Long-Distance Delivery of Bacterial Virulence Factors by Pseudomonas aeruginosa Outer Membrane Vesicles

Abstract

Bacteria use a variety of secreted virulence factors to manipulate host cells, thereby causing significant morbidity and mortality. We report a mechanism for the long-distance delivery of multiple bacterial virulence factors, simultaneously and directly into the host cell cytoplasm, thus obviating the need for direct interaction of the pathogen with the host cell to cause cytotoxicity. We show that outer membrane–derived vesicles (OMV) secreted by the opportunistic human pathogen Pseudomonas aeruginosa deliver multiple virulence factors, including β-lactamase, alkaline phosphatase, hemolytic phospholipase C, and Cif, directly into the host cytoplasm via fusion of OMV with lipid rafts in the host plasma membrane. These virulence factors enter the cytoplasm of the host cell via N-WASP–mediated actin trafficking, where they rapidly distribute to specific subcellular locations to affect host cell biology. We propose that secreted virulence factors are not released individually as naked proteins into the surrounding milieu where they may randomly contact the surface of the host cell, but instead bacterial derived OMV deliver multiple virulence factors simultaneously and directly into the host cell cytoplasm in a coordinated manner. Gram-negative pathogens are responsible for 2 million annual hospital-acquired infections, adding tremendously to U.S. healthcare costs. Pseudomonas aeruginosa, an opportunistic human pathogen, is commonly associated with nosocomial infections, particularly ventilator-associated infections and pseudomonal pneumonia in immunocompromised patients with cystic fibrosis, chronic obstructive pulmonary disease, ventilator-associated pneumonia, community-acquired pneumonia, and bronchiectasis. We have identified the mechanism for a secretion system that Gram-negative bacteria use to strategically deliver toxins to the host to promote bacterial virulence and host colonization, a pathway that we hope to target to develop new therapies to treat P. aeruginosa infections. Our findings have significant implications for the study of Gram-negative bacterial pathogenesis. We propose that secreted virulence factors are not released individually as naked proteins into the surrounding milieu where they may randomly contact the surface of the host cell, but instead bacterial-derived outer membrane vesicles (OMV) deliver multiple virulence factors simultaneously and directly into the host cell cytoplasm in a coordinated manner. This long-distance bacterial communication to the host via OMV is reminiscent of the delivery of signaling proteins and miRNA between eukaryotic cells via exosomes, and may represent a general protein secretion strategy used by both pathogen and host.