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A novel covalent post-translational modification (lysine-NOS-cysteine) was discovered in proteins, initially in the enzyme transaldolase of Neisseria gonorrhoeae ( Ng TAL) [ Nature 2021 , 593 , 460-464], acting as a redox switch. The identification of this novel linkage in solution was unprecedented until now. We present detection of the NOS redox switch in solution using sulfur K-edge X-ray absorption spectroscopy (XAS). The oxidized Ng TAL spectrum shows a distinct shoulder on the low-energy side of the rising edge, corresponding to a dipole-allowed transition from the sulfur 1s core to the unoccupied σ* orbital of the S-O group in the NOS bridge. This feature is absent in the XAS spectrum of reduced Ng TAL, where Lys-NOS-Cys is absent. Our experimental and calculated XAS data support the presence of a NOS bridge in solution, thus potentially facilitating future studies on enzyme activity regulation mediated by the NOS redox switches, drug discovery, biocatalytic applications, and protein design.

The MtrCDE system confers multidrug resistance to Neisseria gonorrhoeae , the causative agent of gonorrhea. Using free and directed molecular dynamics (MD) simulations, we analyzed the interactions between MtrD and azithromycin, a transport substrate of MtrD, and a last-resort clinical treatment for multidrug-resistant gonorrhea. We then simulated the interactions between MtrD and streptomycin, an apparent nonsubstrate of MtrD. Using known conformations of MtrD homologues, we simulated a potential dynamic transport cycle of MtrD using targeted MD techniques (TMD), and we noted that forces were not applied to ligands of interest. In these TMD simulations, we observed the transport of azithromycin and the rejection of streptomycin. In an unbiased, long-time scale simulation of AZY-bound MtrD, we observed the spontaneous diffusion of azithromycin through the periplasmic cleft. Our simulations show how the peristaltic motions of the periplasmic cleft facilitate the transport of substrates by MtrD. Our data also suggest that multiple transport pathways for macrolides may exist within the periplasmic cleft of MtrD.

Three-dimensional full-atom model of the enzyme complex with acetyl-CoA and substrate was constructed on the basis of the primary sequence of amino acid residues of N-acetyl glutamate synthase. Bioinformatics approaches of computer modeling were applied, including multiple sequence alignment, prediction of co-evolutionary contacts, and ab initio folding. On the basis of the results of calculations by classical molecular dynamics and combined quantum and molecular mechanics (QM/MM) methods, the structure of the active site and the reaction mechanism of N-acetylglutamate formation are described. Agreement of the structures of the enzyme-product complexes obtained in computer modeling and in the X-ray studies validates the reliability of modeling predictions.

Aminoacyl-tRNA synthetases (aaRSs) have become viable targets for the development of antimicrobial agents due to their crucial role in protein translation. A series of six amino acids were coupled to the purine-like 7-amino-5-hydroxymethylbenzimidazole nucleoside analogue following an optimized synthetic pathway. These compounds were designed as aaRS inhibitors and can be considered as 1,3-dideazaadenine analogues carrying a 2-hydroxymethyl substituent. Despite our intentions to obtain N 1 -glycosylated 4-aminobenzimidazole congeners, resembling the natural purine nucleosides glycosylated at the N 9 -position, we obtained the N 3 -glycosylated benzimidazole derivatives as the major products, resembling the respective purine N 7 -glycosylated nucleosides. A series of X-ray crystal structures of class I and II aaRSs in complex with newly synthesized compounds revealed interesting interactions of these "base-flipped" analogues with their targets. While the exocyclic amine of the flipped base mimics the reciprocal interaction of the N 3 -purine atom of aminoacyl-sulfamoyl adenosine (aaSA) congeners, the hydroxymethyl substituent of the flipped base apparently loses part of the standard interactions of the adenine N 1 and the N 6 -amine as seen with aaSA analogues. Upon the evaluation of the inhibitory potency of the newly obtained analogues, nanomolar inhibitory activities were noted for the leucine and isoleucine analogues targeting class I aaRS enzymes, while rather weak inhibitory activity against the corresponding class II aaRSs was observed. This class bias could be further explained by detailed structural analysis.

The Gram-negative pathogen Neisseria gonorrhoeae (gonococcus [Gc]) colonizes lysozyme-rich mucosal surfaces. Lysozyme hydrolyzes peptidoglycan, leading to bacterial lysis. Gc expresses two proteins, SliC and NgACP, that bind and inhibit the enzymatic activity of lysozyme. SliC is a surface-exposed lipoprotein, while NgACP is found in the periplasm and also released extracellularly. Purified SliC and NgACP similarly inhibit lysozyme. However, whereas mutation of ngACP increases Gc susceptibility to lysozyme, the sliC mutant is only susceptible to lysozyme when ngACP is inactivated. In this work, we examined how lipidation contributes to SliC expression, cellular localization, and resistance of Gc to killing by lysozyme. To do so, we mutated the conserved cysteine residue (C18) in the N-terminal lipobox motif of SliC, the site for lipid anchor attachment, to alanine. SliC(C18A) localized to soluble rather than membrane fractions in Gc and was not displayed on the bacterial surface. Less SliC(C18A) was detected in Gc lysates compared to the wild-type protein. This was due in part to some release of the C18A mutant, but not wild-type, protein into the extracellular space. Surprisingly, Gc expressing SliC(C18A) survived better than SliC (wild type)-expressing Gc after exposure to lysozyme. We conclude that lipidation is not required for the ability of SliC to inhibit lysozyme, even though the lipidated cysteine is 100% conserved in Gc SliC alleles. These findings shed light on how members of the growing family of lysozyme inhibitors with distinct subcellular localizations contribute to bacterial defense against lysozyme. IMPORTANCE Neisseria gonorrhoeae is one of many bacterial species that express multiple lysozyme inhibitors. It is unclear how inhibitors that differ in their subcellular localization contribute to defense from lysozyme. We investigated how lipidation of SliC, an MliC (membrane-bound lysozyme inhibitor of c-type lysozyme)-type inhibitor, contributes to its localization and lysozyme inhibitory activity. We found that lipidation was required for surface exposure of SliC and yet was dispensable for protecting the gonococcus from killing by lysozyme. To our knowledge, this is the first time the role of lipid anchoring of a lysozyme inhibitor has been investigated. These results help us understand how different lysozyme inhibitors are localized in bacteria and how this impacts resistance to lysozyme., (Copyright © 2020 American Society for Microbiology.)

Neisseria gonorrhoeae responds to host-derived antimicrobials by inducing the expression of the mtrCDE -encoded multidrug efflux pump, which expels microbicides, such as bile salts, fatty acids, and multiple extrinsically administered drugs, from the cell. In the absence of these cytotoxins, the TetR family member MtrR represses the mtrCDE genes. Although antimicrobial-dependent derepression of mtrCDE is clear, the physiological inducers of MtrR are unknown. Here, we report the crystal structure of an induced form of MtrR. In the binding pocket of MtrR, we observed electron density that we hypothesized was N -cyclohexyl-3-aminopropanesulfonic acid (CAPS), a component of the crystallization reagent. Using the MtrR-CAPS structure as an inducer-bound template, we hypothesized that bile salts, which bear significant chemical resemblance to CAPS, are physiologically relevant inducers. Indeed, characterization of MtrR-chenodeoxycholate and MtrR-taurodeoxycholate interactions, both in vitro and in vivo , revealed that these bile salts, but not glyocholate or taurocholate, bind MtrR tightly and can act as bona fide inducers. Furthermore, two residues, W136 and R176, were shown to be important in binding chenodeoxycholate but not taurodeoxycholate, suggesting different binding modes of the bile salts. These data provide insight into a crucial mechanism utilized by the pathogen to overcome innate human defenses. IMPORTANCE Neisseria gonorrhoeae causes a significant disease burden worldwide, and a meteoric rise in its multidrug resistance has reduced the efficacy of antibiotics previously or currently approved for therapy of gonorrheal infections. The multidrug efflux pump MtrCDE transports multiple drugs and host-derived antimicrobials from the bacterial cell and confers survival advantage on the pathogen within the host. Transcription of the pump is repressed by MtrR but relieved by the cytosolic influx of antimicrobials. Here, we describe the structure of induced MtrR and use this structure to identify bile salts as physiological inducers of MtrR. These findings provide a mechanistic basis for antimicrobial sensing and gonococcal protection by MtrR through the derepression of mtrCDE expression after exposure to intrinsic and clinically applied antimicrobials., (Copyright © 2019 Beggs et al.)

Gonorrhea infections are becoming more difficult to treat due to the prevalence of strains exhibiting resistance to antibiotics and new therapeutic approaches are needed. N-acetylmuramyl-L-alanine amidase (AmiC) from Neisseria gonorrhoeae is a hydrolase that functions during cell division by cleaving the bond between the N-acetylmuramyl and L-alanine moieties of peptidoglycan. Inhibiting this enzyme offers the prospect of restoring the efficacy of existing antibiotics as treatments against N. gonorrhoeae. Of its two domains, the C-terminal domain catalyses the hydrolysis reaction and the N-terminal domain (NTD) is believed to target AmiC to its peptidoglycan substrate. Here, we report the 1 H, 13 C, and 15 N resonance assignments of a 131 amino acid NTD construct of AmiC by heteronuclear NMR spectroscopy. The assignments represent the first for N. gonorrhoeae AmiC-NTD, laying the groundwork for detailed examination of its structure and dynamics, and providing a platform for new drug discovery efforts to address antimicrobial-resistant N. gonorrhoeae.

Neisseria gonorrhoeae is a gram-negative obligate human pathogen that contains lipooligosaccharide (LOS) as a major constituent within the outer membrane. LOS plays a major role in pathogenesis by inducing host inflammatory responses and also enabling evasion of host innate immunity through sialylation. Epitopes within LOS are also potential vaccine candidates. In this chapter, we describe a general method based on the Westphal hot phenol extraction process to purify whole LOS from N. gonorrhoeae for structural analyses and for use in in vivo and in vitro biological assays.

The composition of Neisseria peptidoglycan has been of scientific interest for over four decades. Initial investigations focused on discovering the mechanisms causing rising rates of antibiotic resistance in N. gonorrhoeae by determining differences in peptidoglycan composition in penicillin susceptible and resistant strains. The discovery that cytotoxic peptidoglycan fragments are also released by Neisseria furthered the interest in peptidoglycan composition. This method describes the purification, enzymatic degradation, and separation of peptidoglycan fragments by high-performance liquid chromatography (HPLC). It also describes the preparation of samples so that they can be positively identified by mass spectrometry.

Modern DNA recombinant techniques and major advances in genetic engineering have resulted in the development of bacterial expression systems that guarantee an unlimited supply of valuable proteins that have potential clinical or industrial use, but which are often limited by their low natural availability. This chapter provides the reader with a general scheme to clone, express, and purify native histidine (His)-tagged proteins in the desired quantity and quality required for its intended use, and reviews the most important factors affecting the production of recombinant proteins in a soluble form. Alternative methods for purification of insoluble recombinant proteins under denaturing conditions are also discussed. An optimized protocol to successfully purify native Neisseria gonorrhoeae Adhesin Complex Protein (Ng-ACP; NGO1981) is used as a technical example for the processes, which could potentially be applied to any gonococcal recombinant protein of interest.

The Type IV pili are displayed peritrichously on the surfaces of Neisseria gonorrhoeae cells. Here we present protocols for isolating and purifying Type IV pili and dissociating them into PilE pilin subunits. Pilus filaments are isolated from the bacterial cell surface by mechanical shearing and purified by differential precipitation and centrifugation. PilE subunits are extracted by treating the purified pili with detergent to disrupt the hydrophobic interactions holding them together in the filaments. Purified pili and pilin subunits can be used for structural, biophysical, or biochemical characterization and as antigens for antibody production.

Objective: The development of an accurate, sensitive, specific, rapid, reproducible, stable-at-room-temperature and cost-effective diagnostic kit, and a low-cost portable fluorescence detector to fulfil the requirements of diagnostic facilities in developing countries., Methods: We developed the 'Chlamy and Ness CT/NG kit' based on molecular beacons for the detection of Chlamydia trachomatis (CT) and Neisseriagonorrhoeae (NG). Multi-centric evaluation of the CT/NG kit was performed using the commercially available nucleic acid amplification test (NAAT)-based FTD Urethritis basic kit for comparison from December 2014 to November 2016. The stability of the kit reagents at 4 and 37 ˚C and the inter-day reproducibility of results were also analysed., Results: The sensitivity and specificity of the kit were found to be 95.83 and 100.00 % for the detection of C. trachomatis and 93.24 and 99.75 % for N. gonorrhoeae, respectively, when tested against the commercial kit. The positive predictive value (PPV) was 100.00 and 98.57 %, whereas the negative predictive value (NPV) was 99.54 and 98.79 % for C. trachomatis and N. gonorrhoeae, respectively. Analysis of the kappa statistics enhanced the 'inter-rater' κ=0.976 for Chlamydia and κ=0.943 for Neisseria., Conclusion: Our kit was found to be as sensitive and specific as commercially available kits. Its low cost and ease of use will make it suitable for the routine diagnosis of C. trachomatis and N. gonorrhoeae in the resource-limited settings of developing countries.

Sialylation of lacto- N -neotetraose (LNnT) extending from heptose I (HepI) of gonococcal lipooligosaccharide (LOS) contributes to pathogenesis. Previously, gonococcal LOS sialyltransterase (Lst) was shown to sialylate LOS in Triton X-100 extracts of strain 15253, which expresses lactose from both HepI and HepII, the minimal structure required for monoclonal antibody (MAb) 2C7 binding. Ongoing work has shown that growth of 15253 in cytidine monophospho- N -acetylneuraminic acid (CMP-Neu5Ac)-containing medium enables binding to CD33/Siglec-3, a cell surface receptor that binds sialic acid, suggesting that lactose termini on LOSs of intact gonococci can be sialylated. Neu5Ac was detected on LOSs of strains 15253 and an MS11 mutant with lactose only from HepI and HepII by mass spectrometry; deleting HepII lactose rendered Neu5Ac undetectable. Resistance of HepII lactose Neu5Ac to desialylation by α2-3-specific neuraminidase suggested an α2-6 linkage. Although not associated with increased factor H binding, HepII lactose sialylation inhibited complement C3 deposition on gonococci. Strain 15253 mutants that lacked Lst or HepII lactose were significantly attenuated in mice, confirming the importance of HepII Neu5Ac in virulence. All 75 minimally passaged clinical isolates from Nanjing, China, expressed HepII lactose, evidenced by reactivity with MAb 2C7; MAb 2C7 was bactericidal against the first 62 (of 75) isolates that had been collected sequentially and were sialylated before testing. MAb 2C7 effectively attenuated 15253 vaginal colonization in mice. In conclusion, this novel sialylation site could explain the ubiquity of gonococcal HepII lactose in vivo Our findings reinforce the candidacy of the 2C7 epitope as a vaccine antigen and MAb 2C7 as an immunotherapeutic antibody., (Copyright © 2018 American Society for Microbiology.)

The β-barrel assembly machinery (BAM) is a conserved multicomponent protein complex responsible for the biogenesis of β-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., Competing Interests: The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

O-acetylation is a common modification of bacterial glycoconjugates. By modifying oligosaccharide structure and chemistry, O-acetylation has important consequences for biotic and abiotic recognition events and thus bacterial fitness in general. Previous studies of the broad-spectrum O-linked protein glycosylation in pathogenic Neisseria species (including N. gonorrhoeae and N. meningitidis) have revealed O-acetylation of some of their diverse glycoforms and identified the committed acetylase, PglI. Herein, we extend these observations by using mass spectrometry to examine a complete set of all glycan variants identified to date. Regardless of composition, all glycoforms and all sugars in the oligosaccharide are subject to acetylation in a PglI-dependent fashion with the only exception of di-N-acetyl-bacillosamine. Moreover, multiple sugars in a single oligosaccharide could be simultaneously modified. Interestingly, O-acetylation status was found to be correlated with altered chain lengths of oligosaccharides expressed in otherwise isogenic backgrounds. Models for how this unprecedented phenomenon might arise are discussed with some having potentially important implications for the membrane topology of glycan O-acetylation. Together, the findings provide better insight into how O-acetylation can both directly and indirectly govern glycoform structure and diversity., (© The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Development of rapid and multiplexed diagnostic tools is a top priority to address the current epidemic problem of sexually transmitted diseases. Here we introduce a novel nanoplasmonic biosensor for simultaneous detection of the two most common bacterial infections: Chlamydia trachomatis and Neisseria gonorrhoeae. Our plasmonic microarray is composed of gold nanohole sensor arrays that exhibit the extraordinary optical transmission (EOT), providing highly sensitive analysis in a label-free configuration. The integration in a microfluidic system and the precise immobilization of specific antibodies on the individual sensor arrays allow for selective detection and quantification of the bacteria in real-time. We achieved outstanding sensitivities for direct immunoassay of urine samples, with a limit of detection of 300 colony forming units (CFU)/mL for C. trachomatis and 1500CFU/mL for N. gonorrhoeae. The multiplexing capability of our biosensor was demonstrated by analyzing different urine samples spiked with either C. trachomatis or N. gonorrhoeae, and also containing both bacteria. We could successfully detect, identify and quantify the levels of the two bacteria in a one-step assay, without the need for DNA extraction or amplification techniques. This work opens up new possibilities for the implementation of point-of-care biosensors that enable fast, simple and efficient diagnosis of sexually transmitted infections., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Glycomics is a rapidly growing field that focuses on the structure and function of carbohydrates (glycans) in biological systems. Glycan interactions play a major role in infectious disease, at all stages of colonisation and disease progression. Neisseria meningitidis, the cause of meningococcal sepsis and meningitis, and Neisseria gonorrhoeae, which causes the sexually transmitted infection gonorrhoea, are responsible for significant morbidity and mortality worldwide. Neisseria meningitidis displays a range of surface glycosylations including capsule polysaccharide, lipooligosaccharide and O-linked glycoproteins. While N. gonorrhoeae does not have a capsule, it does express both lipooligosaccharide and O-linked glycoproteins. Neisseria gonorrhoeae also has the ability to scavenge host sialic acids, while several N. meningitidis serogroups can synthesise sialic acid. Surface expressed sialic acid is key in serum resistance and survival in the host. On the host side, the pathogenic Neisseria protein adhesins such as Opc and NHBA bind to host glycans for adherence and colonisation of host cells. Essentially, from both the bacterial and host perspective, glycan interactions are fundamental in colonisation and disease of pathogenic Neisseria. The key aspects of glycobiology of the pathogenic Neisseria are reviewed herein., (© FEMS 2017.)

Guanosine 5'-triphosphate (GTP) cyclohydrolase-I (GCYH-I) catalyzes the first step in folic acid biosynthesis in bacteria and plants, biopterin biosynthesis in mammals, and the biosynthesis of 7-deazaguanosine-modified tRNA nucleosides in bacteria and archaea. The type IB GCYH (GCYH-IB) is a prokaryotic-specific enzyme found in many pathogens. GCYH-IB is structurally distinct from the canonical type IA GCYH involved in biopterin biosynthesis in humans and animals, and thus is of interest as a potential antibacterial drug target. We report kinetic and inhibition data of Neisseria gonorrhoeae GCYH-IB and two high-resolution crystal structures of the enzyme; one in complex with the reaction intermediate analog and competitive inhibitor 8-oxoguanosine 5'-triphosphate (8-oxo-GTP), and one with a tris(hydroxymethyl)aminomethane molecule bound in the active site and mimicking another reaction intermediate. Comparison with the type IA enzyme bound to 8-oxo-GTP (guanosine 5'-triphosphate) reveals an inverted mode of binding of the inhibitor ribosyl moiety and, together with site-directed mutagenesis data, shows that the two enzymes utilize different strategies for catalysis. Notably, the inhibitor interacts with a conserved active-site Cys149, and this residue is S-nitrosylated in the structures. This is the first structural characterization of a biologically S-nitrosylated bacterial protein. Mutagenesis and biochemical analyses demonstrate that Cys149 is essential for the cyclohydrolase reaction, and S-nitrosylation maintains enzyme activity, suggesting a potential role of the S -nitrosothiol in catalysis., (© 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.)

Objectives: Resistance to the currently recommended extended-spectrum cephalosporins, which is used to treat Gonorrhea, is increasing continuously and leading to a threat of untreatable infection. It is, therefore, becoming extremely essential to search for new therapeutic strategies to control Gonorrhea. Vaccination may be considered as an effective control measure to control this disease, which is caused by Neisseria gonorrhoeae., Methods: In-silico hierarchical approach was used to help identify candidate proteins of N. gonorrhoeae that might contribute significantly in vaccine research. In contrast to the conventional vaccine research which requires at least 10-12 years, the present approach would reduce the time period drastically and help to identify Potential Universal Vaccine Candidates (PUVCs). These proteins were further analyzed for the presence of T-cell and linear B-cell epitopes, by using HLAPred and ABCpred servers respectively, in order to facilitate the identification of Multi Epitope Peptide Vaccine Constructs., Results: We have identified 23 non-host candidate proteins, using the proteomic information of four sequenced strains of N. gonorrhoeae namely FA 1090, TCDC_NG08107, NCCP11945 and MS11 and labeled them as PUVCs. Since all these identified 23 PUVCs contained both T cell and B cell epitopes, these have been further reiterated as PUVCs which could be used as promising leads for vaccine development., Conclusions: This hierarchical approach is the first comprehensive study to identify potential vaccine candidates which once utilized for vaccine development would surely serve as promising tools for effective control of Gonorrhea., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Regulation of the Neisseria gonorrhoeae pilE gene is ill-defined. In this study, post-transcriptional effects on expression were assessed. In silico analysis predicts the formation of three putative stable stem-loop structures with favourable free energies within the 5' untranslated region of the pilE message. Using quantitative reverse transcriptase PCR analyses, we show that each loop structure forms, with introduced destabilizing stem-loop mutations diminishing loop stability. Utilizing a series of pilE translational fusions, deletion of either loop 1 or loop 2 caused a significant reduction of pilE mRNA resulting in reduced expression of the reporter gene. Consequently, the formation of the loops apparently protects the pilE transcript from degradation. Putative loop 3 contains the pilE ribosomal binding site. Consequently, its formation may influence translation. Analysis of a small RNA transcriptome revealed an antisense RNA being produced upstream of the pilE promoter that is predicted to hybridize across the 5' untranslated region loops. Insertional mutants were created where the antisense RNA is not transcribed. In these mutants, pilE transcript levels are greatly diminished, with any residual message apparently not being translated. Complementation of these insertion mutants in trans with the antisense RNA gene facilitates pilE translation yielding a pilus + phenotype. Overall, this study demonstrates a complex relationship between loop-dependent transcript protection and antisense RNA in modulating pilE expression levels.

Neisseria gonorrhoeae (gonococci) and Neisseria meningitidis (meningococci) are human pathogens that cause gonorrhea and meningococcal meningitis, respectively. Both N. gonorrhoeae and N. meningitidis release a number of small peptidoglycan (PG) fragments, including proinflammatory PG monomers, although N. meningitidis releases fewer PG monomers. The PG fragments released by N. gonorrhoeae and N. meningitidis are generated in the periplasm during cell wall remodeling, and a majority of these fragments are transported into the cytoplasm by an inner membrane permease, AmpG; however, a portion of the PG fragments are released into the extracellular environment through unknown mechanisms. We previously reported that the expression of meningococcal ampG in N. gonorrhoeae reduced PG monomer release by gonococci. This finding suggested that the efficiency of AmpG-mediated PG fragment recycling regulates the amount of PG fragments released into the extracellular milieu. We determined that three AmpG residues near the C-terminal end of the protein modulate AmpG's efficiency. We also investigated the association between PG fragment recycling and release in two species of human-associated nonpathogenic Neisseria: N. sicca and N. mucosa Both N. sicca and N. mucosa release lower levels of PG fragments and are more efficient at recycling PG fragments than N. gonorrhoeae Our results suggest that N. gonorrhoeae has evolved to increase the amounts of toxic PG fragments released by reducing its PG recycling efficiency., Importance: Neisseria gonorrhoeae and Neisseria meningitidis are human pathogens that cause highly inflammatory diseases, although N. meningitidis is also frequently found as a normal member of the nasopharyngeal microbiota. Nonpathogenic Neisseria, such as N. sicca and N. mucosa, also colonize the nasopharynx without causing disease. Although all four species release peptidoglycan fragments, N. gonorrhoeae is the least efficient at recycling and releases the largest amount of proinflammatory peptidoglycan monomers, partly due to differences in the recycling permease AmpG. Studying the interplay between bacterial physiology (peptidoglycan metabolism) and pathogenesis (release of toxic monomers) leads to an increased understanding of how different bacterial species maintain asymptomatic colonization or cause disease and may contribute to efforts to mitigate disease., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Neisseria meningitidis use Type IV pili (T4P) to adhere to endothelial cells and breach the blood brain barrier, causing cause fatal meningitis. T4P are multifunctional polymers of the major pilin protein, which share a conserved hydrophobic N terminus that is a curved extended α-helix, α1, in X-ray crystal structures. Here we report a 1.44 Å crystal structure of the N. meningitidis major pilin PilE and a ∼6 Å cryo-electron microscopy reconstruction of the intact pilus, from which we built an atomic model for the filament. This structure reveals the molecular arrangement of the N-terminal α-helices in the filament core, including a melted central portion of α1 and a bridge of electron density consistent with a predicted salt bridge necessary for pilus assembly. This structure has important implications for understanding pilus biology.

Nucleic acid-based detection of gonorrhea infections typically require a two-step process involving isolation of the nucleic acid, followed by detection of the genomic target often involving polymerase chain reaction (PCR)-based approaches. In an effort to improve on current detection approaches, we have developed a unique two-step microwave-accelerated approach for rapid extraction and detection of Neisseria gonorrhoeae (gonorrhea, GC) DNA. Our approach is based on the use of highly focused microwave radiation to rapidly lyse bacterial cells, release, and subsequently fragment microbial DNA. The DNA target is then detected by a process known as microwave-accelerated metal-enhanced fluorescence (MAMEF), an ultra-sensitive direct DNA detection analytical technique. In the current study, we show that highly focused microwaves at 2.45 GHz, using 12.3-mm gold film equilateral triangles, are able to rapidly lyse both bacteria cells and fragment DNA in a time- and microwave power-dependent manner. Detection of the extracted DNA can be performed by MAMEF, without the need for DNA amplification, in less than 10 min total time or by other PCR-based approaches. Collectively, the use of a microwave-accelerated method for the release and detection of DNA represents a significant step forward toward the development of a point-of-care (POC) platform for detection of gonorrhea infections., Competing Interests: The authors can confirm that they have no financial conflicts of interest or otherwise at this time., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Multidrug and toxic compound extrusion (MATE) transporters contribute to multidrug resistance by extruding different drugs across cell membranes. The MATE transporters alternate between their extracellular and intracellular facing conformations to propel drug export, but how these structural changes occur is unclear. Here we combine site-specific cross-linking and functional studies to probe the movement of transmembrane helices in NorM from Neiserria gonorrheae (NorM-NG), a MATE transporter with known extracellular facing structure. We generated an active, cysteine-less NorM-NG and conducted pairwise cysteine mutagenesis on this variant. We found that copper phenanthroline catalyzed disulfide bond formation within five cysteine pairs and increased the electrophoretic mobility of the corresponding mutants. Furthermore, copper phenanthroline abolished the activity of the five paired cysteine mutants, suggesting that these substituted amino acids come in spatial proximity during transport, and the proximity changes are functionally indispensable. Our data also implied that the substrate-binding transmembrane helices move up to 10 Å in NorM-NG during transport and afforded distance restraints for modeling the intracellular facing transporter, thereby casting new light on the underlying mechanism., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

The RecA recombinase of Escherichia coli has not evolved to optimally promote DNA pairing and strand exchange, the key processes of recombinational DNA repair. Instead, the recombinase function of RecA protein represents an evolutionary compromise between necessary levels of recombinational DNA repair and the potentially deleterious consequences of RecA functionality. A RecA variant, RecA D112R, promotes conjugational recombination at substantially enhanced levels. However, expression of the D112R RecA protein in E. coli results in a reduction in cell growth rates. This report documents the consequences of the substantial selective pressure associated with the RecA-mediated hyperrec phenotype. With continuous growth, the deleterious effects of RecA D112R, along with the observed enhancements in conjugational recombination, are lost over the course of 70 cell generations. The suppression reflects a decline in RecA D112R expression, associated primarily with a deletion in the gene promoter or chromosomal mutations that decrease plasmid copy number. The deleterious effects of RecA D112R on cell growth can also be negated by over-expression of the RecX protein from Neisseria gonorrhoeae. The effects of the RecX proteins in vivo parallel the effects of the same proteins on RecA D112R filaments in vitro. The results indicate that the toxicity of RecA D112R is due to its persistent binding to duplex genomic DNA, creating barriers for other processes in DNA metabolism. A substantial selective pressure is generated to suppress the resulting barrier to growth.

Identification ofNeisseria gonorrhoeaeby the Bruker matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) system may be affected by "B consistency categorization." A supplementary database of 17N. gonorrhoeaemain spectra was constructed. Twelve of 64N. gonorrhoeaeidentifications were categorized with B consistency, which disappeared using the supplementary database. Database extension did not result in misidentification ofNeisseria meningitidis., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Neisseria gonorrhoeae colonizes the genitourinary track, and in these environments, especially in the female host, the bacteria are subjected to low levels of oxygen, and reactive oxygen and nitrosyl species. Here, the biochemical characterization of N. gonorrhoeae Laz is presented, as well as, the solution structure of its soluble domain determined by NMR. N. gonorrhoeae Laz is a type 1 copper protein of the azurin-family based on its spectroscopic properties and structure, with a redox potential of 277±5 mV, at pH7.0, that behaves as a monomer in solution. The globular Laz soluble domain adopts the Greek-key motif, with the copper center located at one end of the β-barrel coordinated by Gly48, His49, Cys113, His118 and Met122, in a distorted trigonal geometry. The edge of the His118 imidazole ring is water exposed, in a surface that is proposed to be involved in the interaction with its redox partners. The heterologously expressed Laz was shown to be a competent electron donor to N. gonorrhoeae cytochrome c peroxidase. This is an evidence for its involvement in the mechanism of protection against hydrogen peroxide generated by neighboring lactobacilli in the host environment., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Initially, pilE transcription in Neisseria gonorrhoeae appeared to be complicated, yet it was eventually simplified into a model where integration host factor activates a single -35/ -10 promoter. However, with the advent of high-throughput RNA sequencing, numerous small pil-specific RNAs (sense as well as antisense) have been identified at the pilE locus as well as at various pilS loci. Using a combination of in vitro transcription, site-directed mutagenesis, Northern analysis and quantitative reverse transcriptase PCR (qRT-PCR) analysis, we have identified three additional non-canonical promoter elements within the pilE gene; two are located within the midgene region (one sense and one antisense), with the third, an antisense promoter, located immediately downstream of the pilE ORF. Using strand-specific qRT-PCR analysis, an inverse correlation exists between the level of antisense expression and the amount of sense message. By their nature, promoter sequences tend to be AT-rich. In Escherichia coli, the small DNA-binding protein H-NS binds to AT-rich sequences and inhibits intragenic transcription. In N. gonorrhoeae hns mutants, pilE antisense transcription was increased twofold, with a concomitant decrease in sense transcript levels. However, most noticeably in these mutants, the absence of H-NS protein caused pilE/pilS recombination to increase dramatically when compared with WT values. Consequently, H-NS protein suppresses pilE intragenic transcription as well as antigenic variation through the pilE/pilS recombination system.

Most bacteria break down a significant portion of their cell wall peptidoglycan during each round of growth and cell division. This process generates peptidoglycan fragments of various sizes that can either be imported back into the cytoplasm for recycling or released from the cell. Released fragments have been shown to act as microbe-associated molecular patterns for the initiation of immune responses, as triggers for the initiation of mutualistic host-microbe relationships, and as signals for cell-cell communication in bacteria. Characterizing these released peptidoglycan fragments can, therefore, be considered an important step in understanding how microbes communicate with other organisms in their environments. In this chapter, we describe methods for labeling cell wall peptidoglycan, calculating the rate at which peptidoglycan is turned over, and collecting released peptidoglycan to determine the abundance and species of released fragments. Methods are described for both the separation of peptidoglycan fragments by size-exclusion chromatography and further detailed analysis by HPLC.

Membrane transporter proteins catalyze the passage of a broad range of solutes across cell membranes, allowing the uptake and efflux of crucial compounds. Because of the difficulty of expressing, purifying, and crystallizing integral membrane proteins, relatively few transporter structures have been elucidated to date. Although every membrane transporter has unique characteristics, structural and mechanistic similarities between evolutionarily diverse transporters have been identified. Here, we compare two recently reported structures of membrane proteins that act as antimicrobial efflux pumps, namely MtrF from Neisseria gonorrhoeae and YdaH from Alcanivorax borkumensis, both with each other and with the previously published structure of a sodium-dependent dicarboxylate transporter from Vibrio cholerae, VcINDY. MtrF and YdaH belong to the p-aminobenzoyl-glutamate transporter (AbgT) family and have been reported as having architectures distinct from those of all other families of transporters. However, our comparative analysis reveals a similar structural arrangement in all three proteins, with highly conserved secondary structure elements. Despite their differences in biological function, the overall "design principle" of MtrF and YdaH appears to be almost identical to that of VcINDY, with a dimeric quaternary structure, helical hairpins, and clear boundaries between the transport and scaffold domains. This observation demonstrates once more that the same secondary transporter architecture can be exploited for multiple distinct transport modes, including cotransport and antiport. Based on our comparisons, we detected conserved motifs in the substrate-binding region and predict specific residues likely to be involved in cation or substrate binding. These findings should prove useful for the future characterization of the transport mechanisms of these families of secondary active transporters.

Neisseria gonorrhoeae, the causative agent of the sexually transmitted infection gonorrhea, is not preventable by vaccination and is rapidly developing resistance to antibiotics. However, the transferrin (Tf) receptor system, composed of TbpA and TbpB, is an ideal target for novel therapeutics and vaccine development. Using a three-dimensional structure of gonococcal TbpA, we investigated two hypotheses, i.e., that loop-derived antibodies can interrupt ligand-receptor interactions in the native bacterium and that the loop 3 helix is a critical functional domain. Preliminary loop-derived antibodies, as well as optimized second-generation antibodies, demonstrated similar modest ligand-blocking effects on the gonococcal surface but different effects in Escherichia coli. Mutagenesis of loop 3 helix residues was employed, generating 11 mutants. We separately analyzed the mutants' abilities to (i) bind Tf and (ii) internalize Tf-bound iron in the absence of the coreceptor TbpB. Single residue mutations resulted in up to 60% reductions in ligand binding and up to 85% reductions in iron utilization. All strains were capable of growing on Tf as the sole iron source. Interestingly, in the presence of TbpB, only a 30% reduction in Tf-iron utilization was observed, indicating that the coreceptor can compensate for TbpA impairment. Complete deletion of the loop 3 helix of TbpA eliminated the abilities to bind Tf, internalize iron, and grow with Tf as the sole iron source. Our studies demonstrate that while the loop 3 helix is a key functional domain, its function does not exclusively rely on any single residue., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

NorM from Neisseria gonorrhoeae (NorM-NG) belongs to the multidrug and toxic compound extrusion (MATE) family of membrane-transport proteins, which can extrude cytotoxic chemicals across cell membranes and confer multidrug resistance. Here, the structure determination of NorM-NG is described, which had been hampered by low resolution (∼ 4 Å), data anisotropy and pseudo-merohedral twinning. The crystal structure was solved using molecular replacement and was corroborated by conducting a difference Fourier analysis. The NorM-NG structure displays an extracellular-facing conformation, similar to that of NorM-NG bound to a crystallization chaperone. The approaches taken to determine the NorM-NG structure and the lessons learned from this study are discussed, which may be useful for analyzing X-ray diffraction data with similar shortcomings.

Many organisms form colonies for a transient period of time to withstand environmental pressure. Bacterial biofilms are a prototypical example of such behavior. Despite significant interest across disciplines, physical mechanisms governing the formation and dissolution of bacterial colonies are still poorly understood. Starting from a kinetic description of motile and interacting cells we derive a hydrodynamic equation for their density on a surface, where most of the kinetic coefficients are estimated from experimental data for N. gonorrhoeae bacteria. We use it to describe the formation of multiple colonies with sizes consistent with experimental observations. Finally, we show how the changes in the cell-to-cell interactions lead to the dissolution of the bacterial colonies. The successful application of kinetic theory to a complex far from equilibrium system such as formation and dissolution of living bacterial colonies potentially paves the way for the physical quantification of the initial stages of biofilm formation.

Background: The sexually transmitted infection gonorrhea remains a public health concern for becoming resistant to drug treatments available. The purpose of this study was to evaluate the usefulness of the matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) to identify and cluster Neisseria gonorrhoeae. From a current monitoring in Italy, as part of the European Gonococcal Antimicrobial Surveillance Programme (EURO-GASP), 93 gonococci collected from 2007 to 2012 susceptible (44 isolates) and resistant (49 isolates) to cefixime were selected. Minimum Inhibitory Concentration (MIC) values for cefixime was assessed by Etest carried out in agreement with the manufacturer's instructions and interpreted referring to European Committee on Antimicrobial Susceptibility testing (EUCAST) clinical breakpoints criteria. Data obtained by N. gonorrhoeae multiantigen sequence typing (NG-MAST) and the dendrogram based on the concatenation of porB and tbpB genes were evaluated. MALDI-TOF MS, to reconfirm gonorrhea identification, analyzed single colonies from freshly grown isolates and applied directly on a ground-steel MALDI target plate. For the MALDI-TOF dendrogram cluster analysis, MSPs (Main Spectrum Profile) from each isolate were created acquiring 5000 shots from 10 technical replicates obtained from bacteria extraction., Results: Molecular typing by NG-MAST showed 28 sequence types (STs); G1407 was the predominant accounting for 75 gonococci. All the 93 gonococci, except one, were correctly identified at species level by MALDI-TOF MS and G1407 isolates were divided into two clusters., Conclusion: MALDI-TOF MS for a real-time detection and cluster analysis of gonorrhea is a promising tool for surveillance purposes. Moreover, additional studies are required to collect more data on the performance of MALDI-TOF MS for gonococci.

Neisseria gonorrhoeae is an obligate human pathogen and the causative agent of the sexually transmitted disease gonorrhea. The control of this disease has been compromised by the increasing proportion of infections due to antibiotic-resistant strains, which are growing at an alarming rate. N. gonorrhoeae MtrF is an integral membrane protein that belongs to the AbgT family of transporters for which no structural information is available. Here, we describe the crystal structure of MtrF, revealing a dimeric molecule with architecture distinct from all other families of transporters. MtrF is a bowl-shaped dimer with a solvent-filled basin extending from the cytoplasm to halfway across the membrane bilayer. Each subunit of the transporter contains nine transmembrane helices and two hairpins, posing a plausible pathway for substrate transport. A combination of the crystal structure and biochemical functional assays suggests that MtrF is an antibiotic efflux pump mediating bacterial resistance to sulfonamide antimetabolite drugs., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Unlabelled: Neisseria meningitidis and Neisseria gonorrhoeae modify the terminal lacto-N-neotetraose moiety of their lipooligosaccharide (LOS) with sialic acid. N. gonorrhoeae LOS sialylation blocks killing by complement, which is mediated at least in part by enhanced binding of the complement inhibitor factor H (FH). The role of LOS sialylation in resistance of N. meningitidis to serum killing is less well defined. Sialylation in each species is catalyzed by the enzyme LOS α-2,3-sialyltransferase (Lst). Previous studies have shown increased Lst activity in N. gonorrhoeae compared to N. meningitidis due to an ~5-fold increase in lst transcription. Using isogenic N. gonorrhoeae strains engineered to express gonococcal lst from either the N. gonorrhoeae or N. meningitidis lst promoter, we show that decreased expression of lst (driven by the N. meningitidis promoter) reduced LOS sialylation as determined by less incorporation of tritium-labeled cytidine monophospho-N-acetylneuraminic acid (CMP-NANA; the donor molecule for sialic acid). Diminished LOS sialylation resulted in reduced rates of FH binding and increased pathway activation compared to N. gonorrhoeae promoter-driven lst expression. The N. meningitidis lst promoter generated sufficient Lst to sialylate N. gonorrhoeae LOS in vivo, and the level of sialylation after 24 h in the mouse genital tract was sufficient to mediate resistance to human serum ex vivo. Despite demonstrable LOS sialylation in vivo, gonococci harboring the N. meningitidis lst promoter were outcompeted by those with the N. gonorrhoeae lst promoter during coinfection of the vaginal tract of estradiol-treated mice. These data highlight the importance of high lst expression levels for gonococcal pathogenesis., Importance: Neisseria gonorrhoeae has become resistant to nearly every therapeutic antibiotic used and is listed as an "urgent threat" by the Centers for Disease Control and Prevention. Novel therapies are needed to combat drug-resistant N. gonorrhoeae. Gonococci express an α-2,3-sialyltransferase (Lst) that can scavenge sialic acid from the host and use it to modify lipooligosaccharide (LOS). Sialylation of gonococcal LOS converts serum-sensitive strains to serum resistance, decreases antibody binding, and combats killing by neutrophils and antimicrobial peptides. Mutant N. gonorrhoeae that lack Lst (cannot sialylate LOS) are attenuated in a mouse model. Lst expression levels differ among N. gonorrhoeae strains, and N. gonorrhoeae typically expresses more Lst than Neisseria meningitidis. Here we examined the significance of differential lst expression levels and determined that the level of LOS sialylation is critical to the ability of N. gonorrhoeae to combat the immune system and survive in an animal model. LOS sialylation may be an ideal target for novel therapies., (Copyright © 2015 Lewis et al.)

Type IV pili (T4P) and T2SS (Type II Secretion System) pseudopili are filaments extending beyond microbial surfaces, comprising homologous subunits called "pilins." In this paper, we presented a new approach to predict pseudo atomic models of pili combining ambiguous symmetric constraints with sparse distance information obtained from experiments and based neither on electronic microscope (EM) maps nor on accurate a priori symmetric details. The approach was validated by the reconstruction of the gonococcal (GC) pilus from Neisseria gonorrhoeae, the type IVb toxin-coregulated pilus (TCP) from Vibrio cholerae, and pseudopilus of the pullulanase T2SS (the PulG pilus) from Klebsiella oxytoca. In addition, analyses of computational errors showed that subunits should be treated cautiously, as they are slightly flexible and not strictly rigid bodies. A global sampling in a wider range was also implemented and implied that a pilus might have more than one but fewer than many possible intact conformations.

The structure and dynamics of Opa proteins, which we report herein, are responsible for the receptor-mediated engulfment of Neisseria gonorrheae or Neisseria meningitidis by human cells and can offer deep understanding into the molecular recognition of pathogen-host receptor interactions. Such interactions are vital to understanding bacterial pathogenesis as well as the mechanism of foreign body entry to a human cell, which may provide insights for the development of targeted pharmaceutical delivery systems. The size and dynamics of the extracellular loops of Opa60 required a hybrid refinement approach wherein membrane and distance restraints were used to generate an initial NMR structural ensemble, which was then further refined using molecular dynamics in a DMPC bilayer. The resulting ensemble revealed that the extracellular loops, which bind host receptors, occupy compact conformations, interact with each other weakly, and are dynamic on the nanosecond time scale. We predict that this conformational sampling is critical for enabling diverse Opa loop sequences to engage a common set of receptors.

The multidrug and toxic compound extrusion transporters extrude a wide variety of substrates out of both mammalian and bacterial cells via the electrochemical gradient of protons and cations across the membrane. The substrates transported by these proteins include toxic metabolites and antimicrobial drugs. These proteins contribute to multidrug resistance in both mammalian and bacterial cells and are therefore extremely important from a biomedical perspective. Although specific residues of the protein are known to be responsible for the extrusion of solutes, mechanistic details and indeed structures of all the conformational states remain elusive. Here, we report the first, to our knowledge, simulation study of the recently resolved x-ray structure of the multidrug and toxic compound extrusion transporter, NorM from Neisseria gonorrhoeae (NorM_NG). Multiple, atomistic simulations of the unbound and bound forms of NorM in a phospholipid lipid bilayer allow us to identify the nature of the drug-protein/ion-protein interactions, and secondly determine how these interactions contribute to the conformational rearrangements of the protein. In particular, we identify the molecular rearrangements that occur to enable the Na(+) ion to enter the cation-binding cavity even in the presence of a bound drug molecule. These include side chain flipping of a key residue, GLU-261 from pointing toward the central cavity to pointing toward the cation binding side when bound to a Na(+) ion. Our simulations also provide support for cation binding in the drug-bound and apo states of NorM_NG., (Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Unlabelled: Phosphoethanolamine (PEA) on Neisseria gonorrhoeae lipid A influences gonococcal inflammatory signaling and susceptibility to innate host defenses in in vitro models. Here, we evaluated the role of PEA-decorated gonococcal lipid A in competitive infections in female mice and in male volunteers. We inoculated mice and men with mixtures of wild-type N. gonorrhoeae and an isogenic mutant that lacks the PEA transferase, LptA. LptA production conferred a marked survival advantage for wild-type gonococci in the murine female genital tract and in the human male urethra. Our studies translate results from test tube to animal model and into the human host and demonstrate the utility of the mouse model for studies of virulence factors of the human-specific pathogen N. gonorrhoeae that interact with non-host-restricted elements of innate immunity. These results validate the use of gonococcal LptA as a potential target for development of novel immunoprophylactic strategies or antimicrobial treatments., Importance: Gonorrhea is one of the most common bacterial sexually transmitted infections, and increasing antibiotic resistance threatens the use of currently available antimicrobial therapies. In this work, encompassing in vitro studies and in vivo studies of animal and human models of experimental genital tract infection, we document the importance of lipid A's structure, mediated by a single bacterial enzyme, LptA, in enhancing the fitness of Neisseria gonorrhoeae. The results of these studies suggest that novel agents targeting LptA may offer urgently needed prevention or treatment strategies for gonorrhea.

β-barrel membrane proteins are essential for nutrient import, signalling, motility and survival. In Gram-negative bacteria, the β-barrel assembly machinery (BAM) complex is responsible for the biogenesis of β-barrel membrane proteins, with homologous complexes found in mitochondria and chloroplasts. Here we describe the structure of BamA, the central and essential component of the BAM complex, from two species of bacteria: Neisseria gonorrhoeae and Haemophilus ducreyi. BamA consists of a large periplasmic domain attached to a 16-strand transmembrane β-barrel domain. Three structural features shed light on the mechanism by which BamA catalyses β-barrel assembly. First, the interior cavity is accessible in one BamA structure and conformationally closed in the other. Second, an exterior rim of the β-barrel has a distinctly narrowed hydrophobic surface, locally destabilizing the outer membrane. And third, the β-barrel can undergo lateral opening, suggesting a route from the interior cavity in BamA into the outer membrane.

Membrane proteins in detergent micelles are large and dynamic complexes that present challenges for solution NMR investigations such as spectral overlap and line broadening. In this study, multiple methods are introduced to facilitate resonance assignment of β-barrel membrane proteins using Opa60 from Neisseria gonorrhoeae as a model system. Opa60 is an eight-stranded β-barrel with long extracellular loops (∼63% of the protein) that engage host receptors and induce engulfment of the bacterium. The NMR spectra of Opa60 in detergent micelles exhibits significant spectral overlap and resonances corresponding to the loop regions had variable line widths, which interfered with a complete assignment of the protein. To assign the β-barrel residues, trypsin cleavage was used to remove much of the extracellular loops while preserving the detergent solubilized β-barrel. The removal of the loop resonances significantly improved the assignment of the Opa60 β-barrel region (97% of the resonances corresponding to the β-barrel and periplasmic turns were assigned). For the loop resonance assignments, two strategies were implemented; modulating temperature and synthetic peptides. Lowering the temperature broadened many peaks beyond detection and simplified the spectra to only the most dynamic regions of the loops facilitating 27 loop resonances to be assigned. To further assign functionally important and unstructured regions of the extracellular loops, a synthetic 20 amino acid peptide was synthesized and had nearly complete spectral overlap with the full-length protein allowing 17 loop resonances to be assigned. Collectively, these strategies are effective tools that may accelerate solution NMR structure determination of β-barrel membrane proteins., (© 2013 The Protein Society.)

Objectives: Identification of genetic targets specific to Neisseria gonorrhoeae for use in molecular detection methods has been a challenge. The porA pseudogene in N gonorrhoeae has been commonly used but recently gonococcal isolates giving a negative result in these PCRs have been reported. Here we describe the characterisation of two such gonococcal isolates received by the reference service at the Health Protection Agency, London, England., Methods: Phenotypic characterisation was achieved using conventional biochemical and immunological tests, matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF-MS), antimicrobial susceptibility testing, serovar determination and detection of meningococcal PorA using monoclonal antibody 4BG4-E7. Genetic species confirmation was determined using commercial and in house PCRs and 16S rRNA gene sequencing. Molecular typing using the N gonorrhoeae multi-antigen sequence typing (NG-MAST) and multilocus sequence typing (MLST) was performed. The DNA sequence of the full-length gonococcal porA pseudogene was determined and compared with published sequences., Results: Both isolates were confirmed, biochemically and immunologically as N gonorrhoeae, but repeatedly gave negative results with two in house real-time PCR assays for the porA pseudogene. Further characterisation of these isolates identified the presence of a meningococcal porA sequence and showed these isolates belong to serovar Bropyst, and to NG-MAST sequence type (ST) 5967 and MLST ST1901., Conclusions: Gonococcal isolates that give false negative results with porA pseudogene PCR assays have now been identified in four countries, three of which are in Europe, and do not appear clonal. This report highlights the genetic diversity of N gonorrhoeae, which remains a challenge for the molecular detection methods.

β-Barrel proteins are present only in the outer membranes of Gram-negative bacteria, chloroplasts and mitochondria. Fungal mitochondria were shown to readily import and assemble bacterial β-barrel proteins, but human mitochondria exhibit certain selectivity. Whereas enterobacterial β-barrel proteins are not imported, neisserial ones are. Of those, solely neisserial Omp85 is integrated into the outer membrane of mitochondria. In this study, we wanted to identify the signal that targets neisserial β-barrel proteins to mitochondria. We exchanged parts of neisserial Omp85 and PorB with their Escherichia coli homologues BamA and OmpC. For PorB, we could show that its C-terminal quarter can direct OmpC to mitochondria. In the case of Omp85, we could identify several amino acids of the C-terminal β-sorting signal as crucial for mitochondrial targeting. Additionally, we found that at least two POTRA (polypeptide-transport associated) domains and not only the β-sorting signal of Omp85 are needed for its membrane integration and function in human mitochondria. We conclude that the signal that directs neisserial β-barrel proteins to mitochondria is not conserved between these proteins. Furthermore, a linear mitochondrial targeting signal probably does not exist. It is possible that the secondary structure of β-barrel proteins plays a role in directing these proteins to mitochondria.

The bacterial human pathogen Neisseria gonorrhoeae expresses a general O-linked protein glycosylation (Pgl) system known to target at least 12 membrane-associated proteins. To facilitate a better understanding of the mechanisms, significance and function of this glycosylation system, we sought to further delineate the target proteome of the Pgl system. To this end, we employed immunoaffinity enrichment of glycoproteins using a monoclonal antibody against the glycan moiety. Enzymatically generated peptides were subsequently analyzed by MS to identify glycopeptides and glycosylation sites. In this way, we increase the total number of known glycoproteins in N. gonorrhoeae to 19. These new glycoproteins are involved in a wide variety of extracytoplasmic functions. By employing collision fragmentation, we mapped nine new glycosylation sites, all of which were serine. No target sequon was readily apparent, although attachment sites were most often localized with regions of low sequence complexity. Moreover, we found that 5 of the proteins were modified with more than one glycan. This work thus confirms and extends earlier observations on the structural features of Neisseria glycoproteins.

Neisseria gonorrhoeae is an obligate pathogen that hijacks iron from the human iron transport protein, holo-transferrin (Fe(2)-Tf), by expressing TonB-dependent outer membrane receptor proteins, TbpA and TbpB. Homologous to other TonB-dependent outer membrane transporters, TbpA is thought to consist of a β-barrel with an N-terminal plug domain. Previous reports by our laboratories show that the sequence EIEYE in the plug domain is highly conserved among various bacterial species that express TbpA and plays a crucial role in iron utilization for gonococci. We hypothesize that this highly conserved EIEYE sequence in the TbpA plug, rich in hard oxygen donor groups, binds with Fe(3+) through the transport process across the outer membrane through the β-barrel. Sequestration of Fe(3+) by the TbpA-plug supports the paradigm that the ferric iron must always remain chelated and controlled throughout the transport process. In order to test this hypothesis here we describe the ability of both the recombinant wild-type plug, and three small peptides that encompass the sequence EIEYE of the plug, to bind Fe(3+). This is the first report of the expression/isolation of the recombinant wild-type TbpA plug. Although CD and SUPREX spectroscopies suggest that a non-native structure is observed for the recombinant plug, fluorescence quenching titrations indicate that the wild-type recombinant TbpA plug binds Fe (3+) with a conditional log K(d) = 7 at pH 7.5, with no evidence of binding at pH 6.3. A recombinant TbpA plug with mutated sequence (NEIEYEN → NEIAAAN) shows no evidence of Fe(3+) binding under our experimental set up. Interestingly, in silico modeling with the wild-type plug also predicts a flexible loop structure for the EIEYE sequence under native conditions which once again supports the Fe(3+) binding hypothesis. These in vitro observations are consistent with the hypothesis that the EIEYE sequence in the wild-type TbpA plug binds Fe(3+) during the outer membrane transport process in vivo., (This journal is © The Royal Society of Chemistry 2012)

ATP-binding cassette (ABC) transporters are integral membrane proteins that carry a variety of substrates across biological membranes at the expense of ATP. The here considered prokaryotic canonical importers consist of three entities: an extracellular solute receptor, two membrane-intrinsic proteins forming a translocation pathway, and two cytoplasmic ATP-binding subunits. The ngo0372-74 and ngo2011-14 gene clusters from the human pathogen Neisseria gonorrhoeae were predicted by sequence homology as ABC transporters for the uptake of cystine and cysteine, respectively, and chosen for structural characterization. The structure of the receptor component Ngo0372 was obtained in a ligand-free "open" conformation and in a "closed" conformation when co-crystallized with L-cystine. Our data provide the first structural information of an L-cystine ABC transporter. Dissociation constants of 21 and 33 nM for L-cystine and L-selenocystine, respectively, were determined by isothermal titration calorimetry. In contrast, L-cystathionine and L-djenkolic acid are weak binders, while no binding was detectable for S-methyl-L-cysteine. Mutational analysis of two residues from the binding pocket, Trp97 and Tyr59, revealed that the latter is crucial for L-cystine binding. The structure of the Ngo2014 receptor was obtained in closed conformation in complex with co-purified L-cysteine. The protein binds L-cysteine with a K(d) of 26 nM. Comparison of the structures of both receptors and analysis of the ligand binding sites shed light on the mode of ligand recognition and provides insight into the tight binding of both substrates. Moreover, since L-cystine limitation leads to reduction in virulence of N. gonorrhoeae, Ngo0372 might be suited as target for an antimicrobial vaccine., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

As a result of a frameshift mutation, the hsdS locus of the NgoAV type IC restriction and modification (RM) system comprises two genes, hsdS(NgoAV1) and hsdS(NgoAV2). The specificity subunit, HsdS(NgoAV), the product of the hsdS(NgoAV1) gene, is a naturally truncated form of an archetypal specificity subunit (208 N-terminal amino acids instead of 410). The presence of a homonucleotide tract of seven guanines (poly[G]) at the 3' end of the hsdS(NgoAV1) gene makes the NgoAV system a strong candidate for phase variation, i.e., stochastic addition or reduction in the guanine number. We have constructed mutants with 6 guanines instead of 7 and demonstrated that the deletion of a single nucleotide within the 3' end of the hsdS(NgoAV1) gene restored the fusion between the hsdS(NgoAV1) and hsdS(NgoAV2) genes. We have demonstrated that such a contraction of the homonucleotide tract may occur in vivo: in a Neisseria gonorrhoeae population, a minor subpopulation of cells appeared to have only 6 guanines at the 3' end of the hsdS(NgoAV1) gene. Escherichia coli cells carrying the fused gene and expressing the NgoAVΔ RM system were able to restrict λ phage at a level comparable to that for the wild-type NgoAV system. NgoAV recognizes the quasipalindromic interrupted sequence 5'-GCA(N(8))TGC-3' and methylates both strands. NgoAVΔ recognizes DNA sequences 5'-GCA(N(7))GTCA-3' and 5'-GCA(N(7))CTCA-3', although the latter sequence is methylated only on the complementary strand within the 5'-CTCA-3' region of the second recognition target sequence.

Two human-specific neisserial pathogens, Neisseria gonorrhoeae and Neisseria meningitidis, require the expression of type IV pili (tfp) for initial attachment to the host during infection. However, the mechanisms controlling the assembly and functionality of tfp are poorly understood. It is known that the gonococcal pilE gene, encoding the major subunit, is positively regulated by IHF, a multifunctional DNA binding protein. A neisserial specific repetitive DNA sequence, termed the Correia repeat-enclosed element (CREE) is situated upstream of three pil loci: pilHIJKX (pilH-X), pilGD, and pilF. CREEs have been shown to contain strong promoters, and some CREE variants contain a functional IHF binding site. CREEs might therefore be involved in the regulation of tfp biogenesis in pathogenic Neisseria. Site-directed and deletion mutagenesis on promoter::cat reporter constructs demonstrated that transcription of pilH-X and pilGD is from a σ(70) promoter and is independent of the CREE. The insertion of a CREE in the pilF promoter region in N. meningitidis generated a functional σ(70) promoter. However, there is also a functional promoter at this position in N. gonorrhoeae, where there is no CREE. These results suggest CREE insertion in these three pil loci does not influence transcription and that IHF does not coordinately regulate tfp biogenesis.