The success of S. aureus as a human pathogen can be attributed to its array of virulence determinants and their precisely-timed regulation. A primary manner in which S. aureus cells control the production of these factors is via the action of secreted proteases. These enzymes are produced alongside toxins and exoenzymes to control their stability, thus facilitating coordinated titration of virulence factors to suit niche specific situations. Consequently, the secreted proteases of S. aureus can be considered master regulators of virulence, that require tight regulation to facilitate infection. As part of our longstanding interest in secreted proteases, we recently discovered a mutation in SAUSA300_1984 that results in impaired proteolytic activity. Further study revealed that deletion of this gene also resulted in ablated production of other secreted virulence factors, whilst demonstrating enhanced production of surface displayed components. These phenotypes were accompanied by a significantly more robust biofilm formed by the mutant strain. Collectively, these findings phenocopy that observed for impaired activity of the central regulator of virulence in S. aureus, the agr system. Transcriptional profiling in the 1984 mutant revealed a decrease in expression for each gene within the agr locus, and a decrease in transcription for downstream effectors of the Agr system. At the same time, known regulators of Agr were found to be unchanged in their activity, suggesting that SAUSA300_1984 influences protease and virulence factor production directly via agr. This hypothesis is supported by the observation that 1984 is located 3.5KB upstream of agr, and belongs to the Abi-domain family proteins, which have been shown to interact with histidine kinases in various Gram-positive bacteria. As such, our hypothesis is that SAUSA300_1984 exerts its influence in S. aureus via interaction with the AgrC sensor kinase. To explore this further, we will: 1. Elucidate the role of SAUSA300_1984 in the regulation of virulence determinants via agr. This will be achieved by quantifying the abundance of Agr system components using mass spec, followed by bacterial-two hybrid to confirm interaction partners. We will also identify domains of function in 1984 using the substituting cysteine accessibility method. 2. Determine the role of additional, uncharacterized Abi- domain proteins in S. aureus on virulence factor production. A previous study has shown that Abi- domain proteins influence production of surface exposed virulence factors in S. aureus. As such, we will identify how these proteins influence virulence factor production using transcriptomic and proteomic approaches, before assessing virulence in murine models of infection. As ABI domain proteins are conserved across Gram-positive pathogens, our findings can be applied to an array of other organisms, deepening our understanding of how virulence factors are regulated. Furthermore, our work on Abi-domain proteins could be used for novel anti-virulence based therapeutic strategies in the future.
Staphylococcus aureus is a highly virulent and widely successful pathogen that is the most common cause of infectious disease and death in the United States. With the continued emergence of multi-drug resistant isolates of this organism (such as MRSA), there is an urgent need to understand the mechanisms by which this deadly pathogen causes disease. This proposal explores this by seeking to understand the function of an ABI-type protein, and its role in S. aureus disease causation.
| Fleeman, Renee M; Debevec, Ginamarie; Antonen, Kirsten et al. (2018) Identification of a Novel Polyamine Scaffold With Potent Efflux Pump Inhibition Activity Toward Multi-Drug Resistant Bacterial Pathogens. Front Microbiol 9:1301 |
