title: Escape of intracellular Shigella from autophagy requires binding to cholesterol through the type III effector, IcsB.
abstract: Type III secretion systems are present in many pathogenic bacteria and mediate the translocation of bacterial effectors into host cells. Identification of host targets of these effectors is crucial for understanding bacterial virulence. IcsB, a type III secretion effector, helps Shigella to evade the host autophagy defense system by binding to the autophagy protein, Atg5. Here, we show that IcsB is able to interact specifically with cholesterol. The cholesterol binding domain (CBD) of IcsB is located between residues 288 and 351. Specific mutations of single tyrosine residues Y297 or Y340 of IcsB by phenylalanine (F) slightly reduced cholesterol binding, whereas deletion of the entire CBD or double mutation Y297F-Y340F strongly abolished interactions with cholesterol. To determine whether Shigella expressing IcsB variants could evade autophagy as effectively as the wild-type Shigella, we infected MDAMC cells stably expressing the autophagy marker LC3 fused to GFP and bacterial autophagosome formation was quantified using fluorescence microscopy. Mutation Y297F or Y340F slightly impaired IcsB function, whereas complete removal of CBD or mutation Y297F-Y340F significantly impaired autophagy evasion. Furthermore, we report that BopA, the counterpart of IcsB in Burkholderia pseudomallei with similar autophagy-evading properties, contains the CBD domain and is also able to bind cholesterol.
authors.affiliation: Laboratoire de Bactériologie Moléculaire, Faculté de Médecine, Université Libre de Bruxelles, Brussels, Belgium.
authors.forename: Christian Aimé
authors.initials: CA
authors.lastname: Kayath
authors.forename: Seamus
authors.initials: S
authors.lastname: Hussey
authors.forename: Nargisse
authors.initials: N
authors.lastname: El hajjami
authors.forename: Karan
authors.initials: K
authors.lastname: Nagra
authors.forename: Dana
authors.initials: D
authors.lastname: Philpott
authors.forename: Abdelmounaaïm
authors.initials: A
authors.lastname: Allaoui
mesh_terms: D001343:Autophagy; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D016957:Burkholderia pseudomallei / Q000276:immunology / Q000378:metabolism; D002460:Cell Line; D002784:Cholesterol / Q000378:metabolism*; D054884:Host-Pathogen Interactions; D006801:Humans; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D011485:Protein Binding; D017384:Sequence Deletion; D012763:Shigella flexneri / Q000276:immunology* / Q000378:metabolism*; D037521:Virulence Factors / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D050505:Mutant Proteins; D037521:Virulence Factors; D002784:Cholesterol