title: Functional characterization of a phospholipase A(2) homolog from Rickettsia typhi.
abstract: Phospholipase A(2) (PLA(2)) has long been proposed to be involved in rickettsial entry into host cells, escape from the phagosome to evade destruction by lysosomal exposure, and lysis of the host cells. However, the corresponding rickettsial gene(s) encoding a protein with PLA(2) activity has not been identified or functionally characterized. Here, we report that the Rickettsia typhi genome possesses two genes encoding patatin-like PLA(2) proteins, RT0590 and RT0522. Sequence analysis of RT0522 and RT0590 reveals the presence of the conserved motifs essential for PLA(2) activity. Transcriptional analysis indicates that RT0522, but not RT0590, is transcribed at all stages of intracellular growth of R. typhi in Vero cells. The differential gene expression pattern of RT0522 at various stages of growth suggests its potential role during R. typhi infection of host cells. In silico, RT0522 is predicted to be noncytoplasmic and its gene does not encode a recognizable signal peptide sequence. However, our data indicate that RT0522 is secreted into the host cytoplasm. In addition, we observe that RT0522 protein expression is cytotoxic to both yeast and Vero cells. Importantly, we demonstrate that recombinant RT0522 possesses phospholipase A activity that requires a eukaryotic host cofactor for activation. Both cytotoxicity and phospholipase A activity associated with RT0522 were reduced by PLA(2) inhibitors. Site-directed mutagenesis of predicted catalytic Ser/Asp residues of RT0522 also eliminates cytotoxicity and phospholipase A activity. To our knowledge, RT0522 is the first protein identified from Rickettsia typhi with functional phospholipase A activity.
authors.affiliation: Department of Microbiology and Immunology, University of Maryland School of Medicine, 685 West Baltimore Street, Room HSF1-380, Baltimore, MD 21201, USA. mrahm001@umaryland.edu
authors.forename: M Sayeedur
authors.initials: MS
authors.lastname: Rahman
authors.forename: Nicole C
authors.initials: NC
authors.lastname: Ammerman
authors.forename: Khandra T
authors.initials: KT
authors.lastname: Sears
authors.forename: Shane M
authors.initials: SM
authors.lastname: Ceraul
authors.forename: Abdu F
authors.initials: AF
authors.lastname: Azad
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002522:Chlorocebus aethiops; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D054467:Phospholipases A2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012286:Rickettsia typhi / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D014709:Vero Cells
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D054467:Phospholipases A2