A novel prolixicin identified in common bed bugs with activity against both bacteria and parasites

Abstract

The hematophagous common bed bug, Cimex lectularius, is not known to transmit human pathogens outside laboratory settings, having evolved various immune defense mechanisms including the expression of antimicrobial peptides (AMPs). We unveil three novel prolixicin AMPs in bed bugs, exhibiting strong homology to the prolixicin of kissing bugs, Rhodnius prolixus, and to diptericin/attacin AMPs. We demonstrate for the first time sex-specific and immune mode-specific upregulation of these prolixicins in immune organs, the midgut and rest of body, following injection and ingestion of Gr+ (Bacillus subtilis) and Gr- (Escherichia coli) bacteria. Synthetic CL-prolixicin2 significantly inhibited growth of E. coli strains and killed or impeded Trypanosoma cruzi, the Chagas disease agent. Our findings suggest that prolixicins are regulated by both IMD and Toll immune pathways, supporting cross-talk and blurred functional differentiation between major immune pathways. The efficacy of CL-prolixicin2 against T. cruzi underscores the potential of AMPs in Chagas disease management.

Keywords: Trypanosoma cruzi; Bed bugs; Chagas disease; Glycine-rich antimicrobial peptides; Prolixicin; Toll and IMD humoral innate immunity.

Figure 1

Comparison of structural features of glycine-rich peptides in bed bugs and other select insects. Sequences were aligned using both MUSCLE (multiple sequence comparison by log expectation; https://www.ebi.ac.uk/Tools/msa/muscle/) and multiple sequence alignment (MSA). Conserved prolines are crimson-highlighted, positively charged side groups (basic residues; lysine (K), arginine (R), histidine (H)) are red-highlighted, negatively charged side groups (acidic residues; glutamate (E), aspartate (D)) are blue-highlighted, and cysteines are yellow-highlighted. The predicted signal-peptide, pro-peptide, and mature-peptide regions are indicated in boxes above MSA, and the cleavage sites between these regions are indicated by upwards and downwards arrows, as indicated in the figure legend. Conserved glycines are grey-highlighted.

Figure 2

Phylogeny of domains of CL-prolixicins identified in bed bugs in comparison with glycine-rich antimicrobial peptides from other insect orders. The bed bug prolixicins are most closely aligned with the prolixicin of the kissing bug Rhodnius prolixus. Glycine-rich sequences were aligned with MUSCLE (https://www.ebi.ac.uk/Tools/msa/muscle/), and alignments were used to build phylogenetic trees using iqtree-2.0-rc2 with substitution models PMB + F + G4. The tree was finalized using FigTree software v. 1.4.4 (http://tree.bio.ed.ac.uk/software/figtree/). Phylogenetic testing included 10,000 replicates of Ultrafast bootstrap (UFBoot), Bayesian inference, and maximum likelihood analyses represented on each branch to provide support for tree branches. The tree scale indicates 0.9 substitutions per site, providing a measure of evolutionary distance.

Figure 3

Comparative transcriptomics and structural analysis of the antimicrobial peptides CL-prolixicin1a & 1b and CL-prolixicin2 in bed bugs. (a) Predicted structures, structural surface characteristics, and pH of CL-prolixicin1a & 1b, and of CL-prolixicin2, using I-TASSER (Iterative Threading ASSEmbly Refinement), visualized using UCSF Chimera. Positively and negatively charged residues are indicated in blue and red, respectively. (b) Secondary structure prediction of the sequences of CL-prolixicin1a (CL-p1a) & 1b (CL-p1b), and of CL-prolixicin2 (CL-p2) predicted by I-TASSER. (c) Changes in gene expression in bed bug midgut and RoB tissues (rest of body containing bodies minus heads and midgut tissues) were quantified after bed bugs ingested sterile blood or blood infected with the Gram-positive bacterium Bacillus subtilis or the Gram-negative bacterium Escherichia coli. The Wald test was used to generate p-values and Log2 fold changes. An asterisk indicates statistically significant changes in gene expression levels (adjusted p-values < 0.05). (d) Map of genes expressing prolixicins in bed bugs. Genes are located on the Chromosome NW_019392897.1. CL-prolixicin1a & 1b (expressed from LOC106664366) and CL-prolixicin2 (expressed from LOC122127760) are in close proximity. Predicted exons are shown as boxes, and introns are illustrated by black lines. The direction of transcription is from left to right.

Figure 4

Tissue- and sex-dependent expression of CL-prolixicin antimicrobial peptides by bed bugs following bacterial injection & ingestion. (a–d) Changes in expression levels of CL-prolixicin1a & 1b (LOC106664366) in the midgut and RoB (Rest of Body: bodies minus heads and midgut tissues) of male and female bed bugs 12 h after intrathoracic injection or ingestion of Gram-negative bacteria (Escherichia coli K12/D31) or Gram-positive bacteria (Bacillus subtilis ATCC 6633). White bars represent data obtained from control bugs that were injected with phosphate buffer saline (PBS) or that ingested sterile blood, evaluated using the ΔΔCT method^,. (e,f) Effect of bed bug sex on changes in expression levels of CL-prolixicin1a & 1b (LOC106664366) in the midgut and RoB. (g,h) Comparison of the mode of infection—bacterial ingestion or injection—on changes in expression levels of CL-prolixicin1a & 1b (LOC106664366) in male and female bed bugs. The relative expression of CL-prolixicin1a & 1b was evaluated using the ΔΔCT method^,. Data from males were used as the second calibrator and were arbitrarily set to 1 (subpanels e,f); the fold-changes of expression in females are shown as purple bars. The effect of bacterial ingestion versus bacterial injection was compared using the formula 2^−ΔCt injected/2^−ΔCt ingested (subpanels g,h). The ingestion-sample data (white bars) representing the calibrator were arbitrarily set to 1, and fold-changes of expression in bacterial injection-sample data are shown as purple bars. Bars represent the mean transcript levels ± 95% CI. Means were compared using the unpaired Student’s t-test (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Figure 5

Expression dynamics of CL-prolixicin2 and effects of synthetic CL-prolixicin2 on the parasite Trypanosoma cruzi. (a,b) Changes in expression levels of CL-prolixicin2 (LOC122127760) in the midgut and RoB (rest of body containing bodies minus heads and midgut tissues) of male bed bugs 12 h after intrathoracic injection or ingestion of Gram-negative bacteria (Escherichia coli) or Gram-positive bacteria (Bacillus subtilis). White bars represent data obtained from control bugs that were injected with phosphate buffer saline (PBS), or that ingested sterile blood. The relative expression of CL-prolixicin1a & 1b (LOC106664366) was evaluated using the DDCT method^,. (c,d) Comparison of expression levels of CL-prolixicin1a & 1b (LOC106664366) and CL-prolixicin2 (LOC122127760) in the midgut and RoB of bed bugs. The effect of bacterial ingestion versus bacterial injection was compared using the formula 2^−ΔCt CL-prolixicin2/2^−ΔCt CL-prolixicin1. The ingestion-sample data (white bars) representing the calibrator were arbitrarily set to 1, and fold-changes in CL-prolixicin2 expression in bacterial injection-sample data are shown as purple bars. In all subpanels, bars represent the mean transcript levels ± 95% CI. Means were compared using the unpaired Student’s t-test (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). (e) Proliferation of T. cruzi over the course of 7 days in response to treatment, or not (control), with CL-prolixicin2 at 25–200 µM. (f) Numbers of dead T. cruzi on days 2 and 7 as a result of exposure to CL-prolixicin2 at 200 µM; note: there were no dead parasites in the control treatment at days 2 and 7. (g) Flagellar oscillations per second of T. cruzi (Y Strain) 7 days after exposure, or not (control), to CL-prolixicin2 at 200 µM. Bars represent the mean transcript levels ± 95% CI. Means were compared using the unpaired Student’s t-test. Different letters above bars in each of subpanels f and g indicate significant differences between samples (p < 0.05). (h) During one complete oscillation, the flagellum moves from “a” to “b” and back to “a”. Per-second oscillations were averaged over 10 s.

Figure 6

Regulatory cross-talk and functional overlap in Toll and IMD signaling pathways of Cimex lectularius. Up-regulation of both CL-defensins and CL-prolixicins in bed bugs in response to the Gram-positive bacterium Bacillus subtilis or the Gram-negative bacterium Escherichia coli ingestion or injection provides further support for functional cross-talk and blurred functional differentiation between the Toll and the IMD pathways in hemipterans.