A short sequence within subdomain N1 of region A of the Staphylococcus aureus MSCRAMM clumping factor A is required for export and surface display.
Clumping factor A (ClfA) is the archetypal fibrinogen-binding surface protein of Staphylococcus aureus and a member of the microbial surface component recognizing adhesive matrix molecules (MSCRAMM) family. An N-terminal signal sequence directs export of the MSCRAMM by the Sec pathway and the C-terminal cell wall-anchoring domain allows covalent attachment of ClfA to peptidoglycan by sortase. Region A of ClfA comprises three independently folded subdomains N1, N2 and N3. Subdomains N2N3 comprise IgG-like folds and promote fibrinogen binding. Nothing is known about the structure or function of subdomain N1. Here we demonstrate an unexpected role for N1 in the export and surface localization of ClfA. Attempted expression of a ClfA variant lacking subdomain N1 resulted in impaired growth of S. aureus and accumulation of ClfA protein in the cytoplasm and cytoplasmic membrane. The presence of residues 211-228 of N1 was required to allow display of ClfA on the bacterial surface. The importance of this region was confirmed when a ClfA variant lacking residues 211-220 was also mislocalized to the cytoplasm and cytoplasmic membrane. However, these residues were not required for export of ClfA lacking the Ser-Asp repeats that link region A to the wall-anchoring domain. Similarly, subdomain N1 of a related MSCRAMM fibronectin-binding protein B was required for export and surface display of the full-length protein, but not a derivative lacking fibronectin-binding repeats. In summary, we demonstrate that residues in the N1 subdomain are required for export and cell wall localization of S. aureus MSCRAMM proteins.
Department of Microbiology, Moyne Institute of Preventive Medicine, School of Genetics and Microbiology, Trinity College Dublin, Dublin 2, Ireland.
Niamh
N
McCormack
Department of Microbiology, Moyne Institute of Preventive Medicine, School of Genetics and Microbiology, Trinity College Dublin, Dublin 2, Ireland.
Timothy J
TJ
Foster
Department of Microbiology, Moyne Institute of Preventive Medicine, School of Genetics and Microbiology, Trinity College Dublin, Dublin 2, Ireland.
Joan A
JA
Geoghegan
D018829:Adhesins, Bacterial / Q000235:genetics / Q000378:metabolism*; D002473:Cell Wall / Q000378:metabolism*; D003030:Coagulase / Q000235:genetics / Q000378:metabolism*; D004252:DNA Mutational Analysis; D010457:Peptidoglycan / Q000378:metabolism; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D017384:Sequence Deletion; D013211:Staphylococcus aureus / Q000235:genetics / Q000378:metabolism*
D018829:Adhesins, Bacterial; C515944:ClfA protein, Staphylococcus aureus; D003030:Coagulase; C509820:MSCRAMM proteins, Staphylococcus; D010457:Peptidoglycan