Research Overview
Neisseria gonorrhoeae (GC) is the etiologic agent of gonorrhea, still one of the most prevalent infectious diseases in the U.S. Untreated infections in women can lead to pelvic inflammatory disease (PID), a significant cause of infertility in this country. N. gonorrhoeae is often isolated along with obligate anaerobes, which presents a conundrum, as the gonococcus has been considered to be an obligate aerobe. We demonstrated that GC is actually a facultative anaerobic that can utilize nitrite as a terminal electron acceptor. An examination of genes that are only expressed anaerobically may reveal new virulence factors that are important in gonococcal pathogenesis.

N. gonorrhoeae grows anaerobically by using the central two reactions of the denitrification pathway, reduction of nitrite by AniA and reduction of nitric oxide by NorB. The gonococcus does not produce energy from these two reactions, but the pathway does enable the organism to maintain an oxidation/reduction balance anaerobically. The presence of AniA and NorB allows the gonococcus to produce and degrade nitric oxide (NO), known to be an important modulator of the host innate immune system and signal transduction pathways. This ability to keep NO at a low level may be responsible for the fact that most gonococcal infections in women are asymptomatic.

We are using a combination of genetic, physiologic, and cell biology techniques to better understand the role of the dentrification pathway in gonococcal virulence. We have elucidated the regulatory pathway for aniA and have identified the regulator of norB. Regulation of these two genes is complex, involving 2 activators and 3 repressors of transcription. We are currently working to identify the anaerobic regulon and the function of anaerobically regulated genes.

We have determined that gonococci establish a NO steady state in the presence of a long half-lived NO donor, reducing the NO level from a pro-inflammatory concentration to an anti-inflammatory concentration. Thus. NO metabolism may enable gonococci to alter the host immune response. NO and its relative peroxynitrite are reactive nitrogen species that are toxic to many bacteria. We have determined that gonococci are highly resistant to the toxic effects of both of these compounds. We are currently working to identify the mechanism of peroxynitrite resistance in N. gonorrhoeae.

• Spence SA, Clark VL, Isabella VM. The role of catalase in gonococcal resistance to peroxynitrite. Microbiology. 2012 Feb; 158(Pt 2):560-70.
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• Isabella VM, Clark VL. Identification of a conserved protein involved in anaerobic unsaturated fatty acid synthesis in Neiserria gonorrhoeae: implications for facultative and obligate anaerobes that lack FabA. Mol Microbiol. 2011 Oct; 82(2):489-501.
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• Isabella VM, Clark VL. Deep sequencing-based analysis of the anaerobic stimulon in Neisseria gonorrhoeae. BMC Genomics. 2011; 12:51.
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• Barth KR, Isabella VM, Clark VL. Biochemical and genomic analysis of the denitrification pathway within the genus Neisseria. Microbiology. 2009 Dec; 155(Pt 12):4093-103.
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• Van Alst NE, Wellington M, Clark VL, Haidaris CG, Iglewski BH. Nitrite reductase NirS is required for type III secretion system expression and virulence in the human monocyte cell line THP-1 by Pseudomonas aeruginosa. Infect Immun. 2009 Oct; 77(10):4446-54.
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• Barth KR, Isabella VM, Wright LF, Clark VL. Resistance to peroxynitrite in Neisseria gonorrhoeae. Microbiology. 2009 Aug; 155(Pt 8):2532-45.
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• Isabella VM, Lapek JD, Kennedy EM, Clark VL. Functional analysis of NsrR, a nitric oxide-sensing Rrf2 repressor in Neisseria gonorrhoeae. Mol Microbiol. 2009 Jan; 71(1):227-39.
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• Barth K, Clark VL. Differences in nitric oxide steady states between arginine, hypoxanthine, uracil auxotrophs (AHU) and non-AHU strains of Neisseria gonorrhoeae during anaerobic respiration in the presence of nitrite. Can J Microbiol. 2008 Aug; 54(8):639-46.
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• Spence JM, Wright L, Clark VL. Laboratory maintenance of Neisseria gonorrhoeae. Curr Protoc Microbiol. 2008 Feb; Chapter 4:Unit 4A.1.
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• Isabella V, Wright LF, Barth K, Spence JM, Grogan S, Genco CA, Clark VL. cis- and trans-acting elements involved in regulation of norB (norZ), the gene encoding nitric oxide reductase in Neisseria gonorrhoeae. Microbiology. 2008 Jan; 154(Pt 1):226-39.
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• Cardinale JA, Clark VL. Determinants of nitric oxide steady-state levels during anaerobic respiration by Neisseria gonorrhoeae. Mol Microbiol. 2005 Oct; 58(1):177-88.
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• Thompson S, Neal S, Clark V. Clinical risk management in obstetrics: eclampsia drills. Qual Saf Health Care. 2004 Apr; 13(2):127-9.
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• Spence JM, Tyler RE, Domaoal RA, Clark VL. L12 enhances gonococcal transcytosis of polarized Hec1B cells via the lutropin receptor. Microb Pathog. 2002 Mar; 32(3):117-25.
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• Householder TC, Fozo EM, Cardinale JA, Clark VL. Gonococcal nitric oxide reductase is encoded by a single gene, norB, which is required for anaerobic growth and is induced by nitric oxide. Infect Immun. 2000 Sep; 68(9):5241-6.
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• Spence JM, Clark VL. Role of ribosomal protein L12 in gonococcal invasion of Hec1B cells. Infect Immun. 2000 Sep; 68(9):5002-10.
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• Lissenden S, Mohan S, Overton T, Regan T, Crooke H, Cardinale JA, Householder TC, Adams P, O'Conner CD, Clark VL, Smith H, Cole JA. Identification of transcription activators that regulate gonococcal adaptation from aerobic to anaerobic or oxygen-limited growth. Mol Microbiol. 2000 Aug; 37(4):839-55.
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• Cardinale JA, Clark VL. Expression of AniA, the major anaerobically induced outer membrane protein of Neisseria gonorrhoeae, provides protection against killing by normal human sera. Infect Immun. 2000 Jul; 68(7):4368-9.
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• Householder TC, Belli WA, Lissenden S, Cole JA, Clark VL. cis- and trans-acting elements involved in regulation of aniA, the gene encoding the major anaerobically induced outer membrane protein in Neisseria gonorrhoeae. J Bacteriol. 1999 Jan; 181(2):541-51.
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• Spence JM, Chen JC, Clark VL. A proposed role for the lutropin receptor in contact-inducible gonococcal invasion of Hec1B cells. Infect Immun. 1997 Sep; 65(9):3736-42.
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• Silver LE, Clark VL. Construction of a translational lacZ fusion system to study gene regulation in Neisseria gonorrhoeae. Gene. 1995 Dec 1; 166(1):101-4.
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• Madden TE, Clark VL, Kuramitsu HK. Revised sequence of the Porphyromonas gingivalis prtT cysteine protease/hemagglutinin gene: homology with streptococcal pyrogenic exotoxin B/streptococcal proteinase. Infect Immun. 1995 Jan; 63(1):238-47.
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• Madden TE, Thompson TM, Clark VL. Expression of Porphyromonas gingivalis proteolytic activity in Escherichia coli. Oral Microbiol Immunol. 1992 Dec; 7(6):349-56.
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• Hoehn GT, Clark VL. Isolation and nucleotide sequence of the gene (aniA) encoding the major anaerobically induced outer membrane protein of Neisseria gonorrhoeae. Infect Immun. 1992 Nov; 60(11):4695-703.
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• Hoehn GT, Clark VL. The major anaerobically induced outer membrane protein of Neisseria gonorrhoeae, Pan 1, is a lipoprotein. Infect Immun. 1992 Nov; 60(11):4704-8.
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• Chen JC, Bavoil P, Clark VL. Enhancement of the invasive ability of Neisseria gonorrhoeae by contact with HecIB, an adenocarcinoma endometrial cell line. Mol Microbiol. 1991 Jun; 5(6):1531-8.
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• Hoehn GT, Clark VL. Distribution of a protein antigenically related to the major anaerobically induced gonococcal outer membrane protein among other Neisseria species. Infect Immun. 1990 Dec; 58(12):3929-33.
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• Klimpel KW, Clark VL. The RNA polymerases of Porphyromonas gingivalis and Fusobacterium nucleatum are unrelated to the RNA polymerase of Escherichia coli. J Dent Res. 1990 Sep; 69(9):1567-72.
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• Palermo-Dilts DA, Silver LE, Clark VL. Distribution of gonococcal lipopolysaccharide biosynthesis genes among strains of Neisseria gonorrhoeae and other neisserial species. Microb Pathog. 1990 Mar; 8(3):227-33.
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• Klimpel KW, Lesley SA, Clark VL. Identification of subunits of gonococcal RNA polymerase by immunoblot analysis: evidence for multiple sigma factors. J Bacteriol. 1989 Jul; 171(7):3713-8.
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• Klimpel KW, Clark VL. The heat shock response of type 1 and type 4 gonococci. Sex Transm Dis. 1989 Jul-Sep; 16(3):141-7.
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• West SE, Clark VL. Genetic loci and linkage associations in Neisseria gonorrhoeae and Neisseria meningitidis. Clin Microbiol Rev. 1989 Apr; 2 Suppl:S92-103.
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• Genco CA, Clark VL. Transfer of a gonococcal beta-lactamase plasmid to conjugation-deficient Neisseria cinerea strains by transformation. J Gen Microbiol. 1988 Dec; 134(12):3277-83.
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• Genco CA, Clark VL. Role of outer-membrane proteins and lipopolysaccharide in conjugation between Neisseria gonorrhoeae and Neisseria cinerea. J Gen Microbiol. 1988 Dec; 134(12):3285-94.
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• Clark VL, Knapp JS, Thompson S, Klimpel KW. Presence of antibodies to the major anaerobically induced gonococcal outer membrane protein in sera from patients with gonococcal infections. Microb Pathog. 1988 Nov; 5(5):381-90.
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• Klimpel KW, Clark VL. Multiple protein differences exist between Neisseria gonorrhoeae type 1 and type 4. Infect Immun. 1988 Apr; 56(4):808-14.
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• Palermo DA, Evans TM, Clark VL. Expression of a cloned lipopolysaccharide antigen from Neisseria gonorrhoeae on the surface of Escherichia coli K-12. Infect Immun. 1987 Nov; 55(11):2844-9.
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• Clark VL, Campbell LA, Palermo DA, Evans TM, Klimpel KW. Induction and repression of outer membrane proteins by anaerobic growth of Neisseria gonorrhoeae. Infect Immun. 1987 Jun; 55(6):1359-64.
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• Campbell LA, Short HB, Young FE, Clark VL. Autoplaquing in Neisseria gonorrhoeae. J Bacteriol. 1985 Oct; 164(1):461-5.
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• Tenover FC, Stein DC, Young FE, Clark VL. Construction and characterization of chimeric beta-lactamase plasmids of Neisseria gonorrhoeae with altered ability to be mobilized during conjugation. Sex Transm Dis. 1985 Apr-Jun; 12(2):76-82.
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• Knapp JS, Clark VL. Anaerobic growth of Neisseria gonorrhoeae coupled to nitrite reduction. Infect Immun. 1984 Oct; 46(1):176-81.
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• Genco CA, Knapp JS, Clark VL. Conjugation of plasmids of Neisseria gonorrhoeae to other Neisseria species: potential reservoirs for the beta-lactamase plasmid. J Infect Dis. 1984 Sep; 150(3):397-401.
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• Stein DC, Silver LE, Clark VL, Young FE. Cloning genes for proline biosynthesis from Neisseria gonorrhoeae: identification by interspecific complementation of Escherichia coli mutants. J Bacteriol. 1984 May; 158(2):696-700.
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• Stein DC, Silver LE, Clark VL, Young FE. Construction and characterization of a new shuttle vector, pLES2, capable of functioning in Escherichia coli and Neisseria gonorrhoeae. Gene. 1983 Nov; 25(2-3):241-7.
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• Stein DC, Clark VL, Young FE. Inhibition of active transport and macromolecular synthesis by pyocin 103 in Neisseria gonorrhoeae. Sex Transm Dis. 1983 Jan-Mar; 10(1):7-13.
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• Stein DC, Young FE, Tenover FC, Clark VL. Characterization of a chimeric beta-lactamase plasmid of Neisseria gonorrhoeae which can function in Escherichia coli. Mol Gen Genet. 1983; 189(1):77-84.
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• Short HB, Clark VL, Kellogg DS, Young FE. Anaerobic survival of clinical isolates and laboratory strains of Neisseria gonorrhoea: use in transfer and storage. J Clin Microbiol. 1982 May; 15(5):915-9.
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• Kolodkin AB, Clark VL, Tenover FC, Young FE. High correlation of the presence of methyladenine in Neisseria gonorrhoeae DNA with the AHU auxotype. Infect Immun. 1982 May; 36(2):586-90.
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• Tenover FC, Clark VL, Young FE. Presence of methyl adenine in the DNA of some strains of Neisseria gonorrhoeae. Biochem Biophys Res Commun. 1980 Aug 29; 95(4):1796-800.
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• Clark VL. Isolation and characterization of an endogenous inhibitor of protein synthesis in Escherichia coli K-12. J Bacteriol. 1979 Jan; 137(1):340-9.
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• Clark VL, Young FE. D-alanine incorporation into macromolecules and effects of D-alanine deprivation on active transport in Bacillus subtilis. J Bacteriol. 1978 Mar; 133(3):1339-50.
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• Clark VL, Young FE. Active transport of D-alanine and related amino acids by whole cells of Bacillus subtilis. J Bacteriol. 1974 Dec; 120(3):1085-92.
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• Clark VL, Peterson DE, Bernlohr RW. Changes in free amino acid production and intracellular amino acid pools of Bacillus licheniformis as a function of culture age and growth media. J Bacteriol. 1972 Nov; 112(2):715-25.
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