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This study examines the alteration in Staphylococcus aureus gene expression following treatment with the type 2 fatty acid synthesis inhibitor AFN-1252. An Affymetrix array study showed that AFN-1252 rapidly increased the expression of fatty acid synthetic genes and repressed the expression of virulence genes controlled by the SaeRS 2-component regulator in exponentially growing cells. AFN-1252 did not alter virulence mRNA levels in a saeR deletion strain or in strain Newman expressing a constitutively active SaeS kinase. AFN-1252 caused a more pronounced increase in fabH mRNA levels in cells entering stationary phase, whereas the depression of virulence factor transcription was attenuated. The effect of AFN-1252 on gene expression in vivo was determined using a mouse subcutaneous granuloma infection model. AFN-1252 was therapeutically effective, and the exposure (area under the concentration-time curve from 0 to 48 h [AUC(0-48)]) of AFN-1252 in the pouch fluid was comparable to the plasma levels in orally dosed animals. The inhibition of fatty acid biosynthesis by AFN-1252 in the infected pouches was signified by the substantial and sustained increase in fabH mRNA levels in pouch-associated bacteria, whereas depression of virulence factor mRNA levels in the AFN-1252-treated pouch bacteria was not as evident as it was in exponentially growing cells in vitro. The trends in fabH and virulence factor gene expression in the animal were similar to those in slower-growing bacteria in vitro. These data indicate that the effects of AFN-1252 on virulence factor gene expression depend on the physiological state of the bacteria.

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.