Numerous factors have, to date, been identified as playing a role in the regulation of Agr activity in Staphylococcus aureus, including transcription factors, antisense RNAs, and host elements. Herein we investigated the product of SAUSA300_ 1984 (termed MroQ), a transmembrane Abi-domain/M79 protease-family protein, as a novel effector of this system. Using a USA300 mroQ mutant, we observed a drastic reduction in proteolysis, hemolysis, and pigmentation that was fully complementable. This appears to result from diminished agr activity, as transcriptional analysis revealed significant decreases in expression of both RNAII and RNAIII in the mroQ mutant. Such effects appear to be direct, rather than indirect, as known agr effectors demonstrated limited alterations in their activity upon mroQ disruption. A comparison of RNA sequencing data sets for both mroQ and agr mutants revealed a profound overlap in their regulomes, with the majority of factors affected being known virulence determinants. Importantly, the preponderance of alterations in expression were more striking in the agr mutant, indicating that MroQ is necessary, but not sufficient, for Agr function. Mechanism profiling revealed that putative residues for metalloprotease activity within MroQ are required for its Agr-controlling effect; however, this was not wielded at the level of AgrD processing. Virulence assessment demonstrated that both mroQ and agr mutants exhibited increased formation of renal abscesses but decreased skin abscess formation alongside diminished dermonecrosis. Collectively, we present the characterization of a novel agr effector in S. aureus which would appear to be a direct regulator, potentially functioning via interaction with the AgrC histidine kinase.

17Numerous factors have to date been identified as playing a role in the regulation of Agr 18 activity in S. aureus, including transcription factors, antisense RNAs, and host elements. 19Herein we investigate the product of SAUSA300_1984 (termed MroQ), a transmembrane 20Abi-domain/M79 protease-family protein, as a novel effector of this system. Using a 21 USA300 mroQ mutant we observed a drastic reduction in proteolysis, hemolysis and 22 pigmentation that was fully complementable. This appears to result from diminished agr 23 activity, as transcriptional analysis revealed significant decreases in expression of both 24 RNAII and RNAIII in the mroQ mutant. Such effects appear to be direct, rather than 25 indirect, as known agr effectors demonstrated limited alterations in their activity upon 26 mroQ disruption. A comparison of RNA-sequencing datasets for both mroQ and agr 27 mutants reveal a profound overlap in their regulomes, with the majority of factors affected 28 being known virulence determinants. Importantly, the preponderance of alterations in 29 expression were more striking in the agr mutant, indicating that MroQ is necessary, but 30 not sufficient, for Agr function. Mechanism profiling revealed that putative residues for 31 metalloprotease activity within MroQ are required for its Agr controlling effect, however 32 this is not wielded at the level of AgrD processing. Virulence assessment demonstrated 33 that mroQ and agr mutants both exhibited increased formation of renal abscesses, but 34 decreased skin abscess formation, alongside diminished dermonecrosis. Collectively, we 35 present the characterization of a novel agr effector in S. aureus, which would appear to 36 be a direct regulator, potentially functioning via interaction with the AgrC histidine kinase. 37Pathogenicity in S. aureus is both diverse and tightly controlled, being highly adaptive to 39 the surrounding environment and available resources (1). In order to survive and persist 40 within the host, this pathogen produces a wide array of virulence factors throughout the 41 different stages of infection (2). For example, during earlier phases of growth, adhesins 42 and other binding proteins are actively synthesized (e.g. Spa, FnbAB), which play a role 43 in colonization (2, 3). In contrast, in later phases, these surface proteins are repressed, 44 whilst secreted toxins, proteases, and exoenzymes are produced to facilitate invasion and 45 dissemination (2, 4). This coordination is facilitated by an array of global regulators of 46 gene expression (2, 5-10). 47 48The agr quorum sensing system is one of the most important regulators of virulence in S. 49 aureus (11). It is a quorum sensing system comprised of two transcriptional units, RNAII 50 and RNAIII, each controlled by its own promoter, P2 and P3, respectively. RNAII encodes 51 the agr operon, which is comprised of four genes, agrBDCA (12). The quorum sensing 52 system functions in three parts: i) a quorum sensing module comprised of AgrB and AgrD; 53ii) a two-component system (TCS) Agr...

No abstract