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Surface-localized spermidine protects the Pseudomonas aeruginosa outer membrane from antibiotic treatment and oxidative stress.

ABSTRACT: Extracellular DNA acts as a cation chelator and induces the expression of antibiotic resistance genes regulated by Mg(2+) levels. Here we report the characterization of novel DNA-induced genes in Pseudomonas aeruginosa that are annotated as homologs of the spermidine synthesis genes speD (PA4773) and speE (PA4774). The addition of sublethal concentrations of DNA and membrane-damaging antibiotics induced expression of the genes PA4773 to PA4775, as shown using transcriptional lux fusions and quantitative RT-PCR. Exogenous polyamine addition prevented DNA- and peptide-mediated gene induction. Mutation of PA4774 resulted in an increased outer membrane (OM) susceptibility phenotype upon polymyxin B, CP10A, and gentamicin treatment. When the membrane-localized fluorescent probe C(11)-BODIPY(581/591) was used as an indicator of peroxidation of membrane lipids, the PA4774::lux mutant demonstrated an increased susceptibility to oxidative membrane damage from H(2)O(2) treatment. Addition of exogenous polyamines protected the membranes of the PA4774::lux mutant from polymyxin B and H(2)O(2) treatment. Polyamines from the outer surface were isolated and shown to contain putrescine and spermidine by using high-performance liquid chromatography and mass spectrometry. The PA4774::lux mutant did not produce spermidine on the cell surface, but genetic complementation restored surface spermidine production as well as the antibiotic and oxidative stress resistance phenotypes of the membrane. We have identified new functions for spermidine on the cell surface and propose that polyamines are produced under Mg(2+)-limiting conditions as an organic polycation to bind lipopolysaccharide (LPS) and to stabilize and protect the outer membrane against antibiotic and oxidative damage.

SUBMITTER: Johnson L

PROVIDER: S-EPMC3272965 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Pseudomonas aeruginosa (PA) is an environmentally ubiquitous, extracellular, opportunistic pathogen, associated with severe infections of immune-compromised host. We demonstrated earlier the presence of both α2,3- and α2,6-linked sialic acids (Sias) on PA (PA(+Sias)) and normal human serum is their source of Sias. PA(+Sias) showed decreased complement deposition and exhibited enhanced association with immune-cells through sialic acid binding immunoglobulin like lectins (Siglecs). Such Sias-siglec-9 interaction between PA(+Sias) and neutrophils helped to subvert host immunity. Additionally, PA(+Sias) showed more resistant to β-lactam antibiotics as reflected in their minimum inhibitory concentration required to inhibit the growth of 50% than PA(-Sias). Accordingly, we have affinity purified sialoglycoproteins of PA(+Sias). They were electrophoresed and identified by matrix-assisted laser desorption-ionization time-of-flight/time-of-flight mass spectrometry analysis. Sequence study indicated the presence of a few α2,6-linked, α2,3-linked, and both α2,3- and α2,6-linked sialylated proteins in PA. The outer membrane porin protein D (OprD), a specialized channel-forming protein, responsible for uptake of β-lactam antibiotics, is one such identified sialoglycoprotein. Accordingly, sialylated (OprD(+Sias)) and non-sialylated (OprD(-Sias)) porin proteins were separately purified by using anion exchange chromatography. Sialylation of purified OprD(+Sias) was confirmed by several analytical and biochemical procedures. Profiling of glycan structures revealed three sialylated N-glycans and two sialylated O-glycans in OprD(+Sias). In contrast, OprD(-Sias) exhibit only one sialylated N-glycans. OprD(-Sias) interacts with β-lactam antibiotics more than OprD(+Sias) as demonstrated by surface plasmon resonance study. Lyposome-swelling assay further exhibited that antibiotics have more capability to penetrate through OprD(-Sias) purified from four clinical isolates of PA. Taken together, it may be envisaged that sialic acids on OprD protein play important role toward the uptake of commonly used antibiotics in PA(+Sias). This might be one of the new mechanisms of PA for β-lactam antibiotic uptake.

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Surface-localized spermidine protects the Pseudomonas aeruginosa outer membrane from antibiotic treatment and oxidative stress.

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