ABSTRACT
Enterococcus faecium is a commensal of the mammalian gastrointestinal tract, but is also found in non-enteric environments where it can grow between 10 °C and 45 °C. E. faecium has recently emerged as a multi-drug resistant nosocomial pathogen. We hypothesized that genes involved in the colonization and infection of mammals exhibit temperature-regulated expression control and we therefore performed a transcriptome analysis of the clinical isolate E. faecium E1162, during mid-exponential growth at 25 °C and 37 °C. One of the genes that exhibited differential expression between 25 °C and 37 °C, was predicted to encode a peptidoglycan-anchored surface protein. The N-terminal domain of this protein is unique to E. faecium and closely related enterococci, while the C-terminal domain is homologous to the Streptococcus agalactiae surface protein BibA. This region of the protein contains proline-rich repeats, leading us to name the protein PrpA for proline-rich protein A. We found that PrpA is a surface-exposed protein which is most abundant during exponential growth at 37 °C in E. faecium E1162. The heterologously expressed and purified N-terminal domain of PrpA was able to bind to the extracellular matrix proteins fibrinogen and fibronectin. In addition, the N-terminal domain of PrpA interacted with both non-activated and activated platelets.
Subject(s)
Bacterial Proteins/metabolism , Blood Platelets/metabolism , Enterococcus faecium/metabolism , Fibrinogen/metabolism , Fibronectins/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Base Sequence , Binding Sites/genetics , Cross Infection/microbiology , Enterococcus faecium/genetics , Enterococcus faecium/ultrastructure , Gene Expression Regulation, Bacterial , Gram-Positive Bacterial Infections/microbiology , Humans , Membrane Proteins/genetics , Membrane Proteins/metabolism , Microscopy, Electron, Transmission , Molecular Sequence Data , Peptidoglycan/metabolism , Promoter Regions, Genetic/genetics , Protein Binding , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Nucleic Acid , TemperatureABSTRACT
The main responses of P2Y(1) ligation are platelet shape change and transient aggregation while P2Y(12) ligation amplifies P2Y(1)-induced aggregation and accelerates aggregation, secretion and thromboxane A(2) production induced by other agonist-receptor complexes. We searched for new targets of P2Y signalling using micro-arrays with 144 peptides representing known phosphosites of protein tyrosine kinases. ADP induced phosphorylation of peptides representing surface receptors, second messenger enzymes and cytoskeletal proteins. Strong phosphorylation was found in peptides representing Ephrin-receptor family members. Blockade of P2Y(1/12) inhibited phosphorylation of EphA4- and EphB1-peptides on micro-arrays. The EphA2/4 inhibitor 2,5-dimethylpyrrolyl benzoic acid derivative interfered with P2Y(1/12)-induced EphA4 phosphorylation, left P2Y(1)-induced aggregation unchanged but inhibited with P2Y(12)-induced secretion, second phase aggregation and thrombus formation on collagen at 1600 s(-1). These results show that platelet EphA4 is an important intermediate in P2Y(12)-induced granule secretion.
Subject(s)
Blood Platelets/enzymology , Phosphoproteins/metabolism , Protein-Tyrosine Kinases/metabolism , Receptor, EphA4/agonists , Receptors, Purinergic P2Y12/metabolism , Secretory Vesicles/enzymology , Adenosine Diphosphate/pharmacology , Blood Platelets/cytology , Blood Platelets/drug effects , Ephrin-A4/agonists , Ephrin-A4/metabolism , Humans , Ligands , Phosphoproteins/agonists , Phosphoproteins/antagonists & inhibitors , Phosphorylation , Platelet Activation/drug effects , Platelet Aggregation/drug effects , Protein Array Analysis , Purinergic P2 Receptor Antagonists/pharmacology , Receptor Cross-Talk , Receptor, EphA4/metabolism , Receptors, Purinergic P2Y1/metabolism , Secretory Vesicles/drug effects , Signal TransductionABSTRACT
OBJECTIVE: The sensitivity of platelets to aggregating agents increases when low-density lipoprotein (LDL) binds to apolipoprotein E receptor 2' (apoER2'), triggering activation of p38MAPK and formation of thromboxane A2. LDL signaling is terminated by PECAM-1 through recruitment and activation of the Ser/Thr protein phosphatase PP2A, but platelets remain unresponsive to LDL when PECAM-1 activation disappears. We report a second mechanism that halts LDL signaling and in addition lowers platelet responsiveness to aggregating agents. METHODS AND RESULTS: After a first stimulation with LDL, platelets remain unresponsive to LDL for 60 minutes, despite normal apoER2' activation by a second dose of LDL. A possible cause is persistent activation of the tyrosine phosphatases SHP-1 and SHP-2, which may not only block a second activation of p38MAPK, PECAM-1, and PP2A by LDL but also seem to reduce aggregation by TRAP, collagen, and ADP. CONCLUSION: These findings reveal that p38MAPK phosphorylation and platelet activation by LDL are suppressed by two mechanisms: (1) short activation of PECAM-1/PP2A, and (2) prolonged activation of SHP-1 and SHP-2. Activation of SHP-1 and SHP-2 is accompanied by reduced responsiveness to aggregating agents, which--if present in vivo--would make LDL an aggregation inhibitor during prolonged contact with platelets.
