Rot and Agr system modulate fibrinogen-binding ability mainly by regulating clfB expression in Staphylococcus aureus NCTC8325

Abstract

Staphylococcus aureus is an important human pathogen that causes a variety of diseases, ranging from localized skin infections to life-threatening systemic infections. The success of S. aureus as a pathogen is partly due to its ability to adhere to a wide range of host tissues by binding to host extracellular matrix proteins such as fibrinogen, fibronectin, and collagen. Staphylococcus aureus expresses two proteins that can bind specifically to fibrinogen, clumping factors A and B (ClfA and ClfB). Repressor of toxins (Rot) is known to be a global regulator of virulence gene expression in S. aureus. The translation of Rot is regulated by the staphylococcal accessory gene regulator (Agr) quorum-sensing system. In this study, we demonstrated that Rot and the Agr system in S. aureus NCTC8325 can affect the bacterial binding ability to human fibrinogen (Fg) under different bacterial growth phases. Our real-time RT-PCR results indicated that both Rot and the Agr system have no significant effect on clfA expression. However, Rot is an activator of clfB, and Agr/RNAIII can regulate clfB expression via Rot. Gel shift data further suggested that Rot might regulate clfB expression by directly binding to the promoter region of clfB. Moreover, Rot and the Agr system exhibited consistent regulatory effects on clfB transcription and bacterial Fg-binding ability, suggesting that Rot and the Agr system might affect bacterial Fg-binding ability mainly through regulating clfB transcription.

Positive Regulation of Staphylococcal Enterotoxin H by Rot (Repressor of Toxin) Protein and Its Importance in Clonal Complex 81 Subtype 1 Lineage-Related Food Poisoning

“…DNA concentration was measured using a NanoDrop 1000 (NanoDrop Technologies, Inc., Wilmington, DE). Rot protein was prepared according to a previously described method (20) using the vectors and primers listed in Tables 2 and 3, respectively. SEH promoter probe and Rot protein were incubated under previously described conditions (20).…”

“…Rot protein was prepared according to a previously described method (20) using the vectors and primers listed in Tables 2 and 3, respectively. SEH promoter probe and Rot protein were incubated under previously described conditions (20). The reaction mixture was electrophoresed with a 6% polyacrylamide gel and 0.5ϫ Tris-borate-EDTA (TBE) buffer under cold conditions.…”

The Staphylococcal Biofilm: Adhesins, Regulation, and Host Response

“…ClfA and ClfB are fibrinogen-binding proteins in S. aureus (40, 44), and both are up-regulated in biofilm growth relative to planktonic (45). Rot and agr affect bacterial binding to fibrinogen by regulating clfB but not clfA ((46), see regulation section).…”

Staphylococcus aureus Aggregation and Coagulation Mechanisms, and Their Function in Host–Pathogen Interactions

“…ClfB also binds to fibrinogen, but unlike ClfA it binds to the α-chain (Walsh et al, 2008). Expression of clfB is under the control of the Agr quorum sensing system and is only expressed in the early exponential phase of growth; consequentially, the contribution of ClfB to clumping is often overshadowed by that of ClfA (Ni Eidhin et al, 1998; Xue et al, 2012). Interestingly, ClfB also binds to cytokeratin 10 and loricrin, facilitating adhesion to nasal epithelial cells (Mulcahy et al, 2012; O’Brien et al, 2002b; Walsh et al, 2004).…”