Vesicle-Mediated Transfer of Virulence Genes from Escherichia coli O157:H7 to Other Enteric Bacteria

Many gram-negative bacteria produce membrane vesicles, suggesting that vesicle production is not purposeless; indeed, studies during the last two decades have presented strong evidence supporting the importance of vesicles. Typical vesicles released from the surfaces of gram-negative bacteria are 50 to 250 nm, spherical, and made up of outer membrane and encapsulated periplasmic components (4, 26). Vesicle components include outer membrane proteins, lipopolysaccharide, periplasmic proteins, phospholipids, DNA, and RNA (9, 12, 15, 22, 34, 40). Vesicles from gram-negative bacteria were reported to fuse to both gram-positive and gram-negative bacteria and in some instances to promote lysis of the target cell (28). Moreover, vesicles may function as an alternative secretory pathway (3, 23) and promote adherence of the parent cell to host cells (17, 32). By virtue of their small size, bilayer protecting envelope, and ability to integrate into the membranes of foreign bacteria and to adhere to or be engulfed by eukaryotic cells, a potential role of vesicles in delivery of virulence factors, including enzymes and toxins, is not unlikely (23). In fact, virulence factors associated with the parent strain, including proteases, phospholipases, autolysin, hemolysins, and Shiga toxins, have been isolated from vesicles (3, 22, 26, 28). Aside from toxic compounds, DNA has also been isolated from vesicles. Vesicles produced by Pseudomonas aeruginosa were reported to contain DNA (22). Vesicles released by Neisseria gonorrhoeae harbor both linear and circular DNA, including 4.2- and 7.1-kb plasmids (12). Chromosomal and bacteriophage-associated virulence genes were detected in Escherichia coli O157:H7 vesicles (26). Moreover, this research demonstrated that DNA was protected from digestion by DNase, suggesting that DNA is packaged within vesicles (26). Bacterial evolution often proceeds by horizontal gene transfer between different genera and species (1, 7). Antibiotic resistance genes and pathogenicity islands have been acquired by a variety of pathogens, including E. coli, Salmonella enterica serovar Typhimurium, Yersinia pestis, Dichelobacter nodosis, and Helicobacter pylori (19). Virulence factors contributing to the pathogenicity of E. coli O157:H7, including Shiga toxins (45, 46) and intimin (31, 44), are encoded on pathogenicity islands in the O157 chromosome and are thought to have been acquired by horizontal transfer. Results of previous studies suggest that vesicles may be involved in the transfer of genetic material among similar bacterial species (8, 12, 26). The hypothesis has been put forth that vesicles influence antibiotic resistance in other bacteria in two ways: by physical dissemination of preformed antibiotic-inactivating enzymes into the recipient periplasm and by delivery of antibiotic resistance plasmids (3, 12). Competent Haemophilus influenzae produces vesicles which are released into the medium when cells are returned to normal growth conditions or a noncompetent state (8). Specific DNA-binding peptides were reported to be present on the surfaces of H. influenzae vesicles (24, 25) and to be associated with vesicles from N. gonorrhoeae (11). Previously, it was reported that vesicles released by E. coli O157:H7 into culture medium contain virulence genes and Shiga toxin (26). In the present study, we demonstrate that E. coli O157:H7 vesicles mediate the transfer of virulence genes, which are subsequently expressed by recipient enteric bacteria. Moreover, the origin of the DNA in E. coli O157:H7 vesicles is elucidated. Observations show that in addition to bacteriophage-associated genes, E. coli O157:H7 vesicles contain plasmids and fragments of chromosomal DNA.