Methicillin-resistant Staphylococcus aureus is estimated to cause more U.S. deaths annually than HIV/AIDS. The emergence of hypervirulent and multidrug-resistant strains has further amplified public health concern and accentuated the need for new classes of antibiotics. RNA degradation is a required cellular process that could be exploited for novel antimicrobial drug development. However, such discovery efforts have been hindered because components of the Gram-positive RNA turnover machinery are incompletely defined. In the current study we found that the essential S. aureus protein, RnpA, catalyzes rRNA and mRNA digestion in vitro. Exploiting this activity, high through-put and secondary screening assays identified a small molecule inhibitor of RnpA-mediated in vitro RNA degradation. This agent was shown to limit cellular mRNA degradation and exhibited antimicrobial activity against predominant methicillin-resistant S. aureus (MRSA) lineages circulating throughout the U.S., vancomycin intermediate susceptible S. aureus (VISA), vancomycin resistant S. aureus (VRSA) and other Gram-positive bacterial pathogens with high RnpA amino acid conservation. We also found that this RnpA-inhibitor ameliorates disease in a systemic mouse infection model and has antimicrobial activity against biofilm-associated S. aureus. Taken together, these findings indicate that RnpA, either alone, as a component of the RNase P holoenzyme, and/or as a member of a more elaborate complex, may play a role in S. aureus RNA degradation and provide proof of principle for RNA catabolism-based antimicrobial therapy.

To investigate the regulatory role of traP (target of RNAIII-activating peptide) in Staphylococcus aureus, we generated traP mutations in the clinical isolates UAMS-1 and USA300. In neither case did mutation of traP affect expression of the accessory gene regulator (agr) or the ability to form a biofilm. We were also unable to confirm that mutation of traP in the prototype 8325-4 laboratory strain RN6390 results in reduced expression of agr, reduced hemolytic activity, or an altered capacity to form a biofilm.There is a growing body of literature indicating that mutation or inhibition of the Staphylococcus aureus regulatory locus designated traP (target of RNAIII-activating peptide) results in reduced expression of the accessory gene regulator (agr), a reduced capacity to form a biofilm, and reduced virulence in animal models of staphylococcal disease (3,5,6,17,23,28). The traP gene reportedly encodes a response regulator (TraP) that is triply phosphorylated as a result of the accumulation of a protein designated RAP (RNAIII-activating peptide), resulting in activation of the agr regulatory system and induction of toxin synthesis (21). A number of studies have also concluded that clinical isolates of S. aureus are responsive to RAP and that a modified peptide derivative designated RIP (RNAIIIinhibiting peptide) can be used to limit phosphorylation of TraP and thereby limit induction of agr, the capacity to form a biofilm, and virulence (1,3,12,14,24).To the extent that both RAP and RIP reportedly function by modulating the activity of TraP, studies indicating that clinical isolates are responsive to RAP and RIP would suggest that the traP regulatory system functions in the same fashion in most if not all S. aureus strains. However, the primary focus to date, particularly in terms of direct mutagenesis studies, has been on derivatives of the prototype 8325-4 laboratory strain such as RN6390, and recent studies from our laboratory have confirmed that regulatory circuits in RN6390 are different than those observed in at least some clinical isolates (9, 11). One such difference is the impact of agr on biofilm formation. Specifically, mutation of agr enhances biofilm formation in RN6390 but has little impact in clinical isolates (7,29). Since TraP reportedly functions through an agr-dependent pathway, we wanted to investigate whether similar, strain-dependent differences also existed with respect to traP. To that end, we mutated traP in two clinical isolates (UAMS-1 and USA300-0114) as well as our version of the prototype laboratory strain RN6390. The impact of mutating traP in all three strains was assessed based on biofilm formation, hemolytic activity, and production of the agr-encoded regulatory molecule RNAIII.

In a previous study, two proteins identified as hyaluronidases were detected in spent media by MS and found to be in greater quantity in the sarA and sarA agr mutant strains when compared with the parent and agr mutant strains of Staphylococcus aureus UAMS-1. In the present study, spent media and total RNA were isolated from UAMS-1 and its regulatory mutants and analysed for hyaluronidase activity and steady-state hyaluronidase (hysA) RNA message levels. Hyaluronidase activity was observed throughout all time points examined regardless of the regulatory effects of sarA and agr but activity was always substantially higher in the sarA and sarA agr mutant strains than in the UAMS-1 parent and agr mutant strains. Northern analysis did not detect hysA message for either the UAMS-1 parent or the agr mutant strains at any time point examined, while steady-state hysA message levels were detected throughout growth for the sarA mutant strain, but only at exponential and early post-exponential growth for the sarA agr mutant strain. An in vitro biofilm plate assay, pre-coated with human plasma as a source of hyaluronic acid, demonstrated no significant increase in biofilm for a sarA mutant strain of S. aureus UAMS-1 defective in hyaluronidase activity when compared with the sarA mutant strain. These data indicate that, while hysA message levels and hyaluronidase activity are elevated in the sarA mutant strains of S. aureus UAMS-1, the increase in activity did not contribute to the biofilm-negative phenotype observed in the sarA mutant strain of S. aureus UAMS-1. INTRODUCTIONStaphylococcus aureus is a Gram-positive bacterium of both community-associated and hospital-acquired diseases that accounts for 14.9 and 18.8 % of all bacterial infections encountered in the clinical setting as either outpatient or inpatient cases, respectively (Styers et al., 2006). Just as important and of great concern is the continual rise in the number of meticillin (oxacillin)-resistant S. aureus (MRSA) encountered in the hospital setting and now in the community (Diekema et al., 2001;Loffler & MacDougall, 2007;Saïd-Salim et al., 2003;Styers et al., 2006). This concern is compounded further by the occurrence of clinical strains resistant to intermediate (Hiramatsu et al., 1997;Srinivasan et al., 2002) and high (Chang et al., 2003;Tenover et al., 2004;Whitener et al., 2004) levels of vancomycin, an antibiotic of last resort for infections involving multiple antibiotic-resistant, hospital-acquired MRSA (Maple et al., 1989).As an aid to the discovery of new potential targets for the development of alternative approaches for the prevention and treatment of staphylococcal diseases, a number of laboratories have utilized whole-genome sequencing and microarray and proteomic technologies to generate comprehensive databases of S. (Hynes & Walton, 2000). For example, the assumption has been that the glycoside hydrolase activity exhibited by hyaluronidase (Mu toxin) and other toxins produced by Clostridium perfringens contributes to virulence by destroying ...