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Subversion of actin dynamics by EspM effectors of attaching and effacing bacterial pathogens

Ana Arbeloa1, Richard R Bulgin, Georgina MacKenzie

  • 1Division of Cell and Molecular Biology, Imperial College London, London SW7 2AZ, UK.

Cellular Microbiology
|March 12, 2008
PubMed

Insights

Bacterial WxxxE effectors, like EspM, manipulate host cell actin by activating the RhoA pathway. These effectors trigger distinct stress fiber formations, crucial for pathogen invasion and host cell manipulation.

Area of Science:

  • Microbiology
  • Cell Biology
  • Biochemistry

Background:

  • Rho GTPases are key regulators of the actin cytoskeleton and are frequently targeted by bacterial virulence factors.
  • Type III secretion system (TSS) effectors, including the WxxxE family, are injected into host cells to manipulate cellular processes.
  • Pathogens like *Shigella*, *E. coli* (EPEC, EHEC), and *Citrobacter rodentium* utilize WxxxE effectors for host cell invasion and pathogenesis.

Purpose of the Study:

  • To characterize the actin cytoskeleton-modulating activities of six WxxxE effectors from attaching and effacing pathogens.
  • To investigate the molecular mechanisms underlying the function of EspM effectors, focusing on RhoA signaling.
  • To determine the structural requirements for WxxxE effector activity.

Main Methods:

  • Expression and purification of WxxxE effector proteins.
  • Microscopy to visualize actin stress fiber formation in host cells.
  • Site-directed mutagenesis to probe the WxxxE motif's role in activity.
  • Biochemical assays including Rhotekin pull-downs and cofilin phosphorylation analysis.
  • Use of dominant-negative constructs and chemical inhibitors to assess pathway dependency.

Main Results:

  • EspM2, TrcA, and EspM1 induced distinct patterns of stress fiber formation (global parallel, localized parallel).
  • EspM3 triggered localized radial stress fibers, demonstrating diverse effector functions.
  • Mutations in the conserved WxxxE motif (Trp to Ala) abolished effector activity, while conservative substitutions retained function.
  • EspM2 and EspM3 activity was dependent on RhoA and ROCK signaling.
  • EspM2 and EspM3 activated RhoA and led to cofilin phosphorylation, indicating modulation of actin dynamics.

Conclusions:

  • The EspM family of WxxxE effectors from attaching and effacing pathogens significantly impacts host cell actin dynamics.
  • These effectors function through the RhoA/ROCK signaling pathway to induce specific stress fiber rearrangements.
  • Understanding these effector mechanisms provides insights into bacterial pathogenesis and host-cell manipulation strategies.

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