The IMD and Toll canonical immune pathways of Triatoma pallidipennis are preferentially activated by Gram-negative and Gram-positive bacteria, respectively, but cross-activation also occurs

Abstract

Background: Antimicrobial peptides (AMPs) participate in the humoral immune response of insects eliminating invasive microorganisms. The immune deficiency pathway (IMD) and Toll are the main pathways by which the synthesis of these molecules is regulated in response to Gram-negative (IMD pathway) or Gram-positive (Toll pathway) bacteria. Various pattern-recognition receptors (PRRs) participate in the recognition of microorganisms, such as pgrp-lc and toll, which trigger signaling cascades and activate NF-κB family transcription factors, such as relish, that translocate to the cell nucleus, mainly in the fat body, inducing AMP gene transcription.

Methods: T. pallidipennis inhibited in Tppgrp-lc, Tptoll, and Tprelish were challenged with E. coli and M. luteus to analyze the expression of AMPs transcripts in the fat body and to execute survival assays.

Results: In this work we investigated the participation of the pgrp-lc and toll receptor genes and the relish transcription factor (designated as Tppgrp-lc, Tptoll, and Tprelish), in the transcriptional regulation of defensin B, prolixicin, and lysozyme B in Triatoma pallidipennis, one of the main vectors of Chagas disease. AMP transcript abundance was higher in the fat body of blood-fed than non-fed bugs. Challenge with Escherichia coli or Micrococcus luteus induced differential increases in AMP transcripts. Additionally, silencing of Tppgrp-lc, Tptoll, and Tprelish resulted in reduced AMP transcription and survival of bugs after a bacterial challenge.

Conclusions: Our findings demonstrated that the IMD and Toll pathways in T. pallidipennis preferentially respond to Gram-negative and Gram-positive bacteria, respectively, by increasing the expression of AMP transcripts, but cross-induction also occurs.

Keywords: IMD pathway; T. pallidipennis; Tppgrp-lc; Tprelish; Tptoll.

Fig. 1

Relative expression of Tppgrp-lc, Tptoll, Tprelish, and antimicrobial peptide transcripts in blood-fed, non-fed, and non-challenged bugs. All transcripts in the fat body of blood-fed bugs showed significantly higher expression (defensin B 3.51-, prolixicin 3.38-, lysozyme B 3.12-, Tppgrp-lc 3.12-, Tptoll 3.08-, and Tprelish 2.63-fold), compared with non-fed groups. Groups with three biological replicas of eight bugs were analyzed. Relative expression (2^−∆∆CT) is the quantified change between transcripts. Asterisks indicate P < 0.0001. Bars represent the mean transcript levels ± 95% CI. All groups were adjusted for β-actin. Upper right: timeline showing the experimental strategy to obtain the fat body of each bug in the non-fed and blood-fed groups

Fig. 2

Relative expression of Tppgrp-lc, Tptoll, and Tprelish and antimicrobial peptide transcripts in fat body of blood-fed and non-fed T. pallidipennis after immune challenge with E. coli and M. luteus. In blood-fed bugs, all transcripts increased significantly after challenge with E. coli or M. luteus. Tppgrp-lc and Tprelish increased more with E. coli than with M. luteus, while Tptoll increased more with M. luteus than with E. challenge. Defensin B and prolixicin expression increased after challenge with E. coli or M. luteus, but these increases were not significant among groups. Lysozyme showed significantly higher expression against E. coli than against M. luteus. In the non-fed groups, the increases were lower than in blood-fed groups. Relative expression (2^−∆∆CT) is the quantified change between transcripts, asterisks indicate P < 0.05, bars represent the mean transcript levels ± 95% CI, points represent the analyzed groups, and a double asterisk with a bar represents the significance (P < 0.05) of expression between the challenged groups, PBS group as control, and this normalized with the blood-fed and unchallenged group adjusted for β-actin. Upper right: timeline showing the experimental strategy to obtain the fat body of each bug in the non-fed and blood-fed groups

Fig. 3

Relative expression of prolixicin, defensin B, and lysozyme B transcripts in the fat body after a challenge with E. coli or M. luteus of Tppgrp-lc-, Tprelish-, and Tptoll-inhibited T. pallidipennis. After inhibiting Tppgrp-lc or Tptoll, the expression of the three AMP transcripts decreased despite the challenge with E. coli or M. luteus. However, in bugs inhibited in Tprelish, lysozyme B and prolixicin transcripts decreased and defensin B transcripts continued to be expressed after challenge. These results confirm the specific participation of Tppgrp-lc and Tptoll in the synthesis of some AMPs after a bacterial challenge. Relative expression (2^−∆∆CT) is the quantified change between transcripts. Significant differences P < 0.05 are indicated, bars represent the mean transcript levels ± 95% CI, and points represent the analyzed groups. Upper right: timeline showing the experimental strategy to obtain the fat body of each bug in blood-fed, inhibited and challenged groups

Fig. 4

Survival of Tppgrp-lc-, Tptoll-, Tprelish-, and Tppgrp-lc/Tptoll-silenced T. pallidipennis after challenge with E. coli and M. luteus. a Bugs inhibited in Tppgrp-lc and challenged with E. coli or M. luteus. b Bugs inhibited in Tptoll and challenged with E. coli. c Bugs inhibited in Tprelish and challenged with E. coli. d Bugs inhibited in Tppgrp-lc/Tptoll and challenged with E. coli or M. luteus. Bugs that received irrelevant dsRNA and were challenged with E. coli or M. luteus or were not challenged served as a control. Statistical analysis of survival analysis was carried out based on Kaplan–Meier plots (log-rank Chi-square test; *P < 0.0001)

Fig. 5

Model proposing the participation of Dorsal transcription factor to compensate for the absence of Tprelish. Although the expression of Tprelish decreased upon inoculation of dsTprelish, defensin B transcript increased, which may be due to the participation of other transcription factors such as Dorsal or other molecules or mechanisms that are activated after challenge with E. coli or M. luteus