Nitric oxide and its derivative peroxynitrites are generated by host defense system to control bacterial infection. However certain Gram positive bacteria including Staphylococcus aureus possess a gene encoding nitric oxide synthase (SaNOS) in their chromosome. In this study it was determined that under normal growth conditions, expression of SaNOS was highest during early exponential phase of the bacterial growth. In oxidative stress studies, deletion of SaNOS led to increased susceptibility of the mutant cells compared to wild-type S. aureus. While inhibition of SaNOS activity by the addition of L-NAME increased sensitivity of the wild-type S. aureus to oxidative stress, the addition of a nitric oxide donor, sodium nitroprusside, restored oxidative stress tolerance of the SaNOS mutant. The SaNOS mutant also showed reduced survival after phagocytosis by PMN cells with respect to wild-type S. aureus. 1. Introduction Staphylococcus aureus is a Gram-positive bacterial pathogen that colonizes anterior nares and mucosal surfaces in humans and is responsible for causing a wide array of diseases from mild skin infections to life-threatening conditions such as bacteremia, pneumonia, and endocarditis [1–4]. The emerging resistant strains of S. aureus exacerbate efforts to control or properly treat staphylococcal infections [5]. The host immune system responds to bacterial infections in a concerted manner to eliminate this pathogen. This involves recruitment of polymorphonuclear leukocytes and macrophages to the site of infection and ingestion of invading bacteria. Uptake of bacteria triggers oxygen-dependent and oxygen-independent microbicidal pathways in the phagocytic cells. The oxygen-dependent pathway generates superoxide anion ( ) that serves as a precursor for additional reactive oxygen species (ROS) such as hydrogen peroxide (H2O2), hydroxyl radical, singlet oxygen, hypochlorous acid (HOCl), and peroxynitrite [6–9]. S. aureus utilizes various strategies to defend itself against host immune attack. It produces antioxidant enzymes such as superoxide dismutase that converts superoxide anion to H2O2, catalase that converts H2O2 to water and oxygen, and alkyl hydroperoxide reductases that detoxify H2O2, peroxynitrites and hydroperoxides [10, 11]. In addition to their ability to protect from host’s oxidants, S. aureus infections impose oxidative stress in a host [12]. During infection with a methicillin resistant S. aureus strain, host neutrophils respond by an increase in nitric oxide production [12]. Nitric oxide (NO) is a free radical synthesized by nitric
References
[1]
J. R. Mediavilla, L. Chen, B. Mathema, and B. N. Kreiswirth, “Global epidemiology of community-associated methicillin resistant Staphylococcus aureus (CA-MRSA),” Current Opinion in Microbiology, vol. 15, no. 5, pp. 588–595, 2012.
[2]
E. Stenehjem and D. Rimland, “MRSA nasal colonization burden and risk of MRSA infection,” American Journal of Infection Control, 2012.
[3]
R. R. Watkins, M. Z. David, and R. A. Salata, “Current concepts on the virulence mechanisms of meticillin-resistant Staphylococcus aureus,” Journal of Medical Microbiology, vol. 61, part 9, pp. 1179–1193, 2012.
[4]
F. D. Lowy, “Medical progress: Staphylococcus aureus infections,” The New England Journal of Medicine, vol. 339, no. 8, pp. 520–532, 1998.
[5]
A. M. Rivera and H. W. Boucher, “Current concepts in antimicrobial therapy against select gram-positive organisms: methicillin-resistant Staphylococcus aureus, penicillin-resistant pneumococci, and vancomycin-resistant enterococci,” Mayo Clinic Proceedings, vol. 86, no. 12, pp. 1230–1243, 2011.
[6]
F. C. Fang, “Antimicrobial reactive oxygen and nitrogen species: concepts and controversies,” Nature Reviews Microbiology, vol. 2, no. 10, pp. 820–832, 2004.
[7]
C. K. Ferrari, P. C. Souto, E. L. Fran？a, and A. C. Honorio-Fran？a, “Oxidative and nitrosative stress on phagocytes' function: from effective defense to immunity evasion mechanisms,” Archivum Immunologiae et Therapia Experimentalis, vol. 59, no. 6, pp. 441–448, 2011.
[8]
J. MacMicking, Q. W. Xie, and C. Nathan, “Nitric oxide and macrophage function,” Annual Review of Immunology, vol. 15, pp. 323–350, 1997.
[9]
J. M. Voyich, K. R. Braughton, D. E. Sturdevant et al., “Insights into mechanisms used by Staphylococcus aureus to avoid destruction by human neutrophils,” Journal of Immunology, vol. 175, no. 6, pp. 3907–3919, 2005.
[10]
F. R. DeLeo, B. A. Diep, and M. Otto, “Host defense and pathogenesis in Staphylococcus aureus infections,” Infectious Disease Clinics of North America, vol. 23, no. 1, pp. 17–34, 2009.
[11]
G. Y. Liu, “Molecular pathogenesis of Staphylococcus aureus infection,” Pediatric Research, vol. 65, no. 5, part 2, pp. 71R–77R, 2009.
[12]
S. P. Chakraborty, P. Pramanik, and S. Roy, “Staphylococcus aureus infection induced oxidative imbalance in neutrophils: possible protective role of nanoconjugated vancomycin,” ISRN Pharmacol, vol. 2012, Article ID 435214, 2012.
[13]
J. Kopincova, A. Puzserova, and I. Bernatova, “Biochemical aspects of nitric oxide synthase feedback regulation by nitric oxide,” Interdisciplinary Toxicology, vol. 4, no. 2, pp. 63–68, 2011.
[14]
S. Mariotto, M. Menegazzi, and H. Suzuki, “Biochemical aspects of nitric oxide,” Current Pharmaceutical Design, vol. 10, no. 14, pp. 1627–1645, 2004.
[15]
B. R. Crane, “The enzymology of nitric oxide in bacterial pathogenesis and resistance,” Biochemical Society Transactions, vol. 36, part 6, pp. 1149–1154, 2008.
[16]
L. E. Bird, J. Ren, J. Zhang et al., “Crystal structure of SANOS, a bacterial nitric oxide synthase oxygenase protein from Staphylococcus aureus,” Structure, vol. 10, no. 12, pp. 1687–1696, 2002.
[17]
A. Brunel, J. Santolini, and P. Dorlet, “Electron paramagnetic resonance characterization of tetrahydrobiopterin radical formation in bacterial nitric oxide synthase compared to mammalian nitric oxide synthase,” Biophysical Journal, vol. 103, no. 1, pp. 109–117, 2012.
[18]
K. Pant, A. M. Bilwes, S. Adak, D. J. Stuehr, and B. R. Crane, “Structure of a nitric oxide synthase heme protein from Bacillus subtilis,” Biochemistry, vol. 41, no. 37, pp. 11071–11079, 2002.
[19]
I. Gusarov and E. Nudler, “NO-mediated cytoprotection: instant adaptation to oxidative stress in bacteria,” Proceedings of the National Academy of Sciences of the United States of America, vol. 102, no. 39, pp. 13855–13860, 2005.
[20]
K. Shatalin, I. Gusarov, E. Avetissova et al., “Bacillus anthracis-derived nitric oxide is essential for pathogen virulence and survival in macrophages,” Proceedings of the National Academy of Sciences of the United States of America, vol. 105, no. 3, pp. 1009–1013, 2008.
[21]
N. M. van Sorge, F. C. Beasley, I. Gusarov, et al., “Methicillin-resistant Staphylococcus aureus bacterial nitric oxide synthase affects antibiotic sensitivity and skin abscess development,” The Journal of Biological Chemistry, 2013.
[22]
M. J. Horsburgh, J. L. Aish, I. J. White, L. Shaw, J. K. Lithgow, and S. J. Foster, “δb modulates virulence determinant expression and stress resistance: characterization of a functional rsbU strain derived from Staphylococcus aureus 8325-4,” Journal of Bacteriology, vol. 184, no. 19, pp. 5457–5467, 2002.
[23]
D. A. Mead, E. Szczesna-Skorupa, and B. Kemper, “Single-stranded DNA “blue” t7 promoter plasmids: a versatile tandem promoter system for cloning and protein engineering,” Protein Engineering, Design and Selection, vol. 1, no. 1, pp. 67–74, 1986.
[24]
V. K. Singh, D. S. Hattangady, E. S. Giotis et al., “Insertional inactivation of branched-chain α-keto acid dehydrogenase in Staphylococcus aureus leads to decreased branched-chain membrane fatty acid content and increased susceptibility to certain stresses,” Applied and Environmental Microbiology, vol. 74, no. 19, pp. 5882–5890, 2008.
[25]
V. K. Singh, S. Utaida, L. S. Jackson, R. K. Jayaswal, B. J. Wilkinson, and N. R. Chamberlain, “Role for dnaK locus in tolerance of multiple stresses in Staphylococcus aureus,” Microbiology, vol. 153, no. 9, pp. 3162–3173, 2007.
[26]
J. Augustin, R. Rosenstein, B. Wieland et al., “Genetic analysis of epidermin biosynthetic genes and epidermin-negative mutants of Staphylococcus epidermidis,” European Journal of Biochemistry, vol. 204, no. 3, pp. 1149–1154, 1992.
[27]
V. K. Singh, M. Syring, A. Singh, K. Singhal, A. Dalecki, and T. Johansson, “An insight into the significance of the DnaK heat shock system in Staphylococcus aureus,” International Journal of Medical Microbiology, vol. 302, no. 6, pp. 242–252, 2012.
[28]
V. K. Singh, R. K. Jayaswal, and B. J. Wilkinson, “Cell wall-active antibiotic induced proteins of Staphylococcus aureus identified using a proteomic approach,” FEMS Microbiology Letters, vol. 199, no. 1, pp. 79–84, 2001.
[29]
S. J. Collins, “The HL-60 promyelocytic leukemia cell line: proliferation, differentiation, and cellular oncogene expression,” Blood, vol. 70, no. 5, pp. 1233–1244, 1987.
[30]
C. Tarella, D. Ferrero, and E. Gallo, “Induction of differentiation of HL-60 cells by dimethyl sulfoxide: evidence for a stochastic model not linked to the cell division cycle,” Cancer Research, vol. 42, no. 2, pp. 445–449, 1982.
