<i>Rickettsia typhi</i> Possesses Phospholipase A₂ Enzymes that Are Involved in Infection of Host Cells

<div><p>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 <i>Rickettsia prowazekii</i> and <i>R. typhi</i>. However, <i>pat2</i> is not encoded in all <i>Rickettsia</i> genomes; yet another uncharacterized patatin (Pat1) is indeed ubiquitous. Here, evolutionary analysis of both patatins across 46 <i>Rickettsia</i> genomes revealed 1) <i>pat1</i> and <i>pat2</i> loci are syntenic across all genomes, 2) both Pat1 and Pat2 do not contain predicted Sec-dependent signal sequences, 3) <i>pat2</i> has been pseudogenized multiple times in rickettsial evolution, and 4) ubiquitous <i>pat1</i> forms two divergent groups (<i>pat1A</i> and <i>pat1B</i>) with strong evidence for recombination between <i>pat1B</i> and plasmid-encoded homologs. In light of these findings, we extended the characterization of <i>R. typhi</i> 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 <i>R. typhi</i> 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 <i>R. typhi</i> infection, antibodies to both proteins were used to pretreat rickettsiae. Subsequent invasion and plaque assays both indicated a significant decrease in <i>R. typhi</i> infection compared to that by pre-immune IgG. Furthermore, antibody-pretreatment of <i>R. typhi</i> blocked/delayed phagosomal escapes. Together, these data suggest both enzymes are involved early in the infection process. Collectively, our study suggests that <i>R. typhi</i> utilizes two evolutionary divergent patatin phospholipases to support its intracellular life cycle, a mechanism distinguishing it from other rickettsial species.</p>