Characterization of Listeria monocytogenes pathogenesis in a strain expressing perfringolysin O in place of listeriolysin O.
Listeriolysin O (LLO) is a pore-forming cytolysin that enables Listeria monocytogenes to escape from a host cell vacuole. The structural gene for the related cytolysin perfringolysin O (pfo) was cloned downstream from the promoter for hly, the gene encoding LLO, both on a plasmid and on the L. monocytogenes chromosome. Both strains secreted active PFO, although regulation was not identical to that of LLO. The chromosomal PFO-expressing strain was characterized for intracellular growth and cell-to-cell spread. It escaped from a host cell vacuole with 64% efficiency compared with the wild type as determined by immunofluorescent staining of bacteria for F-actin, a marker for entry into the cytoplasm. In addition, it replicated intracellularly with a doubling time similar to that of the wild type for 5 h, after which growth was aborted because of a cytotoxic effect on the host cell and influx of extracellular gentamicin. The chromosomal PFO strain was able to plaque in mouse L2 fibroblasts, but it did so at 20% efficiency compared with the wild type and the plaques were significantly smaller. Both strains expressing PFO were completely avirulent in mice. These results indicate that PFO can mediate escape from a host cell vacuole but cannot complement an hly deletion strain for virulence.
Jones
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Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia 19104-6076.
Portnoy
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DA
D000818:Animals; D001427:Bacterial Toxins / Q000235:genetics*; D001433:Bacteriolysis; D001483:Base Sequence; D002450:Cell Communication; D002876:Chromosomes, Bacterial; D003001:Cloning, Molecular; D005347:Fibroblasts; D006360:Heat-Shock Proteins / Q000235:genetics*; D006460:Hemolysin Proteins / Q000235:genetics*; D008089:Listeria monocytogenes / Q000235:genetics / Q000254:growth & development / Q000472:pathogenicity*; D008088:Listeriosis / Q000209:etiology; D008264:Macrophages; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D014617:Vacuoles / Q000502:physiology; D014774:Virulence / Q000235:genetics
D001427:Bacterial Toxins; D006360:Heat-Shock Proteins; D006460:Hemolysin Proteins; C020452:Clostridium perfringens theta-toxin; C038705:hlyA protein, Listeria monocytogenes