A role for ActA in epithelial cell invasion by Listeria monocytogenes.
We assessed the role of the actin-polymerizing protein, ActA, in host cell invasion by Listeria monocytogenes. An in frame DeltaactA mutant was constructed in a hyperinvasive strain of prfA* genotype, in which all genes of the PrfA-dependent virulence regulon, including actA, are highly expressed in vitro. Loss of ActA production in prfA* bacteria reduced entry into Caco-2, HeLa, MDCK and Vero epithelial cells to basal levels. Reintroduction of actA into the DeltaactA prfA* mutant fully restored invasiveness, demonstrating that ActA is involved in epithelial cell invasion. ActA did not contribute to internalization by COS-1 fibroblasts and Hepa 1-6 hepatocytes. Expression of actA in Listeria innocua was sufficient to promote entry of this non-invasive species into epithelial cell lines, but not into COS-1 and Hepa 1-6 cells, indicating that ActA directs an internalization pathway specific for epithelial cells. Scanning electron microscopy of infected Caco-2 human enterocytes suggested that this pathway involves microvilli. prfA* bacteria, but not wild-type bacteria (which express PrfA-dependent genes very weakly in vitro) or prfA* DeltaactA bacteria, efficiently invaded differentiated Caco-2 cells via their apical surface. Microvilli played an active role in the phagocytosis of the prfA* strain, and actA was required for their remodelling into pseudopods mediating bacterial uptake. Thus, ActA appears to be a multifunctional virulence factor involved in two important aspects of Listeria pathogenesis: actin-based motility and host cell tropism and invasion.
Suárez
M
M
Grupo de Patogénesis Molecular Bacteriana, Unidad de Microbiología e Inmunología, Facultad de Veterinaria, Universidad Complutense, 28040 Madrid, Spain.
González-Zorn
B
B
Vega
Y
Y
Chico-Calero
I
I
Vázquez-Boland
J A
JA
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D001483:Base Sequence; D019556:COS Cells; D018938:Caco-2 Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D004269:DNA, Bacterial; D004285:Dogs; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism / Q000382:microbiology; D015870:Gene Expression; D006367:HeLa Cells; D006801:Humans; D008089:Listeria monocytogenes / Q000302:isolation & purification / Q000378:metabolism / Q000502:physiology* / Q000648:ultrastructure; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D008855:Microscopy, Electron, Scanning / Q000379:methods; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010454:Peptide Termination Factors; D015534:Trans-Activators; D014709:Vero Cells
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D008565:Membrane Proteins; D010454:Peptide Termination Factors; D015534:Trans-Activators; C072980:actA protein, Listeria monocytogenes