SarA based novel therapeutic candidate against Staphylococcus aureus associated with vascular graft infections

Abstract

Staphylococcus aureus is a common pathogen seen in prosthetic vascular graft, leading to high morbidity and mortality. The virulence genes for severity of infections are under the control of global regulators. Staphylococcal accessory regulator A (SarA) a known master controller of biofilm formation is an attractive target for the drug development. A structure based screening of lead compounds was employed for the identification of novel small molecule inhibitors targeted to interact to the DNA binding domain of the transcriptional activator, SarA and hinder its response over the control of genes that up-regulate the phenotype, biofilm. The top-hit SarA selective inhibitor, 4-[(2,4-diflurobenzyl)amino] cyclohexanol (SarABI) was further validated in-vitro for its efficacy. The SarABI was found to have MBIC50value of 200 μg/ml and also down-regulated the expression of the RNA effector, (RNAIII), Hemolysin (hld), and fibronectin-binding protein (fnbA). The anti-adherence property of SarABI on S. aureus invasion to the host epithelial cell lines (Hep-2) was examined where no significant attachment of S. aureus was observed. The SarABI inhibits the colonization of MDR S. aureus in animal model experiment significantly cohere to the molecular docking studies and in vitro experiments. So, we propose that the SarABI could be a novel substitute to overcome a higher degree of MDR S. aureus colonization on vascular graft.

Keywords: SarA; Staphylococcus aureus; molecular docking; multi drug resistance; quorum sensing; vascular graft associated infection; virulence gene expression.

Figure 1

Determination of minimum biofilm inhibitory concentration (MBIC). Cultures treated with various concentrations of SarABI were analyzed after 24 h comparing the effects of expressing a biofilm in S. aureus ATCC 25923. The static biofilm assay was performed with TSB medium, dotted line shown the difference between control and experimental groups. Statistical analysis was performed using the Two-Way ANOVA and Values of P < 0.001 were considered significant. Asterisk (^*) indicates non detectable expression of biofilm.

Figure 2

Colony forming unit assays of clinical isolates and mutant strains of S. aureus. The strains were incubated for 24 h (2 × 10⁷ cfu/ml) with the addition of 200 μg/ml and 1 mg/ml SarABI in tryptic soy agar plates which does not affect the viability of clinical isolates compared to control. A similar effect was observed in mutant laboratory strains (ALC355, ALC637, ALC638), dotted line shown the difference between control and experimental groups. Statistical analysis was performed using the Two-Way ANOVA and Values of P < 0.001 were considered significant.

Figure 3

Synergistic in comparison to direct lysis of rabbit blood erythrocytes by S. aureus with SarABI. Culture supernatants were grown in presence (200 μg/ml and 1 mg/ml, respectively) or absence of SarABI, culture filtered and incubated with a 2% solution of defibrinated rabbit blood for 30 min at 37°C. Hemolysis was measured by determining OD₅₄₀nm using spectrophotometer and % lysis calculated from lysed erythrocyte standards, dotted line shown the difference between control and experimental groups. Statistical analysis was performed using the Two-Way ANOVA and Values of P < 0.001 were considered significant. SarABI significantly decreases the expression of hemolysin in all experimental groups. Asterisk (^*) indicates no weak expression of hemolysin.

Figure 4

Effective concentration of SarABI reduced for the adherence of Staphylococcus aureus. (A) FITC labeled ATCC 25923 cells (10⁹ CFU) were applied to glass slide with 100 μl of PBS, (B) clinical isolate SA1061, (C) ALC637 (sarA::Tn917LTV1), (D) ALC355 (Δagr::tetM), (E) ALC638 (Δagr::tetM sarA::Tn917LTV1) effective concentration of SarABI were used for the adherence. Cells were incubated for 24 h at 37°C, unbound cells were removed by PBS washing and adherent cells observed under the fluorescent microscopy 5x.

Figure 5

(A) The effect of SarABI on S. aureus attachment to polystyrene In vitro: Cell attachment compared with the clinical isolates and the mutant backgrounds ALC355 (Δagr::tetM), ALC637 (ΔsarA::Tn917LTV1) single mutants, and the ALC638 (Δagr::tetM, Δsar::Tn917LTV1 double mutants) treated with SarABI. S. aureus cultures grown in 96-well microtiter plates were pretreated with SarABI (200 μg/ml and 1 mg/ml respectively) and incubated for 24 h. Values show mean numbers of biofilm formation/well, and error bars indicate range, dotted line shown the difference between control and experimental groups. Statistical analysis was performed using the Two-Way ANOVA and values of P < 0.001 were considered significant. SarABI significantly decreases the expression of biofilm in all experimental groups. Asterisk (^*) indicates no detectable expression of biofilm. (B) Quantitative analysis of biofilm development on coverslips by S. aureus strains treated with SarABI using CLSM shows biomass, roughness coefficient, thickness and minimal colony at the surface and biofilm production by S. aureus as assessed by three dimensional images compared with untreated biofilms. Statistical analysis was performed using the Two-Way ANOVA and values of P < 0.001 were considered significant. Asterisk (^*) indicates drastically decline fluorescent intensity.

Figure 6

The validation of virulence gene expression level using quantitative RT-PCR. S. aureus were grown overnight at 37°C with (TSA, treated S. auerus) or without (CSA, control S. aureus) treated of 1 mg/ml SarABI in tryptic soy broth. Expression pattern of the fnbA, hld, and RNAIII were diminished in cells treated with SarABI in comparison to without treated. Statistical analysis was performed using the Two-Way ANOVA and values of P < 0.001 were considered significant. Asterisk (^*) indicates significantly low expression of genes.

Figure 7

Quantitative microbiological evaluation experiments shows SarABI reduced in-vivo infection. Bacteria (2 × 10⁷CFUs) were incubated with SarABI for challenge the animals. After 14 day incubation, the graft was removed, and the number of bacteria were determined on blood agar plates. The graph depicts group-I (control), group-II (DC, Diseased control infected with ATCC 25923), group-III (CI, S. aureus clinical isolate with SarABI), group-IV (AT, ATCC 25923 with SarABI), group-V (AG, agr::tetM with SarABI), group-VI (SA, sar::Tn917LTV1 with SarABI) and group-VII (DM, agr::tetM, sar::Tn917LTV1 with SarABI). The low and effective doses of SarABI were used for studied, dotted line shown the difference between control and experimental groups. Statistical analysis was performed using the One-Way ANOVA and values of P < 0.001 were considered significant. Asterisk (^*) indicates no detectable bacteria, suggesting <10 CFUs/ml.

Figure 8

Hematoxylene and eosin stained sections of tissues from implant site. Skin ulcer and necrotic debris was observed in disease control group. Disease control group (B, I) also exhibits a marked tissue reaction with predominant macrophages, fibroblasts, lymphocytes and neutrophils along with collagen deposition. A fibrous capsule and granulation tissue formation with angiogenesis was predominantly present in the sections. (I,II) represents the 200 μg/ml and 1 mg/ml SarABI coated vascular grafts experimental groups respectively. Representative samples from different groups treated with SarABI do not show any significant difference from saline control (A, H), DC (B, I), CI (C, J), AT (D, K), ALC355 (E, L), ALC637 (F, M) and ALC638 (G, N).

Figure 9

Effect of SarABI on adherence of S. aureus to HEp-2 cell lines. The graph depicts Control (with normal saline), DC (Diseased control contaminated with ATCC 25923), CI (S. aureus clinical isolate with SarABI), AT (ATCC 25923 with SarABI), ALC355 (agr::tetM with SarABI), ALC637 (sar::Tn917LTV1 with SarABI) and ALC638 (agr::tetM, sar::Tn917LTV1 with SarABI). The effective concentration of SarABI synergistically diminished the adherence of S. aureus to HEp-2 cell lines. (A) Data revealed the significantly reduce in biomass, roughness coefficient thickness and colony at substrate compared with untreated samples. (B) Three-dimensional reconstructed renderings of the S. aureus adherence to HEp-2 cells on coverslip. The effective doses of SarABI were used for studied. Statistical analysis was performed using the Two-Way ANOVA and values of P < 0.001 were considered significant. Asterisk (^*) indicates significant result.