Streptococcus suis sortase A is Ca2+ independent and is inhibited by acteoside, isoquercitrin and baicalin

Abstract

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.

Transpeptidation-Mediated Assembly of Tripartite Split Green Fluorescent Protein for Label-Free Assay of Sortase Activity

“…Since sortase is considered as an ideal target for novel antivirulence drug design and development, sortase inhibition assay, as well as sortase activity assay, is very important for drug screening. 12 Here, two potent inhibitors (quercetin and curcumin 42 ) of SrtA were chosen to investigate whether the method can be used for SrtA inhibitor screening. The inhibition efficiency (IE) was determined by the following equation,…”

“…It then catalyzes the formation of an amide bond between the carboxyl group of the T and the cell wall precursor molecule lipid II, creating the LPETG-containing proteins that can promote bacteria adhere to host cells and tissues. − Nowadays, drug resistance of pathogens becomes a worldwide health problem, due to excessive use and abuse of antibiotics . As a virulence-related molecule, sortase is not necessary for bacterial growth and its inhibitors exert little selective pressure on pathogens to develop drug resistance . Consequently, sortase is considered as an ideal target for novel antivirulence drug design and development and has attracted a lot of interest.…”

“…11 As a virulence-related molecule, sortase is not necessary for bacterial growth and its inhibitors exert little selective pressure on pathogens to develop drug resistance. 12 Consequently, sortase is considered as an ideal target for novel antivirulence drug design and development and has attracted a lot of interest. Probing sortase activity and screening its inhibitors are of great significance to not only fundamental biological research but also pharmaceutical development and medical diagnostics.…”

The pharmacokinetic property and pharmacological activity of acteoside: A review

“…Specifically, K111 forms a salt bridge with D180. AC has been identified as a novel SrtA inhibitor with an IC 50 value of 36.3 μM, and the endogenous SrtA activity indicates 86 % inhibition against S. aureus and 98 % against Streptococcus suis [130] . However, whether the discrepancy is associated with Ca 2+ or the salt bridge is still needed to be further elucidated.…”

Pull the plug: Anti‐angiogenesis potential of natural products in gastrointestinal cancer therapy

“…Isoquercetin (IQ), as a monoglucoside of the most abundant natural flavonoid compound quercetin, widely exists in fruits, vegetables and cereals. Isoquercetin has antioxidant (Valentová, Vrba, Bancířová, Ulrichová, & Křen, 2014), antiviral (Chen, Xie, et al, 2017), immunomodulatory (Valentová et al, 2016) and other effects. In addition, studies have confirmed that IQ has anti‐tumor effects on several cancers including GC (Orfali et al, 2016).…”