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Background Gonorrhea, caused by the bacterium Neisseria gonorrhoeae, is a globally prevalent sexually transmitted infection. The dynamics of gonococcal population biology have been poorly defined due to a lack of resolution in strain typing methods. Methods In this study, we assess how the core genome can be used to improve our understanding of gonococcal population structure compared with current typing schemes. Results A total of 1668 loci were identified as core to the gonococcal genome. These were organized into a core genome multilocus sequence typing scheme (N gonorrhoeae cgMLST v1.0). A clustering algorithm using a threshold of 400 allelic differences between isolates resolved gonococci into discrete and stable core genome groups, some of which persisted for multiple decades. These groups were associated with antimicrobial genotypes and non-overlapping NG-STAR and NG-MAST sequence types. The MLST-STs were more widely distributed among core genome groups. Conclusions Clustering with cgMLST identified globally distributed, persistent, gonococcal lineages improving understanding of the population biology of gonococci and revealing its population structure. These findings have implications for the emergence of antimicrobial resistance in gonococci and how this is associated with lineages, some of which are more predisposed to developing antimicrobial resistance than others.

Neisseria gonorrhoeae causes the sexually transmitted disease gonorrhea exclusively in humans and uses multiple strategies to infect, including acquisition of host sialic acids that cap and mask lipooligosaccharide termini, while restricting complement activation. We hypothesized that gonococci selectively target human anti-inflammatory sialic acid-recognizing Siglec receptors on innate immune cells to blunt host responses and that pro-inflammatory Siglecs and SIGLEC pseudogene polymorphisms represent host evolutionary adaptations to counteract this interaction. N.gonorrhoeae can indeed engage multiple human but not chimpanzee CD33rSiglecs expressed on innate immune cells and in the genitourinary tract--including Siglec-11 (inhibitory) and Siglec-16 (activating), which we detected for the first time on human cervical epithelium. Surprisingly, in addition to LOS sialic acid, we found that gonococcal porin (PorB) mediated binding to multiple Siglecs. PorB also bound preferentially to human Siglecs and not chimpanzee orthologs, modulating host immune reactions in a human-specific manner. Lastly, we studied the distribution of null SIGLEC polymorphisms in a Namibian cohort with a high prevalence of gonorrhea and found that uninfected women preferentially harbor functional SIGLEC16 alleles encoding an activating immune receptor. These results contribute to the understanding of the human specificity of N.gonorrhoeae and how it evolved to evade the human immune defense.

Chlamydia trachomatis is the most frequently detected sexually transmitted bacterial pathogen in the world. Attempts to control these infections with screening programs and antibiotics have failed and, therefore, a vaccine is the best approach to control this epidemic. The Chlamydia major outer membrane protein (MOMP) is the most protective subunit vaccine so far tested. Protection induced by MOMP is, in part, dependent on its tertiary structure. We have previously described new recombinant antigens composed of the Neisseria lactamica PorB engineered to express the variable domains (VD) from Chlamydia muridarum MOMP. Here we tested antigens containing each individual MOMP VD and different VD combinations. Following immunization, mice were challenged intranasally with C. muridarum. Our results show that three constructs, PorB/VD1–3, PorB/VD1–4, and PorB/VD1–2–4, elicited high serum IgG titers in vivo, significant IFN-γ levels upon T cells re-stimulation in vitro, and evidence of protective immunity in vivo. PorB/VD1–3, PorB/VD1–4, and PorB/VD1–2–4 immunized mice lost less body weight, had lighter lungs, and decreased numbers of inclusion forming units (IFUs) in lungs than other PorB/VD construct tested and mock PBS-immunized mice. These results suggest that this approach may be a promising alternative to the use of MOMP in a Chlamydia vaccine.

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., 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.

There is an increasingly severe trend of antibiotic-resistant Neisseria gonorrhoeae strains worldwide and new therapeutic strategies are needed against this sexually-transmitted pathogen. Despite the urgency, progress towards a gonococcal vaccine has been slowed by a scarcity of suitable antigens, lack of correlates of protection in humans and limited animal models of infection. N. gonorrhoeae gene expression levels in the natural human host does not reflect expression in vitro, further complicating in vitro-basedvaccine analysis platforms. We designed a novel candidate antigen selection strategy (CASS), based on a reverse vaccinology-like approach coupled with bioinformatics. We utilized the CASS to mine gonococcal proteins expressed during human mucosal infection, reported in our previous studies, and focused on a large pool of hypothetical proteins as an untapped source of potential new antigens. Via two discovery and analysis phases (DAP), we identified 36 targets predicted to be immunogenic, membrane-associated proteins conserved in N. gonorrhoeae and suitable for recombinant expression. Six initial candidates were produced and used to immunize mice. Characterization of the immune responses indicated cross-reactive antibodies and serum bactericidal activity against different N. gonorrhoeae strains. These results support the CASS as a tool for the discovery of new vaccine candidates.

Neisseria gonorrhoeae is rapidly developing antimicrobial resistance. There is an urgent need for an effective gonococcal vaccine. In this study we examined epidemiological and clinical factors associated with gonorrhea in a cohort of women exposed to men with gonococcal urethritis attending the National Center for STD Control clinic in Nanjing, China, to understand the natural history and the risk factors for gonorrhea in this vulnerable population. This analysis will help identify the best target populations for vaccination, which is essential information for the development of vaccine strategies. We observed that 75% of the women in our cohort yielded a N. gonorrhoeae positive culture (infected women) and reported multiple sexual exposures to their infected partner. Infected women were younger than exposed but uninfected women. Contrary to the general belief that gonorrhea is asymptomatic in most women, 68% of the infected women acknowledged symptoms during their STD clinic visit, and overt inflammatory responses were detected upon medical examination in 88% of subjects. Other sexually transmitted infections were detected in 85% of subjects. This study confirmed that N. gonorrhoeae infections are underdiagnosed in women and, consequentially, untreated. Thus, our analysis reinforces the need to establish strategies for gonococcal prevention through the determination of the target population for a gonococcal vaccine.

PorB is a pan-Neisserial major outer membrane protein with a trimeric β-barrel structure. Each monomer presents eight periplasmic turns and eight surface exposed loop regions with sequence variability. PorB induces activation of host cell responses via a TLR2-dependent mechanism likely mediated by electrostatic interactions between TLR2 and PorB surface exposed loops. Variability in the loop amino acid sequence is known to influence cell responses to PorB in vitro, particularly for the residues in L5 and L7. In this work, the sequence of the porB gene and the electrostatic surface charges of PorB from 35 invasive meningococcal isolates belonging to the main clonal complexes identified in Italy and from five carriage genomes available on the website http://pubmlst.org/neisseria/ were examined. Analysis of the porB encoding regions from the invasive meningococci has identified four new alleles and a potential association between porB alleles, serogroup, and clonal complexes. Through computer-based modeling and analysis of the electrostatic surface charges of PorB from these strains, loop charge segregation between PorB from invasive serogroups B and C was observed. Specifically, loops 1, 4, and 7 were negatively charged and L2 and L8 were mostly neutral in serogroup B isolates, while an overall homogeneous positive surface charge was present in PorB from invasive serogroup C strains. A higher PorB sequence variability was observed among carriage genomes, and a general prevalence of negative loop surface charges. The surface charge differences in PorB from serogroups B and C invasive and carriage strains may, in part, influence the outcomes of Neisseriae interactions with host cells. © 2016 IUBMB Life, 68(6):488-495, 2016.

Recent emergence of antimicrobial resistance of Neisseria gonorrhoeae worldwide has resulted in limited therapeutic choices for treatment of infections caused by this organism. We performed global transcriptomic analysis of N. gonorrhoeae in subjects with gonorrhea who attended a Nanjing, China, sexually transmitted infection (STI) clinic, where antimicrobial resistance of N. gonorrhoeae is high and increasing. We found that N. gonorrhoeae transcriptional responses to infection differed in genital specimens taken from men and women, particularly antibiotic resistance gene expression, which was increased in men. These sex-specific findings may provide a new approach to guide therapeutic interventions and preventive measures that are also sex specific while providing additional insight to address antimicrobial resistance of N. gonorrhoeae., Neisseria gonorrhoeae is a bacterial pathogen responsible for the sexually transmitted infection gonorrhea. Emergence of antimicrobial resistance (AMR) of N. gonorrhoeae worldwide has resulted in limited therapeutic choices for this infection. Men who seek treatment often have symptomatic urethritis; in contrast, gonococcal cervicitis in women is usually minimally symptomatic, but may progress to pelvic inflammatory disease. Previously, we reported the first analysis of gonococcal transcriptome expression determined in secretions from women with cervical infection. Here, we defined gonococcal global transcriptional responses in urethral specimens from men with symptomatic urethritis and compared these with transcriptional responses in specimens obtained from women with cervical infections and in vitro-grown N. gonorrhoeae isolates. This is the first comprehensive comparison of gonococcal gene expression in infected men and women. RNA sequencing analysis revealed that 9.4% of gonococcal genes showed increased expression exclusively in men and included genes involved in host immune cell interactions, while 4.3% showed increased expression exclusively in women and included phage-associated genes. Infected men and women displayed comparable antibiotic-resistant genotypes and in vitro phenotypes, but a 4-fold higher expression of the Mtr efflux pump-related genes was observed in men. These results suggest that expression of AMR genes is programed genotypically and also driven by sex-specific environments. Collectively, our results indicate that distinct N. gonorrhoeae gene expression signatures are detected during genital infection in men and women. We propose that therapeutic strategies could target sex-specific differences in expression of antibiotic resistance genes. IMPORTANCE Recent emergence of antimicrobial resistance of Neisseria gonorrhoeae worldwide has resulted in limited therapeutic choices for treatment of infections caused by this organism. We performed global transcriptomic analysis of N. gonorrhoeae in subjects with gonorrhea who attended a Nanjing, China, sexually transmitted infection (STI) clinic, where antimicrobial resistance of N. gonorrhoeae is high and increasing. We found that N. gonorrhoeae transcriptional responses to infection differed in genital specimens taken from men and women, particularly antibiotic resistance gene expression, which was increased in men. These sex-specific findings may provide a new approach to guide therapeutic interventions and preventive measures that are also sex specific while providing additional insight to address antimicrobial resistance of N. gonorrhoeae.

Neisseria gonorrhoeaeis a bacterial pathogen responsible for the sexually transmitted infection, gonorrhea. Emergence of antimicrobial resistance (AMR) ofN. gonorrhoeaeworldwide has resulted in limited therapeutic choices for this infection. Men who seek treatment often have symptomatic urethritis; in contrast, gonococcal cervicitis in women is usually minimally symptomatic, but may progress to pelvic inflammatory disease. Previously, we reported the first analysis of gonococcal transcriptome expression determined in secretions from women with cervical infection. Here, we defined gonococcal global transcriptional responses in urethral specimens from men with symptomatic urethritis and compared these with transcriptional responses in specimens obtained from women with cervical infections, and ofin vitro-grown N. gonorrhoeaeisolates. This is the first comprehensive comparison of gonococcal gene expression in infected men and women. RNA sequencing analysis revealed that 9.4% of gonococcal genes showed increased expression exclusively in men and included genes involved in host immune cell interactions and 4.3% showed increased expression exclusively in women and included phage-associated genes. Infected men and women displayed comparable antibiotic resistant-genotypes andin vitro–phenotypes, but a 4-fold higher expression of the Mtr efflux pump-related genes was observed in men. These results suggest that expression of AMR genes is programmed genotypically, and also driven by sex-specific environments. Collectively, our results indicate that distinctN. gonorrhoeaegene expression signatures are detected during genital infection in men and women. We propose that therapeutic strategies could target sex specific differences in expression of antibiotic resistance genes.

Several bacterial pathogens persist and survive in the host by modulating host cell death pathways. We previously demonstrated that Neisseria 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 by N. gonorrhoeae has also been reported in other cell types. The mechanisms by which N. gonorrhoeae modulates cell death are not clear, although a role for the inhibitor of apoptosis-2 (cIAP2) has been proposed. In this study, we confirmed that N. gonorrhoeae induces production of cIAP2 in human cervical epithelial cells. High levels of intracellular cIAP2 were detected early after N. gonorrhoeae stimulation, 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 in N. gonorrhoeae -stimulated epithelial cells resulted in increased cell death and interleukin-1β (IL-1β) production. Collectively these results indicate that N. gonorrhoeae stimulation of human endocervical epithelial cells induces the release of cIAP2, an essential regulator of cell death and immune signaling.

Bacterial porins are major outer membrane proteins that function as essential solute transporters between the bacteria and the extracellular environment. Structural features of porins are also recognized by eukaryotic cell receptors involved in innate and adaptive immunity. To better investigate the function of porins, proper refolding is necessary following purification from inclusion bodies [1, 2]. Using a single-step size exclusion chromatographic method, we have purified three major porins from pathogenic bacteria, the OmpP2 (P2) from Haemophilus influenzae, FomA from Fusobacterium nucleatum and PorB from Neisseria meningitidis, at high yield and report their unique solute transport activity with size exclusion limit. Furthermore, we have optimized their purification method and achieved improvement of their thermostability for facilitating functional and structural analyses.

Chlamydia is the most common cause of bacterial sexually transmitted infections worldwide. While infections resolve with antibiotic treatment, this is often neglected in women due to frequent asymptomatic infections, leading to disease progression and severe sequelae (pelvic inflammatory disease, ectopic pregnancy, infertility). Development of a vaccine against Chlamydia is crucial. Whole organism-based vaccines have short-lived activity, serovar/subgroup-specific immunity and can cause adverse reactions in vaccinated subjects. The Chlamydia major outer membrane protein (MOMP) is a prime candidate for a subunit vaccine. MOMP contains four regions of sequence variability (variable domains, VDs) with B-cell and T-cell epitopes that elicit protective immunity. However, barriers for developing a MOMP-based vaccine include solubility, yield and refolding. We have engineered novel recombinant antigens in which the VDs are expressed into a carrier protein structurally similar to MOMP and suitable for recombinant expression at a high yield in a correctly folded and detergent-free form. Using a carrier such as the PorB porin from the human commensal organism N. lactamica, we show that PorB/VD chimeric proteins are immunogenic, antigenic and cross-reactive with MOMP. VDs are unique for each serovar but if combined in a single vaccine, a broad coverage against the major Chlamydia serovars can be ensured.

Neisseria gonorrhoeae is one of the most prevalent sexually transmitted infections worldwide. This obligate human pathogen has been extensively studied in vitro, where bacterial factors that are known to contribute to gonococcal disease and their regulation are relatively well defined. However, these in vitro experimental conditions only loosely replicate the host specific environment encountered by the bacteria in vivo. We recently reported on the complete gonococcal transcriptome expressed during natural human mucosal infection using RNA-seq analysis. Gene transcripts expressed in vivo (in vivo expressed factors) included genes encoding antibiotic resistance determinants, and a large number of hypothetical genes. A comparison of the gonococcal transcriptome expressed in vivo with the corresponding strain grown in vitro identified sets of genes regulated by infection, including those regulated by iron and the transcriptional regulatory protein Fur. We highlight here the role of Fur and gonococcal-specific regulatory processes important for infection and pathogenicity. We have determined that the genes controlled by Fur follow the same expression pattern in vivo as described previously in vitro, confirming Fur's regulatory role during infection. Collectively, these studies provide new insights into how bacterial fitness and pathogenicity are modulated during human mucosal infection., This article reviews how combining transcriptomics on pathogens from naturally infected hosts and other mechanistic studies can improve our understanding the genes involved in bacterial pathogenesis and how they are regulated.

Vaccine efficacy is optimized by addition of immune adjuvants. However, although adjuvants have been used for over a century, to date, only few adjuvants are approved for human use, mostly aimed at improving vaccine efficacy and antigen-specific protective antibody production. The mechanism of action of immune adjuvants is diverse, depending on their chemical and molecular nature, ranging from non-specific effects (i.e., antigen depot at the immunization site) to specific activation of immune cells leading to improved host innate and adaptive responses. Although the detailed molecular mechanism of action of many adjuvants is still elusive, the discovery of Toll-like receptors (TLRs) has provided new critical information on immunostimulatory effect of numerous bacterial components that engage TLRs. These ligands have been shown to improve both the quality and the quantity of host adaptive immune responses when used in vaccine formulations targeted to infectious diseases and cancer that require both humoral and cell-mediated immunity. The potential of such TLR adjuvants in improving the design and the outcomes of several vaccines is continuously evolving, as new agonists are discovered and tested in experimental and clinical models of vaccination. In this review, a summary of the recent progress in development of TLR adjuvants is presented.

Among all Neisseriae species, Neisseria meningitidis and Neisseria gonorrhoeae are the only human pathogens, causative agents of bacterial meningitis and gonorrhoea, respectively. PorB, a pan-Neisseriae trimeric porin that mediates diffusive transport of essential molecules across the bacterial outer membrane, is also known to activate host innate immunity via Toll-like receptor 2 (TLR2)-mediated signaling. The molecular mechanism of PorB binding to TLR2 is not known, but it has been hypothesized that electrostatic interactions contribute to ligand/receptor binding. Strain-specific sequence variability in the surface-exposed loops of PorB which are potentially implicated in TLR2 binding, may explain the difference in TLR2-mediated cell activation in vitro by PorB homologs from the commensal Neisseriae lactamica and the pathogen N. meningitidis. Here, we report a comparative structural analysis of PorB from N. meningitidis serogroup B strain 8765 (63% sequence homology with PorB from N. meningitidis serogroup W135) and a mutant in which amino acid substitutions in the extracellular loop 7 lead to significantly reduced TLR2-dependent activity in vitro. We observe that this mutation both alters the loop conformation and causes dramatic changes of electrostatic surface charge, both of which may affect TLR2 recognition and signaling.

Neisseria meningitidis (NM) is an opportunistic gram-negative human pathogen that colonizes the human nasopharyngeal epithelium. Asymptomatic carriage is common, but some meningococcal strains can invade nasopharyngeal epithelial cells and proceed to cause severe and often fatal infections. Invasion is predominantly driven by expression of bacterial virulence factors and host cell cognate receptors for bacterial recognition. Porins are among the Neisserial components involved in host cell activation and bacterial internalization processes. Similar to other virulence factors, porins present antigenic and structure variability among strains. Such sequence variability in the surface-exposed loop regions has been correlated to bacterial invasiveness and to variability in host cell responses via Toll-like receptor 2 (TLR2). Here, we examined whether TLR2 signaling by porins influences recovery of intracellular Neisseriae from epithelial cells in vitro. Our results show that TLR2 stimulation, either by the organism or exogenously, generally enhances Neisseriae internalization by epithelial cells. TLR2-driven intracellular signaling via ERK1/2, JNK and particularly NF-κB plays a role in this process. Based on these results, it is possible that expression of porin sequence variants that strongly induce TLR2 activation may be a mechanism to enhance the invasive features of pathogenic Neisseriae strains.

PorB is a pan-Neisserial major outer membrane protein with a trimeric β-barrel structure. Each monomer presents eight periplasmic turns and eight surface exposed loop regions with sequence variability. PorB induces activation of host cell responses via a TLR2-dependent mechanism likely mediated by electrostatic interactions between TLR2 and PorB surface exposed loops. Variability in the loop amino acid sequence is known to influence cell responses to PorB in vitro, particularly for the residues in L5 and L7. In this work, the sequence of the porB gene and the electrostatic surface charges of PorB from 35 invasive meningococcal isolates belonging to the main clonal complexes identified in Italy and from five carriage genomes available on the website http://pubmlst.org/neisseria/ were examined. Analysis of the porB encoding regions from the invasive meningococci has identified four new alleles and a potential association between porB alleles, serogroup, and clonal complexes. Through computer-based modeling and analysis of the electrostatic surface charges of PorB from these strains, loop charge segregation between PorB from invasive serogroups B and C was observed. Specifically, loops 1, 4, and 7 were negatively charged and L2 and L8 were mostly neutral in serogroup B isolates, while an overall homogeneous positive surface charge was present in PorB from invasive serogroup C strains. A higher PorB sequence variability was observed among carriage genomes, and a general prevalence of negative loop surface charges. The surface charge differences in PorB from serogroups B and C invasive and carriage strains may, in part, influence the outcomes of Neisseriae interactions with host cells. © 2016 IUBMB Life, 68(6):488-495, 2016.

In vivo anti-polysaccharide Ig responses to isolated polysaccharide (PS) are T cell independent, rapid, and fail to generate memory. However, little is known regarding PS-specific Ig responses to intact Gram-positive and Gram-negative extracellular bacteria. We previously demonstrated that intact heat-killed Streptococcus pneumoniae, a Gram-positive bacterium, elicited a rapid primary pneumococcal capsular PS (PPS) response in mice that was dependent on CD4+ T cells, B7-dependent costimulation, and CD40–CD40L interactions. However, this response was ICOS independent and failed to generate a boosted PPS-specific secondary IgG response. In the current study, we analyzed the murine meningococcal type C PS (MCPS)-specific Ig response to i.p.-injected intact, heat-killed Neisseria meningitidis, serogroup C (MenC), a Gram-negative bacterium. In contrast to S. pneumoniae, the IgG anti-MCPS response to MenC exhibited delayed primary kinetics and was highly boosted after secondary immunization, whereas the IgG anti-MCPS response to isolated MCPS was rapid, without secondary boosting, and consisted of only IgG1 and IgG3, as opposed to all four IgG isotypes in response to intact MenC. The secondary, but not primary, IgG anti-MCPS response to MenC was dependent on CD4+ T cells, CD40L, CD28, and ICOS. The primary and secondary IgG anti-MCPS responses were lower in TLR4-defective (C3H/HeJ) but not TLR2−/− or MyD88−/− mice, but secondary boosting was still observed. Of interest, coimmunization of S. pneumoniae and MenC resulted in a boosted secondary IgG anti-PPS response to S. pneumoniae. Our data demonstrate that the nature of the in vivo anti-PS response is markedly influenced by the composition and/or architecture of the bacterial subcapsular domain.

The human airway epithelium is constantly exposed to microbial products from colonizing organisms. Regulation of Toll-like receptor (TLR) expression and specific interactions with bacterial ligands is thought to mitigate exacerbation of inflammatory processes induced by the commensal flora in these cells. The genus Neisseria comprises pathogenic and commensal organisms that colonize the human nasopharynx. Neisseria lactamica is not associated with disease, but N. meningitidis occasionally invades the host, causing meningococcal disease and septicemia. Upon colonization of the airway epithelium, specific host cell receptors interact with numerous Neisseria components, including the PorB porin, at the immediate bacterial-host cell interface. This major outer membrane protein is expressed by all Neisseria strains, regardless of pathogenicity, but its amino acid sequence varies among strains, particularly in the surface-exposed regions. The interaction of Neisseria PorB with TLR2 is essential for driving TLR2/TLR1-dependent cellular responses and is thought to occur via the porin's surface-exposed loop regions. Our studies show that N. lactamica PorB is a TLR2 ligand but its binding specificity for TLR2 is different from that of meningococcal PorB. Furthermore, N. lactamica PorB is a poor inducer of proinflammatory mediators and of TLR2 expression in human airway epithelial cells. These effects are reproduced by whole N. lactamica organisms. Since the responsiveness of human airway epithelial cells to colonizing bacteria is in part regulated via TLR2 expression and signaling, commensal organisms such as N. lactamica would benefit from expressing a product that induces low TLR2-dependent local inflammation, likely delaying or avoiding clearance by the host.

Meningococcal porin PorB is an inhibitor of apoptosis induced via the intrinsic pathway in various cell types. This effect is attributed to prevention of mitochondrial depolarization and of subsequent release of proapoptotic mitochondrial factors. To determine whether apoptosis is globally inhibited by PorB, we compared the intrinsic and extrinsic pathways in HeLa cells. Interestingly, PorB does not prevent extrinsic apoptosis induced by tumor necrosis factor alpha plus cycloheximide, suggesting a unique mitochondrial pathway specificity. Several intracellular factors regulated by NF-κB, including members of the Bcl-2 family and of the inhibitor of apoptosis (IAP) family, play major roles in controlling apoptosis, and some of them are thought to contribute to the antiapoptotic effect of the gonococcal porin, PIB. However, most of the members of the Bcl-2 family and the IAP family are not induced by meningococcal PorB in HeLa cells, with the exception of Bfl-1/A1. Interestingly, PorB does not induce NF-κB activation in HeLa cells, likely due to a lack of Toll-like receptor 2 (TLR2) expression in these cells. Bfl-1/A1 expression is also regulated by CBF1, a nuclear component of the Notch signaling pathway, independent of NF-κB activation. Since HeLa cells are protected by PorB from intrinsic apoptosis events, regardless of TLR2 and NF-κB expression, the possibility of a contribution of alternative signaling pathways to this effect cannot be excluded. In this paper, we describe an initial dissection of the cascade of cellular events involved in the antiapoptotic effect of PorB in the absence of TLR2.

Several microbial pathogens can modulate the host apoptotic response to infection, which may contribute to immune evasion. Various studies have reported that infection with the sexually transmitted disease pathogen Neisseria gonorrhoeae can either inhibit or induce apoptosis. N. gonorrhoeae infection initiates at the mucosal epithelium, and in women, cells from the ectocervix and endocervix are among the first host cells encountered by this pathogen. In this study, we defined the antiapoptotic effect of N. gonorrhoeae infection in human endocervical epithelial cells (End/E6E7 cells). We first established that N. gonorrhoeae strain FA1090B failed to induce cell death in End/E6E7 cells. Subsequently, we demonstrated that stimulation with N. gonorrhoeae protected these cells from staurosporine (STS)-induced apoptosis. Importantly, only End/E6E7 cells incubated with live bacteria and in direct association with N. gonorrhoeae were protected from STS-induced apoptosis, while heat-killed and antibiotic-killed bacteria failed to induce protection. Stimulation of End/E6E7 cells with live N. gonorrhoeae induced NF-κB activation and resulted in increased gene expression of the NF-κB-regulated antiapoptotic genes bfl-1 , cIAP-2 , and c-FLIP . Furthermore, cIAP-2 protein levels also increased in End/E6E7 cells incubated with gonococci. Collectively, our results indicate that the antiapoptotic effect of N. gonorrhoeae in human endocervical epithelial cells results from live infection via expression of host antiapoptotic proteins. Securing an intracellular niche through the inhibition of apoptosis may be an important mechanism utilized by N. gonorrhoeae for microbial survival and immune evasion in cervical epithelial cells.

The Spherizone process technology, which is based on a multizone circulating reactor (MZCR), represents a significant innovation in polypropylene (PP) production. In comparison to traditional multi-step technologies, the MZCR provides a step change in the polymer homogeneity of the final product, obtained by continuously circulating the growing polymeric granules between two interrelated zones where different gas-phase compositions are realized. In addition to traditional Z-N catalysts, metallocene catalysts are also being introduced in PP manufacture: single-site catalysts allow differentiated products with outstanding property combinations. Due to its versatility and sustainability, PP continues to be utilized in a large range of applications. Innovative polymer structures featuring diverse comonomers exhibit improved properties (i.e., stiffness-impact balance, aesthetics, transparency, processability), which reinforce the capability of PP to replace traditional materials such as wood, paper, glass, and metals, as well as standard polymer products.

Gonorrhea is a highly prevalent disease resulting in significant morbidity worldwide, with an estimated 106 cases reported annually. Neisseria gonorrhoeae, the causative agent of gonorrhea, colonizes and infects the human genital tract and often evades host immune mechanisms until successful antibiotic treatment is used. The alarming increase in antibiotic-resistant strains of N. gonorrhoeae, the often asymptomatic nature of this disease in women and the lack of a vaccine directed at crucial virulence determinants have prompted us to perform transcriptome analysis to understand gonococcal gene expression patterns during natural infection. We sequenced RNA extracted from cervico-vaginal lavage samples collected from women recently exposed to infected male partners and determined the complete N. gonorrhoeae transcriptome during infection of the lower genital tract in women. On average, 3.19% of total RNA isolated from female samples aligned to the N. gonorrhoeae NCCP11945 genome and 1750 gonococcal ORFs (65% of all protein-coding genes) were transcribed. High expression in vivo was observed in genes encoding antimicrobial efflux pumps, iron response, phage production, pilin structure, outer membrane structures and hypothetical proteins. A parallel analysis was performed using the same strains grown in vitro in a chemically defined media (CDM). A total of 140 genes were increased in expression during natural infection compared to growth in CDM, and 165 genes were decreased in expression. Large differences were found in gene expression profiles under each condition, particularly with genes involved in DNA and RNA processing, iron, transposase, pilin and lipoproteins. We specifically interrogated genes encoding DNA binding regulators and iron-scavenging proteins, and identified increased expression of several iron-regulated genes, including tbpAB and fbpAB, during infection in women as compared to growth in vitro, suggesting that during infection of the genital tract in women, the gonococcus is exposed to an iron deplete environment. Collectively, we demonstrate that a large portion of the gonococcal genome is expressed and regulated during mucosal infection including genes involved in regulatory functions and iron scavenging.

PurposeThe purpose of this paper is to explore the links between customer loyalty attitude, customer loyalty behaviours (measured by customer purchase behaviours) and profitability. The aim is to define a conceptual framework within which to analyse the relationships between attitudes, behaviour, and profitability of the customers.Design/methodology/approachReference was made to earlier studies which argued that loyal customers constitute competitive asset of business organizations. Several authors noted that customers generally vary in terms of loyalty behaviours and attitudes and highlighted that differences about customers' loyalty levels affect a firm's profitability results. Customer loyalty, its antecedents and outcomes, and the links between customer satisfaction, customer loyalty and profitability have been analyzed at a customer level.FindingsThe results showed support for all but one of the five hypotheses, the exception being H2.Originality/valueThe results of the study provide evidence that a Loyalty Index can give managers an adequate support for market segmentation. This means that actual market segment strategies, based on geographical, demographical and/or psychographic variables, should take into account also loyalty measurement models.

Neisserial porins have been shown to act as B cell mitogens and immune adjuvants. PorA and PorB are the major outer membrane porin proteins of the human pathogen Neisseria meningitidis. We have shown that the mechanism of the immunopotentiating capability of porin involves up-regulation of the T cell costimulatory ligand, CD86. Due to neisserial porin’s ability to activate B cells and potentiate immune responses, we hypothesized that porin also employs the potent immune stimulatory function of dendritic cells (DC). We examined the ability of purified N. meningitidis PorB to induce maturation of murine splenic and bone marrow-derived DC. PorB treatment induced DC maturation, as demonstrated by increased expression of CD86 and class I and II MHC molecules. In addition, PorB not only enhanced the allostimulatory activity of DC, but also augmented the ability of DC to stimulate T cells in an Ag-specific manner. PorB-matured DC secreted the inflammatory cytokine IL-6, which may have implications for the adjuvant property of porin. Induction of IL-6 by PorB is also significant because IL-6 is one of a number of cytokines produced during infection with N. meningitidis and may be involved in the inflammatory process observed during infection and disease. We previously demonstrated the requirement of MyD88 and TLR2 for PorB-induced B cell activation. In the present study, MyD88 and TLR2 were also essential for PorB-induced DC activation. This work is significant for elucidating the mechanism(s) of neisserial porin’s immune stimulatory activity.

The immunopotentiating activity of neisserial porins, the major outer membrane protein of the pathogenic Neisseria, is mediated by its ability to stimulate B cells and up-regulate the surface expression of B7-2. This ability is dependent on MyD88 and Toll-like receptor (TLR)2 expression, as demonstrated by a lack of a response by B cells from MyD88 or TLR2 knockout mice to the porins. Using previously described TLR2-dependent reporter constructs, these results were confirmed and were shown to be due to induction of NF-κB nuclear translocation. This is the first demonstration of known vaccine adjuvant to stimulate immune cells via TLR2.

Toll-Like Receptor (TLR) signaling represents one of the best studied pathways to implement defense mechanisms against invading microbes in humans as well as in animals. TLRs respond to specific microbial ligands and to danger signals produced by the host during infection, and initiate downstream cascades that activate both innate and adaptive immunity. TLRs are expressed by professional immune cells and by the large majority of non-hematopoietic cells, including epithelial cells. In epithelial tissues, TLR functions are particularly important because these sites are constantly exposed to microorganisms, due to their location at the host interface with the environment. While at these sites, specific defense mechanisms and inflammatory responses are initiated via TLR signaling against pathogens, suppression or lack of TLR activation is also observed in response to the commensal microbiota. The mechanisms by which TLR signaling is regulated in mucosal epithelial cells include differential expression and levels of TLRs (and their signaling partners), their cellular localization and positioning within the tissue in a fashion that favors responses to pathogens while dampening responses to commensals and maintaining tissue homeostasis in physiologic conditions. In this review, the expression and activation of TLRs in mucosal epithelial cells of several sites of the human body are examined. Specifically, the oral cavity, the ear canal and eye, the airways, the gut and the reproductive tract are discussed, along with how site-specific host defense mechanisms are implemented via TLR signaling.

Preliminary clinical evidence suggests that Helicobacter pylori may be associated with diarrhea through its vacuolating toxin (VacA). To establish whether VacA induces intestinal secretion, epithelial damage, or both, purified pH-activated VacA was added to Caco-2 cell monolayers mounted in Ussing chambers, and electrical parameters were monitored. Mucosal addition of VacA induced an increase in short circuit current, consistent with enterotoxic effect. The effect was time- and dose-dependent and saturable. It was not found if the toxin was not pH-activated, added to the serosal side, or preheated. In cells preloaded with the Ca2+ buffering compound BAPTA/AM or with the Cl- channel inhibitor 5-nitro-2-3-(3-phenylpropylamino)benzoic acid, short circuit current did not change, indicating that VacA induces activation of Ca2+-dependent Cl- channels. VacA did not show cytopathic effects, as judged by tissue resistance. These results support the hypothesis that H. pylori may be associated with diarrhea through production of VacA.

We have previously shown that infection of mice with H. pylori can be prevented by oral immunization with H. pylori antigens given together with E. coli heat-labile enterotoxin (LT) as adjuvant. Since LT cannot be used in humans because of its unacceptable toxicity, we investigated whether protection of mice could be achieved by co-administration of antigens with non-toxic LT mutants. Here we show that CD1/SPF mice are protected against infection after oral vaccination with either purified H. pylori antigens (native and recombinant VacA, urease and CagA), or whole-cell vaccine formulations, given together with the non-toxic mutant LTK63 as a mucosal adjuvant. Furthermore we show that such protection is antigen-specific since immunization with recombinant or native VacA plus LTK63 conferred protection against infection by an H. pylori Type I strain, which expresses VacA, but not against challenge with a Type II strain which is not able to express this antigen. These results show that: (1) protection against H. pylori can be achieved in the mouse model of infection using subunit recombinant constructs plus non-toxic mucosal adjuvants; and (2) this mouse model is an useful tool in testing H. pylori vaccine formulations for eventual use in humans.

Vaccines formulated with the Chlamydia muridarum native major outer membrane protein (nMOMP) have so far been shown to elicit the most robust protection against this pathogen. nMOMP is a membrane protein and therefore, detergents are used to keep it in solution. Detergents however, have toxic effects. To address this limitation, we tested a nMOMP proteosome vaccine and compared its ability to elicit protection against nMOMP solubilized in the detergent Z3-14. The two preparations were formulated with or without CpG+Montanide (C/M). As a control antigen we used ovalbumin. Mice vaccinated with nMOMP developed strong humoral and cell mediated Chlamydia-specific immune responses. Based on the IgG2a/IgG1 levels in serum and amounts of IFN-γ in splenocytes supernatants the immune responses were predominantly Th1-biased. The animals were subsequently challenged intranasally with 2×10(3)Chlamydia inclusion forming units (IFU) and the course of the infection was followed for 10 days when the mice were euthanized. Based on changes in body weight, weight of the lungs and number of IFU recovered from the lungs, mice immunized with nMOMP-Ps and nMOMP+Z3-14 adjuvanted with C/M showed the most robust protection. In summary, nMOMP-Ps should be considered as Chlamydia vaccine candidates.

Cytotoxic strains of Helicobacter pylori cause an intense vacuolar degeneration of cells, due to the enlargement of late endosomes in the presence of membrane permeant weak bases. Bafilomycins, specific inhibitors of the vacuolar-type (V-) ATPase proton pump, prevent vacuole formation. The presence of the V-ATPase on vacuolar membranes was demonstrated by immunofluorescence with a monoclonal antibody (MAb) specific for the human 116-kDa regulatory subunit. The V-ATPase co-localised with the late endosomal marker rab7 on vacuolar membranes. In contrast, the early recycling endosomal compartment was not altered by the VacA cytotoxin, although it was endowed with the V-ATPase. Endocytosis of a MAb against the 116-kDa regulatory subunit of V-ATPase blocked endosomal acidification in HeLa cells and prevented VacA action. These results indicate that selective swelling of late endosomes, due to accumulation of osmotically active weak bases driven by the V-ATPase, is essential for vacuole formation.

Growth of Helicobacter pylori in liquid culture requires the addition of media supplements that often interfere with subsequent purification of bacterial antigens. In order to determine whether cyclodextrins can substitute for conventional H. pylori growth supplements, we cultured H. pylori in the presence of five commercially available cyclodextrins. The effect of these compounds on the production of the vacuolating cytotoxin antigen was evaluated. Several cyclodextrins supported flourishing growth and permitted the consistent production of vacuolating cytotoxin. These data suggest that Brucella broth supplemented with cyclodextrins is an improved medium for bacterial culture and industrial production of H. pylori antigens.

Porphyromonas gingivalis is a primary etiological agent of chronic periodontal disease, an infection-driven chronic inflammatory disease that leads to the resorption of tooth-supporting alveolar bone. We previously reported that TLR2 is required for P. gingivalis–induced alveolar bone loss in vivo, and our in vitro work implicated TNF as a key downstream mediator. In this study, we show that TNF-deficient (Tnf−/−) mice are resistant to alveolar bone loss following oral infection with P. gingivalis, and thus establish a central role for TNF in experimental periodontal disease. Using bone marrow–derived macrophages (BMDM) from wild-type and gene-specific knockout mice, we demonstrate that the initial inflammatory response to P. gingivalis in naive macrophages is MyD88 dependent and requires cooperative signaling of TLR2 and TLR4. The ability of P. gingivalis to activate cells via TLR2 or TLR4 was confirmed in TLR2- or TLR4-transformed human embryonic kidney cells. Additional studies using bacterial mutants demonstrated a role for fimbriae in the modulation of TLR-mediated activation of NF-κB. Whereas both TLR2 and TLR4 contributed to TNF production in naive macrophages, P. gingivalis preferentially exploited TLR2 in endotoxin-tolerant BMDM to trigger excessive TNF production. We found that TNF induced surface TLR2 expression and augmented TLR-induced cytokine production in P. gingivalis–stimulated BMDM, establishing a previously unidentified TNF-dependent feedback loop. Adoptive transfer of TLR2-expressing macrophages to TLR2-deficient mice restored the ability of P. gingivalis to induce alveolar bone loss in vivo. Collectively, our results identify a TLR2- and TNF-dependent macrophage-specific mechanism underlying pathogen-induced inflammatory bone loss in vivo.

Chlamydia trachomatis is the most common sexually transmitted bacterial pathogen and the etiologic agent of blinding trachoma. Intracellular signaling pathways leading to host cell inflammation and innate immunity to Chlamydia include those mediated by Toll-like receptors (TLRs) and nucleotide binding oligomerization domain 1 (Nod1) protein. In epithelial cells, TLR-dependent signaling contributes to local immune responses via induction of inflammatory mediators. There is evidence that TLR3, TLR4, and, particularly, TLR2 are critical for Chlamydia -mediated host cell activation and pathology. Despite the importance of TLR2, major chlamydial TLR2 antigens have not been identified so far. Numerous bacterial porins are known TLR2 agonists, i.e., porins from Neisseriae , Shigella , Salmonella , Haemophilus influenzae , and Fusobacterium nucleatum , which share structural and functional similarities with the chlamydial major outer membrane protein (MOMP), a strong antigen candidate for a potential vaccine against C. trachomatis . We describe the ability of purified, detergent-free MOMP to signal via TLR2 in vitro in TLR-overexpressing cells and TLR2-competent human reproductive tract epithelial cell lines. Using MOMP formed in pure protein micelles (proteosomes), we show the induction of TLR2-dependent interleukin-8 (IL-8) and IL-6 secretion in vitro , the involvement of TLR1 as a TLR2 coreceptor, and the activation of both NF-κB and mitogen-activated protein (MAP) kinase intracellular pathways. Interestingly, MOMP proteosomes induce cytokine secretion in endocervical epithelial cells (End/E6E7) but not in urethral epithelial cells (THUECs). A detailed understanding of the TLR2-dependent molecular mechanisms that characterize the effect of MOMP proteosomes on host cells may provide new insights for its successful development as an immunotherapeutic target against Chlamydia .

Toll-like receptors (TLRs) play a major role in host mucosal and systemic defense mechanisms by recognizing a diverse array of conserved pathogen-associated molecular patterns (PAMPs). TLR2, with TLR1 and TLR6, recognizes structurally diverse bacterial products such as lipidated factors (lipoproteins and peptidoglycans) and nonlipidated proteins, i.e., bacterial porins. PorB is a pan-neisserial porin expressed regardless of organisms' pathogenicity. However, commensal Neisseria lactamica organisms and purified N. lactamica PorB (published elsewhere as Nlac PorB) induce TLR2-dependent proinflammatory responses of lower magnitude than N. meningitidis organisms and N. meningitidis PorB (published elsewhere as Nme PorB). Both PorB types bind to TLR2 in vitro but with different apparent specificities. The structural and molecular details of PorB-TLR2 interaction are only beginning to be unraveled and may be due to electrostatic attraction. PorB molecules have significant strain-specific sequence variability within surface-exposed regions (loops) putatively involved in TLR2 interaction. By constructing chimeric recombinant PorB loop mutants in which surface-exposed loop residues have been switched between N. lactamica PorB and N. meningitidis PorB, we identified residues in loop 5 and loop 7 that influence TLR2-dependent cell activation using HEK cells and BEAS-2B cells. These loops are not uniquely responsible for PorB interaction with TLR2, but NF-κB and MAP kinases signaling downstream of TLR2 recognition are likely influenced by a hypothetical “TLR2-binding signature” within the sequence of PorB surface-exposed loops. Consistent with the effect of purified PorB in vitro , a chimeric N. meningitidis strain expressing N. lactamica PorB induces lower levels of interleukin 8 (IL-8) secretion than wild-type N. meningitidis , suggesting a role for PorB in induction of host cell activation by whole bacteria.

Many bacterial components selectively activate immune and nonhematopoietic target cells via Toll-like receptor (TLR) signaling; modulation of such host responses defines the immune adjuvant properties of these bacterial products. For example, the outer membrane protein porins fromNeisseria,Salmonella, andShigellaare known TLR2 agonists with established systemic and mucosal immune adjuvanticity. Early work indicated that the FomA porin fromFusobacterium nucleatumhas immune adjuvant activity in mice. Using a purified recombinant FomA, we have verified its immune stimulatory properties and have defined a role for TLR2 signaling in itsin vitroandin vivoactivity. FomA induces interleukin 8 (IL-8) secretion and NF-κB-dependent luciferase activity in HEK cells expressing TLR2, IL-6 secretion, and cell surface upregulation of CD86 and major histocompatibility complex (MHC) II in primary B cells from wild-type mice, but it fails to activate cells from TLR2 knockout mice. Accordingly, the immune adjuvant activity of FomA is also TLR2 dependent. In a mouse model of immunization with ovalbumin (OVA), FomA induces enhanced production of OVA-specific IgM and IgG, including IgG1 and IgG2b antibodies, as well as enhanced secretion of IL-10 and IL-6, consistent with a Th2-type adjuvant effect. We also observe a moderate production of anti-FomA antibodies, suggesting that FomA is also immunogenic, a quality that is also TLR2 dependent. Therefore, modulation of host immune responses by FomA may be effective for targeting general host immunity not only to pathogens (as a novel TLR2 adjuvant) but also toF. nucleatumitself (as an antigen), expanding its use as a self-adjuvanted antigen in an immunization strategy against polymicrobial infections, including those byF. nucleatum.

Toll-like receptors (TLRs) are recognition molecules for multiple pathogens, including bacteria, viruses, fungi, and parasites. TLR2 forms heterodimers with TLR1 and TLR6, which is the initial step in a cascade of events leading to significant innate immune responses, development of adaptive immunity to pathogens and protection from immune sequelae related to infection with these pathogens. This review will discuss the current status of TLR2 mediated immune responses by recognition of pathogen-associated molecular patterns (PAMPS) on these organisms. We will emphasize both canonical and non-canonical responses to TLR2 ligands with emphasis on whether the inflammation induced by these responses contributes to the disease state or to protection from diseases.

The process of cellular apoptosis is mediated by a number of microbial pathogens to modulate host defense mechanisms. Inhibition of apoptosis is thought to favor microbial survival, replication or immune evasion, while induction of apoptosis is likely to promote escape of the organisms from host cells. Several studies have reported that infection with Neisseria spp. can inhibit or reduce apoptotic cell death, thus allowing adaptation, intracellular replication, and immune evasion, events that are likely to spread infection. In this chapter, various techniques are described for direct measurement of host cell responses to infection with Neisseria meningitidis and to treatment with pure Neisseria porins, the major proteins found in the outer membrane of the pathogen.