Vesiculation from Pseudomonas aeruginosa under SOS

Abstract

Bacterial infections can be aggravated by antibiotic treatment that induces SOS response and vesiculation. This leads to a hypothesis concerning association of SOS with vesiculation. To test it, we conducted multiple analyses of outer membrane vesicles (OMVs) produced from the Pseudomonas aeruginosa wild type in which SOS is induced by ciprofloxacin and from the LexA noncleavable (lexAN) strain in which SOS is repressed. The levels of OMV proteins, lipids, and cytotoxicity increased for both the treated strains, demonstrating vesiculation stimulation by the antibiotic treatment. However, the further increase was suppressed in the lexAN strains, suggesting the SOS involvement. Obviously, the stimulated vesiculation is attributed by both SOS-related and unrelated factors. OMV subproteomic analysis was performed to examine these factors, which reflected the OMV-mediated cytotoxicity and the physiology of the vesiculating cells under treatment and SOS. Thus, SOS plays a role in the vesiculation stimulation that contributes to cytotoxicity.

Figure 1

The bacterial SOS response. The response is triggered by DNA-damage antibiotics. This response is controlled by the RecA-LexA interplay, in which LexA represses the SOS genes. DNA damage activates RecA to simulate autocatalytic cleavage of LexA so that the SOS genes are depressed and expressed. X, the mutation rendering LexA noncleavable.

Figure 2

Microscopy of P. aeruginosa under the ciprofloxacininduced SOS response. The wild-type (PAO1) and the LexA noncleavable (lexAN) strains were grown in LB with shaking for 2 hrs, and then ciprofloxacin (CPX) was added (1 μg/mL). The culture continued for 6 hrs. (a) PAO1 without and (b) with CPX. (c) PAO1 with 5 μg/mL CPX. (d) lexAN without and (e) with CPX. (f) lexAN with 5 μg/mL CPX. Arrows in (c) and (f) show damaged and lysed cells. Cell bar, 5 μm. Inset (b) shows transmission electron microscopy of OMVs from the treated wild-type cultures. Inset (e) shows lexAN OMVs. OMV bar, 0.1 μm.

Figure 3

OMV production from P. aeruginosa under the ciprofloxacin-induced SOS response. (a) Growth of the wild-type (PAO1) and the LexA noncleavable strains. Both strain were grown in LB with shaking for 2 hrs, and then ciprofloxacin (CPX) was added. The culture continued for 6 hrs. OMVs were isolated from the wild-type and the lexAN cells treated with or without ciprofloxacin (CPX). (b) Quantification of OMV proteins by Bradford (n = 7) and (c) OMV lipids by weight (n = 3). (*P < 0.05).

Figure 4

OMV-mediated macrophage cytotoxicity is SOS dependant. OMVs isolated from the wild-type (wt) and the LexA noncleavable (lexAN) cells as indicated were incubated with macrophage. Top: phase-contrast microscopy of macrophage incubated with OMVs at 1.3 μg/mL after a 1 hr incubation. Bar, 20 μm. Bottom: cytotoxicity was measured as the levels of released cytosolic lactate dehydrogenase after 4 hr incubation. It was set 0 for the macrophage-only control. OMV concentrations: grey, 1.3 μg/mL; doted, 0.65 μg/mL; blank, 0.325 μg/mL. (*P < 0.05, n = 4).

Figure 5

The SDS-PAGE based proteomic analyses of OMV proteins from the SOS-induced and the un-induced cells. CPX, ciprofloxacin. N, LexA noncleavable; and wt, wild-type. Illustrated to the right were the proteins known to associate with OMVs, cytotoxicity, and SOS (See text and Tables for details).