Abstract
The long-standing proposal that phospholipase A2 (PLA2) enzymes are involved in rickettsial infection of host cells has been given support by the recent characterization of a patatin phospholipase (Pat2) with PLA2 activity from the pathogens Rickettsia prowazekii and R. typhi. However, pat2 is not encoded in all Rickettsia genomes; yet another uncharacterized patatin (Pat1) is indeed ubiquitous. Here, evolutionary analysis of both patatins across 46 Rickettsia genomes revealed 1) pat1 and pat2 loci are syntenic across all genomes, 2) both Pat1 and Pat2 do not contain predicted Sec-dependent signal sequences, 3) pat2 has been pseudogenized multiple times in rickettsial evolution, and 4) ubiquitous pat1 forms two divergent groups (pat1A and pat1B) with strong evidence for recombination between pat1B and plasmid-encoded homologs. In light of these findings, we extended the characterization of R. typhi Pat1 and Pat2 proteins and determined their role in the infection process. As previously demonstrated for Pat2, we determined that 1) Pat1 is expressed and secreted into the host cytoplasm during R. typhi infection, 2) expression of recombinant Pat1 is cytotoxic to yeast cells, 3) recombinant Pat1 possesses PLA2 activity that requires a host cofactor, and 4) both Pat1 cytotoxicity and PLA2 activity were reduced by PLA2 inhibitors and abolished by site-directed mutagenesis of catalytic Ser/Asp residues. To ascertain the role of Pat1 and Pat2 in R. typhi infection, antibodies to both proteins were used to pretreat rickettsiae. Subsequent invasion and plaque assays both indicated a significant decrease in R. typhi infection compared to that by pre-immune IgG. Furthermore, antibody-pretreatment of R. typhi blocked/delayed phagosomal escapes. Together, these data suggest both enzymes are involved early in the infection process. Collectively, our study suggests that R. typhi utilizes two evolutionary divergent patatin phospholipases to support its intracellular life cycle, a mechanism distinguishing it from other rickettsial species.
Highlights
Bacterial species of the genus Rickettsia (Alphaproteobacteria: Rickettsiales: Rickettsiaceae) are Gram-negative, obligate intracellular bacteria with a life cycle typically involving arthropod vectors and vertebrate hosts [1]
We demonstrate across 46 Rickettsia genomes that genes encoding two phospholipase A2 (PLA2) enzymes have divergent evolutionary histories and discordant selective constraints, with pat2 deleted from most other non-typhus group (TG) rickettsiae genomes
As previously determined for Pat2, we show here that R. typhi Pat1 is secreted into the host cell cytoplasm during infection and requires a host cofactor for enzymatic activity
Summary
Bacterial species of the genus Rickettsia (Alphaproteobacteria: Rickettsiales: Rickettsiaceae) are Gram-negative, obligate intracellular bacteria with a life cycle typically involving arthropod vectors and vertebrate hosts [1]. Some members of the genus Rickettsia are serious human pathogens, such as the agents of epidemic typhus (R. prowazekii) and Rocky Mountain Spotted Fever (R. rickettsii) [1]. The obligate intracellular life cycle of Rickettsia spp. involves entry into host cells by phagocytosis (or induced phagocytosis for non-phagocytic cell types), rapid escape from the phagocytic vacuole into the host cytoplasm to evade phagosome-lysosome fusion, replication within the host cytoplasm, and exit from the host cell by actin-mediated motility (e.g., Spotted Fever Group rickettsiae) or lysis of host cells (e.g., TG rickettsiae) [1,10]. Very little is known regarding the molecular mechanisms of rickettsial intracellular growth and pathogenesis due to limited tools and approaches for genetic manipulation [12,13]
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