Outer membrane machinery and alginate synthesis regulators control membrane vesicle production in Pseudomonas
Yosuke Tashiro1, Ryosuke Sakai, Masanori Toyofuku
1Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan.
Abstract:
The opportunistic human bacterial pathogen Pseudomonas aeruginosa produces membrane vesicles (MVs) in its surrounding environment. Several features of the P. aeruginosa MV production mechanism are still unknown. We previously observed that depletion of Opr86, which has a role in outer membrane protein (OMP) assembly, resulted in hypervesiculation. In this study, we showed that the outer membrane machinery and alginate synthesis regulatory machinery are closely related to MV production in P. aeruginosa. Depletion of Opr86 resulted in increased expression of the periplasmic serine protease MucD, suggesting that the accumulation of misfolded OMPs in the periplasm is related to MV production. Indeed, the mucD mutant showed a mucoid phenotype and the mucD mutation caused increased MV production. Strains with the gene encoding alginate synthetic regulator AlgU, MucA, or MucB deleted also caused altered MV production. Overexpression of either MucD or AlgW serine proteases resulted in decreased MV production, suggesting that proteases localized in the periplasm repress MV production in P. aeruginosa. Deletion of mucD resulted in increased MV proteins, even in strains with mutations in the Pseudomonas quinolone signal (PQS), which serves as a positive regulator of MV production. This study suggests that misfolded OMPs may be important for MV production, in addition to PQS, and that these regulators act in independent pathways.
Insights
Misfolded outer membrane proteins (OMPs) and alginate synthesis regulators influence Pseudomonas aeruginosa membrane vesicle (MV) production. Periplasmic proteases repress MV production, acting independently of the Pseudomonas quinolone signal (PQS).
Area of Science:
- Microbiology
- Bacterial Pathogenesis
- Cell Biology
Background:
- Pseudomonas aeruginosa, an opportunistic pathogen, generates membrane vesicles (MVs).
- The precise mechanisms regulating P. aeruginosa MV production remain incompletely understood.
- Previous work indicated that Opr86 depletion, affecting outer membrane protein (OMP) assembly, leads to increased MV production.
Purpose of the Study:
- To investigate the relationship between outer membrane machinery, alginate synthesis regulation, and P. aeruginosa MV production.
- To elucidate the role of misfolded OMPs and periplasmic proteases in MV biogenesis.
- To determine how these factors interact with the Pseudomonas quinolone signal (PQS) pathway in regulating MV production.
Main Methods:
- Genetic manipulation of P. aeruginosa strains, including gene deletions and overexpression.
- Analysis of outer membrane protein (OMP) assembly and periplasmic protein expression.
- Assessment of membrane vesicle (MV) production and protein content.
- Investigation of alginate synthesis regulatory pathways (AlgU, MucA, MucB) and protease activity (MucD, AlgW).
Main Results:
- Depletion of Opr86 increased MucD expression, linking misfolded OMPs to MV production.
- Mutations in mucD, algU, mucA, or mucB significantly altered MV production.
- Overexpression of MucD or AlgW proteases decreased MV production, indicating a repressive role.
- Deletion of mucD enhanced MV protein content, even in PQS pathway mutants.
- Misfolded OMPs and PQS appear to regulate MV production through independent pathways.
Conclusions:
- Misfolded OMPs, regulated by periplasmic proteases like MucD, are key contributors to P. aeruginosa MV production.
- Alginate synthesis regulatory machinery also plays a role in modulating MV biogenesis.
- These pathways are distinct from the PQS-mediated regulation of MV production.
- Understanding these mechanisms offers insights into P. aeruginosa virulence and intercellular communication.
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