Exploring the Influence of an Intracellular Aminopeptidase on S. aureus Virulence

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DESCRIPTION (provided by applicant): Staphylococcus aureus is a highly virulent and widely successful pathogen, which is speculated to be the most common cause of human disease. Currently, it is a leading agent of both community and hospital- acquired infections worldwide, causing a variety of ailments in a plethora of ecological niches within the host. With the advent of antibiotic resistance, and the emergence of clinical isolates resistant to last resort antibiotis, a thorough exploration of the pathogenic mechanisms employed by this organism is urgently required. Our group has previously published a number of reports documenting the role of secreted proteases as virulence factors in S. aureus infections. However, until now, the intracellular proteolytic enzymes of this dangerous bacterium have not been explored as virulence affecting entities. During a screen in our lab on the impact of such enzymes, we identified a mutant in an intracellular leucine aminopeptidase (LAP, pepZ) that strongly influences the pathogenic potential of S. aureus. Specifically we demonstrated significant attenuation of pepZ mutants using laboratory and clinical strains, localized and systemic infections, and human and murine models of disease. These findings should not be undersold: few intracellular aminopeptidases have ever been shown to contribute to bacterial virulence; making the S. aureus LAP highly unique. Critically, it should be noted that the observed attenuation (i) does not result from a simple growth defect; and (ii) is not a common feature for other aminopeptidase in S. aureus. Enzymatically, aminopeptidases cleave N-terminal amino acids from protein substrates. Thus, our central hypothesis is that pepZ mutant cells fail to process key cellular targets, leading to decreased fitness, and pathogenesis. This is supported by preliminary data that reveals alterations in intracellular and extracellular proteomes upon pepZ disruption. Accordingly, we will explore the contribution of LAP to S. aureus disease causation by 1. Identifying LAP Targets within S. aureus Cells: This will be achieved by mapping the extended substrate specificity of LAP, before identifying pathophysiological substrates using proteomic tools. The affinity and reaction kinetics of LAP interaction with identified substrates will also be explored. We will then 2. Determine the influence of LAP and PrsA on virulence determinant secretion: Preliminary data suggests that secretomes are markedly affected in pepZ mutants, and that this might be mediated by LAP targeting the PrsA foldase. As PrsA is known to mediate protein secretion and virulence in a variety of Gram-positive pathogens, we will explore the influence of LAP on PrsA processing and trafficking, before assessing the role of both proteins in virulence determinant secretion. We contend that this analysis will provide a unique insight into a novel enzyme, enable us to better understand the pathogenic properties of S. aureus, and may aid in the rational development of new therapeutic treatments.