Sortase A Mediated Bioconjugation of Common Epitopes Decreases Biofilm Formation in Staphylococcus aureus

Abstract

Staphylococcus aureus is one of the most notorious pathogens and is frequently associated with nosocomial infections imposing serious risk to immune-compromised patients. This is in part due to its ability to colonize at the surface of indwelling medical devices and biofilm formation. Combating the biofilm formation with antibiotics has its own challenges like higher values of minimum inhibitory concentrations. Here, we describe a new approach to target biofilm formation by Gram positive bacteria. Sortase A is a transpeptidase enzyme which is responsible for tagging of around ∼22 cell surface proteins onto the outer surface. These proteins play a major role in the bacterial virulence. Sortase A recognizes its substrate through LPXTG motif. Here, we use this approach to install the synthetic peptide substrates onS. aureus. Sortase A substrate mimic, 6His-LPETG peptide was synthesized using solid phase peptide chemistry. Incorporation of the peptide on the cell surface was measured using ELISA. Effect of peptide incubation on Staphylococcus aureus biofilm was also studied. 71.1% biofilm inhibition was observed with 100 μM peptide while on silicon coated rubber latex catheter, 45.82% inhibition was observed. The present work demonstrates the inability of surface modified S. aureus to establish biofilm formation thereby presenting a novel method for attenuating its virulence.

Keywords: LPXTG; S. aureus; Sortase A; biofilm; infection.

FIGURE 1

(A) Reversed phase HPLC chromatogram of the 6His-LPETG peptide with retention time 8.717 min. (B) MALDI-TOF analysis peaks of 6His-LPETG peptide. 6His-LPETG peptide conjugation on cell surface of (C) S. aureus and (D) E. faecalis as measured by ELISA done in triplicates. P< 0.05 was considered as significant. Error bars are mean ± SEM. *p < 0.05, ***p < 0.001.

FIGURE 2

0.1% Crystal Violet assay with S. aureus using (A) 6His-LPETG, (B) 6His, and (C) FLAG-LPETG at indicated concentrations. One hundred micro meter Trans-chalcone was used as positive control in crystal violet assay. Peptide was incubated with 1/100 diluted S. aureus cell culture at indicated concentrations and absorbance (OD₆₀₀) was measured after 24 h. 0.1% Crystal Violet assay with E. faecalis using (D) 6His-LPETG and (E) FLAG-LPETG at indicated concentrations. (F) S. aureus cell viability assay in the presence of 6His-LPETG. All the experiments weredone is triplicates and P< 0.05 was considered as significant. Error bars are mean ± SEM. *p < 0.05, **P < 0.01, ***p < 0.001.

FIGURE 3

Representative SEM image of S. aureus biofilm formation using different concentrations of synthetic peptide. Images were acquired at 6000X magnification, (A) Negative Control (B) 100 μM Transchalcone used as positive control, 6His-LPETG peptide at (C) 10 μM and (D) 100 μM. (E) Biofilm formation by S. aureus as assayed by crystal violet, growing on silicone coated latex catheter disks in the presence of 6His-LPETG. Experiment was done is triplicates and P < 0.05 was considered as significant. Error bars are mean ± SEM. *p < 0.05.