Characterization of a defensin from the sand fly Phlebotomus duboscqi induced by challenge with bacteria or the protozoan parasite Leishmania major

Abstract

Antimicrobial peptides are major components of the innate immune response of epithelial cells. In insect vectors, these peptides may play a role in the control of gut pathogens. We have analyzed antimicrobial peptides produced by the sand fly Phlebotomus duboscqi, after challenge by injected bacteria or feeding with bacteria or the protozoan parasite Leishmania major. A new hemolymph peptide with antimicrobial activity was identified and shown to be a member of the insect defensin family. Interestingly, this defensin exhibits an antiparasitic activity against the promastigote forms of L. major, which reside normally within the sand fly midgut. P. duboscqi defensin could be induced by both hemolymph or gut infections. Defensin mRNA was induced following infection by wild-type L. major, and this induction was much less following infections with L. major knockout mutants that survive poorly in sand flies, due to specific deficiencies in abundant cell surface glycoconjugates containing phosphoglycans (including lipophosphoglycan). The ability of gut pathogens to induce gut as well as fat body expression of defensin raises the possibility that this antimicrobial peptide might play a key role in the development of parasitic infections.

FIG. 1.

RP-HPLC of hemolymph of P. duboscqi adult insects (around 400 insects) inoculated with E. carotovora subsp. carotovora. The antimicrobial activity of defensin, detected by a solid growth inhibition zone assay, is expressed by black bars, representing activity (in millimeters) against M. luteus. Elution was performed with a gradient of ACN (dotted line), and the absorbance was measured at 214 nm (solid line). The inset shows the spectrum obtained by MALDI-TOF MS of the fraction with activity against gram-positive organisms, corresponding to the sand fly defensin.

FIG. 2.

The nucleotide and deduced amino acid sequences of P. duboscqi defensin cDNA. Amino acids are presented as single-letter codes above each codon. The sequence coding for the 40-amino-acid mature peptide is shown in boldface type. The stop codon (TAA) is indicated by an asterisk, and the putative polyadenylation consensus signal (AATAAA) is underlined.

FIG. 3.

Multisequence alignment of P. duboscqi defensin with representative defensins of different insect vectors. Defensins of Dipteran insects from the suborders Nematocera and Brachycera and insects from the order Hemiptera are shown. Gaps are introduced to optimize the alignment. Conserved residues are shown in bold and cysteine residues are underlined.

FIG. 4.

MS analysis of sand fly defensin induction in gut tissue (A) 24 h after per os infection with bacteria (E. carotovora subsp. carotovora) and (B) 4 days after per os infection with L. major. The results shown (A and B) represent data from two separate experiments. For bacterial infections, defensin was detected in individual sand flies (10 insects tested for each protocol); for parasitic infections, defensin was detected in a pool of 10 midguts (three pools for each protocol).

FIG. 5.

Assessment of defensin transcription in whole bodies of P. duboscqi with Q-PCR after bacterial infections. Naïve (uninfected insects), bacterium-pricked insects at 24 h (Bact-24H) and per os bacterium-infected insects at 24 h are the three protocols studied. The values determined with naïve insects were arbitrarily given a value of 1, and other treatments are expressed as a difference in transcript numbers. Each bar represents an average of six assessments by Q-PCR.

FIG. 6.

(A) Defensin transcription in whole bodies of P. duboscqi after ingestion of a negative blood meal or amastigote forms of L. major (LV561). (B) Defensin transcription in whole bodies of P. duboscqi after Leishmania infection with promastigote forms of WT (LV39) and two mutants deficient in the LPG surface molecule (lpg1⁻ and lpg2⁻). (C) Rates and intensities of infections in P. duboscqi females presented in panel B. Infections were classified into three categories: light (<100 parasites/gut), moderate (100 to 1,000 parasites/gut), heavy (>1,000 parasites/gut).