A Novel Aza-Derivative Inhibits agr Quorum Sensing Signaling and Synergizes Methicillin-Resistant Staphylococcus aureus to Clindamycin

Abstract

Increasing antibiotic resistance and diminishing pharmaceutical industry investments have increased the need for molecules that can treat infections caused by dangerous pathogens such as methicillin-resistant Staphylococcus aureus (MRSA). Quorum Sensing (QS) is a signaling mechanism that regulates bacterial virulence in pathogens. A report demonstrating that the anti-inflammatory drug Diflunisal reduces MRSA virulence factors’ expression prompted us to design, synthesize and test 16 aza-analogs as inhibitors of S. aureus virulence factors controlled by the accessory gene regulator (agr) QS system. At first, we evaluated by qRT-PCR the activity of compounds on rnaIII expression, a QS related gene. Azan-7 was the most active molecule tested and it did not show cytotoxic activity in human cell lines. Moreover, we demonstrated that it did not affect bacterial proliferation. Regulation of MRSA virulence genes by Azan-7 was investigated using qRT-PCR and RNAseq. Azan-7 significantly reduced hla, psmα, hysA, agrA, cap1A, and cap1C gene expression. In silico docking demonstrated that Azan-7 binds the response regulator AgrA. This data was confirmed by electrophoretic mobility shift assay (EMSA) reporting that Azan-7 binding to AgrA protein strongly reduced the AgrA-DNA complex formation at the P3 promoter region involved in the regulation of rnaIII transcription. Azan-7 inhibited MRSA-mediated haemolysis, reduced survival of the pathogen at low pH levels, and increased macrophage killing. In addition, Azan-7 enhanced MRSA susceptibility to clindamycin both in planktonic growth and biofilm. Azan-7 did not induce resistance over 10 days in culture. It was equally active against all the AgrA MRSA subtypes encountered among clinical isolates, but it was not active against Staphylococcus epidermidis, although the AgrA proteins show an approximate 80% homology. These results demonstrate that Azan-7 inhibits the expression of MRSA virulence factors by interfering in the QS and synergizes MRSA biofilm with clindamycin, indicating the compound as a promising candidate for the treatment of MRSA infections.

Attenuating the virulence of the superbug Staphylococcus aureus by sodium bicarbonate, dexamethasone, and ascorbic acid

“…Additionally, Abbas et al showed that diclofenac had a signi cant down-regulation effect on the relative expression of virulence regulating genes SarA, AgrA, hla, FnbA, IcaA, SigB, and CrtM in S. aureus [63]. In another study, parallel with the current results, azan-7 (a novel aza-derivative) signi cantly reduced hla and agrA gene expression in MRSA [73]. Moreover, in accordance with our results, luteolin (a avonoid of plant origin), and alpha-cyperone (a Chinese medicinal herb) inhibit hla and agrA gene expression in S. aureus [74].…”

A novel phenolic derivative inhibits AHL-dependent quorum sensing signaling in Pseudomonas aeruginosa

“…PAO1 was cultured in LB with DMSO or supplemented with the selected molecules for 24 h at 37°C. Then cultures were centrifuged, the medium discarded and total RNA was isolated and purified using the GRS Total RNA Kit—Bacteria (#GK16.0100, GRISP Research Solution) with mechanical and enzymatic cellular disruption, according to the manufacturer’s instructions and as previously described ( Berbabè et al, 2021 ). A treatment with DNase I was used to remove contaminating DNA.…”

“…The distance movement was calculated using the freely available software ImageJ. Plates inoculated with not-treated PAO1 were used as the positive control ( Berbabè et al, 2021 ).…”

Accessory Gene Regulator (agr) Allelic Variants in Cognate Staphylococcus aureus Strain Display Similar Phenotypes

“…The a ccessory g ene r egulator ( agr ) quorum-sensing system, a globe transcriptional regulator of Staphylococcus aureus , plays a key role in its pathogenesis and resistance ( Bernabè et al, 2021 ) and has been intensively studied to aid drug and vaccine development ( Tan et al, 2018 ). The agr locus comprises two adjacent transcripts, RNAII and RNAIII, which are controlled by P2 and P3 promoters, respectively ( Ji et al, 1995 ).…”