The Insect Innate Response to Rickettsial Infection: Characterization of Pathogen Detection and Effector Response in Ctenocephalides felis Using a Model System

Abstract

Rickettsia spp. are gram-negative, pleiomorphic, aerobic, obligate intracellular bacteria. Some members of the genus Rickettsia are the causative agents of significant human diseases such as epidemic typhus and Rocky Mountain spotted fevers. For unknown reasons, arthropod vectors of Rickettsia, such as the cat flea, Ctenocephalides felis, fail to clear but do not succumb to rickettsial infection. The genome of C. felis has not yet been sequenced and little is known about its immune response to bacterial infection; however, cDNA libraries developed from the midguts of R. typhi-infected and uninfected C. felis have identified numerous transcripts involved in antimicrobial defense and host interaction, including the peptidoglycan recognition protein, PGRP-LB, and the antimicrobial peptide (AMP), defensin. The full-length sequence of the C. felis predicted pgrp was determined by Rapid Amplification of cDNA Ends (RACE) and predicted to contain 190 residues, an N-terminal signal sequence, and a PGRP domain. The expression of PGRP-SA, LF, LB, and SD in Drosophila S2 cells was also investigated and found to vary depending on the R. typhi strain that infected the cell. The C. felis putative defensin function as an AMP was evaluated against Gram-positive Streptococcus pyogenes and Gram-negative Escherichia coli and Francisella novicida using CFU assays, and that against Rickettsia typhi and Rickettsia montanensis using real time RT-PCR, immunofluoresence assays, and immunoblotting. While C. felis defensin was found to have an antimicrobial effect on E. coli, it did not appear to function as an AMP against F. novicida. It was hypothesized that the observed lack of a strong antimicrobial effect against the various bacteria could be due to the concentrations of defensin used. At these potentially too high concentrations, the protein could aggregate and precipitate, which would prevent their function as an AMP. When the C. felis defensin concentrations were lowered for antimicrobial assays against E. coli and S. pyogenes, C. felis defensin displayed an antimicrobial effect against both bacterium. Defensin also appears to function as an AMP against R. typhi and R. montanensis as evidenced by the leakage of the rickettsial cytoplasmic elongation factor thermostable (EF-Ts) into the supernatant, which is fatal to the bacteria.