The Role of SSR42 in Virulence Factor Regulation in S. aureus - ABSTRACT SSR42 is a 1,233 nt regulatory RNA bearing no ORFs, that exhibits enhanced stability during stationary growth and maintains extensive nucleotide sequence conservation (>98% across diverse strains). Characterization of SSR42 by others has shown that SSR42 mutants are impaired in their ability to lyse rabbit- and sheep- erythrocytes, have reduced survival upon challenge by neutrophils, and engender limited intracellular toxicity towards HeLa cells. Additionally, an SSR42 mutant has significantly decrease bacterial burden in a murine model of skin abscess formation. Herein, we demonstrate that an SSR42 mutant also has impaired hemolytic activity towards human erythrocytes, limited cytolytic activity towards human leukocytes, and increased proteolytic capacity. In addition, SSR42 also regulates numerous secreted virulence factors known to be central to S. aureus infection. Furthermore, our SSR42 mutant has a decreased ability to survive and disseminate in the liver, heart, lungs, and spleen of mice during sepsis, and decreased viability during murine pneumonia, all by several orders of magnitude. Previous work by our group revealed that SSR42 is found only in S. aureus strains. Not only this, but it is the most highly expressed, S. aureus-specific transcript in stationary phase cells. Indeed, only RNAIII is present in more copies after 15h growth. This means that 20% of the total RNA (minus rRNA) in stationary phases cells belongs to SSR42, and 45% to SSR42 and RNAIII combined. The importance of the Agr system, and thus RNAIII, is clear and undeniable; thus the paucity of knowledge for how SSR42 mediates its role, and is itself controlled, demonstrates a major knowledge gap in S. aureus pathogenesis research. Accordingly, in the present application we will dissect the role and regulation of this important, yet understudied component of the S. aureus regulatory machinery by: Aim #1: Determining the Mechanism by which SSR42 Regulates Virulence Factor Production: We will perform RNA EMSAs coupled with mutational genetics to explore SSR42 mediated RNA:RNA interaction. We will then determine the importance of such events in the S. aureus cell. Aim #2: Defining the Impact of SSR42 on S. aureus Virulence Factors Expression in vivo: Next, we will perform a longitudinal study of virulence factor production by S. aureus during both localized and disseminated disease to understand the impact of SSR42 on the severity and timing of infection. This will be achieved using cutting edge mass-spectrometry to detect virulence factors in infected tissues. Aim #3: Dissecting the Hierarchical Control of SSR42: Given the powerful nature of SSR42 as a regulator, and its profound ability to impact infection, we consider it of primary importance to understand how it too is controlled. Herein we have generated a testable map of SSR42 regulation using a novel high-throughput screen which we will interrogate via classical genetic approaches, providing a comprehensive understanding of SSR42 regulation.
