Virginia L. Clark

Gonococcal Nitric Oxide Reductase Is Encoded by a Single Gene, norB, Which Is Required for Anaerobic Growth and Is Induced by Nitric Oxide

ABSTRACT The gene encoding a nitric oxide reductase has been identified inNeisseria gonorrhoeae. The norB gene product shares significant identity with the nitric oxide reductases inRalstonia eutropha and Synechocystis sp. and, like those organisms, the gonococcus lacks a norC homolog. The gonococcal norB gene was found to be required for anaerobic growth, but the absence of norB did not dramatically decrease anaerobic survival. In a wild-type background, induction of norB expression was seen anaerobically in the presence of nitrite but not anaerobically without nitrite or aerobically. norB expression is not regulated by FNR or NarP, but a functional aniA gene (which encodes an anaerobically induced outer membrane nitrite reductase) is necessary for expression. When aniA is constitutively expressed,norB expression can be induced both anaerobically and aerobically, but only in the presence of nitrite, suggesting that nitric oxide, which is likely to be produced by AniA as a product of nitrite reduction, is the inducing agent. This was confirmed with the use of the nitric oxide donor, spermine-nitric oxide complex, in ananiA null background both anaerobically and aerobically. NorB is important for gonococcal adaptation to an anaerobic environment, a physiologically relevant state during gonococcal infection. The presence of this enzyme, which is induced by nitric oxide, may also have implications in immune evasion and immunomodulation in the human host.

Deep sequencing-based analysis of the anaerobic stimulon in Neisseria gonorrhoeae

BackgroundMaintenance of an anaerobic denitrification system in the obligate human pathogen, Neisseria gonorrhoeae, suggests that an anaerobic lifestyle may be important during the course of infection. Furthermore, mounting evidence suggests that reduction of host-produced nitric oxide has several immunomodulary effects on the host. However, at this point there have been no studies analyzing the complete gonococcal transcriptome response to anaerobiosis. Here we performed deep sequencing to compare the gonococcal transcriptomes of aerobically and anaerobically grown cells. Using the information derived from this sequencing, we discuss the implications of the robust transcriptional response to anaerobic growth.ResultsWe determined that 198 chromosomal genes were differentially expressed (~10% of the genome) in response to anaerobic conditions. We also observed a large induction of genes encoded within the cryptic plasmid, pJD1. Validation of RNA-seq data using translational-lacZ fusions or RT-PCR demonstrated the RNA-seq results to be very reproducible. Surprisingly, many genes of prophage origin were induced anaerobically, as well as several transcriptional regulators previously unknown to be involved in anaerobic growth. We also confirmed expression and regulation of a small RNA, likely a functional equivalent of fnrS in the Enterobacteriaceae family. We also determined that many genes found to be responsive to anaerobiosis have also been shown to be responsive to iron and/or oxidative stress.ConclusionsGonococci will be subject to many forms of environmental stress, including oxygen-limitation, during the course of infection. Here we determined that the anaerobic stimulon in gonococci was larger than previous studies would suggest. Many new targets for future research have been uncovered, and the results derived from this study may have helped to elucidate factors or mechanisms of virulence that may have otherwise been overlooked.

Presence of antibodies to the major anaerobically induced gonococcal outer membrane protein in sera from patients with gonococcal infections.

Anaerobically grown Neisseria gonorrhoeae induces and represses the synthesis of outer membrane proteins. One of the anaerobically induced proteins, Pan 1, reacted strongly on Western blots with sera from patients with uncomplicated gonococcal infection, pelvic inflammatory disease, and disseminated gonococcal infection, but not with normal human serum. The pattern of reactivity of the sera against Pan 1 from several gonococcal strains suggested that the protein was antigenically heterogeneous, containing both common and unique epitopes. Staphylococcus aureus V8 protease digestion of Pan 1 from four gonococcal strains revealed the presence of common peptides, with one strain also containing some unique peptides and lacking others. The class of the antibody reactive with gonococcal outer membrane antigens was examined; anti-Pan 1 antibody was found to be IgG or IgM, but not IgA. The IgM antibody present reacted predominantly with Pan 1. These data indicate that the Pan 1 protein is expressed in vivo and strongly suggest that N. gonorrhoeae can grow anaerobically in vivo.

Functional analysis of NsrR, a nitric oxide‐sensing Rrf2 repressor in Neisseria gonorrhoeae

Nitric oxide (NO) has been shown to be an important component of the human immune response, and as such, it is important to understand how pathogenic organisms respond to its presence. In Neisseria gonorrhoeae, recent work has revealed that NsrR, an Rrf2‐type transcriptional repressor, can sense NO and control the expression of genes responsible for NO metabolism. A highly pure extract of epitope‐tagged NsrR was isolated and mass spectroscopic analysis suggested that the protein contained a [2Fe−2S] cluster. NsrR/DNA interactions were thoroughly analysed in vitro. Using EMSA analysis, NsrR::FLAG was shown to interact with predicted operators in the norB, aniA and nsrR upstream regions with a Kd of 7, 19 and 35 nM respectively. DNase I footprint analysis was performed on the upstream regions of norB and nsrR, where NsrR was shown to protect the predicted 29 bp binding sites. The presence of exogenously added NO inhibited DNA binding by NsrR. Alanine substitution of C90, C97 or C103 in NsrR abrogated repression of norB::lacZ and inhibited DNA binding, consistent with their presumed role in co‐ordination of a NO‐sensitive Fe–S centre required for DNA binding.

Identification of transcription activators that regulate gonococcal adaptation from aerobic to anaerobic or oxygen-limited growth

Analysis of the Neisseria gonorrhoeae DNA sequence database revealed the presence of two genes, one encoding a protein predicted to be 37.5% identical (50% similar) in amino acid sequence to the Escherichia coli FNR protein and the other encoding a protein 41% and 42% identical (54 and 51% sequence similarity) to the E. coli NarL and NarP proteins respectively. Both genes have been cloned into E. coli and insertionally inactivated in vitro. The mutated genes have been transformed into gonococci and recombined into the chromosome. The fnr mutation totally abolished and the narP mutation severely diminished the ability of gonococci to: (i) grow anaerobically; (ii) adapt to oxygen‐limited growth; (iii) initiate transcription from the aniA promoter (which directs the expression of a copper‐containing nitrite reductase, AniA, in response to the presence of nitrite); and (iv) reduce nitrite during growth in oxygen‐limited media. The product of nitrite reduction was identified to be nitrous oxide. Immediately upstream of the narL/narP gene is an open reading frame that, if translated, would encode a homologue of the E. coli nitrate‐ and nitrite‐sensing proteins NarX and NarQ. As transcription from the aniA promoter was not activated during oxygen‐limited growth in the presence of nitrate, the gonococcal two‐component regulatory system is designated NarQ–NarP rather than NarX–NarL. As far as we are aware, this is the first well‐documented example of a two‐component regulatory system working in partnership with a transcription activator in pathogenic neisseria. A 45 kDa c‐type cytochrome that was synthesized during oxygen‐limited, but not during oxygen sufficient, growth was identified as a homologue of cytochrome c peroxidases (CCP) of other bacteria. The gene for this cytochrome, designated ccp, was located, and its regulatory region was cloned into the promoter probe vector pLES94. Transcription from the ccp promoter was repressed during aerobic growth and induced during oxygen‐limited growth and was totally FNR dependent, suggesting that the gonococcal FNR protein is a transcription activator of at least two genes. However, unlike AniA, synthesis of the CCP homologue was insensitive to the presence of nitrite during oxygen‐limited growth.

Role of Ribosomal Protein L12 in Gonococcal Invasion of Hec1B Cells

ABSTRACT Previous studies led to the development of a model of contact-induced enhanced gonococcal invasion of human reproductive cells that utilizes the lutropin receptor (LHr) as both the induction signal for conversion to this enhanced-gonococcal-invasion phenotype (Inv+ GC) and as the specific Inv+ GC uptake mechanism. This model proposes that gonococci express a surface feature that mimics human chorionic gonadotropin (hCG), the cognate ligand for LHr, and that this structure is responsible for the specific and productive interaction of GC with LHr. In this report, we identify a 13-kDa gonococcal protein with immunological similarities to hCG. The antiserum reactivity is specific since interaction with the 13-kDa gonococcal protein can be blocked by the addition of highly purified hCG. This gonococcal “hCG-like” protein, purified from two-dimensional gels and by immunoprecipitation, was determined by N-terminal sequencing to be the ribosomal protein L12. We present evidence that gonococcal L12 is membrane associated and surface exposed in gonococci, as shown by immunoblot analysis of soluble and insoluble gonococcal protein and antibody adsorption studies with fixed GC. Using highly purified recombinant gonococcal L12, we show that preincubation of Inv− GC with micromolar amounts of rL12 leads to a subsequent five- to eightfold increase in invasion of the human endometrial cell line, Hec1B. In addition, nanomolar concentrations of exogenous L12 inhibits gonococcal invasion to approximately 70% of the level in controls. Thus, we propose a novel cellular location for the gonococcal ribosomal protein L12 and concomitant function in LHr-mediated gonococcal invasion of human reproductive cells.

CONSTRUCTION OF A TRANSLATIONAL LACZ FUSION SYSTEM TO STUDY GENE REGULATION IN NEISSERIA GONORRHOEAE

A translational lacZ reporter system to study gene regulation in Neisseria gonorrhoeae (Ng) was developed. The pUC18-based vector pLES94 transforms Ng and recombines into the Ng chromosome at the site of the proAB genes. The vector contains a restriction site for cloning promoters that will result in a lacZ gene fusion. Initial cloning and characterization of promoters can be done in Escherichia coli. The vector contains both ApR and CmR genes, however the ApR gene is lost when the insert combines into the Ng chromosome. This system gives single copy expression of the fusion and does not result in the inactivation of the gene of interest.

Enhancement of the invasive ability of Neisseria gonorrhoeae by contact with HeclB, an adeno‐carcinoma endometrial cell line

Since Neisseria gonorrhoeae is an obligate pathogen, there is no animal model for identification of virulence factors for this bacterium. An alternative model for assessment of gonococcal virulence is invasion of the adenocarcinoma endometrial cell line, HeclB. Pre‐incubation of gonococci with glutaraldehyde‐fixed HeclB cells eliminated the six‐ to eight‐hour lag in entry of bacteria into a fresh HeIIB monolayer seen with unpreincubated gonococci or gonococci pre‐incubated in tissue‐culture medium alone. Gonococci tightly bound to fixed HecIB cells were more Invasive than cells free in the tissue‐culture medium, suggesting that actual contact with HecIB cells was required for the enhancement of invasive ability. Chloramphenicol addition during the preincubation prevented the enhanced invasion. Preincubated gonococci were not more adherent to HecIB cells, suggesting that a stage in invasion after binding of gonococci to HecIB cells was enhanced. The enhanced invasion occurred only when gonococci were preincubated with HecIB ceils and not with HEp‐2, HeLa, Chang or CHO cells. This eukaryotic cell specificity for induction of enhanced invasion may indicate a role for invasion in gonococcal infection of the endometrium.

Nitrite Reductase NirS Is Required for Type III Secretion System Expression and Virulence in the Human Monocyte Cell Line THP-1 by Pseudomonas aeruginosa

ABSTRACT The nitrate dissimilation pathway is important for anaerobic growth in Pseudomonas aeruginosa. In addition, this pathway contributes to P. aeruginosa virulence by using the nematode Caenorhabditis elegans as a model host, as well as biofilm formation and motility. We used a set of nitrate dissimilation pathway mutants to evaluate the virulence of P. aeruginosa PA14 in a model of P. aeruginosa-phagocyte interaction by using the human monocytic cell line THP-1. Both membrane nitrate reductase and nitrite reductase enzyme complexes were important for cytotoxicity during the interaction of P. aeruginosa PA14 with THP-1 cells. Furthermore, deletion mutations in genes encoding membrane nitrate reductase (ΔnarGH) and nitrite reductase (ΔnirS) produced defects in the expression of type III secretion system (T3SS) components, extracellular protease, and elastase. Interestingly, exotoxin A expression was unaffected in these mutants. Addition of exogenous nitric oxide (NO)-generating compounds to ΔnirS mutant cultures restored the production of T3SS phospholipase ExoU, whereas nitrite addition had no effect. These data suggest that NO generated via nitrite reductase NirS contributes to the regulation of expression of selected virulence factors in P. aeruginosa PA14.

Genetic loci and linkage associations in Neisseria gonorrhoeae and Neisseria meningitidis.

The purpose of this review is to present the currently available information on genetic loci that have been identified in Neisseria gonorrhoeae and Neisseria meningitidis. We will include genes that were identified by mutations that alter the phenotype of the organism, if the gene was transferred into another genetic background, and genes whose products were well characterized, even if the genes were not identified by mutation. We also will include genes that were cloned and identified either by complementation of Escherichia coli mutations or by identification of the gene product.