Transcription of Antimicrobial Peptides in Galleria mellonella Larvae Infected with Rickettsia rickettsii
Location
Session 1 (Room 1300)
Session Format
Oral Presentation
Your Campus
Statesboro Campus- Henderson Library, April 20th
Academic Unit
Department of Biology
Research Area Topic:
MBI - Molecular Biology Initiative
Co-Presenters and Faculty Mentors or Advisors
Marina E. Eremeeva MD PhD
Abstract
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's 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.
Program Description
The purpose of this study was to determine if a Rickettsia rickettsii infection modulates transcriptional expression of antimicrobial peptides produced as a part of Galleria mellonella larva innate immune response.
Creative Commons License
This work is licensed under a Creative Commons Attribution 4.0 License.
Presentation Type and Release Option
Presentation (File Not Available for Download)
Start Date
4-20-2022 11:00 AM
End Date
4-20-2022 12:00 PM
Recommended Citation
Hervey, William E., "Transcription of Antimicrobial Peptides in Galleria mellonella Larvae Infected with Rickettsia rickettsii" (2022). GS4 Georgia Southern Student Scholars Symposium. 65.
https://digitalcommons.georgiasouthern.edu/research_symposium/2022/2022/65
Transcription of Antimicrobial Peptides in Galleria mellonella Larvae Infected with Rickettsia rickettsii
Session 1 (Room 1300)
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's 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.
