Oligopeptide Targeting Sortase A as Potential Anti-infective Therapy for Staphylococcus aureus

Abstract

Sortase A (SrtA)-catalyzed anchorage of surface proteins in most Gram-positive bacteria is indispensable for their virulence, suggesting that this transpeptidase is a promising target for antivirulence therapy. Here, an oligopeptide, LPRDA, was identified as an effective inhibitor of SrtA via virtual screening based on the LPXTG substrate sequence, and it was found to inhibit SrtA activity in vitro and in vivo (IC₅₀ = 10.61 μM) by competitively occupying the active site of SrtA. Further, the oligopeptide treatment had no anti-Staphylococcus aureus activity, but it provided protection against S. aureus-induced mastitis in a mouse model. These findings indicate that the oligopeptide could be used as an effective anti-infective agent for the treatment of infection caused by S. aureus or other Gram-positive bacteria via the targeting of SrtA.

Keywords: Sortase A; Staphylococcus aureus; antivirulence; mastitis; oligopeptide.

FIGURE 1

Mutation of srtA in Staphylococcus aureus USA 300. (A) The intact srtA gene in S. aureus USA 300 was disrupted by recombination deletion with a Janus cassette. (B) The srtA mutation was confirmed by PCR based on the chromosomal DNA sequences in S. aureus USA 300 and its SrtA mutant strain ΔSrtA. PCR products for the parent strain USA 300 (lane 1) and its mutant strain ΔSrtA (lane 2).

FIGURE 2

Inhibition of SrtA activity both in vitro and in vivo by the oligopeptide. (A) The molecular structure of the oligopeptide, LPRDA. (B) The inhibitory effect of the oligopeptide on SrtA activity. The fluorescent peptide substrate Dabcyl-QALPTTGEE-Edans was mixed with purified SrtA preincubated with various concentrations of the oligopeptide, and the fluorescence intensity of the reaction system was determined at emission and excitation wavelengths of 350 and 520 nm, respectively (n = 3). (C) The growth of S. aureus USA 300 cultured in TSB in the presence of increasing concentrations of the oligopeptide was monitored by measuring the OD at 600 nm every 60 min. Oligopeptide treatment inhibits the binding of S. aureus to fibronectin (D) and the formation of S. aureus biofilm (E) (n = 3) (^∗p < 0.05, ^∗∗p < 0.01). (F) Inhibition of S. aureus invasion into J774 cells by the oligopeptide. J774 cells were infected with S. aureus at 37°C for 1 h, and the total colony-forming units (CFU) for lysed cells was evaluated (n = 3) (^∗p < 0.05, ^∗∗p < 0.01). (G) The presence of protein A on the cell envelopes of S. aureus treated with or without the oligopeptide and its SrtA mutant was viewed under a confocal laser scanning microscope following staining with an FITC-conjugated antibody. Results are reported as mean ± SEM.

FIGURE 3

Oligopeptide provides protection against S. aureus mastitis in a mouse model. Lactating BALB/c mice were infected by injecting the canal glands with 5 × 10⁷ CFU bacterial cells and treatment with the oligopeptide or PBS at the time of infection. Gross pathological changes (A) and histopathological analysis (B) of the mammary gland tissues at 48 h post-infection (n = 10). Significant alleviation of the pathological abnormalities was observed in the infected mice that received 50 mg/kg oligopeptide compared with the control mice treated with PBS. (C) Oligopeptide reduces the inflammatory response in infected mice. The levels of cytokines, including IL-1β, IL-6, and TNF-α, in the mammary gland tissues of infected mice were evaluated by ELISA (n = 10) (^∗p < 0.05, ^∗∗∗p < 0.001). (D) The effect of the oligopeptide on the bacterial burden in infected mice. Mammary gland tissues were collected, homogenized, and plated on TSB agar plates for assessment of the bacterial burden (n = 5) (^∗p < 0.05, ^∗∗p < 0.01). Results are reported as mean ± SEM.

FIGURE 4

Determination of the 3D structure of the SrtA-oligopeptide complex using a molecular modeling method. (A) The structure of SrtA-oligopeptide. (B) The RMSD calculated for the backbone atoms of the protein during MD simulations of the SrtA-oligopeptide is presented. (C) Decomposition of the binding energy on a per-residue basis at the binding sites of the SrtA-oligopeptide complex.

FIGURE 5

Determination of the binding sites for the oligopeptide on SrtA and its inhibitory mechanism. (A) The predicted binding mode of SrtA with the oligopeptide is shown, with labeling of key residues in the binding sites. (B) The catalytic activity of SrtA and its derivative. The activity of SrtA mutants was calculated by comparison with the wild type SrtA activity that was set to 100%. (C) Influences of the oligopeptide on the activities of SrtA and its derivatives. SrtA was incubated with various concentrations of the oligopeptide, and the fluorescent peptide substrate Dabcyl-QALPTTGEE-Edans was added for determination of SrtA protein activity, as described in Figure 1B (n = 3). Results are reported as mean ± SEM.