Stress-induced outer membrane vesicle production by Pseudomonas aeruginosa

Abstract

As an opportunistic Gram-negative pathogen, Pseudomonas aeruginosa must be able to adapt and survive changes and stressors in its environment during the course of infection. To aid survival in the hostile host environment, P. aeruginosa has evolved defense mechanisms, including the production of an exopolysaccharide capsule and the secretion of a myriad of degradative proteases and lipases. The production of outer membrane-derived vesicles (OMVs) serves as a secretion mechanism for virulence factors as well as a general bacterial response to envelope-acting stressors. This study investigated the effect of sublethal physiological stressors on OMV production by P. aeruginosa and whether the Pseudomonas quinolone signal (PQS) and the MucD periplasmic protease are critical mechanistic factors in this response. Exposure to some environmental stressors was determined to increase the level of OMV production as well as the activity of AlgU, the sigma factor that controls MucD expression. Overexpression of AlgU was shown to be sufficient to induce OMV production; however, stress-induced OMV production was not dependent on activation of AlgU, since stress caused increased vesiculation in strains lacking algU. We further determined that MucD levels were not an indicator of OMV production under acute stress, and PQS was not required for OMV production under stress or unstressed conditions. Finally, an investigation of the response of P. aeruginosa to oxidative stress revealed that peroxide-induced OMV production requires the presence of B-band but not A-band lipopolysaccharide. Together, these results demonstrate that distinct mechanisms exist for stress-induced OMV production in P. aeruginosa.

Fig 1

Treatments with stressors induce OMV production in PA14 independent of AlgU and MucD levels. OMVs were collected and quantitated from cultures of PA14/pLW127 treated with 250 μg/ml d-cycloserine (A), 4 μg/ml polymyxin B (B), or 250 μM hydrogen peroxide (H₂O₂) (C). OMV yields were averaged and normalized to untreated controls (untreated) to calculate fold change. (D) OMVs were collected and quantitated from cultures of PA14 pLW127 grown at 25°C and shifted to 37°C or 39°C. OMV yields were averaged and normalized to a culture maintained at 25°C (25°C). AlgU promoter activity was measured in cultures (37°C; OD₆₀₀, 0.9 to 1.1) of PA14/pLW127 treated with 250 μg/ml d-cycloserine (E), 4 μg/ml polymyxin B (F), or 250 μM hydrogen peroxide (H₂O₂) (G) using a β-galactosidase assay. Values were averaged and normalized to untreated controls (untreated) to calculate fold change. (H) AlgU promoter activity was measured in cultures (OD₆₀₀, 0.9 to 1.1) of PA14/pLW127 grown at 25°C and shifted to 37°C or 39°C. Values were averaged and normalized to a culture maintained at 25°C to calculate fold change. Periplasmic MucD expression in PA14/pLW127 15 to 30 min after treatment with or without 250 μg/ml d-cycloserine (I), 4 μg/ml polymyxin B (J), or 250 μM hydrogen peroxide (H₂O₂) (K) was determined by densitometry. Values were averaged and normalized to the untreated samples to calculate fold change. *, P ≤ 0.05; **, P ≤ 0.01 (n ≥ 3).

Fig 2

AlgU expression in PA14 is sufficient but not necessary for OMV production. (A) OMVs were collected and quantitated from cultures of PA14 ΔalgU/pMF54 (ΔalgU + vector) and PA14 ΔalgU/pIM001 (ΔalgU + AlgU) that had both been supplemented with 1 mM IPTG at mid-log phase. The difference was not significant (P = 0.068). (B) OMVs were collected and quantitated from cultures of PA14 ΔalgD/pMF54 (ΔalgD + vector) and PA14 ΔalgD/pIM001 (ΔalgD + AlgU) that had both been supplemented with 200 μM IPTG at mid-log phase. (C) OMVs were collected and quantitated from cultures of PA14 ΔalgU (ΔalgU) treated with 250 μM hydrogen peroxide (ΔalgU + H₂O₂). OMV yield was normalized to that of the vector-containing (A and B) or untreated culture (C) to calculate fold change. *, P < 0.05; **, P ≤ 0.01 (n = 3).

Fig 3

Overexpression of MucD increases OMV production by PAO1, but not PA14. (A) Periplasmic MucD expression in cultures of PA14/pMF54 (Vector + In) or PA14/pLW112 (MucD + In) induced with 1 mM IPTG was measured by densitometry of immunoblotted samples and the values normalized to the vector control to calculate fold change. (B) OMVs were collected and quantitated from cultures of PA14/pMF54 (Vector + In) and PA14/pLW112 (MucD +In) both induced with IPTG at mid-log phase, and OMV yield was normalized to the induced vector control (Vector) to calculate fold change. (C) OMVs were collected and quantitated from cultures of PAO1/pMF54 (PAO1 + In) and PAO1/pLW112 (PAO1/MucD +In) both induced with IPTG at mid-log phase, and OMV yield normalized to the induced vector control (PAO1 + In) to calculate fold change. *, P ≤ 0.05; **, P ≤ 0.01 (n = 3).

Fig 4

MucD overexpression does not decrease oxidative stress induction of OMVs in PA14. OMVs were collected and quantitated from cultures of PA14/pMF54 (Vector + H2O2) and PA14/pLW112 (MucD + H2O2), both induced with IPTG and treated with 250 μM hydrogen peroxide at mid-log phase. OMV yield was normalized to the induced vector control (Vector + H2O2) to calculate fold change. **, P ≤ 0.01 (n = 3).

Fig 5

PQS is not required for constitutive or stress-induced OMV production in PA14. (A) PA14 and PA14 ΔpqsA were grown to late log phase in LB broth, and the cell-free supernatants were precipitated using ammonium sulfate. OMVs were purified from the concentrated supernatant by density gradient fractionation. OMV production was determined by FM4-64 quantitation of lipid in the OMV fractions (FM4-64/CFU). (B) PA14 and PA14 ΔpqsA were grown overnight in BHI broth and the cell-free supernatants were ultracentrifuged to isolate OMVs. OMV production was determined by FM4-64 quantitation of lipid in the OMV fractions (FM4- 64/CFU). (C to E) OMVs were collected and quantitated from cultures of PA14 ΔpqsA treated with 250 μg/ml d-cycloserine (C), 4 μg/ml polymyxin B (D), or 1 mM hydrogen peroxide (H₂O₂) (E). OMV yield was normalized to untreated controls (ΔpqsA) to calculate fold change. **, P ≤ 0.01 (n ≥ 3).

Fig 6

B-band LPS is required for H₂O₂-induced OMV formation in PA14. OMVs were collected and quantitated from untreated cultures of PA14, PA14 ΔwbpM (B-band mutant), PA14 Δrmd (A-band mutant), and PA14 ΔwapR (A- and B-band mutant) and from cultures treated with 250 μM hydrogen peroxide (ΔwbpM + H2O2, Δrmd + H2O2, ΔwapR + H2O2). OMV yield was normalized to that of an untreated culture of PA14 (PA14). *, P ≤ 0.05; **, P ≤ 0.01 (n ≥ 3).