Methylthioadenosine/S-adenosylhomocysteine nucleosidase (Pfs) of Staphylococcus aureus is essential for the virulence independent of LuxS/AI-2 system

Abstract

Staphylococcus aureus is a major cause of infectious morbidity and mortality in both community and hospital settings. The bacterium continues to cause diverse invasive, life-threatening infections, such as pneumonia, endocarditis, and septicemia. Methylthioadenosine/S-adenosylhomocysteine nucleosidase (Pfs) is predicted to be an important enzyme involved in methylation reactions, polyamine synthesis, vitamin synthesis, and quorum sensing pathways. For the first time, we demonstrate that Pfs is essential for the virulence of S. aureus. The pfs mutant strain, as compared to the isogenic wild type, displayed a decreased production of extracellular proteases, which was correlated with a dramatic decrease in the expression of the sspABC operon and a moderate decrease of aur expression. The mouse model of sepsis and subcutaneous abscesses indicated that the pfs mutant strain displayed highly impaired virulence compared to the isogenic wild type. The decreased virulence of the pfs mutant strain is in correspondence with its decreased proliferation in vivo, indicated with a real-time analysis in the transparent system of zebrafish embryos. These phenotypes of the pfs mutant strain are LuxS/AI-2 independent despite the essential role pfs plays in AI-2 production. Our data suggest that Pfs is a potential novel target for anti-infection therapy.

Introduction

Staphylococcus aureus can induce diverse invasive, life-threatening infections, such as pneumonia, endocarditis and septicaemia (Bubeck Wardenburg et al., 2007, Panizzi et al., 2011), and is a major cause of infectious morbidity and mortality in both community and hospital settings (Boucher and Corey, 2008, David and Daum, 2010). The worldwide emergence of antibiotic resistant strains continues unabated, along with an overall increase in the number of infections worldwide (Fridkin et al., 2005, Jain et al., 2011), highlighting the urgent need for new agents for the treatment of S. aureus infection. Non-conventional anti-infective approaches have been explored that are non-lethal to bacteria, where the potential to develop resistance is assumed to be less significant (Maresso and Schneewind, 2008, Wyatt et al., 2010).

As an activated group donor, S-adenosylmethionine (SAM) is essential in a broad array of metabolic reactions, such as methylation reactions, polyamine synthesis, SAM radical-mediated vitamin synthesis, and N-acyl-homoserine lactone (autoinducer-1) synthesis (Parveen and Cornell, 2011) (Fig. 1). Methylthioadenosine (MTA) (Pajula and Raina, 1979), S-adenosylhomocysteine (SAH) (Simms and Subbaramaiah, 1991), and 5′-deoxyadenosine (5′dADO) (Choi-Rhee and Cronan, 2005) are product inhibitors of these reactions, and MTA/SAH nucleosidase (Pfs) is the enzyme that catalyses their irreversible hydrolytic deadenylation reaction in bacteria (Della Ragione et al., 1985). Methylthioadenosine/S-adenosylhomocysteine nucleosidase is widespread among bacteria (Winzer et al., 2002). Sun and co-workers showed that 51 out of 138 bacterial species with completely sequenced genomes possess cytoplasmic MTA/SAH nucleosidase (Sun et al., 2004). It was reported that the inhibition of MTA/SAH nucleosidase activity led to an accumulation of MTA and SAH within bacterial cells (Heurlier et al., 2009) and ended into the inhibition of SAM-dependent synthase activities. In addition, Pfs is involved in the recycling pathway of adenine, sulphur, and methionine, and it also produces the universal quorum-sensing signal, autoinducer-2 (AI-2) (Heurlier et al., 2009).

On the basis of its importance in a wide array of metabolic reactions and as an enzyme present in most bacterial species but absent in humans, Pfs is an attractive target for developing new classes of broad-spectrum inhibitors for the treatment of bacterial infections (Parveen and Cornell, 2011). An evaluation of the substrate analogs and transition state analogs effective against MTA/SAH nucleosidases of Borrelia burgdorferi, which uniquely expresses three homologous functional enzymes (Fraser et al., 1997, Parveen et al., 2006, Parveen and Leong, 2000), led to the identification of compounds that either inhibited the growth of these spirochaetes or showed bactericidal activities (Cornell et al., 2009). Using transition state analogs, the role of Pfs inhibitors has been explored in pathogenic strains of Vibrio cholera and Escherichia coli, where they can inhibit AI-2 production and reduce biofilm formation, but with minimal effect on bacterial growth (Gutierrez et al., 2009). These results indicate that the inhibition of this enzyme can affect the physiological activities of different bacteria. New inhibitors against Pfs are currently being explored for the development of potential novel broad-spectrum antimicrobials. However, despite this, the importance of Pfs independent of AI-2 remains generally underappreciated. Up to now, nothing is known about the role Pfs plays in the virulence of bacteria.

In S. aureus, the structure of Pfs was determined in a complex with the transition-state analog formycin A at a 1.7 Å resolution (Siu et al., 2008). It was highly conserved in the active-site residues and revealed an identical mode of inhibitor binding with available E. coli Pfs structures (Siu et al., 2008). Furthermore, it was confirmed that Pfs of S. aureus displays MTA and SAH nucleosidase activity (Siu et al., 2008). Besides this, the biological role of Pfs in the Staphylococcaceae family remains unknown.

AI-2, shared by both Gram-positive and Gram-negative bacteria, is generally considered to be a universal language for intraspecies and interspecies communication (Vendeville et al., 2005). S. aureus possesses a functional luxS gene, which has been proved to be essential for AI-2 production (Doherty et al., 2006, Winzer et al., 2002). In addition, S. aureus LuxS/AI-2 system has been reported to regulate a range of behaviors, such as virulence-associated traits (Zhao et al., 2010), biofilm formation (Yu et al., 2012), and susceptibility to cell wall synthesis inhibitor antibiotics (Xue et al., 2013).

In this study, we aim to prove that Pfs is essential for the virulence of S. aureus. A molecular genetics approach of targeted mutagenesis was used, and the pfs mutant strain was compared to the isogenic wild type strain with respect to the pathogenesis-related traits. It was found that the pfs mutation decreased the extracellular proteases expression of S. aureus. And the animal models of mouse and zebrafish were used to confirm that Pfs contributes to the pathogenicity of S. aureus. In addition, our results show that despite the essential role pfs plays in AI-2 production, these phenotypes of the pfs mutant strain is LuxS/AI-2 independent.