Historically, LpxC inhibitors, which target the UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase catalyzing the rate-limiting step in lipid A biosynthesis, have a hydroxamate zinc-chelating head group that targets the active site and can participate in non-specific metal chelation. We synthesized novel molecules with a 2-(1S-hydroxymethyl)-imidazole head group to target the Neisseria gonorrhoeae LpxC enzyme. The gonococcal LpxC structure was generated with AlphaFold, and relative affinities of PF-04753299, CHIR-090, and our novel inhibitors were compared in docking simulations. The MIC of the most potent novel inhibitor for human challenge and multidrug-resistant N. gonorrhoeae was less than 1 μg/mL, intermediate to PF-04753299 and CHIR-090 MICs. Potency in bactericidal assays, which were designed to reflect in vivo conditions, and the ability to inhibit induction of TNF-α in THP-1 monocytes infected with gonococci, reflected relative MIC values. Cytotoxicity of the inhibitors was minimal in hemolysis and lactate dehydrogenase release assays. We showed that treating gonococci with the most potent novel inhibitor reduced lipooligosaccharide expression. Alternatives to differences in affinity for LpxC that could affect potency, including iron binding via the hydroxamate moieties and variable permeability, were explored utilizing synergism experiments and mutational analyses. We isolated spontaneous mutants resistant to the inhibitors and demonstrated that this resistance could be transferred by DNA-mediated transformation. DNA sequence analysis indicated that resistance was not due to lpxC alterations. Overall, the non-hydroxamate inhibitors exhibited high efficacy and low cytotoxicity. Furthermore, exploration of multiple antibacterial mechanisms led us to develop a promising dual therapy approach that could inform strategies to combat multidrug resistance.IMPORTANCENeisseria gonorrhoeae is a major pathogen worldwide and the second most commonly reported cause of sexually transmitted infection in the USA. A total of 601,319 cases of gonorrhea were reported to the Centers for Disease Control and Prevention (CDC) in 2023. The bacteria typically infect the urogenital tract, but infections also occur in the eye, throat, and rectum. The CDC has classified gonorrhea as an urgent public health threat, as there is no vaccine and the bacteria have developed resistance to all but one class of antibiotic. We developed novel inhibitors of an enzyme that plays a critical role in the synthesis of lipooligosaccharide, an important bacterial cell surface toxin, that have high efficacy and low cytotoxicity. Importantly, we found that the inhibitors may have relatively low propensity to engender resistance and, furthermore, we identified a potential dual therapy approach utilizing the novel inhibitors that could have clinical applicability.

Neisseria gonorrhoeae is a human-exclusive pathogen that causes gonorrhea. Gonococci (GC) initiate female infections by colonizing the cervix, which can remain asymptomatic, cause cervicitis, or ascend to the upper female reproductive tract (FRT), leading to severe tissue damage. The FRT undergoes sex hormone-mediated changes during the menstrual cycle, which have long been implicated in the vulnerability to GC infection. One of the major changes is the increase and decrease in the production of the gel-forming mucin MUC5B by the endocervix in response to the level of estradiol (E2). This study examined the impact of sex hormones on GC infection of the human cervix, utilizing a human cervical tissue explant model. Tissue explants were treated without and with E2 alone or in combination with progesterone (E2+P4) to mimic various menstrual cycle phases. Treatment of E2 or E2+P4 enhanced GC colonization at the endocervix exclusively, but did not affect epithelial transmigration. While both treatments increased the number of GC microcolonies, E2+P4 increased GC colony size on the endocervical epithelium. These increases were independent of GC host receptors, carcinoembryonic antigen-related cell adhesion molecules. GC effectively diffused through cervical mucus to interact with the cervical epithelium under all hormone conditions and through mucin hydrogels with different MUC5B and MUC5AC compositions. Mucus gels collected from cervical explants and animal mucin mixtures enhanced GC aggregation in vitro . GC diffusion through mucin-hydrogels and aggregation in the presence of cervical mucus or animal mucins decreased as the MUC5B concentration increased. Our results suggest that female sex hormones promote GC colonization at the human endocervix by modulating the cervical mucus production, regulating women’s susceptibility to GC infection, and further reveal the ability of GC to evade the mucus defense barrier for infection. Author Summary Neisseria gonorrhoeae is a bacterial pathogen that primarily infects the human genital and female reproductive tracts, causing gonorrhea. While this bacterium can infect both men and women, the infection can lead to severe and permanent damage to women’s reproductive systems. Currently, the relationship of gonococcal infection with the menstrual cycle is unknown. Here, we utilize a human cervical tissue explant model that mimics gonococcal infection in women to examine the impact of female sex hormones that drive the menstrual cycle on gonococcal infection. We found that estrogen alone or in combination with progesterone enhanced gonococcal colonization, increasing both the number and size of bacterial microcolonies on the cervical luminal surface, through regulating mucus production. Gonococci effectively penetrate through mucus layers to reach cervical epithelial cells and also prefer to aggregate with each other in the presence of mucus. Our results reveal that hormone-regulated mucus production changes the vulnerability of women to gonococcal infection, and that gonococci convert the mucus defense barrier into a colonization facilitator. ### Competing Interest Statement The authors have declared no competing interest.

Gonorrhea, caused by the human-restricted pathogen Neisseria gonorrhoeae, is a commonly reported sexually transmitted infection. Since most infections in women are asymptomatic, the true number of infections is likely much higher than reported. How gonococci (GC) colonize women’s cervixes without triggering symptoms remains elusive. Using a human cervical tissue explant model, we found that GC inoculation increased the local secretion of both proinflammatory (IL-1β and TNF-α) and antiinflammatory (IL-10) cytokines during the first 24 hours of infection. Cytokine induction required GC expression of Opa isoforms that bind the host receptors carcinoembryonic antigen-related cell adhesion molecules (CEACAMs). GC inoculation induced NF-κB activation in both cervical epithelial and subepithelial cells. However, inhibition of NF-κB activation, which reduced GC-induced IL-1β and TNF-α, did not affect GC colonization. Neutralizing IL-10 or blocking IL-10 receptors by antibodies reduced GC colonization by increasing epithelial shedding and epithelial cell-cell junction disassembly. Inhibition of the CEACAM downstream signaling molecule SHP1/2, which reduced GC colonization and increased epithelial shedding, decreased GC-induced IL-10 secretion. These results show that GC induce local secretion of IL-10, a potent antiinflammatory cytokine, at the cervix by engaging the host CEACAMs to prevent GC-colonizing epithelial cells from shedding, providing a potential mechanism for GC asymptomatic colonization in women.

Catanionic surfactant vesicles (SVs) composed of sodium dodecylbenzenesulfonate (SDBS) and cetyltrimethylammonium tosylate (CTAT) have potential applications as targeted drug delivery systems, vaccine platforms, and diagnostic tools. To facilitate these applications, we evaluated various methods to attach proteins to the surface of SDBS/CTAT vesicles. Acid phosphatase from wheat germ was used as a model protein. Acid phosphatase was successfully conjugated to vesicles enriched with a Triton-X 100 derivative containing an unsaturated ester. Enzymatic activity of acid phosphatase attached to vesicles was assessed using an acid phosphatase assay. Results from the acid phosphatase assay indicated that 15 ± 3% of the attached protein remained functional but the presence of vesicles interferes with the assay. DLS and zeta potential results correlated with the protein functionalization studies. Acid phosphatase functionalized vesicles had an average diameter of 175 ± 85 nm and an average zeta potential of -61 ± 5 mV in PBS. As a control, vesicles enriched with Triton-X 100 were prepared and analyzed by DLS and zeta potential measurements. Triton X-100 enriched vesicles had an average diameter of 140 ± 67 nm and an average zeta potential of -49 ± 2 mV in PBS. Functionalizing the surface of SVs with proteins may be a key step in developing vesicle-based technologies. For drug delivery, antibodies could be used as targeting molecules; for vaccine formulation, functionalizing the surface with spike proteins may produce novel vaccine platforms.

Abstract The cervix is the gate to the upper female reproductive tract (FRT), and the mucosal surface is the first line of defense against pathogens. The sexually transmitted pathogen Neisseria gonorrhoeae (GC) infects from the lower tract and does not induce inflammation until GC ascends through the cervix to the upper FRT, suggesting immune evasion. We examined local immune responses in the human cervix to GC infection during the first 24 hours using a human tissue explant model. GC inoculation increased the secretion of the pro-inflammatory cytokines IL-1β, TNF-α, and TNF-β, the anti-inflammatory cytokines IL-10 and LIF, and the chemokines IL-8, CXCL1, CXCL2, and CCL3. GC expressing opacity-associated protein Opa52 (binds to CEACAM) enhanced anti-inflammatory cytokines production. The elevated cytokine production was concurrent with increased nuclear staining of NF-κB p65. RNAseq analysis found increases in mRNA levels of cytokines and NF-κB pathway genes in GC-inoculated cervical tissues, compared to no GC controls. Inhibiting NF-κB activation by Bay reduced GC-induced secretion of pro-inflammatory but not anti-inflammatory cytokines. While macrophages and T-cells were abundant in the human cervix, colonization of GC expressing Opa52 did not recruit the immune cells, despite the induction of chemokines and cytokines. However, colonization of Opa-deleted GC recruited macrophages to the subepithelium of the cervical transformational zone and increased the number of CD3+ T-cells contacting Langerhans cells in the ectocervical epithelium. Together these results suggest that GC infection modulates the balance of the cervical cytokine environment towards anti-inflammatory to suppress local inflammatory responses. Supported by grants from NIH RO1 AI141894 and RO1 AI123340

Gonorrhea is the second most common sexually transmitted infection, which is primarily localized but can be disseminated systemically. The mechanisms by which a localized infection becomes a disseminated infection are unknown. We used five pairs of Neisseria gonorrhoeae isolates from the cervix/urethra (localized) and the blood (disseminated) of patients with disseminated gonococcal infection to examine the mechanisms that confine gonococci to the genital tract or enable them to disseminate to the blood. Multilocus sequence analysis found that the local and disseminated isolates from the same patients were isogenic. When culturing in vitro, disseminated isolates aggregated significantly less and transmigrated across a polarized epithelial monolayer more efficiently than localized isolates. While localized cervical isolates transmigrated across epithelial monolayers inefficiently, those transmigrated bacteria self-aggregated less and transmigrated more than cervical isolates but comparably to disseminating isolates. The local cervical isolates recruited the host receptors of gonococcal Opa proteins carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) on epithelial cells. However, the transmigrated cervical isolate and the disseminated blood isolates recruit CEACAMs significantly less often. Our results collectively suggest that switching off the expression of CEACAM-binding Opa(s), which reduces self-aggregation, promotes gonococcal dissemination.

Phagemid particles based on the Neisseria gonorrhoeae filamentous phage NgoΦ6 were used as a vaccine delivery system. We demonstrate that the host proteins incorporated into/associated with these particles can be encoded by chromosomal genes of the host bacterium or from plasmids able to replicate as an autonomous entity in the phagemid host. Phagemid particles were prepared from three types of cells, namely, Salmonella enterica ser. Typhimurium [pBSKS::Φ6fm(ST)] containing phagemid genome as an autonomous plasmid, Haemophilus influenzae Rd containing phagemid [pBSKS::Φ6fm(Hin)] integrated into the chromosome, and S. enterica ser. Typhimurium [pMPMT6::Φ6fm(ST)] containing an additional plasmid, pE1 HCV, encoding the Hepatitis C virus envelope glycoprotein E1. Approximately 200 μg of purified phage particles was used to immunize rabbits. The phagemid particles prepared from these three strains all elicited a large amount of IgG antibodies that were able to recognize bacterial host cells and proteins, as determined by ELISA and FACS analysis. The amount of specific anti-S. enterica ser. Typhimurium, anti-H. influenzae, and anti-E1 HCV antibodies elicited by vaccination was 170 μg/ml for anti-Salmonella, 80 μg/ml for anti-H. influenzae, and 65 μg/ml for anti-E1 HCV. Taken in toto, these data suggest that classical phage display methods have underestimated the potential for filamentous phage as a novel immunogen delivery system.

Neisseria gonorrhoeae (GC) establishes infection in women from the cervix, lined with heterogeneous epithelial cells from non-polarized stratified at the ectocervix to polarized columnar at the endocervix. We have previously shown that GC differentially colonize and transmigrate across the ecto and endocervical epithelia. However, whether and how GC invade into heterogeneous cervical epithelial cells is unknown. This study examined GC entry of epithelial cells with various properties, using human cervical tissue explant and non-polarized/polarized epithelial cell line models. While adhering to non-polarized and polarized epithelial cells at similar levels, GC invaded into non-polarized more efficiently than polarized epithelial cells. The enhanced GC invasion in non-polarized epithelial cells was associated with increased ezrin phosphorylation, F-actin and ezrin recruitment to GC adherent sites, and the elongation of GC-associated microvilli. Inhibition of ezrin phosphorylation inhibited F-actin and ezrin recruitment and microvilli elongation, leading to a reduction in GC invasion. The reduced GC invasion in polarized epithelial cells was associated with non-muscle myosin II-mediated F-actin disassembly and microvilli denudation at GC adherence sites. Surprisingly, intraepithelial GC were only detected inside epithelial cells shedding from the cervix by immunofluorescence microscopy, but not significantly in the ectocervical and the endocervical regions. We observed similar ezrin and F-actin recruitment in exfoliated cervical epithelial cells but not in those that remained in the ectocervical epithelium, as the luminal layer of ectocervical epithelial cells expressed ten-fold lower levels of ezrin than those beneath. However, GC inoculation induced F-actin reduction and myosin recruitment in the endocervix, similar to what was seen in polarized epithelial cells. Collectively, our results suggest that while GC invade non-polarized epithelial cells through ezrin-driven microvilli elongation, the apical polarization of ezrin and F-actin inhibits GC entry into polarized epithelial cells.

Identification of antigens is important for vaccine production. We tested extraction protocols using cetyltrimethylammonium tosylate (CTAT) and sodium dodecylbenzenesulfonate (SDBS) to formulate surfactant vesicles (SVs) containing components from Neisseria gonorrhoeae. Carbohydrate and protein assays demonstrated that protein and carbohydrates were incorporated into the vesicle leaflet. Depending on the extraction protocol utilized, 100–400 µg of protein/mL of SVs solution was obtained. Gel electrophoresis followed by silver staining demonstrated that SV extracts contained lipooligosaccharide and a subset of bacterial proteins and lipoproteins. Western blotting and mass spectral analysis indicated that the majority of the proteins were derived from the outer membrane. Mass spectrometric and bioinformatics analysis of SVs identified 29 membrane proteins, including porin and opacity-associated protein. Proteins embedded in the SVs leaflet could be degraded by the addition of trypsin or proteinase K. Our data showed that the incorporation of CTAT and SDBS into vesicles eliminated their toxicity as measured by a THP-1 killing assay. Incorporation of gonococcal cell surface components into SVs reduced toxicity as compared to the whole cell extracts, as measured by cytokine induction, while retaining the immunogenicity. This process constitutes a general method for extracting bacterial surface components and identification of antigens that might be included in vaccines.

Gonorrhea, caused by Neisseria gonorrhoeae, is a common sexually transmitted infection and an urgent public health problem. Humans are the exclusive host, and the genital tract with heterogeneous epithelia is the primary niche of this bacterium, creating unique challenges for understanding its pathogenesis. The cervical tissue explant model that we have developed enabled us to show that the properties of the epithelial cells in the female reproductive tract are the main factors driving gonococcal adaptation. Gonococcal variants that colonize strongly and penetrate poorly, thereby causing asymptomatic infection, survive better in the cervix. Gonococci adapt to different epithelial cell types by varying their surfaces and modulating distinct epithelial cell-cell adhesion complexes through manipulation of host cell signaling. These findings provide critical new insights on the mechanisms by which N. gonorrhoeae adapts to the human mucosal surface and causes asymptomatic infection.

Gonorrhea, caused by Neisseria gonorrhoeae , is a common sexually transmitted infection and an urgent public health problem. Humans are the exclusive host, and the genital tract with heterogeneous epithelia is the primary niche of this bacterium, creating unique challenges for understanding its pathogenesis. The cervical tissue explant model that we have developed enabled us to show that the properties of the epithelial cells in the female reproductive tract are the main factors driving gonococcal adaptation. Gonococcal variants that colonize strongly and penetrate poorly, thereby causing asymptomatic infection, survive better in the cervix. Gonococci adapt to different epithelial cell types by varying their surfaces and modulating distinct epithelial cell-cell adhesion complexes through manipulation of host cell signaling. These findings provide critical new insights on the mechanisms by which N. gonorrhoeae adapts to the human mucosal surface and causes asymptomatic infection.

All Neisseria gonorrhoeae strains contain multiple copies of integrated filamentous phage genomes with undefined structures. In this study, we sought to characterize the capsid proteins of filamentous N. gonorrhoeae bacteriophage NgoΦ6 and phagemids propagated in different bacteria. The data demonstrate that purified phage contain phage-encoded structural proteins and bacterial host proteins; host proteins consistently copurified with the phage particles. The bacterial host proteins associated with the phage filament (as identified by mass spectrometry) tended to be one of the predominant outer membrane components of the host strain, plus minor additional host proteins. We were able to copurify a functional ß-lactamase, a phagemid-encoded protein, with phage filaments. We used protein modeling and immunological analysis to identify the major phage encoded structural proteins. The antigenic properties of these proteins depended on the bacterium where the phages were propagated. Polyclonal antibodies against N. gonorrhoeae phage NgoΦ6 recognized phage-encoded proteins if the phage was propagated in N. gonorrhoeae or H. influenzae cells but not if it was propagated in Salmonella or E. coli. We show that the phage filaments isolated from gonococci and Haemophilus are glycosylated, and this may explain the antigenic diversity seen. Taken en toto, the data demonstrate that while the neisserial filamentous phage are similar to other Inovirus with respect to overall genomic organization, their ability to closely associate with host proteins suggests that they have unique surface properties and are secreted by a here-to-fore unknown secretory pathway.

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-β1, which inhibits epithelial shedding. GC selectively penetrate into the squamocolumnar junction (TZ) and endocervical epithelia by inducing β-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-β1 activation and inhibiting β-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.

The emergence of antibiotic resistant Neisseria gonorrhoeae (GC) is a worldwide health threat and highlights the need to identify individuals who fail treatment. This Gram-negative bacterium causes gonorrhea exclusively in humans. During infection, it is able to form aggregates and/or biofilms. The minimum inhibitory concentration (MIC) test is used for to determine susceptibility to antibiotics and to define appropriate treatment. However, the mechanism of the eradication in vivo and its relationship to laboratory results are not known. A method that examines how GC aggregation affects antibiotic susceptibility and shows the relationship between aggregate size and antibiotic susceptibility was developed. When GC aggregate, they are more resistant to antibiotic killing, with bacteria in the center surviving ceftriaxone treatment better than those in the periphery. The data indicate that N. gonorrhoeae aggregation can reduce its susceptibility to ceftriaxone, which is not reflected using the standard agar plate-based MIC methods. The method used in this study will allow researchers to test bacterial susceptibility under clinically relevant conditions.

Antibiotic resistance in Neisseria gonorrhoeae (GC) has become an emerging threat worldwide and heightens the need for monitoring treatment failures. N. gonorrhoeae, a gram-negative bacterium responsible for gonorrhea, infects humans exclusively and can form aggregates during infection. While minimal inhibitory concentration (MIC) tests are often used for determining antibiotic resistance development and treatment, the knowledge of the true MIC in individual patients and how it relates to this laboratory measure is not known. We examined the effect of aggregation on GC antibiotic susceptibility and the relationship between bacterial aggregate size and their antibiotic susceptibility. Aggregated GC have a higher survival rate when treated with ceftriaxone than non-aggregated GC, with bacteria in the core of the aggregates surviving the treatment. GC lacking opacity-associated protein or pili, or expressing a truncated lipooligosaccharide, three surface molecules that mediate GC-GC interactions, reduce both aggregation and ceftriaxone survival. This study demonstrates that the aggregation of N. gonorrhoeae can reduce the susceptibility to antibiotics, and suggests that antibiotic utilization can select for GC surface molecules that promote aggregation which in turn drive pathogen evolution. Inhibiting aggregation may be a potential way of increasing the efficacy of ceftriaxone treatment, consequently reducing treatment failure.

Glycomics lags substantially behind proteomics and genomics in its ability to decipher and synthesize complex glycans. The slow progress in deciphering glycan interactions at a molecular level is in large part due to the absence of a functional system to express, on a large scale, carbohydrates of known structure, in the context of a biologically relevant assay system. Here, the characterization of glycan‐functionalized catanionic surfactant vesicles (CVs) as a platform for glycan synthesis is described, and it is demonstrated that the resulting glycan‐functionalized CVs can serve as a scaffold for the interrogation of protein‐glycan interactions. It is demonstrated that Neisseria gonorrhoeae lipooligosaccharide (LOS) glycosyltransferase LgtE, an enzyme that catalyzes the addition of galactose onto a terminal glucose found on LOS, can be used to biochemically modify LOS or glucose functionalized CVs. CVs are characterized by differential lectin binding using flow cytometry. LgtE activity is measured on whole cells and LOS functionalized vesicles and found to have approximately the same biochemical properties. It is further demonstrated that CVs can be inkjet printed. This paper presents proof‐of‐concept that glycan‐functionalized catanionic vesicles can be used to create a high‐specificity and high‐throughput glycan array that will allow for the investigation of a variety of protein–glycan interactions.

Colonization and penetration of the epithelium is the infection-initiating route of mucosal pathogens. The epithelium counteracts infection by eliciting host cell responses while maintaining the mucosal barrier function. The obligate human sexually transmitted bacterium Neisseria gonorrhoeae, or gonococcus (GC) infects the female reproductive tract primarily from the endocervical epithelium. Due to lack of an infection model that mimics all aspects of human infections in the female reproductive tract, GC pathogenesis is poorly understood. This protocol takes advantage of the viability and functional integrity of human cervical tissues propagated in culture to generate an ex vivo infection model. This tissue model maintains the nature of the infection target and environment without any manipulation such as immortalization of epithelial cells by viruses. Using immunofluorescence microscopy, the interaction of GC with the endocervical epithelium was analyzed.