Rho GTPase activity modulates Pseudomonas aeruginosa internalization by epithelial cells

Area of Science:

• Microbiology
• Cell Biology
• Bacterial Pathogenesis

Background:

• *Pseudomonas aeruginosa* is a Gram-negative pathogen that invades host epithelial cells.
• Bacterial internalization pathways are actin-dependent and involve Rho family GTPases.
• Epithelial cell polarization affects bacterial internalization, suggesting a role for Rho GTPases.

Purpose of the Study:

• To investigate the role of Rho family GTPases in *Pseudomonas aeruginosa* internalization into epithelial cells.
• To determine if RhoA, Rac1, or Cdc42 mediate bacterial uptake.
• To elucidate the mechanism by which *P. aeruginosa* invades epithelial cells.

Main Methods:

• *Pseudomonas aeruginosa* strain PA103 internalization assays in Madin-Darby canine kidney (MDCK) cells.
• Stimulation of Rho family GTPases using cytotoxic necrotizing factor 1 (CNF-1).
• Expression of constitutively active and dominant-negative Rho GTPase alleles.
• Inhibition of actin polymerization with latrunculin A.
• Measurement of endogenous Rho GTP levels during bacterial infection.

Main Results:

• Activation of Rho family GTPases by CNF-1 stimulates *P. aeruginosa* internalization.
• Constitutively active RhoA (RhoAV14) increases bacterial uptake, while Rac1 and Cdc42 do not.
• RhoAV14-mediated uptake is actin-dependent and not solely due to tight junction disruption.
• Infection with invasive *P. aeruginosa* increases endogenous Rho GTP levels, while non-invasive strains decrease them.
• Experimental inhibition of Rho does not prevent bacterial internalization.

Conclusions:

• RhoA activation, but not inhibition, promotes *P. aeruginosa* internalization.
• RhoA plays a complex role in bacterial invasion, potentially involving both promotion and regulation.
• The findings provide insights into the host cell mechanisms exploited by *P. aeruginosa* for invasion.