title: Avoidance of autophagy mediated by PlcA or ActA is required for Listeria monocytogenes growth in macrophages.
abstract: Listeria monocytogenes is a facultative intracellular pathogen that escapes from phagosomes and grows in the cytosol of infected host cells. Most of the determinants that govern its intracellular life cycle are controlled by the transcription factor PrfA, including the pore-forming cytolysin listeriolysin O (LLO), two phospholipases C (PlcA and PlcB), and ActA. We constructed a strain that lacked PrfA but expressed LLO from a PrfA-independent promoter, thereby allowing the bacteria to gain access to the host cytosol. This strain did not grow efficiently in wild-type macrophages but grew normally in macrophages that lacked ATG5, a component of the autophagy LC3 conjugation system. This strain colocalized more with the autophagy marker LC3 (42% ± 7%) at 2 h postinfection, which constituted a 5-fold increase over the colocalization exhibited by the wild-type strain (8% ± 6%). While mutants lacking the PrfA-dependent virulence factor PlcA, PlcB, or ActA grew normally, a double mutant lacking both PlcA and ActA failed to grow in wild-type macrophages and colocalized more with LC3 (38% ± 5%). Coexpression of LLO and PlcA in a PrfA-negative strain was sufficient to restore intracellular growth and decrease the colocalization of the bacteria with LC3. In a cell-free assay, purified PlcA protein blocked LC3 lipidation, a key step in early autophagosome biogenesis, presumably by preventing the formation of phosphatidylinositol 3-phosphate (PI3P). The results of this study showed that avoidance of autophagy by L. monocytogenes primarily involves PlcA and ActA and that either one of these factors must be present for L. monocytogenes growth in macrophages.
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California, USA.
authors.forename: Gabriel
authors.initials: G
authors.lastname: Mitchell
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California, USA Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, California, USA.
authors.forename: Liang
authors.initials: L
authors.lastname: Ge
authors.affiliation: Department of Chemistry, Boston College, Chestnut Hill, Massachusetts, USA.
authors.forename: Qiongying
authors.initials: Q
authors.lastname: Huang
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California, USA.
authors.forename: Chen
authors.initials: C
authors.lastname: Chen
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California, USA.
authors.forename: Sara
authors.initials: S
authors.lastname: Kianian
authors.affiliation: Department of Chemistry, Boston College, Chestnut Hill, Massachusetts, USA.
authors.forename: Mary F
authors.initials: MF
authors.lastname: Roberts
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California, USA Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, California, USA.
authors.forename: Randy
authors.initials: R
authors.lastname: Schekman
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California, USA School of Public Health, University of California, Berkeley, Berkeley, California, USA portnoy@berkeley.edu.
authors.forename: Daniel A
authors.initials: DA
authors.lastname: Portnoy
mesh_terms: D000818:Animals; D001343:Autophagy / Q000276:immunology*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D017353:Gene Deletion; D057131:Immune Evasion; D008089:Listeria monocytogenes / Q000254:growth & development* / Q000276:immunology*; D008264:Macrophages / Q000382:microbiology*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008810:Mice, Inbred C57BL; D010738:Type C Phospholipases / Q000235:genetics / Q000378:metabolism*; D037521:Virulence Factors / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D008565:Membrane Proteins; D037521:Virulence Factors; C072980:actA protein, Listeria monocytogenes; D010738:Type C Phospholipases