The SiaABC threonine phosphorylation pathway controls biofilm formation in response to carbon availability in Pseudomonas aeruginosa

PLoS One. 2020 Nov 6;15(11):e0241019. doi: 10.1371/journal.pone.0241019. eCollection 2020.

Abstract

The critical role of bacterial biofilms in chronic human infections calls for novel anti-biofilm strategies targeting the regulation of biofilm development. However, the regulation of biofilm development is very complex and can include multiple, highly interconnected signal transduction/response pathways, which are incompletely understood. We demonstrated previously that in the opportunistic, human pathogen P. aeruginosa, the PP2C-like protein phosphatase SiaA and the di-guanylate cyclase SiaD control the formation of macroscopic cellular aggregates, a type of suspended biofilms, in response to surfactant stress. In this study, we demonstrate that the SiaABC proteins represent a signal response pathway that functions through a partner switch mechanism to control biofilm formation. We also demonstrate that SiaABCD functionality is dependent on carbon substrate availability for a variety of substrates, and that upon carbon starvation, SiaB mutants show impaired dispersal, in particular with the primary fermentation product ethanol. This suggests that carbon availability is at least one of the key environmental cues integrated by the SiaABCD system. Further, our biochemical, physiological and crystallographic data reveals that the phosphatase SiaA and its kinase counterpart SiaB balance the phosphorylation status of their target protein SiaC at threonine 68 (T68). Crystallographic analysis of the SiaA-PP2C domain shows that SiaA is present as a dimer. Dynamic modelling of SiaA with SiaC suggested that SiaA interacts strongly with phosphorylated SiaC and dissociates rapidly upon dephosphorylation of SiaC. Further, we show that the known phosphatase inhibitor fumonisin inhibits SiaA mediated phosphatase activity in vitro. In conclusion, the present work improves our understanding of how P. aeuruginosa integrates specific environmental conditions, such as carbon availability and surfactant stress, to regulate cellular aggregation and biofilm formation. With the biochemical and structural characterization of SiaA, initial data on the catalytic inhibition of SiaA, and the interaction between SiaA and SiaC, our study identifies promising targets for the development of biofilm-interference drugs to combat infections of this aggressive opportunistic pathogen.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Biofilms / drug effects
  • Biofilms / growth & development*
  • Carbon / metabolism*
  • Crystallography, X-Ray
  • Fumonisins / pharmacology
  • Humans
  • Microscopy, Electron, Scanning
  • Models, Molecular
  • Molecular Docking Simulation
  • Molecular Dynamics Simulation
  • Phosphoprotein Phosphatases / chemistry
  • Phosphoprotein Phosphatases / genetics
  • Phosphoprotein Phosphatases / metabolism
  • Phosphorylation
  • Protein Kinases / chemistry
  • Protein Kinases / genetics
  • Protein Kinases / metabolism
  • Pseudomonas aeruginosa / genetics
  • Pseudomonas aeruginosa / pathogenicity
  • Pseudomonas aeruginosa / physiology*
  • Signal Transduction
  • Threonine / metabolism*

Substances

  • Bacterial Proteins
  • Fumonisins
  • Threonine
  • fumonisin B1
  • Carbon
  • Protein Kinases
  • Phosphoprotein Phosphatases

Grants and funding

The work of Janosch Klebensberger, Wee Han Poh, Julien Lescar, Jianqing Lin, Scott A. Rice were partially supported by a joint mobility grant funded by the Federal Ministry of Education and Research in Germany (FKZ: 01DP17004) and the Ministry of Trade and Industry in Singapore (SGP-PROG3-023. 2017). We acknowledge financial support from the Singapore Centre for Environmental Life Sciences Engineering, whose research is supported by the National Research Foundation Singapore, Ministry of Education, Nanyang Technological University and National University of Singapore, under its Research Centre of Excellence Programme as well as funding from the Ministry of Education (AcRF tier 1 grant RG154/14). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.