and characterize the N. gonorrhoeae host-glycan binding profile (glycointeractome), which revealed numerous interactions, including high-affinity binding to mannosyl glycans. We identify gonococcal potential mannose-binding proteins and show that N. gonorrhoeae uses mannosyl glycans expressed on the surface of cervical and urethral epithelia to facilitate adherence. Furthermore, a mannose-binding lectin or a mannoside compound was able to reduce this adherence. By characterizing the glycointeractome of N. gonorrhoeae, we were able to elucidate a novel mechanism used by this important pathogen to interact with human cells, and this interaction could be exploited to develop novel therapeutics to treat antibiotic-resistant gonorrhea.

In Vitro Models of Eye Infection with Neisseria gonorrhoeae.

Prophylaxis with silver nitrate and later antibiotics has significantly reduced the cases of infant blindness from gonococcal infection at birth to the point where it has all but been forgotten in the developed world as the devastating disease that it was in the pre-antibiotic era. As a result, while it is known that the bacteria are transmitted to the eyes during passage through the infected birth canal, little is known about Neisseria gonorrhoeae colonization of the eye and the establishment and progression of keratitis. Treatment failures due to rising antimicrobial resistance necessitate investigations into all aspects of gonococcal disease, including eye infections, so that new treatment strategies can be developed. Here we present models for N. gonorrhoeae eye infection using excised bovine corneas and coculture of gonococci with primary human corneal epithelial cells. These models can be used to explore the interactions of the bacteria with corneal tissues and cells and to investigate novel therapeutics against infection.

Female Mouse Model of Neisseria gonorrhoeae Infection.

Mouse models of infection are important tools in the study of infectious disease or host the development of products to prevent or treat infections. The estradiol-treated mouse model of Neisseria gonorrhoeae genital tract infection has proved to be a valuable system for determining the importance of gonococcal factors that mediate evasion of host innate effectors in vivo or host gonococcal adaptation to hormonally driven host factors in females. Examination of mechanisms that Neisseria gonorrhoeae uses to subvert the host immune response also has been greatly aided by this whole model system, as have studies on the consequence of antibiotic resistance mutations on gonococcal fitness in vivo and the search for new antibiotics to treat antibiotic-resistant infections. The strict human specificity of N. gonorrhoeae limits the ability of experimental murine infection to mimic human infection. However, in recent years, the development of transgenic mice and protocols for supplementing mice with human factors has improved animal modeling of gonorrhea. To date, however, because the mouse estrous cycle is much shorter than the human reproductive cycle, all reported gonorrhea mouse models require treatment with estradiol and antibiotics to maintain an estrus-like state and suppress the overgrowth of inhibitory commensal flora that occurs under the influence of estrogen to allow sustained N. gonorrhoeae infection. In this chapter, we detail the methods used to (1) prepare the mice for experimental infection with N. gonorrhoeae, (2) inoculate mice and quantitatively culture vaginal swabs for noncompetitive and competitive infection experiments, and (3) monitor the host innate immune response to infection.

Neisseria gonorrhoeae ciprofloxacin-resistant strains were associated with Chlamydia trachomatis coinfection.

Aim: This study aims to characterize circulating strains to predict their relationship with sexually transmitted microorganisms, Chlamydia trachomatis, HIV, HCV, Treponema pallidum, HPV, Mycoplasmas, in an Italian multiethnic area, which has revealed a recent increase of Neisseria gonorrhoeae first-line antibiotic resistance. Materials & methods: We performed N. gonorrhoeae multiantigen sequence typing and the N. gonorrhoeae sequence typing for antimicrobial resistance. Results: We identified mutations in genes conferring resistance to cephalosporins, macrolides, fluoroquinolones through por and tbpB loci, and we reported new combinations of already known alleles. N. gonorrhoeae resistance to ciprofloxacin was associated with C. trachomatis coinfection. Conclusion: This study's data proved the utility of a routine N. gonorrhoeae molecular characterization to monitor the evolution of antibiotic resistance and to detect the most effective clinical treatment.

The Detroit 562 Pharyngeal Immortalized Cell Line Model for the Assessment of Infectivity of Pathogenic Neisseria sp.

Neisseria meningitidis and Neisseria gonorrhoeae are obligate pathogens of the human host. Due to their adaptation to the human host, many factors required for infection are specialized for the human host to the point that natural infection processes are difficult to replicate in animal models. Immortalized human cell lines have been used to identify the host factors necessary for successful colonization of human mucosal surfaces. One such model is the Detroit 562 pharyngeal immortalized cell monolayer model which is used to measure the rate of attachment to and invasion of N. meningitidis and N. gonorrhoeae into epithelial cells. The methodology of this assay, as well as the maintenance of Detroit 562 cells necessary for the experiment, will be described.

Neisseria gonorrhoeae evades autophagic killing by downregulating CD46-cyt1 and remodeling lysosomes.

The Gram-negative human pathogen N. gonorrhoeae (Ngo) quickly attaches to epithelial cells, and large numbers of the bacteria remain on the cell surface for prolonged periods. Ngo invades cells but few viable intracellular bacteria are recovered until later stages of infection, leading to the assumption that Ngo is a weak invader. On the cell surface, Ngo quickly recruits CD46-cyt1 to the epithelial cell cortex directly beneath the bacteria and causes its cleavage by metalloproteinases and Presenilin/γSecretease; how these interactions affect the Ngo lifecycle is unknown. Here, we show Ngo induces an autophagic response in the epithelial cell through CD46-cyt1/GOPC, and this response kills early invaders. Throughout infection, the pathogen slowly downregulates CD46-cyt1 and remodeling of lysosomes, another key autophagy component, and these activities ultimately promote intracellular survival. We present a model on the dynamics of Ngo infection and describe how this dual interference with the autophagic pathway allows late invaders to survive within the cell.

Outer membrane vesicles from Neisseria gonorrhoeae target PorB to mitochondria and induce apoptosis.

Neisseria gonorrhoeae causes the sexually transmitted disease gonorrhoea by evading innate immunity. Colonizing the mucosa of the reproductive tract depends on the bacterial outer membrane porin, PorB, which is essential for ion and nutrient uptake. PorB is also targeted to host mitochondria and regulates apoptosis pathways to promote infections. How PorB traffics from the outer membrane of N. gonorrhoeae to mitochondria and whether it modulates innate immune cells, such as macrophages, remains unclear. Here, we show that N. gonorrhoeae secretes PorB via outer membrane vesicles (OMVs). Purified OMVs contained primarily outer membrane proteins including oligomeric PorB. The porin was targeted to mitochondria of macrophages after exposure to purified OMVs and wild type N. gonorrhoeae. This was associated with loss of mitochondrial membrane potential, release of cytochrome c, activation of apoptotic caspases and cell death in a time-dependent manner. Consistent with this, OMV-induced macrophage death was prevented with the pan-caspase inhibitor, Q-VD-PH. This shows that N. gonorrhoeae utilizes OMVs to target PorB to mitochondria and to induce apoptosis in macrophages, thus affecting innate immunity.

A rare ocular complication of neisseria gonorrhoeae.

BACKGROUND: Spontaneous formation of subconjunctival filtering blebs is rare and has been observed after penetrating ocular surgery, in association with connective tissue diseases and inflammatory conditions. METHOD: We describe here a case of a spontaneous formation of subconjunctival filtering bleb as sequelae of gonococcal conjunctivitis in an adult patient. RESULTS: Spontaneous filtering bleb is a rare complication of gonococcal conjunctivitis. In this case, the patient presented with a recent history of gonococcal conjunctivitis treated with moxifloxacin eye drops and intravenous cephalothin for a week. On the first exam, there was superior corneal thinning with surrounding conjunctival hyperemia and edema in the right eye, but after the patient lost the follow-up for 2 weeks, the corneal thinning had become a pseudopterygium. After another week, we observed a focal elevation on the superior conjunctiva that had the appearance of a bleb, although the anterior chamber remained deep. The filtering bleb was confirmed by ultrasound biomicroscopy examination (UBM). CONCLUSION: This case is relevant to the clinical practice because filtering blebs should be considered as a complication of gonococcal conjunctivitis because they pose a threat to the eye when substantial leakage occurs.