Mutation of traP in Staphylococcus aureus Has No Impact on Expression of agr or Biofilm Formation

Abstract

To investigate the regulatory role of traP (target of RNAIII-activating peptide) in Staphylococcus aureus, we generated traP mutations in the clinical isolates UAMS-1 and USA300. In neither case did mutation of traP affect expression of the accessory gene regulator (agr) or the ability to form a biofilm. We were also unable to confirm that mutation of traP in the prototype 8325-4 laboratory strain RN6390 results in reduced expression of agr, reduced hemolytic activity, or an altered capacity to form a biofilm.There is a growing body of literature indicating that mutation or inhibition of the Staphylococcus aureus regulatory locus designated traP (target of RNAIII-activating peptide) results in reduced expression of the accessory gene regulator (agr), a reduced capacity to form a biofilm, and reduced virulence in animal models of staphylococcal disease (3,5,6,17,23,28). The traP gene reportedly encodes a response regulator (TraP) that is triply phosphorylated as a result of the accumulation of a protein designated RAP (RNAIII-activating peptide), resulting in activation of the agr regulatory system and induction of toxin synthesis (21). A number of studies have also concluded that clinical isolates of S. aureus are responsive to RAP and that a modified peptide derivative designated RIP (RNAIIIinhibiting peptide) can be used to limit phosphorylation of TraP and thereby limit induction of agr, the capacity to form a biofilm, and virulence (1,3,12,14,24).To the extent that both RAP and RIP reportedly function by modulating the activity of TraP, studies indicating that clinical isolates are responsive to RAP and RIP would suggest that the traP regulatory system functions in the same fashion in most if not all S. aureus strains. However, the primary focus to date, particularly in terms of direct mutagenesis studies, has been on derivatives of the prototype 8325-4 laboratory strain such as RN6390, and recent studies from our laboratory have confirmed that regulatory circuits in RN6390 are different than those observed in at least some clinical isolates (9, 11). One such difference is the impact of agr on biofilm formation. Specifically, mutation of agr enhances biofilm formation in RN6390 but has little impact in clinical isolates (7,29). Since TraP reportedly functions through an agr-dependent pathway, we wanted to investigate whether similar, strain-dependent differences also existed with respect to traP. To that end, we mutated traP in two clinical isolates (UAMS-1 and USA300-0114) as well as our version of the prototype laboratory strain RN6390. The impact of mutating traP in all three strains was assessed based on biofilm formation, hemolytic activity, and production of the agr-encoded regulatory molecule RNAIII.

A Nonsense Mutation in agrA Accounts for the Defect in agr Expression and the Avirulence of Staphylococcus aureus 8325-4 traP :: kan

“…This mutation is responsible for the failure of the traP mutant to express agr, for its avirulence in the murine abscess model (5), and for the identity of the traP transcriptome with that of agr (7). These findings are entirely consistent with two other reports (15,20) demonstrating that traP mutations have no effect on agr expression or virulence. …”

“…Finally, we also transduced the traP-4 mutation from NB8 to the agr ϩ 8325-4(NB)-s, and again, all of 10 Km r transductants were fully hemolytic and indistinguishable from the recipient strain. As noted above, in-frame traP deletion mutations also had no detectable effect on agr expression (20), ruling out the possibility that a polar effect of the inserted kan cassette may have been responsible for the traP mutant phenotype. The reported complementation, therefore, is explainable by the lack of any agr defect in strain NB8.…”

“…All of the Km r transductants were fully hemolytic and fully proteolytic, indistinguishable from the recipient strain [8325-4(NB)-s] on SBA or on casein agar, ruling out the possibility that the effects of traP are strain specific. We note that in-frame traP deletions have recently been constructed in several clinical strains as well as in 8325-4(RN) by Tsang et al (20) and that none of these had any effect on agr activity. Typical results are shown in Fig.…”

Impact of sarA on Daptomycin Susceptibility of Staphylococcus aureus Biofilms In Vivo

“…Daptomycin susceptibility in the context of an established biofilm was assessed using a catheter-associated model of biofilm formation (42). Briefly, 1-cm segments of fluorinated ethylene propylene (FEP) catheters (14 gauge; Introcan Safety, B. Braun, Bethlehem, PA) first were coated with human plasma (1) and then placed in the wells of a 12-well microtiter plate containing 2 ml of TSB supplemented with glucose and sodium chloride (biofilm medium) as previously described (1,38,42). Each well then was inoculated with the test strain at an optical density at 600 nm of 0.05.…”

Impact of sarA on Antibiotic Susceptibility of Staphylococcus aureus in a Catheter-Associated In Vitro Model of Biofilm Formation

“…Studies focusing on the staphylococci have led to the identification of many potential targets (24). However, in almost every case, there are conflicting reports about the roles of different genes and gene products in biofilm formation (12,24,35).The single exception is the staphylococcal accessory regulator (sarA), the mutation of which has been shown to limit biofilm formation in both S. aureus and S. epidermidis (2,7,15,25,28,31,32,33,34,37). This is true with respect to both in vitro and in vivo models of biofilm formation (3, 6).…”