Commensal Neisseria cinerea outer membrane vesicles as a platform for the delivery of meningococcal and gonococcal antigens to the immune system.

An affordable, accessible, and broadly protective vaccine is required to tackle the re-occurring bacterial meningococcal epidemics in Sub-Saharan Africa as well as an effective control of multi-drug resistant strains of gonococcus. Outer membrane vesicles (OMVs) secreted from Gram-negative bacteria represent an attractive platform for antigen delivery to the immune system and therefore for development of multi-component vaccines. In this study, we describe the generation of modified OMVs (mOMVs) from commensal biosafety-level 1 (BSL-1) Neisseria cinerea ATCC® 14685TM, which is phylogenetically close to the pathogenic bacteria Neisseria meningitidis and Neisseria gonorrhoeae. mOMVs were prepared from N. cinerea engineered to express heterologous antigens from N. meningitidis (factor H binding protein (fHbp) and Neisseria Heparin Binding Antigen (NHBA-2)) and from N. gonorrhoeae (NHBA-542). Mice immunised with the mOMVs produced antibodies against fHbp and NHBA. The work indicates that mOMV from N. cinerea can be used as a platform to induce immune responses against antigens involved in the protective immune response against meningococcal and gonococcal diseases.

Versatile and efficient genome editing with Neisseria cinerea Cas9.

The CRISPR/Cas9 system is a versatile genome editing platform in biotechnology and therapeutics. However, the requirement of protospacer adjacent motifs (PAMs) limits the genome targeting scope. To expand this repertoire, we revisited and engineered a compact Cas9 orthologue derived from Neisseria cinerea (NcCas9) for efficient genome editing in mammal cells. We demonstrated that NcCas9 generates genome editing at target sites with N4GYAT (Y = T/C) PAM which cannot be recognized by existing Cas9s. By optimizing the NcCas9 architecture and its spacer length, editing efficacy of NcCas9 was further improved in human cells. In addition, the NcCas9-derived Base editors can efficiently generate base conversions. Six anti-CRISPR (Acr) proteins were identified as off-switches for NcCas9. Moreover, NcCas9 successfully generated efficient editing of mouse embryos by microinjection of NcCas9 mRNA and the corresponding sgRNA. Thus, the NcCas9 holds the potential to broaden the CRISPR/Cas9 toolsets for efficient gene modifications and therapeutic applications.

Type VI secretion system killing by commensal Neisseria is influenced by expression of type four pili.

Type VI Secretion Systems (T6SSs) are widespread in bacteria and can dictate the development and organisation of polymicrobial ecosystems by mediating contact dependent killing. In Neisseria species, including Neisseria cinerea a commensal of the human respiratory tract, interbacterial contacts are mediated by Type four pili (Tfp) which promote formation of aggregates and govern the spatial dynamics of growing Neisseria microcolonies. Here, we show that N. cinerea expresses a plasmid-encoded T6SS that is active and can limit growth of related pathogens. We explored the impact of Tfp on N. cinerea T6SS-dependent killing within a colony and show that pilus expression by a prey strain enhances susceptibility to T6SS compared to a non-piliated prey, by preventing segregation from a T6SS-wielding attacker. Our findings have important implications for understanding how spatial constraints during contact-dependent antagonism can shape the evolution of microbial communities.

Commensal Neisseria cinerea impairs Neisseria meningitidis microcolony development and reduces pathogen colonisation of epithelial cells.

It is increasingly being recognised that the interplay between commensal and pathogenic bacteria can dictate the outcome of infection. Consequently, there is a need to understand how commensals interact with their human host and influence pathogen behaviour at epithelial surfaces. Neisseria meningitidis, a leading cause of sepsis and meningitis, exclusively colonises the human nasopharynx and shares this niche with several other Neisseria species, including the commensal Neisseria cinerea. Here, we demonstrate that during adhesion to human epithelial cells N. cinerea co-localises with molecules that are also recruited by the meningococcus, and show that, similar to N. meningitidis, N. cinerea forms dynamic microcolonies on the cell surface in a Type four pilus (Tfp) dependent manner. Finally, we demonstrate that N. cinerea colocalises with N. meningitidis on the epithelial cell surface, limits the size and motility of meningococcal microcolonies, and impairs the effective colonisation of epithelial cells by the pathogen. Our data establish that commensal Neisseria can mimic and affect the behaviour of a pathogen on epithelial cell surfaces.

Meningococcal Disease-Associated Prophage-Like Elements Are Present in Neisseria gonorrhoeae and Some Commensal Neisseria Species.

Neisseria spp. possess four genogroups of filamentous prophages, termed Nf1 to 4. A filamentous bacteriophage from the Nf1 genogroup termed meningococcal disease-associated phage (MDA φ) is associated with clonal complexes of Neisseria meningitidis that cause invasive meningococcal disease. Recently, we recovered an isolate of Neisseria gonorrhoeae (ExNg63) from a rare case of gonococcal meningitis, and found that it possessed a region with 90% similarity to Nf1 prophages, specifically, the meningococcal MDA φ. This led to the hypothesis that the Nf1 prophage may be more widely distributed amongst the genus Neisseria. An analysis of 92 reference genomes revealed the presence of intact Nf1 prophages in the commensal species, Neisseria lactamica and Neisseria cinerea in addition to the pathogen N. gonorrhoeae. In N. gonorrhoeae, Nf1 prophages had a restricted distribution but were present in all representatives of MLST ST1918. Of the 160 phage integration sites identified, only one common insertion site was found between one isolate of N. gonorrhoeae and N. meningitidis. There was an absence of any obvious conservation of the receptor for prophage entry, PilE, suggesting that the phage may have been obtained by natural transformation. An examination of the restriction modification systems and mutated mismatch repair systems with prophage presence suggested that there was no obvious preference for these hosts. A timed phylogeny inferred that N. meningitidis was the donor of the Nf1 prophages in N. lactamica and N. gonorrhoeae. Further work is required to determine whether Nf1 prophages are active and can act as accessory colonization factors in these species.

Neonatal Conjunctivitis Caused by Neisseria cinerea: A Case of Mistaken Identity.

We report a case of a 3-day-old boy with Neisseria cinerea conjunctivitis, originally misidentified as Neisseria gonorrhoeae conjunctivitis. Neonates are at increased risk for disseminated gonococcal infection, and physicians should be cognizant of N cinerea and its potential to be mistaken for N gonorrhoeae.

Neisseria cinerea with High Ceftriaxone MIC Is a Source of Ceftriaxone and Cefixime Resistance-Mediating penA Sequences in Neisseria gonorrhoeae.

Mosaic penA alleles have caused most of the cephalosporin resistance in Neisseria gonorrhoeae, but their evolution is mostly unknown. The penA gene from Neisseria cinerea strain AM1601 (ceftriaxone MIC, 1.0 µg/ml) caused ceftriaxone resistance (MIC, 1 µg/ml) in a ceftriaxone-susceptible gonococcal strain. The 3'-terminal half of AM1601 penA was almost identical to that of the ceftriaxone-resistant gonococcal GU140106 and FC428 strains. N. cinerea can serve as a reservoir of ceftriaxone resistance-mediating penA sequences that can be transferred to gonococci.

Neisseria cinerea bacteremia in a patient receiving eculizumab: a case report.

Neisseria cinerea is a commensal species capable of colonizing the upper respiratory tract of humans. Bacteremia associated with this organism is very uncommon, with only seven previous cases described. We report a case of Neisseria cinerea bacteremia in a patient maintained on indefinite eculizumab therapy. It is the eighth reported case of Neisseria cinerea bacteremia and represents the first case in a patient receiving eculizumab.

Neisseria cinerea isolates can adhere to human epithelial cells by type IV pilus-independent mechanisms.

In pathogenic Neisseria species the type IV pili (Tfp) are of primary importance in host-pathogen interactions. Tfp mediate initial bacterial attachment to cell surfaces and formation of microcolonies via pilus-pilus interactions. Based on genome analysis, many non-pathogenic Neisseria species are predicted to express Tfp, but aside from studies on Neisseria elongata, relatively little is known about the formation and function of pili in these organisms. Here, we have analysed pilin expression and the role of Tfp in Neisseria cinerea. This non-pathogenic species shares a close taxonomic relationship to the pathogen Neisseria meningitidis and also colonizes the human oropharyngeal cavity. Through analysis of non-pathogenic Neisseria genomes we identified two genes with homology to pilE, which encodes the major pilin of N. meningitidis. We show which of the two genes is required for Tfp expression in N. cinerea and that Tfp in this species are required for DNA competence, similar to other Neisseria. However, in contrast to the meningococcus, deletion of the pilin gene did not impact the association of N. cinerea to human epithelial cells, demonstrating that N. cinerea isolates can adhere to human epithelial cells by Tfp-independent mechanisms.

Meningitis and Bacteremia Due to Neisseria cinerea following a Percutaneous Rhizotomy of the Trigeminal Ganglion.

Neisseria cinerea is a human commensal. The first known case of meningitis and bacteremia due to Neisseria cinerea following percutaneous glycerol instillation of the trigeminal ganglion is reported. Conventional phenotypic methods and complete 16S RNA gene sequencing accurately identified the pathogen. Difficulties in differentiation from pathogenic neisseriae are discussed.

Comparative genomics of Neisseria meningitidis: core genome, islands of horizontal transfer and pathogen-specific genes.

To better understand Neisseria meningitidis genomes and virulence, microarray comparative genome hybridization (mCGH) data were collected from one Neisseria cinerea, two Neisseria lactamica, two Neisseria gonorrhoeae and 48 Neisseria meningitidis isolates. For N. meningitidis, these isolates are from diverse clonal complexes, invasive and carriage strains, and all major serogroups. The microarray platform represented N. meningitidis strains MC58, Z2491 and FAM18, and N. gonorrhoeae FA1090. By comparing hybridization data to genome sequences, the core N. meningitidis genome and insertions/deletions (e.g. capsule locus, type I secretion system) related to pathogenicity were identified, including further characterization of the capsule locus, bioinformatics analysis of a type I secretion system, and identification of some metabolic pathways associated with intracellular survival in pathogens. Hybridization data clustered meningococcal isolates from similar clonal complexes that were distinguished by the differential presence of six distinct islands of horizontal transfer. Several of these islands contained prophage or other mobile elements, including a novel prophage and a transposon carrying portions of a type I secretion system. Acquisition of some genetic islands appears to have occurred in multiple lineages, including transfer between N. lactamica and N. meningitidis. However, island acquisition occurs infrequently, such that the genomic-level relationship is not obscured within clonal complexes. The N. meningitidis genome is characterized by the horizontal acquisition of multiple genetic islands; the study of these islands reveals important sets of genes varying between isolates and likely to be related to pathogenicity.

Ciprofloxacin treatment of bacterial peritonitis associated with chronic ambulatory peritoneal dialysis caused by Neisseria cinerea.

Bacterial peritonitis is a well-recognized complication of chronic ambulatory peritoneal dialysis (CAPD) in patients with end-stage renal failure. We present a case of peritonitis due to an unusual pathogen, Neisseria cinerea, unresponsive to the standard intraperitoneal (i.p.) vancomycin and gentamicin, which responded rapidly to oral ciprofloxacin.

Bacterial activation of mast cells.

Mast cells often are found in a perivascular location but especially in mucosae, where they may response to various stimuli. They typically associate with immediate hypersensitive responses and are likely to play a critical role in host defense. In this chapter, a common airway pathogen, Moraxella catarrhalis, and a commensal bacterium, Neiserria cinerea, are used to illustrate activation of human mast cells. A human mast cell line (HMC-1) derived from a patient with mast cell leukemia was activated with varying concentrations of heat-killed bacteria. Active aggregation of bacteria over mast cell surfaces was detected by scanning electron microscopy. The activation of mast cells was analyzed by nuclear factor-kappaB (NF-kappaB) activation and cytokine production in culture supernatants. Both M. catarrhalis and N. cinerea induce mast cell activation and the secretion of two key inflammatory cytokines, interleukin-6 and MCP-1. This is accompanied by NF-kappaB activation. Direct bacterial contact with mast cells appears to be essential for this activation because neither cell-free bacterial supernatants nor bacterial lipopolysaccharide induce cytokine secretion.

Critical aspects of analysis of Micrococcus luteus, Neisseria cinerea, and Pseudomonas fluorescens by means of capillary electrophoresis.

Within the frame of our study we investigated Microccocus luteus, Neisseria cinerea, and Pseudomonas fluorescens by means of capillary zone electrophoresis (CZE). They form chains and clusters on a different scale, which can be reflected in the electropherograms. A low buffer concentration of Tris-borate and Na2EDTA containing a polymeric matrix of 0.0125% poly(ethylene) oxide (PEO) was used. Key factors were the standardization and optimization of CE conditions, buffer solution, and pretreatment of bacterial samples, which are not transferable to different bacterial strains, in general. The different compositions of the cell wall of on the one hand Gram-positive (M. luteus) and Gram-negative (N. cinerea) cocci and on the other hand Gram-negative, rod-shaped bacteria (P. fluorescens), are probably responsible for the different pretreatment conditions.

Functional diversity of three different DsbA proteins from Neisseria meningitidis.

The genome of Neisseria meningitidis serogroup B strain MC58 contains three genes - nmb0278, nmb0294 and nmb0407 - encoding putative homologues of DsbA, a periplasmic thiol disulphide oxidoreductase protein-folding catalyst of the Dsb protein family. DsbA assists the folding of periplasmic and membrane proteins in diverse organisms. While all three cloned genes complemented the DTT sensitivity of dsbA-null Escherichia coli, they showed different activities in folding specific target proteins in this background. NMB0278 protein was the most active in complementing defects in motility and alkaline phosphatase activity, while NMB0294 was the most active in folding periplasmic MalF. NMB0407 showed the weakest activity in all assays. It is extremely unusual for organisms to contain more than one chromosomal dsbA. Among the members of the genus Neisseria, only the meningococcus carries all three of these genes. Strains of Neisseria gonorrhoeae, Neisseria lactamica, Neisseria cinerea and Neisseria polysaccharea contained only homologues of nmb0278 and nmb0407, while Neisseria flava, Neisseria subflava and Neisseria flavescens carried only nmb0294. It is speculated that the versatility of the meningococcus in surviving in different colonizing and invasive disease settings may be derived in part from an enhanced potential to deploy outer-membrane proteins, a consequence of carrying an extended repertoire of protein-folding catalysts.

Moraxella catarrhalis induces mast cell activation and nuclear factor kappa B-dependent cytokine synthesis.

Human mast cells are often found perivascularly and at mucosal sites and may play crucial roles in the inflammatory response. Recent studies have suggested a prominent role for mast cells in host defense. In this study, we analyzed the effects of a common airway pathogen, Moraxella catarrhalis and a commensal bacterium, Neiserria cinerea, on activation of human mast cells. Human mast cell leukemia cells (HMC-1) were activated with either phorbol myristate acetate (PMA) and calcium ionophore or with varying concentrations of heat-killed suspensions of bacteria. Supernatants were assayed for the cytokines interleukin-4 (IL-4), granulocyte macrophage colony stimulating factor (GM-CSF), IL-6, IL-8, IL-13 and monocyte chemotactic protein-1 (MCP-1). Nuclear proteins were isolated and assayed by electrophoretic mobility shift assay (EMSA) for nuclear factor kappaB (NF-kappaB) nuclear binding activity. In some experiments, NF-kappaB inhibitor, Bay-11 was added to determine functional significance. Both M. catarrhalis and N. cinerea induced mast cell activation and selective secretion of two key inflammatory cytokines, IL-6 and MCP-1. This was accompanied by NF-kappaB activation. Neither spun bacterial supernatants nor bacterial lipopolysaccharide induced cytokine secretion, suggesting need for direct bacterial contact with mast cells. Scanning electron microscopy revealed active aggregation of bacteria over mast cell surfaces. The NF-kappaB inhibitor, Bay-11, inhibited expression of MCP-1. These findings suggest the possibility of direct interactions between human mast cells and common bacteria and provide evidence for a novel role for human mast cells in innate immunity.