Contribution of the nos-pdt Operon to Virulence Phenotypes in Methicillin-Sensitive Staphylococcus aureus

Abstract

Nitric oxide (NO) is emerging as an important regulator of bacterial stress resistance, biofilm development, and virulence. One potential source of endogenous NO production in the pathogen Staphylococcus aureus is its NO-synthase (saNOS) enzyme, encoded by the nos gene. Although a role for saNOS in oxidative stress resistance, antibiotic resistance, and virulence has been recently-described, insights into the regulation of nos expression and saNOS enzyme activity remain elusive. To this end, transcriptional analysis of the nos gene in S. aureus strain UAMS-1 was performed, which revealed that nos expression increases during low-oxygen growth and is growth-phase dependent. Furthermore, nos is co-transcribed with a downstream gene, designated pdt, which encodes a prephenate dehydratase (PDT) enzyme involved in phenylalanine biosynthesis. Deletion of pdt significantly impaired the ability of UAMS-1 to grow in chemically-defined media lacking phenylalanine, confirming the function of this enzyme. Bioinformatics analysis revealed that the operon organization of nos-pdt appears to be unique to the staphylococci. As described for other S. aureus nos mutants, inactivation of nos in UAMS-1 conferred sensitivity to oxidative stress, while deletion of pdt did not affect this phenotype. The nos mutant also displayed reduced virulence in a murine sepsis infection model, and increased carotenoid pigmentation when cultured on agar plates, both previously-undescribed nos mutant phenotypes. Utilizing the fluorescent stain 4-Amino-5-Methylamino-2',7'-Difluorofluorescein (DAF-FM) diacetate, decreased levels of intracellular NO/reactive nitrogen species (RNS) were detected in the nos mutant on agar plates. These results reinforce the important role of saNOS in S. aureus physiology and virulence, and have identified an in vitro growth condition under which saNOS activity appears to be upregulated. However, the significance of the operon organization of nos-pdt and potential relationship between these two enzymes remains to be elucidated.

Evidence for Nitric Oxide Synthase Activity in Staphylococcus xylosus Mediating Nitrosoheme Formation

“…The S. xylosus C2a Δ nos mutant displayed higher colony pigmentation than the wild-type strain after prolonged cultivation on agar medium, as observed for the S. aureus Δ nos strain UAMS-1 (Sapp et al, 2014). S. aureus produced the intermediary yellow carotenoid 4,4′-diaponeurosporene, which, after prolonged cultivation, is converted to the yellow-orange end-product staphyloxanthin (Wieland et al, 1994).…”

“…The Δ nos mutant was unable to produce NO under aerobic conditions. The loss of NOS activity in S. aureus UAMS-1 mutant led to NO production of 65% as evaluated using fluorescent stain DAF-FM diacetate after growth on agar plates (Sapp et al, 2014), while the deletion of nos in S. aureus UAMS1182 completely abolished NO production after growth under aerobic conditions (van Sorge et al, 2013). These results revealed that some Staphylococcus strains are able to produce NO by other pathways under limited oxygenation.…”

“…NOS is involved in the production of NO by Bacillus subtilis (Gusarov and Nudler, 2005; Gusarov et al, 2008), Bacillus anthracis (Shatalin et al, 2008), Bacillus cereus (Montgomery et al, 2010), Geobacillus stearothermophilus (Kabir et al, 2008), Streptomyces turgidiscabies (Kers et al, 2004), Deinococcus radiodurans (Patel et al, 2009), and Staphylococcus aureus (van Sorge et al, 2013). S. aureus NOS is involved in resistance to oxidative stress and antibiotics, and virulence (Vaish and Singh, 2013; van Sorge et al, 2013; Sapp et al, 2014). In all sequenced S. aureus genomes, the nos gene is part of a cluster containing another gene encoding a prephenate dehydratase, which is involved in phenylalanine biosynthesis.…”

“…In all sequenced S. aureus genomes, the nos gene is part of a cluster containing another gene encoding a prephenate dehydratase, which is involved in phenylalanine biosynthesis. This genetic organization appears to be unique to staphylococcal genomes (Sapp et al, 2014). The nos genes have been identified in many staphylococci (van Sorge et al, 2013; Sánchez-Mainar et al, 2014; Sapp et al, 2014), but NOS-mediated NO production has only been characterized in the pathogenic S. aureus .…”

“…This genetic organization appears to be unique to staphylococcal genomes (Sapp et al, 2014). The nos genes have been identified in many staphylococci (van Sorge et al, 2013; Sánchez-Mainar et al, 2014; Sapp et al, 2014), but NOS-mediated NO production has only been characterized in the pathogenic S. aureus . The contribution of the NOS activity of S. xylosus to formation of nitrosomyoglobin remains to be demonstrated.…”