Development and validation of multiplex assays for mouse and human IgG and IgA to Neisseria gonorrhoeae antigens.

There is an urgent need for vaccines against Neisseria gonorrhoeae (Ng), the causative agent of gonorrhea. Vaccination with an outer-membrane vesicle (OMV)-based Neisseria meningitidis (Nm) vaccine provides some protection from Ng; however, the mechanisms underlying this cross-protection are unknown. To address this need, we developed multiplexed bead-based assays for the relative quantification of human and mouse IgG and IgA against Ng antigens. The assays were evaluated for analyte independence, dilutional linearity, specificity, sensitivity, intra- and inter-assay variability, and robustness to sample storage conditions. The assay was then used to test samples from mice and humans immunized with an Nm-OMV vaccine.

Development of a Tag/Catcher-mediated capsid virus-like particle vaccine presenting the conserved Neisseria gonorrhoeae SliC antigen that blocks human lysozyme.

Virus-like particles (VLPs) are promising nanotools for the development of subunit vaccines due to high immunogenicity and safety. Herein, we explored the versatile and effective Tag/Catcher-AP205 capsid VLP (cVLP) vaccine platform to address the urgent need for the development of an effective and safe vaccine against gonorrhea. The benefits of this clinically validated cVLP platform include its ability to facilitate unidirectional, high-density display of complex/full-length antigens through an effective split-protein Tag/Catcher conjugation system. To assess this modular approach for making cVLP vaccines, we used a conserved surface lipoprotein, SliC, that contributes to the Neisseria gonorrhoeae defense against human lysozyme, as a model antigen. This protein was genetically fused at the N- or C-terminus to the small peptide Tag enabling their conjugation to AP205 cVLP, displaying the complementary Catcher. We determined that SliC with the N-terminal SpyTag, N-SliC, retained lysozyme-blocking activity and could be displayed at high density on cVLPs without causing aggregation. In mice, the N-SliC-VLP vaccines, adjuvanted with AddaVax or CpG, induced significantly higher antibody titers compared to controls. In contrast, similar vaccine formulations containing monomeric SliC were non-immunogenic. Accordingly, sera from N-SliC-VLP-immunized mice also had significantly higher human complement-dependent serum bactericidal activity. Furthermore, the N-SliC-VLP vaccines administered subcutaneously with an intranasal boost elicited systemic and vaginal IgG and IgA, whereas subcutaneous delivery alone failed to induce vaginal IgA. The N-SliC-VLP with CpG (10 µg/dose) induced the most significant increase in total serum IgG and IgG3 titers, vaginal IgG and IgA, and bactericidal antibodies.

Comprehensive Bioinformatic Assessments of the Variability of Neisseria gonorrhoeae Vaccine Candidates.

A protective vaccine is the only viable way to stop the spread of gonorrhea in the face of rising antibiotic resistance. However, the notorious phase and antigenic variation of Neisseria gonorrhoeae surface proteins remains one of the challenges in vaccine development. To facilitate vaccine advancement efforts, we carried out comprehensive bioinformatic analyses of sequence variation by comparing 34 gonorrhea antigen candidates among >5,000 clinical N. gonorrhoeae isolates deposited in the Neisseria PubMLST database. Eight protein antigens showed exceptional conservation by having a single allele variant distributed in >80% of isolates. An additional 18 vaccine candidates were represented by ≤3 alleles in >50% of N. gonorrhoeae isolates globally. Phylogenetic analyses highlighted closely related antigen variants and additionally showed that AniA and FetB were the closest between N. gonorrhoeae and N. meningitidis Up to 44% of N. meningitidis alleles for both antigens have premature stop codons, suggesting differential expression. Mapping polymorphisms to the available three-dimensional structures of 12 antigens revealed low-frequency surface polymorphisms. PorB and TbpB possessed numerous high-prevalence polymorphic sites. While TbpA was also highly variable, conserved loops were nonetheless identified. A high degree of sequence conservation, the distribution of a single antigen variant among N. gonorrhoeae strains globally, or low-frequency sequence polymorphisms in surface loops make ACP, AniA, BamA, BamE, MtrE, NspA, NGO0778, NGO1251, NGO1985, OpcA, PldA, Slam2, and ZnuD promising candidates for a gonorrhea vaccine. Finally, the commonly used N. gonorrhoeae FA1090 strain emerges as a vaccine prototype, as it carries antigen sequence types identical to the most broadly distributed antigen variants.IMPORTANCENeisseria gonorrhoeae, the Gram-negative bacterium responsible for the sexually transmitted infection gonorrhea, is categorized as a high-priority pathogen for research and development efforts. N. gonorrhoeae's "superbug" status, its high morbidity, and the serious health impact associated with gonorrhea highlight the importance of vaccine development. One of the longstanding barriers to developing an effective vaccine against N. gonorrhoeae is the remarkable variability of surface-exposed antigens. In this report, we addressed this roadblock by applying extensive bioinformatic analyses to 34 gonorrhea antigen candidates among >5,000 clinical N. gonorrhoeae isolates. Our studies are important, as they reveal promising, conserved gonorrhea vaccine candidates and aid structural vaccinology. Moreover, these approaches are broadly applicable to other infectious diseases where surface antigen variability impedes successful vaccine design.

A novel gonorrhea vaccine composed of MetQ lipoprotein formulated with CpG shortens experimental murine infection.

Bacterial surface lipoproteins are emerging as attractive vaccine candidates due to their biological importance and the feasibility of their large-scale production for vaccine manufacturing. The global prevalence of gonorrhea, resistance to antibiotics, and serious consequences to reproductive and neonatal health necessitate development of effective vaccines. Reverse vaccinology identified the surface-displayed L-methionine binding lipoprotein MetQ (NGO2139) and its homolog GNA1946 (NMB1946) as gonococcal and meningococcal vaccine candidates, respectively. Here, we assessed the suitability of MetQ for inclusion in a gonorrhea vaccine by examining MetQ conservation, its function inNeisseria gonorrhoeae (Ng) pathogenesis, and its ability to induce protective immune responses using a female murine model of lower genital tract infection. In-depth bioinformatics, phylogenetics and mapping the most prevalent Ng polymorphic amino acids to the GNA1946 crystal structure revealed remarkable MetQ conservation: ~97% Ng isolates worldwide possess a single MetQ variant. Mice immunized with rMetQ-CpG (n = 40), a vaccine containing a tag-free version of MetQ formulated with CpG, exhibited robust, antigen-specific antibody responses in serum and at the vaginal mucosae including IgA. Consistent with the activity of CpG as a Th1-stimulating adjuvant, the serum IgG1/IgG2a ratio of 0.38 suggested a Th1 bias. Combined data from two independent challenge experiments demonstrated that rMetQ-CpG immunized mice cleared infection faster than control animals (vehicle, p < 0.0001; CpG, p = 0.002) and had lower Ng burden (vehicle, p = 0.03; CpG, p < 0.0001). We conclude rMetQ-CpG induces a protective immune response that accelerates bacterial clearance from the murine lower genital tract and represents an attractive component of a gonorrhea subunit vaccine.

Bioinformatics Workflow for Gonococcal Proteomics.

High-throughput quantitative proteomics unravels secrets of Neisseria gonorrhoeae biology by profiling proteome responses to environmental and endogenous cues and opens translational research paths through identification of vaccine candidates, drug targets/virulence factors, and biomarkers. Bioinformatics tools and databases are indispensable for downstream analysis of proteomic datasets to generate biologically meaningful outcomes. In this chapter, we present a workflow for proteomic data analysis with emphasis on publicly available resources, software systems, and tools that predict protein subcellular localization (CELLO, PSORTb v3.0, SOSUI-GramN, SignalP 4.1, LipoP 1.0, TMHMM 2.0) and functional annotation (EggNOG-mapper 4.5.1., DAVID v6.8, and KEGG) of N. gonorrhoeae proteins. This computational step-by-step procedure may help to foster new hypotheses and to provide insights into the structure-function relationship of proteins.

Phenotypic MicroArray Screening of Neisseria gonorrhoeae in Chemically Defined Liquid Medium.

Phenotype MicroArrays (PMs) provide a considerable benefit to the evaluation of potential vaccine/drug targets and the assessment of hypothetical protein function. Nearly 2000 conditions can be screened relatively quickly either to search for phenotypes associated with the loss of a protein or to understand metabolic differences between closely related bacterial isolates. The fastidious organism Neisseria gonorrhoeae presents an experimental challenge for phenotypic screening due to its nutrient restrictions and its autolytic activity upon reaching the stationary phase of growth. These limitations can be mitigated by modulating screening parameters. In this chapter, we describe a technique optimized for the phenotypic screening of N. gonorrhoeae FA1090 and isogenic mutant strains. Inoculum size and culturing times have been adjusted for growth in chemically defined, protein-free Graver-Wade liquid medium in the 96-well microtiter plate format employed by the PMs. With the conditions presented, highly reproducible gonococcal growth is achieved, and autolysis prior to the experimental endpoint is minimized.

Neisseria gonorrhoeae MlaA influences gonococcal virulence and membrane vesicle production.

The six-component maintenance of lipid asymmetry (Mla) system is responsible for retrograde transport of phospholipids, ensuring the barrier function of the Gram-negative cell envelope. Located within the outer membrane, MlaA (VacJ) acts as a channel to shuttle phospholipids from the outer leaflet. We identified Neisseria gonorrhoeae MlaA (ngo2121) during high-throughput proteomic mining for potential therapeutic targets against this medically important human pathogen. Our follow-up phenotypic microarrays revealed that lack of MlaA results in a complex sensitivity phenome. Herein we focused on MlaA function in cell envelope biogenesis and pathogenesis. We demonstrate the existence of two MlaA classes among 21 bacterial species, characterized by the presence or lack of a lipoprotein signal peptide. Purified truncated N. gonorrhoeae MlaA elicited antibodies that cross-reacted with a panel of different Neisseria. Little is known about MlaA expression; we provide the first evidence that MlaA levels increase in stationary phase and under anaerobiosis but decrease during iron starvation. Lack of MlaA resulted in higher cell counts during conditions mimicking different host niches; however, it also significantly decreased colony size. Antimicrobial peptides such as polymyxin B exacerbated the size difference while human defensin was detrimental to mutant viability. Consistent with the proposed role of MlaA in vesicle biogenesis, the ΔmlaA mutant released 1.7-fold more membrane vesicles. Comparative proteomics of cell envelopes and native membrane vesicles derived from ΔmlaA and wild type bacteria revealed enrichment of TadA-which recodes proteins through mRNA editing-as well as increased levels of adhesins and virulence factors. MlaA-deficient gonococci significantly outcompeted (up to 16-fold) wild-type bacteria in the murine lower genital tract, suggesting the growth advantage or increased expression of virulence factors afforded by inactivation of mlaA is advantageous in vivo. Based on these results, we propose N. gonorrhoeae restricts MlaA levels to modulate cell envelope homeostasis and fine-tune virulence.

Quantitative Proteomics of the 2016 WHO Neisseria gonorrhoeae Reference Strains Surveys Vaccine Candidates and Antimicrobial Resistance Determinants.

The sexually transmitted disease gonorrhea (causative agent: Neisseria gonorrhoeae) remains an urgent public health threat globally because of its reproductive health repercussions, high incidence, widespread antimicrobial resistance (AMR), and absence of a vaccine. To mine gonorrhea antigens and enhance our understanding of gonococcal AMR at the proteome level, we performed the first large-scale proteomic profiling of a diverse panel (n = 15) of gonococcal strains, including the 2016 World Health Organization (WHO) reference strains. These strains show all existing AMR profiles - established through phenotypic characterization and reference genome publication - and are intended for quality assurance in laboratory investigations. Herein, these isolates were subjected to subcellular fractionation and labeling with tandem mass tags coupled to mass spectrometry and multi-combinatorial bioinformatics. Our analyses detected 904 and 723 common proteins in cell envelope and cytoplasmic subproteomes, respectively. We identified nine novel gonorrhea vaccine candidates. Expression and conservation of new and previously selected antigens were investigated. In addition, established gonococcal AMR determinants were evaluated for the first time using quantitative proteomics. Six new proteins, WHO_F_00238, WHO_F_00635c, WHO_F_00745, WHO_F_01139, WHO_F_01144c, and WHO_F_01126, were differentially expressed in all strains, suggesting that they represent global proteomic AMR markers, indicate a predisposition toward developing or compensating gonococcal AMR, and/or act as new antimicrobial targets. Finally, phenotypic clustering based on the isolates' defined antibiograms and common differentially expressed proteins yielded seven matching clusters between established and proteome-derived AMR signatures. Together, our investigations provide a reference proteomics data bank for gonococcal vaccine and AMR research endeavors, which enables microbiological, clinical, or epidemiological projects and enhances the utility of the WHO reference strains.

Reductive Metabolism of Xanthohumol and 8-Prenylnaringenin by the Intestinal Bacterium Eubacterium ramulus.

SCOPE: The intestinal microbiota transforms a wide range of available substrates, including polyphenols. Microbial catabolites of polyphenols can contribute in significant ways to the health-promoting properties of their parent polyphenols. This work aims to identify intestinal metabolites of xanthohumol (XN), a prenylated flavonoid found in hops (Humulus lupulus) and beer, as well as to identify pathways of metabolism of XN in the gut. METHODS AND RESULTS: To investigate intestinal metabolism, XN and related prenylated flavonoids, isoxanthohumol (IX), and 8-prenylnaringenin (8PN) were added to growing cultures of intestinal bacteria, Eubacterium ramulus and E. limosum. Liquid chromatography coupled with mass spectrometry was used to identify metabolites of the flavonoids from the cultures. The metabolic capacity of E. limosum appears to be limited to O-demethylation. Evidence from the study indicates that E. ramulus hydrogenates XN to form α,ß-dihydroxanthohumol (DXN) and metabolizes the potent phytoestrogen 8PN into the chalcones, O-desmethylxanthohumol (DMX) and O-desmethyl-α,ß-dihydroxanthohumol (DDXN). CONCLUSION: Microbial metabolism is likely to affect both activity and toxicity of XN and derivatives. This study along with others highlights that attention should be focused on metabolites, in particular, products of intestinal microbial metabolism.

Proteomics, Bioinformatics and Structure-Function Antigen Mining For Gonorrhea Vaccines.

Expanding efforts to develop preventive gonorrhea vaccines is critical because of the serious health consequences combined with the prevalence and the dire possibility of untreatable gonorrhea. Reverse vaccinology, which includes genome and proteome mining, has proven successful in the discovery of vaccine candidates against many pathogenic bacteria. Here, we describe proteomic applications including comprehensive, quantitative proteomic platforms and immunoproteomics coupled with broad-ranging bioinformatics that have been applied for antigen mining to develop gonorrhea vaccine(s). We further focus on outlining the vaccine candidate decision tree, describe the structure-function of novel proteome-derived antigens as well as ways to gain insights into their roles in the cell envelope, and underscore new lessons learned about the fascinating biology of Neisseria gonorrhoeae.

Lipid-Modified Azurin of Neisseria gonorrhoeae Is Not Surface Exposed and Does Not Interact With the Nitrite Reductase AniA.

Lipid-modified cupredoxin azurin (Laz) is involved in electron transport in Neisseria and proposed to act as an electron donor to the surface-displayed nitrite reductase AniA. We identified Laz in Neisseria gonorrhoeae cell envelopes and naturally elaborated membrane vesicles in proteomic investigations focused on discovering new vaccine and therapeutic targets for this increasingly difficult to treat pathogen. Its surface exposure in N. meningitidis suggested Laz could be a vaccine candidate for N. gonorrhoeae. Here we characterized the localization, expression, and role of Laz within the gonococcal cell envelope and challenged the hypothesis that Laz and AniA interact. While we demonstrate that Laz indeed shows some good features of a vaccine antigen, such as stable expression, high conservation, and ability to elicit antibodies that cross-react with a diverse panel of Neisseria, it is not a surface-displayed lipoprotein in the gonococcus. This discovery eliminates Laz as a gonorrhea vaccine candidate, further highlighting the necessity of examining homologous protein localization between closely related species. Absence of Laz slightly altered cell envelope integrity but was not associated with growth defects in vitro, including during anoxia, implicating the presence of other electron pathways to AniA. To further dissect the implied AniA-Laz interaction, we utilized biolayer interferometry and optimized and executed chemical cross-linking coupled with immunoblotting to covalently link interacting protein partners in living gonococci. This method, applied for the first time in N. gonorrhoeae research to interrogate protein complexes, was validated by the appearance of the trimer form of AniA, as well as by increased formation of the ß-barrel assembly machinery complex, in the presence of cross-linker. We conclude that Laz is not an electron donor to AniA based on their distinct subcellular localization, discordant expression during infection of the female mouse lower genital tract, and lack of interaction in vivo and in vitro.

PubMLST for Antigen Allele Mining to Inform Development of Gonorrhea Protein-Based Vaccines.

Neisseria gonorrhoeae (Ng) is a human-specific pathogen and the etiological agent of gonorrhea, a sexually transmitted infection with a significant global health burden. While often asymptomatic, untreated gonorrhea can lead to pelvic inflammatory disease, ectopic pregnancy, infertility, and increased transmission/acquisition of HIV. A protective gonorrhea vaccine may be the only way to control disease transmission in the future due to the inexorable development of antibiotic resistance. Subunit antigens are proven candidates for vaccine development due to their safety, cost-effectiveness, and rapid preparation. To inform protein-based gonorrhea vaccine design by including different antigen variants, herein we present bioinformatics mining of alleles and single nucleotide/amino acid polymorphisms using DNA/protein sequences of all Ng isolates deposited into the PubMLST database and MtrE and BamA as model antigens. We also present phylogenetic analyses that can be performed using sequence data to gain insights into the evolutionary relationships between the polymorphisms found among the population of isolates using a convenient tool: Molecular Evolutionary Genetics Analysis (MEGA) software. Finally, we perform antigen polymorphism mapping onto the MtrE and BamA structures. This methodology can be applied for rational vaccine design to increase vaccine coverage and cross-protection by heteroligand presentation achieved via inclusion of diverse antigen variants and is relevant to over 100 different species and genera deposited into the PubMLST family of databases.

Transkingdom network reveals bacterial players associated with cervical cancer gene expression program.

Cervical cancer is the fourth most common cancer in women worldwide with human papillomavirus (HPV) being the main cause the disease. Chromosomal amplifications have been identified as a source of upregulation for cervical cancer driver genes but cannot fully explain increased expression of immune genes in invasive carcinoma. Insight into additional factors that may tip the balance from immune tolerance of HPV to the elimination of the virus may lead to better diagnosis markers. We investigated whether microbiota affect molecular pathways in cervical carcinogenesis by performing microbiome analysis via sequencing 16S rRNA in tumor biopsies from 121 patients. While we detected a large number of intra-tumor taxa (289 operational taxonomic units (OTUs)), we focused on the 38 most abundantly represented microbes. To search for microbes and host genes potentially involved in the interaction, we reconstructed a transkingdom network by integrating a previously discovered cervical cancer gene expression network with our bacterial co-abundance network and employed bipartite betweenness centrality. The top ranked microbes were represented by the families Bacillaceae, Halobacteriaceae, and Prevotellaceae. While we could not define the first two families to the species level, Prevotellaceae was assigned to Prevotella bivia. By co-culturing a cervical cancer cell line with P. bivia, we confirmed that three out of the ten top predicted genes in the transkingdom network (lysosomal associated membrane protein 3 (LAMP3), STAT1, TAP1), all regulators of immunological pathways, were upregulated by this microorganism. Therefore, we propose that intra-tumor microbiota may contribute to cervical carcinogenesis through the induction of immune response drivers, including the well-known cancer gene LAMP3.

Utilization of Vibrio cholerae as a Model Organism to Screen Natural Product Libraries for Identification of New Antibiotics.

The development of antibiotic-resistant bacteria requires increasing research efforts in drug discovery. Vibrio cholerae can be utilized as a model gram-negative enteric pathogen in high- and medium-throughput screening campaigns to identify antimicrobials with different modes of action. In this chapter, we describe methods for the optimal growth of V. cholerae in 384-well plates, preparation of suitable microtiter natural product sample libraries, as well as their screening using measurements of bacterial density and activity of type II secretion-dependent protease as readouts. Concomitant LC-MS/MS profiling and spectral data networking of assay sample libraries facilitate dereplication of putative known and/or nuisance compounds and efficient prioritization of samples containing putative new natural products for further investigation.

Methods for Assessments of Collagenolytic Activity of the Vibrio cholerae Extracellular Proteases, Purification of Secreted Collagenase VchC, and Extraction of Type I Collagen from Fish Skin.

Secreted proteases have been linked to facilitating the survival of Vibrio cholerae in different environmental niches. Examination of protease activity using various measures is critical to determine the substrate specificity of a given proteolytic enzyme and its biological function. Here, we describe methods to assess collagenolytic activity of the V. cholerae extracellular proteases using synthetic and natural substrates in quantitative and qualitative assays. Purification of the secreted V. cholerae collagenase VchC and extraction of type I collagen from fish skin are also described.

SliC is a surface-displayed lipoprotein that is required for the anti-lysozyme strategy during Neisseria gonorrhoeae infection.

Lysozymes are nearly omnipresent as the first line of immune defense against microbes in animals. They exert bactericidal action through antimicrobial peptide activity and peptidoglycan hydrolysis. Gram-negative bacteria developed several weapons to battle lysozymes, including inhibitors of c-type lysozymes in the MliC/PliC family and the Neisseria adhesin complex protein (ACP). Until the recent discovery of ACP, no proteinaceous lysozyme inhibitors were reported for the genus Neisseria, including the important human pathogen N. gonorrhoeae. Here, we describe a previously unrecognized gonococcal virulence mechanism involving a protein encoded by the open reading frame ngo1063 that acts to counteract c-type Iysozyme and provides a competitive advantage in the murine model of gonorrhea. We named this protein SliC as a surface-exposed lysozyme inhibitor of c-type lysozyme. SliC displays low overall primary sequence similarity to the MliC/PliC inhibitors, but we demonstrate that it has a parallel inhibitory mechanism. Our studies provide the first evidence that bacterial proteinaceous lysozyme inhibitors protect against host lysozyme during infection based on lack of attenuation of the ΔsliC mutant in lysozyme knock-out mice, and that the conserved residues involved in lysozyme inhibition, S83 and K103, are functionally indispensable during infection in wild type mice. Recombinant SliC completely abrogated the lytic activity of human and chicken c-type lysozymes, showing a preference towards human lysozyme with an IC50 of 1.85 µM and calculated KD value of 9.2 ± 1.9 µM. In contrast, mutated SliC bearing S83A and K103A substitutions failed to protect fluorescein-labeled cell-wall from lysozyme-mediated hydrolysis. Further, we present data revealing that SliC is a surface-displayed lipoprotein released in membrane vesicles that is expressed throughout all phases of growth, in conditions relevant to different niches of the human host, and during experimental infection of the murine genital tract. SliC is also highly conserved and expressed by diverse gonococcal isolates as well as N. meningitidis, N. lactamica, and N. weaveri. This study is the first to highlight the importance of an anti-lysozyme strategy to escape the innate immune response during N. gonorrhoeae infection.

Jizanpeptins, Cyanobacterial Protease Inhibitors from a Symploca sp. Cyanobacterium Collected in the Red Sea.

Jizanpeptins A-E (1-5) are micropeptin depsipeptides isolated from a Red Sea specimen of a Symploca sp. cyanobacterium. The planar structures of the jizanpeptins were established using NMR spectroscopy and mass spectrometry and contain 3-amino-6-hydroxy-2-piperidone (Ahp) as one of eight residues in a typical micropeptin motif, as well as a side chain terminal glyceric acid sulfate moiety. The absolute configurations of the jizanpeptins were assigned using a combination of Marfey's methodology and chiral-phase HPLC analysis of hydrolysis products compared to commercial and synthesized standards. Jizanpeptins A-E showed specific inhibition of the serine protease trypsin (IC50 = 72 nM to 1 µM) compared to chymotrypsin (IC50 = 1.4 to >10 µM) in vitro and were not overtly cytotoxic to HeLa cervical or NCI-H460 lung cancer cell lines at micromolar concentrations.

Structural and functional insights into the role of BamD and BamE within the ß-barrel assembly machinery in Neisseria gonorrhoeae.

The ß-barrel assembly machinery (BAM) is a conserved multicomponent protein complex responsible for the biogenesis of ß-barrel outer membrane proteins (OMPs) in Gram-negative bacteria. Given its role in the production of OMPs for survival and pathogenesis, BAM represents an attractive target for the development of therapeutic interventions, including drugs and vaccines against multidrug-resistant bacteria such as Neisseria gonorrhoeae The first structure of BamA, the central component of BAM, was from N. gonorrhoeae, the etiological agent of the sexually transmitted disease gonorrhea. To aid in pharmaceutical targeting of BAM, we expanded our studies to BamD and BamE within BAM of this clinically relevant human pathogen. We found that the presence of BamD, but not BamE, is essential for gonococcal viability. However, BamE, but not BamD, was cell-surface-displayed under native conditions; however, in the absence of BamE, BamD indeed becomes surface-exposed. Loss of BamE altered cell envelope composition, leading to slower growth and an increase in both antibiotic susceptibility and formation of membrane vesicles containing greater amounts of vaccine antigens. Both BamD and BamE are expressed in diverse gonococcal isolates, under host-relevant conditions, and throughout different phases of growth. The solved structures of Neisseria BamD and BamE share overall folds with Escherichia coli proteins but contain differences that may be important for function. Together, these studies highlight that, although BAM is conserved across Gram-negative bacteria, structural and functional differences do exist across species, which may be leveraged in the development of species-specific therapeutics in the effort to combat multidrug resistance.