Transcription of Antimicrobial Peptides in Galleria mellonella Larvae Infected with Rickettsia rickettsii

Rickettsia rickettsii is the causative agent of Rocky Mountain spotted fever. This obligately intracellular, gram-negative bacterium causes a potentially fatal illness with flu-like symptoms and rash. We have previously shown that Galleria mellonella larvae (GML) are susceptible to infection by R. rickettsii exhibiting dose-dependent responses including death. The purpose of this study was to determine if R. rickettsii infection modulates transcriptional expression of antimicrobial peptides (AMP) produced as a part of the insect\u27s innate defense response. Methods: 5th instar GML were injected with 107 plaque-forming units (PFU) of R. rickettsii. Infected and control GML were observed daily, and their health scores were tabulated using a standard health metric. GML hemolymph and fat bodies were collected at various time points throughout infection and processed to extract DNA and RNA, respectively. Rickettsia DNA copy numbers were determined using the OmpA gene PCR assay and normalized to the corresponding total DNA concentration. Total GML RNA from fat bodies was used to synthesize cDNA, which was tested for transcripts of GML AMP genes using gene-specific qPCR assays developed in our laboratory. Transcriptional AMP gene expression was calculated using the 2^-ΔΔCt method and GML actin gene as the housekeeping gene. Results: R. rickettsii infection of GML caused time-dependent melanization and decreased motility starting at 2 hours and 10% lethality at 72 hours. Transcriptional changes were demonstrated for four AMP genes. Transcription of GML genes encoding pro-phenoloxidase, galliomycin and gallerimycin had elevated levels at two time points, 2 and 48 hours of infection while transcription of gloverin was continuously upregulated between 2 and 48 hours and declined at 72 hours of infection. Conclusion: Our data provide insight into innate immune responses of GML to R. rickettsii. Further studies are necessary to determine relevance of the AMP expression to innate immune responses in ticks and mammals