Streptococcus suis sortase A is Ca2+ independent and is inhibited by acteoside, isoquercitrin and baicalin.
Sortase A (SrtA) has long been recognized as an ideal drug target for therapeutic agents against Gram-positive pathogens. However, the SrtA of Streptococcus suis (Ss-SrtA), an important zoonotic agent, has not been studied. In this study, the enzymatic properties of Ss-SrtA were investigated, and inhibition of Ss-SrtA by natural products was evaluated. Ss-SrtA was expressed and purified. The purified recombinant Ss-SrtA had maximal activity at pH 6.0-7.5, 45°C, and showed a Km of 6.7 μM for the hydrolysis of substrate abz-LPATG-dnp. Different from Staphylococcus aureus SrtA (Sa-SrtA) which is stimulated by Ca2+, Ss-SrtA was observed to be Ca2+ independent. Structural analysis showed that salt bridges formed between K111 and D180 in Ss-SrtA replaced the function of Ca2+ in Sa-SrtA to stabilize the substrate-binding cleft. Site-directed mutagenesis identified H126, C192 and R200 as the key residues of Ss-SrtA active site. To discover potential inhibitors, the percent inhibition of sortase activity by natural products was measured. Among these selected natural products, acteoside, isoquercitrin and baicalin were discovered as novel SrtA inhibitors, with IC50 values of 36.3 ± 1.3 μM, 100.0 ± 1.3 μM and 85.4 ± 1.5 μM, respectively. The inhibitory effects of these three natural products were further confirmed on endogenous Sa-SrtA. Using a previously established S. aureus model with a fluorescent-labeled Sa-SrtA substrate, acteoside, isoquercitrin, and baicalin showed 86%, 28% and 45% inhibition on endogenous Sa-SrtA activity, respectively. Overall, these findings shed new light on enzymatic properties, Ca2+-independent catalytic mechanism and potential inhibitors of Ss-SrtA.
Division of Bacterial Diseases, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Xiangfang District, Harbin, PR China.
Fuguang
F
Chen
Division of Bacterial Diseases, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Xiangfang District, Harbin, PR China.
Fang
F
Xie
College of Animal Science and Veterinary Medicine, Shenyang Agricultural University, Shenyang, PR China.
Baoling
B
Yang
College of Veterinary Medicine, Northeast Agricultural University, Xiangfang District, Harbin, PR China.
Chengcheng
C
Wang
Division of Bacterial Diseases, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Xiangfang District, Harbin, PR China.
Siguo
S
Liu
Division of Bacterial Diseases, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Xiangfang District, Harbin, PR China.
Yueling
http://orcid.org/0000-0002-5619-8410
Y
Zhang
D000595:Amino Acid Sequence; D019881:Aminoacyltransferases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D020134:Catalytic Domain; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004353:Drug Evaluation, Preclinical; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005419:Flavonoids / Q000494:pharmacology*; D005960:Glucosides / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010636:Phenols / Q000494:pharmacology*; D011794:Quercetin / Q000031:analogs & derivatives* / Q000494:pharmacology; D017011:Streptococcus suis / Q000201:enzymology*; D013696:Temperature; D059808:Polyphenols
D019881:Aminoacyltransferases; D001426:Bacterial Proteins; D002118:Calcium; D003546:Cysteine Endopeptidases; D004791:Enzyme Inhibitors; D005419:Flavonoids; D005960:Glucosides; D010636:Phenols; D011794:Quercetin; C016527:isoquercitrin; C038044:baicalin; C058956:acteoside; D059808:Polyphenols; C120542:sortase A