Macrophages To Legionella Pneumophila Encoding Ubiquitin Ligase Biochemical Properties And Biological Functions Of Research

Legionella pneumophila, the causative agent of Legionnaires' pneumonia, grows in a membrane-bound vacuole within host cells, upon uptake by alveolar macrophages. Through manipulating the secretory and endocytic traffic pathway of the host cell, Legionella promotes close association of their replication compartment with specific host organelles. This intimate touch facilitates membrane material exchange between the Legionella-containing vacuole (LCV) and host organelles, and prevents from fusion with the lysosome. The LCV recruits early secretory vesicles from the host endoplasmic reticulum (ER) and transforms its vacuole into a ribosome-studded compartment that morphologically resembles rough ER. Within this specialized vacuole, L. pneumophila replicates to a high density, which results into ultimate lysis of the host cell, release bacteria of new generation and subsequent infection of neighboring macrophages. Intracellular growth of L. pneumophila requires an intact typeⅣsecretion system (T4SS), encoded by the dot/icm gene clusters. This T4SS mediates translocation of bacterial effector molecules into the host cell cytosol. Mutations in dot/icm genes lead to defective targeting of the LCV into an endocytic compartment and an inability to associate with the rough ER. Little is known about the identity and function of the translocated substrates of the L. pneumophila Dot/Icm system.Here we describe and characterize three candidate effectors (1pg2830, 1pg2144 and 1pg0171) that harbor eukaryotic specific ubiquitin ligase activity. Lpg2830 undergoes selfubiquitination in vivo or in vitro in the presence of E1, ATP, appropriate E2 and ubiquitin molecule. Intriguingly, the linkage of ubiquitin chains formed between in vivo and in vitro appear ro be be differerent. Lpg2144 and lpg0171 interacts with the eukaryotic Cullin 1 and Skp 1 and forms a SCF-like E3 ligase complex when expressed in the mammalian cells. Sequence examination and confocal immunofluorescence studies suggest that all three putative Legionella E3 liages have a distinct functional C-terminal membrane targeting signal including the eukaryotic specific CAAX motif. Protein interaction studies with IcmW, the chaperon of the TFSS, and infection of U937 monocytes confirm that all three proteins are indeed secreted by the Legionella typeⅣsecretion system (TFSS). In the intracellular growth assay, neither single mutation in the three genes nor lpg2144 and lpg0171 double mutation has any effect in the intracellular multiplication of Legionella. Despite the little effect in the intracellular growth, the biochemical characterization of the three effector candidates highlights their potential roles in hijacking the host ubiquitin system for Legionella pathogenesis.