Rickettsia rickettsii encodes a secretory lipase that facilitates intracytosolic colonization in host cells.

The key cellular processes required for rickettsial obligate intracellular lifestyle, include internalization by phagocytosis, regulation of intracellular trafficking, and evasion of lysosomal destruction to establish an intracytosolic replication niche, remain poorly defined. Recent reports showed that rickettsial phospholipases play an important role in vacuolar escape, but their functions are dispensable depending on the host cell-type. Here, we report the identification of a highly conserved putative lipase containing a Serine hydrolase motif (GXSXG), named RLip (Rickettsia Lipase). Our work reveals that RLip expression is cytotoxic to yeast cells, a genetically tractable heterologous model system. We demonstrate that RLip possesses lipase enzymatic activity and show a lipid specificity towards phosphoinositide (PI)(3), PI(3,4,5)P3, and PI(3,4)P2, and to a lesser extent PI(4,5)P2. Further, we found that RLip expression is induced during infection of pathogenic R. rickettsii, while its expression is low or undetectable for R. parkeri (mild-pathogenic) and R. montanensis (non-pathogenic), respectively, during host invasion. Intriguingly, RLip is highly enriched in the cytoplasmic fraction of host cells, however, minimally retained by the rickettsiae themselves, suggesting RLip is synthesized during infection and then secreted into the host cell cytoplasm. Neutralization of RLip activity, by antibody-blocking, significantly abrogated R. rickettsii escape from bactericidal phagolysosomal fusion, suggesting RLip plays a critical role in facilitating the intracytosolic colonization of pathogenic R. rickettsii. ImportanceArthropod-borne rickettsial diseases are on the rise globally, presenting a perilous threat to humans and livestock. However, our inadequate understanding on how Rickettsia manipulates cellular processes, including the evasion of lysosomal destruction, has impaired the development of effective therapeutic interventions. Here, we identify of a conserved putative lipase containing a Serine hydrolase motif, named RLip (Rickettsia Lipase). Our work demonstrates that RLip possesses lipase enzymatic activity and is enriched in the cytoplasm of host cells, while minimally retained by the bacteria itself. Neutralization of RLip activity abrogated R. rickettsii escape from bactericidal phagolysosomal fusion. In sum, our data support a mechanism by which pathogenic R. rickettsii employs RLip to escape from bactericidal phagolysosomal fusion in order to colonize the host.