MroQ is a Novel Abi-domain Protein That Influences Virulence Gene Expression in Staphylococcus aureus via Modulation of Agr Activity

Abstract

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...

Lipoprotein N -Acylation in Staphylococcus aureus Is Catalyzed by a Two-Component Acyl Transferase System

“…Many bacteria encode multiple CPBPs, with S. aureus NCTC8325 containing at least six other CPBP enzymes in addition to LnsB ( 62 ). Of these, MroQ is a suspected protease that processes auto-inducing peptide ( 63 , 64 ). There is phenotypic evidence for roles of four other staphylococcal CPBPs in maintaining cell envelope integrity ( 65 ) and in the expression of cell wall-attached surface proteins with YSIRK peptide signals ( 66 ).…”

Analysis of the CodY RNome reveals RsaD as a stress‐responsive riboregulator of overflow metabolism in Staphylococcus aureus

“…The current thought is that reduced respiration due to NO exposure or other stresses results in the accumulation of reduced menaquinone and triggers SrrAB activity (Mashruwala & Boyd, ; Mashruwala, Guchte, & Boyd, ). Another recent study examined global changes in transcript abundance in an ∆ agr mutant and measured an increase in rsaD expression (Marroquin et al, ), indicating that RsaD production is controlled by population density. The mechanisms by which this integration is achieved remain unexplored but are the current focus of our lab.…”

“…However, in Gram‐positive bacteria like S. aureus , regulatory RNAs are still an emerging field; only a relatively small number of the known sRNAs have established biological functions (Broach, Weiss, & Shaw, ; Guillet, Hallier, & Felden, ; Mars, Nicolas, Denham, & Dijl, ; Quereda & Cossart, ). For instance, RNAIII—the sRNA effector of the accessory gene regulator (Agr) quorum sensing system—adjusts toxin expression in response to population density via the regulation of many direct and indirect targets (Balaban & Novick, ; Boisset et al, ; Cheung, Wang, Khan, Sturdevant, & Otto, ; Marroquin et al, ; Novick et al, ; Queck et al, ; Recsei et al, ). Other sRNAs in S. aureus have been discovered to mediate responses to changing environmental conditions (Nielsen et al, ), prompt the synthesis of virulence‐associated factors (Manna et al, ; Romilly et al, ; Zapf et al, ), influence antibiotic resistance (Eyraud, Tattevin, Chabelskaya, & Felden, ), and adjust metabolism during growth (Bohn et al, ; Geissmann et al, ).…”

cydA, spdC, and mroQ are novel genes involved in the plasma coagulation of Staphylococcus aureus

“…The possible mechanism involves hydrolysis of auto‐inducing peptide by mroQ coding synthase, resulting in the inactivation of agrC . This subsequently inactivated the function of downstream RNAII and RNAIII, and the regulated genes, including coagulase, showed no expression 23,24 …”