wnregulation or inhibition of the inflammatory responses. Previous studies have indicated that SzP in S. zooepidemicus shares similar antiphagocytic characteristics with the M proteins in the Lancefield groups A and G streptococci. SzP of S. zooepidemicus is a 40.1-kD surface-anchored protein that elicits serum opsonic and protective responses in mice and horses. We cloned SzP gene of S. zooepidemicus ATCC35246 and investigated the mechanism by which they conferred antiphagocytic effects against the host immune responses. 6 Mechanism of M-Like Protein in Antiphagocytosis Initial attempt to identify SzP interaction partners using the classical yeast two-hybrid system was not successful. Classical Y2H requires both proteins to interact in the nucleus and the nuclear MedChemExpress 5-ROX environment may cause certain proteins to fold improperly and thus abolish their interactions with other proteins. Next, we used the split-ubiquitin yeast two-hybrid system to detect SzP interaction partners. This system does not require protein interactions to occur in the nucleus and is therefore ideal for cytosolic proteins as well as membrane proteins. The C-terminal of SzP is anchored to the membrane while the rest of the protein is highly hydrophilic. We conjectured that the SzP/TRX interaction occurred at the cytosolic side of the plasma membrane. The current study identified the interaction between SzP and TRX. We further provided three lines of evidence to verify that SzP was associated with TRX; An interaction between SzP and TRX was screened and confirmed in the split-ubiquitin yeast twohybrid system. SzP and TRX were co-immunoprecipitated from pAcGFP1-SzP and pProLabel-TRX transfected HEK293 cells, and the result was detected with ProLabel-tag enzyme activity and confirmed with western-blot. FH was able to bind with SzP and TRX, as well as the SzP/TRX complex. This suggested that the binding of SzP to TRX was biologically relevant. TRX plays a variety of redox-related roles that are conserved from E. coli to humans. It is a small, ubiquitous, multifunctional protein that contains a redox-active disulfide/dithiol within its active site, -Cys-Gly-Pro-Cys-. TRX mutants with Cys32/ Cys35 to Ser mutations were still able to interact with SzP. Furthermore, both oxidized and reduced forms of TRX were able to interact with SzP. This suggested that the PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/22189475 redox active site was not the site of SzP/TRX interaction. It was found that oxidization leads to TRX dimerization through a disulfide bond between Cys73 in both monomers. Therefore, the residues necessary for SzP interaction were not hindered in the dimeric form of TRX. It was interesting to note that the activity of TRX was not inhibited by SzP in vitro. These results could explain why SzP/TRX interaction did not inhibit TRX activity. To better understand how surface protein SzP could contribute to the pathogenecity of S. zooepidemicus, we investigated the interaction of S. zooepidemicus with Raw264.7 cells, using the wild strain ATCC35246 and SzP-knockout strain attenuated in virulence. We employed flow cytometry to detect the recruitment of TRX to the surface of viable S. zooepidemicus. 
This recruitment was mediated by SzP, since SzPknockout strain was unable to recruit TRX to the surface. We also found that both the S. zooepidemicus wild type and the SzP-knockout strain were efficiently phagocytized by macrophages when TRX was absent, though the wild strain was harder to be ingested than the SzP-knockout strain. We belie
