Abstract
Rickettsia parkeri, an intracellular bacterium transmitted by Amblyomma maculatum ticks, causes a febrile illness associated with eschar formation in humans. As a less virulent member of the spotted fever group within the Rickettsia genus, R. parkeri serves as an ideal model for studying interactions between rickettsial pathogens, their vectors, and hosts. Our previous research showed that R. parkeri modulates mitochondrial-dependent apoptosis in tick cells, enhancing its intracellular survival and replication; however, the underlying mechanisms remain unclear. To investigate further, we employed a Himar1-based transposon mutagenesis system to identify R. parkeri genes involved in apoptosis. Using the modified plasmid pLoxHimar, we introduced a transposon into the R. parkeri Tate's Hell strain and selected a mutant with an insertion in RPATATE_1266, a hypothetical protein with homology to exopolyphosphatase/guanosine pentaphosphate phosphohydrolase (Ppx/Gppa) family proteins, using mCherry fluorescence and spectinomycin/streptomycin resistance. The mutant genotype and single insertion site were confirmed by insertion site sequencing and inverse PCR. We found that the RPATATE_1266 mutant had reduced infection rates, growth, and plaque formation compared to wild-type R. parkeri in tick and mammalian cells. Moreover, the mutant showed upregulation of genes associated with ppx/gppa gene regulation networks (GRNs) and exhibited a marked inhibition of apoptosis in tick cells, including decreased expression of apoptosis-related genes, reduced mitochondrial membrane potential, and less DNA fragmentation. Restoration of RPATATE_1266 resulted in significant recovery of later-phase apoptosis, as measured by Caspase 3/7 activity. These findings suggest that RPATATE_1266 is a critical regulator of apoptosis in tick cells and could be a potential target for controlling R. parkeri infection and replication. This research enhances our understanding of how vector responses to pathogen infection influence pathogen replication and transmission. IMPORTANCE: Rickettsia parkeri infections, though less severe than other rickettsioses, are becoming increasingly significant due to the expanding geographic range of their tick vector and their role in shaping our understanding of rickettsial biology. Advancing knowledge of the molecular mechanisms that regulate R. parkeri infection and replication is important for the field of vector-pathogen interactions. This study identifies the RPATATE_1266 gene (a hypothetical protein with homology to exopolyphosphatase/guanosine pentaphosphate phosphohydrolase [Ppx/Gppa] family proteins) as a key regulator of mitochondrial-dependent apoptosis in tick cells, a process critical for rickettsial intracellular survival. By elucidating the role of this gene, we provide new insights into the molecular interactions between rickettsial pathogens and their vectors. These findings not only enhance our understanding of pathogen-vector dynamics but also highlight potential directions for developing future strategies to manage rickettsial diseases beyond those caused by R. parkeri.