title: Conserved Pro-Glu (PE) and Pro-Pro-Glu (PPE) protein domains target LipY lipases of pathogenic mycobacteria to the cell surface via the ESX-5 pathway.
abstract: The type VII secretion system ESX-5 is a major pathway for export of PE and PPE proteins in pathogenic mycobacteria. These mycobacteria-specific protein families are characterized by conserved N-terminal domains of 100 and 180 amino acids, which contain the proline-glutamic acid (PE) and proline-proline-glutamic acid (PPE) motifs after which they are named. Here we investigated secretion of the triacylglycerol lipase LipY, which in fast-growing mycobacteria contains a signal sequence, but in slow-growing species appears to have replaced the signal peptide with a PE or PPE domain. Selected LipY homologues were expressed in wild-type Mycobacterium marinum and its corresponding ESX-5 mutant, and localization of the proteins was investigated by immunoblotting and electron microscopy. Our study shows that Mycobacterium tuberculosis PE-LipY (LipY(tub)) and M. marinum PPE-LipY (LipY(mar)) are both secreted to the bacterial surface in an ESX-5-dependent fashion. After transport, the PE/PPE domains are removed by proteolytic cleavage. In contrast, Mycobacterium gilvum LipY, which has a signal sequence, is not transported to the cell surface. Furthermore, we show that LipY(tub) and LipY(mar) require their respective PE and PPE domains for ESX-5-dependent secretion. The role of the PE domain in ESX-5 secretion was confirmed in a whole cell lipase assay, in which wild-type bacteria expressing full-length LipY(tub), but not LipY(tub) lacking its PE domain, were shown to hydrolyze extracellular lipids. In conclusion, both PE and PPE domains contain a signal required for secretion of LipY by the ESX-5 system, and these domains are proteolytically removed upon translocation.
authors.affiliation: Department of Medical Microbiology and Infection Control, VU University Medical Centre, 1081 BT Amsterdam, The Netherlands.
authors.forename: Maria H
authors.initials: MH
authors.lastname: Daleke
authors.forename: Alessandro
authors.initials: A
authors.lastname: Cascioferro
authors.forename: Karin
authors.initials: K
authors.lastname: de Punder
authors.forename: Roy
authors.initials: R
authors.lastname: Ummels
authors.forename: Abdallah M
authors.initials: AM
authors.lastname: Abdallah
authors.forename: Nicole
authors.initials: N
authors.lastname: van der Wel
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Peters
authors.forename: Joen
authors.initials: J
authors.lastname: Luirink
authors.forename: Riccardo
authors.initials: R
authors.lastname: Manganelli
authors.forename: Wilbert
authors.initials: W
authors.lastname: Bitter
mesh_terms: D020816:Amino Acid Motifs; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D058947:Bacterial Secretion Systems / Q000502:physiology*; D008049:Lipase / Q000235:genetics / Q000378:metabolism*; D019910:Mycobacterium marinum / Q000201:enzymology*; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D021382:Protein Sorting Signals / Q000502:physiology*; D017434:Protein Structure, Tertiary; D013045:Species Specificity
chemical_list: D001426:Bacterial Proteins; D058947:Bacterial Secretion Systems; D021382:Protein Sorting Signals; D008049:Lipase