Epithelial Cell NOD1/IRGM Recruits STX17 to Neisseria gonorrhoeae-Containing Endosomes to Initiate Lysosomal Degradation.

Neisseria gonorrhoeae establishes tight interactions with mucosal epithelia through activity of its type IV pilus, while pilus retraction forces activate autophagic responses toward invading gonococci. Here we studied pilus-independent epithelial cell responses and showed that pilus-negative gonococci residing in early and late endosomes are detected and targeted by nucleotide-binding oligomerization domain 1 (NOD1). NOD1 subsequently forms a complex with immunity-related guanosine triphosphatase M (IRGM) and autophagy-related 16-like 1 (ATG16L1) to activate autophagy and recruit microtubule-associated protein light chain 3 (LC3) to the intracellular bacteria. IRGM furthermore directly recruits syntaxin 17 (STX17), which is able to form tethering complexes with the lysosome. Importantly, IRGM-STX17 interactions are enhanced by LC3 but were still observed at lower levels in an LC3 knockout cell line. These findings demonstrate key roles for NOD1 and IRGM in the sensing of intracellular N gonorrhoeae and subsequent directing of the bacterium to the lysosome for degradation.

Moenomycin is broadly active against multidrug-resistant Neisseria gonorrhoeae and clears an infection from a murine vaginal tract infection model.

OBJECTIVES: Ceftriaxone therapy for gonorrhoea has become under increasing pressure due to waning susceptibility levels and emergence of high-level resistant strains such as the FC428 clone. Moenomycin was recently identified to display potent anti-gonococcal activity against some reference strains. Therefore, the aim of this study was to investigate moenomycin in vitro and in vivo antimicrobial activity. METHODS: Moenomycin in vitro antimicrobial activity was investigated against 575 clinical isolates, including strains associated with the FC428 clone, using the agar dilution method. Moenomycin in vivo activity was investigated in a mouse vaginal tract gonococcal infection model. RESULTS: The moenomycin MIC range for the strain collection was 0.004-0.06 mg/L, with a MIC50 of 0.016 mg/L and a MIC90 of 0.03 mg/L. The correlation between moenomycin and ceftriaxone susceptibility levels was poor (R = 0.13), while the fractional inhibitory concentration index (FICI) resulted in indifference for all tested strains. Therefore, development of cross-resistance between moenomycin and ceftriaxone is unlikely for N. gonorrhoeae. Determination of the moenomycin mode of activity against N. gonorrhoeae by time-kill assays showed that moenomycin is bactericidal, with over 104-fold inactivation observed after 4 h exposure. Finally, an intramuscular moenomycin dose of 10 mg/kg given on 2 consecutive days was able to clear a gonococcal infection in a mouse vaginal tract infection model within 1-3 days after the second dose, which was significantly faster than for mice treated with the vehicle control (P < 0.0001). CONCLUSIONS: Moenomycin displays potent in vitro and in vivo antimicrobial activity against N. gonorrhoeae, warranting further exploration as alternative therapy.

Cyclic-di-GMP stimulates keratinocyte innate immune responses and attenuates methicillin-resistant Staphylococcus aureus colonization in a murine skin wound infection model.

BACKGROUND: Staphylococcus aureus is a leading cause for morbidity and mortality associated with skin and burn wound infections. Therapeutic options for methicillin-resistant S. aureus (MRSA) have dwindled and therefore alternative treatments are urgently needed. In this study, the immuno-stimulating and anti-MRSA effects of cyclic di-guanosine monophosphate (c-di-GMP), a uniquely bacterial second messenger and immuno-modulator, were investigated in HaCaT human epidermal keratinocytes and a murine skin wound infection model. RESULTS: Stimulation of HaCaT cells with 125 µM c-di-GMP for 12 h prior to MRSA challenge resulted in a 20-fold reduction in bacterial colonization compared with untreated control cells, which was not the result of a direct c-di-GMP toxic effect, since bacterial viability was not affected by this dose in the absence of HaCaT cells. C-di-GMP-stimulated or MRSA-challenged HaCaT cells displayed enhanced secretion of the antimicrobial peptides human ß-defensin 1 (hBD-1), hBD-2, hBD-3 and LL-37, but for hBD1 and LL-37 the responses were additive in a c-di-GMP-dose-dependent manner. Secretion of the chemokines CXCL1 and CXCL8 was also elevated after stimulation of HaCaT cells with lower c-di-GMP doses and peaked at a dose of 5 µM. Finally, pre-treatment of mice with a 200 nmol dose of c-di-GMP 24 h before a challenge with MRSA in skin wound infection model resulted in a major reduction (up to 1,100-fold by day 2) in bacterial CFU counts recovered from challenged skin tissue sections compared PBS-treated control animals. Tissue sections displayed inflammatory cell infiltration and enhanced neutrophil influx in the c-di-GMP pre-treated animals, which might account for the reduced ability of MRSA to colonize c-di-GMP pre-treated mice. CONCLUSIONS: These results demonstrate that c-di-GMP is a potent immuno-modulator that can stimulate anti-MRSA immune responses in vivo and might therefore be a suitable alternative prophylactic or therapeutic agent for MRSA skin or burn wound infections.

Mycoplasma hominis bloodstream infection and persistent pneumonia in a neurosurgery patient: a case report.

BACKGROUND: Mycoplasma hominis is typically associated with a urogenital tract infection, while its association with bacteremia and pneumonia is rare and therefore easily overlooked. Here we report a M. hominis bloodstream infection and pneumonia in a surgical patient. CASE PRESENTATION: A 56-year-old male with symptoms of pneumonia underwent microsurgery and decompressive craniectomy after a left basal ganglia hemorrhage. The patient recovered well from surgery, but pulmonary symptoms progressively worsened, with antimicrobial therapies seemingly ineffective. Culturing of bilateral blood samples resulted in pin-point-sized colonies on blood agar plates, which were subsequently identified as M. hominis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Furthermore, sequencing of bronchoalveolar lavage samples also identified M. hominis as the main pathogen responsible for the pulmonary symptoms. The M. hominis strain was ciprofloxacin resistant, but susceptible to doxycycline and moxifloxacin. Doxycycline and moxifloxacin were subsequently used in a successful combination therapy that finally alleviated the patient's fever and resulted in absorption of pleural effusion. At 1-month follow-up, following complaints of dysuria, a prostate abscess containing M. hominis was detected as the likely primary source of infection. The abscess was successfully drained and treated with doxycycline. CONCLUSIONS: Mycoplasma hominis should be considered as a source of bloodstream infections and pneumonia, particularly when the response to standard antimicrobial therapy is limited. In this case, effective antimicrobial therapy was only commenced after identification of M. hominis and antimicrobial susceptibility testing.

Gonococcal Adaptation to Palmitic Acid Through farAB Expression and FadD Activity Mutations Increases In Vivo Fitness in a Murine Genital Tract Infection Model.

Neisseria gonorrhoeae is a bacterial pathogen that colonizes mucosal epithelia that are rich in antimicrobial molecules such as long-chain fatty acids. Here we studied the mechanisms involved in palmitic acid resistance and their impact on in vivo biological fitness in a murine genital tract infection model. A stable palmitic acid-resistant derivative was obtained by serial passage with incremental palmitic acid concentrations. This derivative outcompeted its parent strain for colonization and survival in the murine infection model. Subsequent whole-genome sequencing resulted in the identification of the 3 resistance-related SNPs ihfAC5T, fadDC772T, and farAG-52T (promoter) that were verified for resistance against palmitic acid. Subsequent characterization of the associated resistance determinants showed that ihfAC5T and farAG-52T induced gene expression of the FarAB efflux pump, whereas fadDC772T increased the maximum enzyme activity of the FadD long-chain fatty acid-coenzyme A ligase. Our results highlight the mechanisms involved in gonococcal adaptation to the murine host environment.

Role of Base Excision Repair in Listeria monocytogenes DNA Stress Survival During Infections.

BACKGROUND: Base excision repair (BER), consisting mostly of lesion-specific DNA glycosylases and apurinic/apyrimidinic (AP) endonucleases, is one of the most important DNA repair mechanisms for repair of single nucleobase lesions generated by reactive oxygen and nitrogen species as part of an immune response against bacterial infections. However, few studies have addressed the contribution of BER to bacterial virulence and Listeria monocytogenes BER has thus far remained completely uncharacterized. METHODS: Analysis of the L. monocytogenes EGDe genome identified 7 DNA glycosylases (MutM, MutY, Nth, Tag, Mpg, Ung, and Ung2) and 2 apurinic/apyrimidinic endonucleases (Xth and Nfo) as part of BER. Markerless in-frame deletion mutants were generated for all 9 genes, and mutants were tested for DNA damage survival, mutagenesis, and the ability to colonize a mouse model of infection. RESULTS: Distinct lesion-specific phenotypes were identified for all deletion mutants. Importantly, the Δnth, ΔmutY, and Δnfo mutants were significantly attenuated for virulence in the mouse model and showed much lower colonization of the liver and spleen or were unable to compete with the wild-type strain during in vivo competition assays. CONCLUSIONS: Our results highlight the importance of BER for L. monocytogenes virulence and survival of DNA-damaging insults during host colonization.

High percentage of the ceftriaxone-resistant Neisseria gonorrhoeae FC428 clone among isolates from a single hospital in Hangzhou, China.

OBJECTIVES: Ceftriaxone is currently the last-remaining empirical antimicrobial therapy for treatment of gonorrhoea. However, the high-level ceftriaxone-resistant gonococcal FC428 clone has shown transmission in China in recent years. Therefore, the aim of this study was to analyse ceftriaxone resistance among a collection of recent clinical isolates, with a specific focus on prevalence of the FC428 clone. METHODS: A total of 70 consecutive gonococcal isolates were collected between May and October 2019 from a single hospital in Hangzhou, China, and analysed for antimicrobial susceptibility by the agar dilution method. STs were determined by PCR and sequences and isolates related to the FC428 clone were further characterized by WGS and phylogenetic analysis. RESULTS: Ceftriaxone resistance (MIC >0.125 mg/L) was observed in 21 (30%) isolates, while 14 (20%) isolates displayed a ceftriaxone MIC of 0.125 mg/L. Importantly, seven (10%) isolates were related to the gonococcal FC428 clone based on the presence of mosaic penA allele 60.001, displaying identical or closely related STs, and phylogenetic analysis after WGS. These seven isolates displayed high-level ceftriaxone resistance (MIC = 1 mg/L) and all associated gonorrhoea cases resulted in treatment failure because oral cephalosporins were initially prescribed. Subsequent re-treatment with a higher dose (2 g) of IV ceftriaxone appeared to be successful because all patients returning for test-of-cure became culture-negative. CONCLUSIONS: Here, we report a high percentage of the internationally spreading gonococcal FC428 clone among clinical isolates from a single hospital in Hangzhou, China. A high dose of ceftriaxone is currently the only recommended and effective therapy.

Sustained transmission of the ceftriaxone-resistant Neisseria gonorrhoeae FC428 clone in China.

BACKGROUND: Ceftriaxone resistance in Neisseria gonorrhoeae has become an imminent threat to effective control of gonorrhoea globally. In recent years, the ceftriaxone-resistant FC428 clone has shown international dissemination. After our first report of the FC428 clone in China in 2016, we now describe another six cases of FC428-related ceftriaxone-resistant N. gonorrhoeae isolates from 2017 and 2018. OBJECTIVES: To identify the phenotypic and molecular characteristics of newly reported ceftriaxone-resistant isolates in China and to investigate the relationship between these isolates and FC428 clones reported globally. METHODS: Antimicrobial susceptibility to ceftriaxone, cefixime, azithromycin, spectinomycin, penicillin, ciprofloxacin and tetracycline was determined by the agar dilution method. N. gonorrhoeae multi-antigen sequence typing (NG-MAST), MLST and N. gonorrhoeae sequence typing for antimicrobial resistance (NG-STAR) were performed for genotyping and SNPs extracted from whole-genome sequences were used for phylogenetic analysis. RESULTS: All isolates were resistant to ceftriaxone, cefixime, penicillin, tetracycline and ciprofloxacin, but were susceptible to azithromycin and spectinomycin. NG-MAST, MLST and NG-STAR genotyping showed that all isolates shared identical or similar STs (<10 bp difference) to FC428 (NG-MAST ST3435, MLST ST1903, NG-STAR ST233) and contained the same mosaic penA allele 60.001. Phylogenetic analysis showed the Chinese isolates spreading in the whole phylogenetic tree and fully mixed with other international isolates. Half of the Chinese isolates were more closely related (<100 SNPs) to Japanese isolates than other international isolates. CONCLUSIONS: The newly reported cases in China were related to the internationally spreading FC428 clone. These isolates might have played a central role in international transmission of the FC428 clone. High ceftriaxone doses (1-2 g) still provide effective therapy.

Human Papillomavirus 11 Early Protein E6 Activates Autophagy by Repressing AKT/mTOR and Erk/mTOR.

Low-risk human papillomaviruses (LR-HPVs) are the causative agents of genital warts, which are a widespread sexually transmitted disease. How LR-HPVs affect autophagy and the specific proteins involved are unknown. In the current study, we investigated the impact of LR-HPV11 early protein 6 (E6) on the activity of the autophagy pathway. We transfected an HPV11 E6 (11E6) plasmid into HaCaT cells, H8 cells, and NHEK cells and established a stable cell line expressing the HPV11 E6 protein. The differences in autophagy activity and upstream regulatory pathways compared with those in the parent cell lines were investigated using a Western blot analysis of the total and phosphorylated protein levels and confocal microscopy of immunostained cells and cells transfected with an mCherry-green fluorescent protein-LC3 expression plasmid. We used short hairpin RNA (shRNA) to knock down 11E6 and showed that these effects require continued 11E6 expression. Compared with its expression in the control cells, the expression of HPV11 E6 in the cells activated the autophagy pathway. The increased autophagy activity was the result of the decreased phosphorylation levels of the canonical autophagy repressor mammalian target of rapamycin (mTOR) at its Ser2448 position (the mTOR complex 1 [mTORC1] phosphorylation site) and decreased AKT and Erk phosphorylation. Therefore, these results indicate that HPV11 E6 activates autophagy through the AKT/mTOR and Erk/mTOR pathways. Our findings provide novel insight into the relationship between LR-HPV infections and autophagy and could help elucidate the pathogenic mechanisms of LR-HPV.IMPORTANCE We transfected an HPV11 E6 plasmid into HaCaT cells, H8 cells, and NHEK cells and established a stable cell line expressing the HPV11 E6 protein. Then, we confirmed that HPV11 E6 induces autophagy by suppressing the AKT/mTOR and Erk/mTOR pathways. In contrast to the high-risk HPV E6 genes, HPV11 E6 did not affect the expression of p53. To the best of our knowledge, this study represents the first direct in-depth investigation of the relationship between the LR-HPV E6 gene and autophagy, which may help to reveal the pathogenesis of LR-HPV infection.

A Subpopulation of Intracellular Neisseria gonorrhoeae Escapes Autophagy-Mediated Killing Inside Epithelial Cells.

The bacterial pathogen Neisseria gonorrhoeae is able to transmigrate across the mucosal epithelia following the intracellular route and cause disseminated infections. It is currently unknown whether the autophagy pathway is able target intracellular N. gonorrhoeae for destruction in autolysosomes or whether this bacterium is able to escape autophagy-mediated killing. In this study, we demonstrate that during the early stage of epithelial cell invasion, N. gonorrhoeae is targeted by the autophagy pathway and sequestered into double-membrane autophagosomes that subsequently fuse with lysosomes for destruction. However, a subpopulation of the intracellular gonococci is able to escape early autophagy-mediated killing. N. gonorrhoeae is subsequently able to inhibit this pathway, allowing intracellular survival and exocytosis. During this stage, N. gonorrhoeae activates the autophagy repressor mammalian target of rapamycin complex 1 and inhibits autophagosome maturation and lysosome fusion. Thus, our results provide novel insight into the interactions between N. gonorrhoeae and the autophagy pathway during invasion and transcytosis of epithelial cells.

Neisseria gonorrhoeae 23S rRNA A2059G mutation is the only determinant necessary for high-level azithromycin resistance and improves in vivo biological fitness.

Objectives: The global emergence of Neisseria gonorrhoeae isolates displaying high-level azithromycin resistance is a major concern for the currently recommended azithromycin/ceftriaxone dual therapy. N. gonorrhoeae high-level azithromycin resistance has been associated with an A2059G mutation in 23S rRNA. Here we investigated the specific contribution of this 23S rRNA A2059G mutation to high-level azithromycin resistance and its impact on biological fitness. Methods: A2059G/G2059A alleles were specifically cloned into all four genomic copies of 23S rDNA of an azithromycin-susceptible isolate and a high-level azithromycin-resistant isolate. WT and mutant strains were subsequently investigated for azithromycin susceptibility using the agar dilution method. In addition, their biological fitness was studied by comparative liquid growth in the presence of hydrophobic and amphipathic compounds, by competition assays in a mouse vaginal tract infection model and by competition assays for invasion and intracellular survival. Results: Azithromycin susceptibility analyses showed that the 23S rRNA A2059G mutation is the only genetic determinant required for N. gonorrhoeae to display the high-level azithromycin resistance phenotype. Further analysis of biological fitness showed that strains containing 2059G outcompeted isogenic strains containing 2059A for colonization in the mouse vaginal tract infection model and for invasion of HeLa cervical epithelial cells. Furthermore, the A2059G mutation enhanced growth in the presence of lithocholic acid or Triton X-100. Conclusions: Our findings that the 23S rRNA A2059G mutation is sufficient for high-level azithromycin resistance and that this mutation generally enhanced the biological fitness of N. gonorrhoeae have important implications for the currently recommended treatment policies and antimicrobial stewardship programmes.

Increasing prevalence of Neisseria gonorrhoeae with decreased susceptibility to ceftriaxone and resistance to azithromycin in Hangzhou, China (2015-17).

Objectives: Development of resistance in Neisseria gonorrhoeae to ceftriaxone monotherapy or ceftriaxone plus azithromycin dual therapy is a global public health concern. The aim of this study was to analyse the trend in antimicrobial resistance in Hangzhou, China, over the period 2015-17. Methods: In total, 379 clinical isolates were collected from seven hospitals and antimicrobial susceptibility was determined using the agar dilution method. Isolates showing resistance to ceftriaxone, azithromycin or cefixime were analysed for the presence of resistance determinants. STs were determined with the N. gonorrhoeae multiantigen sequence typing (NG-MAST) method and phylogenetic analysis and strain clustering was determined using porB and tbpB sequences. Results: Ceftriaxone resistance, decreased susceptibility to ceftriaxone and azithromycin resistance were observed in 3%, 17% and 21% of the isolates, respectively. This resulted in 5% of the isolates showing both decreased susceptibility to ceftriaxone and azithromycin resistance. Importantly, resistance levels to ceftriaxone and azithromycin increased over the study period, resulting in 5% ceftriaxone resistance, 27% decreased susceptibility to ceftriaxone and 35% azithromycin resistance in 2017 and 11% of the isolates showing both decreased susceptibility to ceftriaxone and azithromycin resistance. Phylogenetic and cluster analysis showed the emergence and expansion in 2017 of a clonally related cluster containing strains with high abundance of decreased susceptibility to ceftriaxone and/or cefixime, which was related to the presence of the mosaic penA allele X. Co-resistance to azithromycin was also observed in this cluster. Conclusions: Our findings have major implications for the future reliability of ceftriaxone monotherapy and ceftriaxone plus azithromycin dual therapy in China.

Development of a dual-antimicrobial counterselection method for markerless genetic engineering of bacterial genomes.

Markerless genetic engineering of bacterial genomes is commonly performed by two-step homologous recombination methods using vectors carrying flanking regions of the target gene for site-specific vector integration and counterselection markers to provide positive selection pressure on the second recombination step resulting in vector excision. Here, we provide the proof-of-principle of a novel counterselection method that exploits antagonistic activities between bactericidal and bacteriostatic antibiotics and which can provide selection pressure on the second recombination step by selective killing of bacteria retaining the antibiotic selection marker. This method was optimized for the bacterial pathogens Listeria monocytogenes and Neisseria meningitidis by screening for antagonistic activities between the bactericidal aminoglycosides kanamycin, streptomycin, and gentamicin in combination with the bacteriostatic antibiotics chloramphenicol and erythromycin. The largest difference in selective killing of both L. monocytogenes and N. meningitidis containing an antibiotic selection marker versus wild-type bacteria was observed for the combination of erythromycin, gentamicin, and ermC as antibiotic selection marker. Therefore, this combination was used to generate two markerless deletion mutants for both L. monocytogenes and N. meningitidis. After applying the dual-antimicrobial selection pressure on cultures during the second recombination step, surviving colonies were replica plated on agar with and without erythromycin. On average, 12-13% of the randomly selected bacterial colonies had lost the selection marker due to a second recombination event and approximately half of these colonies were the desired markerless in-frame deletion mutants. Therefore, this method proved to be easy and fast and should be applicable to a wide variety of bacterial species.

Identification and Characterization of the Neisseria gonorrhoeae MscS-Like Mechanosensitive Channel.

Mechanosensitive channels are ubiquitous in bacteria and provide an essential mechanism to survive sudden exposure to a hypo-osmotic environment by the sensing and release of increased turgor pressure. No mechanosensitive channels have thus far been identified and characterized for the human-specific bacterial pathogen Neisseria gonorrhoeae In this study, we identified and characterized the N. gonorrhoeae MscS-like mechanosensitive channel (Ng-MscS). Electrophysiological analyses by the patch clamp method showed that Ng-MscS is stretch activated and contains pressure-dependent gating properties. Further mutagenesis studies of critical residues forming the hydrophobic vapor lock showed that gain-of-function mutations in Ng-MscS inhibited bacterial growth. Subsequent analysis of the function of Ng-MscS in N. gonorrhoeae by osmotic down-shock assays revealed that the survival of Ng-mscS deletion mutants was significantly reduced compared with that of wild-type strains, while down-shock survival was restored upon the ectopic complementation of mscS Finally, to investigate whether Ng-MscS is important for N. gonorrhoeae during infections, competition assays were performed by using a murine vaginal tract infection model. Ng-mscS deletion mutants were outcompeted by N. gonorrhoeae wild-type strains for colonization and survival in this infection model, highlighting that Ng-MscS contributes to in vivo colonization and survival. Therefore, Ng-MscS might be a promising target for the future development of novel antimicrobials.

The closo-Dodecaborate Dianion Fused with Oxazoles Provides 3D Diboraheterocycles with Selective Antimicrobial Activity.

The synthesis and application of icosahedral boron cluster compounds has been studied extensively since their discovery several decades ago; however, two aspects of their chemistry have received little attention: The possibility to form inorganic/organic fused boraheterocycles and their potential to act as antimicrobial agents. This work comprises the preparation of a class of 3D diborabenzoxazole analogues with the closo-dodecaborate in place of the benzene moiety. The presented synthetic procedures provide access to a wide range of diboraheterocycles under mild conditions. These 3D heterocycles exhibit strong and selective antimicrobial activity against Neisseria gonorrhoeae, a widespread bacterial pathogen that has shown increasing incidences of multidrug resistance and for which the development of new antimicrobial compounds is urgently needed.

Defining a protective epitope on factor H binding protein, a key meningococcal virulence factor and vaccine antigen.

Mapping of epitopes recognized by functional monoclonal antibodies (mAbs) is essential for understanding the nature of immune responses and designing improved vaccines, therapeutics, and diagnostics. In recent years, identification of B-cell epitopes targeted by neutralizing antibodies has facilitated the design of peptide-based vaccines against highly variable pathogens like HIV, respiratory syncytial virus, and Helicobacter pylori; however, none of these products has yet progressed into clinical stages. Linear epitopes identified by conventional mapping techniques only partially reflect the immunogenic properties of the epitope in its natural conformation, thus limiting the success of this approach. To investigate antigen-antibody interactions and assess the potential of the most common epitope mapping techniques, we generated a series of mAbs against factor H binding protein (fHbp), a key virulence factor and vaccine antigen of Neisseria meningitidis. The interaction of fHbp with the bactericidal mAb 12C1 was studied by various epitope mapping methods. Although a 12-residue epitope in the C terminus of fHbp was identified by both Peptide Scanning and Phage Display Library screening, other approaches, such as hydrogen/deuterium exchange mass spectrometry (MS) and X-ray crystallography, showed that mAb 12C1 occupies an area of ∼1,000 Å(2) on fHbp, including >20 fHbp residues distributed on both N- and C-terminal domains. Collectively, these data show that linear epitope mapping techniques provide useful but incomplete descriptions of B-cell epitopes, indicating that increased efforts to fully characterize antigen-antibody interfaces are required to understand and design effective immunogens.

Two cross-reactive monoclonal antibodies recognize overlapping epitopes on Neisseria meningitidis factor H binding protein but have different functional properties.

Factor H binding protein (fHbp) is one of the main antigens of the 4-component meningococcus B (4CMenB) multicomponent vaccine against disease caused by serogroup B Neisseria meningitidis (MenB). fHbp binds the complement down-regulating protein human factor H (hfH), thus resulting in immune evasion. fHbp exists in 3 variant groups with limited cross-protective responses. Previous studies have described the generation of monoclonal antibodies (mAbs) targeting variant-specific regions of fHbp. Here we report for the first time the functional characterization of two mAbs that recognize a wide panel of fHbp variants and subvariants on the MenB surface and that are able to inhibit fHbp binding to hfH. The antigenic regions targeted by the two mAbs were accurately mapped by hydrogen-deuterium exchange mass spectrometry (HDX-MS), revealing partially overlapping epitopes on the N terminus of fHbp. Furthermore, while none of the mAbs had bactericidal activity on its own, a synergistic effect was observed for each of them when tested by the human complement serum bactericidal activity (hSBA) assay in combination with a second nonbactericidal mAb. The bases underlying fHbp variant cross-reactivity, as well as inhibition of hfH binding and cooperativity effect observed for the two mAbs, are discussed in light of the mapped epitopes.

Nonfunctional variant 3 factor H binding proteins as meningococcal vaccine candidates.

Neisseria meningitidis is a human-specific pathogen and leading cause of meningitis and septicemia. Factor H binding protein (fHbp), a virulence factor which protects N. meningitidis from innate immunity by binding the human complement regulator factor H (fH) with high affinity, is also a key antigen in vaccines being developed to prevent meningococcal disease. fHbp can be divided into three variant groups (V1, V2, and V3) that elicit limited immunological cross-reactivity. The interaction of fH with fHbp could impair the immunogenicity of this antigen by hindering access to the antigenic epitopes in fHbp, providing the rationale for the development of nonfunctional fHbps as vaccine candidates. Here, we characterized the two nonfunctional V3 fHbps, fHbp(T286A) and fHbp(E313A), which each contains a single amino acid substitution that leads to a marked reduction in affinity for fH without affecting the folding of the proteins. The immunogenicity of the nonfunctional fHbps was assessed in transgenic mice expressing a single chimeric fH containing domains of human fH involved in binding to fHbp. No differences in anti-V3 fHbp antibody titers were elicited by the wild-type V3 fHbp, V3 fHbp(T286A), and V3 fHbp(E313A), demonstrating that the nonfunctional fHbps retain their immunogenicity. Furthermore, the nonfunctional V3 fHbps elicit serum bactericidal activity that is equivalent to or higher than that observed with the wild-type protein. Our findings provide the basis for the rational design of next-generation vaccines containing nonfunctional V3 fHbps.

Design and evaluation of meningococcal vaccines through structure-based modification of host and pathogen molecules.

Neisseria meningitis remains a leading cause of sepsis and meningitis, and vaccines are required to prevent infections by this important human pathogen. Factor H binding protein (fHbp) is a key antigen that elicits protective immunity against the meningococcus and recruits the host complement regulator, fH. As the high affinity interaction between fHbp and fH could impair immune responses, we sought to identify non-functional fHbps that could act as effective immunogens. This was achieved by alanine substitution of fHbps from all three variant groups (V1, V2 and V3 fHbp) of the protein; while some residues affected fH binding in each variant group, the distribution of key amino underlying the interaction with fH differed between the V1, V2 and V3 proteins. The atomic structure of V3 fHbp in complex with fH and of the C-terminal barrel of V2 fHbp provide explanations to the differences in the precise nature of their interactions with fH, and the instability of the V2 protein. To develop transgenic models to assess the efficacy of non-functional fHbps, we determined the structural basis of the low level of interaction between fHbp and murine fH; in addition to changes in amino acids in the fHbp binding site, murine fH has a distinct conformation compared with the human protein that would sterically inhibit binding to fHbp. Non-functional V1 fHbps were further characterised by binding and structural studies, and shown in non-transgenic and transgenic mice (expressing chimeric fH that binds fHbp and precisely regulates complement system) to retain their immunogenicity. Our findings provide a catalogue of non-functional fHbps from all variant groups that can be included in new generation meningococcal vaccines, and establish proof-in-principle for clinical studies to compare their efficacy with wild-type fHbps.