Rhodnius prolixus Hemolymph Immuno-Physiology: Deciphering the Systemic Immune Response Triggered by Trypanosoma cruzi Establishment in the Vector Using Quantitative Proteomics

Abstract

Understanding the development of Trypanosoma cruzi within the triatomine vector at the molecular level should provide novel targets for interrupting parasitic life cycle and affect vectorial competence. The aim of the current study is to provide new insights into triatomines immunology through the characterization of the hemolymph proteome of Rhodnius prolixus, a major Chagas disease vector, in order to gain an overview of its immune physiology. Surprisingly, proteomics investigation of the immunomodulation of T. cruzi-infected blood reveals that the parasite triggers an early systemic response in the hemolymph. The analysis of the expression profiles of hemolymph proteins from 6 h to 24 h allowed the identification of a broad range of immune proteins expressed already in the early hours post-blood-feeding regardless of the presence of the parasite, ready to mount a rapid response exemplified by the significant phenol oxidase activation. Nevertheless, we have also observed a remarkable induction of the immune response triggered by an rpPGRP-LC and the overexpression of defensins 6 h post-T. cruzi infection. Moreover, we have identified novel proteins with immune properties such as the putative c1q-like protein and the immunoglobulin I-set domain-containing protein, which have never been described in triatomines and could play a role in T. cruzi recognition. Twelve proteins with unknown function are modulated by the presence of T. cruzi in the hemolymph. Determining the function of these parasite-induced proteins represents an exciting challenge for increasing our knowledge about the diversity of the immune response from the universal one studied in holometabolous insects. This will provide us with clear answers for misunderstood mechanisms in host-parasite interaction, leading to the development of new generation strategies to control vector populations and pathogen transmission.

Keywords: Chagas disease; antiparasitic response; insect immunity; proteins expression; triatomines.

Figure 1

(A) Venn diagram showing the distribution of the identified proteins of the hemolymph at 6 h and 24 h post-infection. Intersections display protein expression specificity to each condition (Table S1); (B) Representation of the distribution of total hemolymph proteome between intracellular and extracellular proteins. Extracellular proteins are recognized using OutCyte prediction tool either by the presence of a predicted signal peptide using the SignalP algorithm, transmembrane or potential unconventional protein secretions (UPS) from intracellular proteins. Numbers in brackets indicate the percentage of proteins in each category.

Figure 2

Functional annotation of R. prolixus hemolymph proteins. The proteins have been classified according to their molecular function (A) and biological process; (B) according to Gene Ontology. Exhaustive information about the identified proteins is provided in Table S1.

Figure 3

Circular histogram illustrating the distribution of R. prolixus secreted hemolymph proteins. The height of each bar is proportional to the LFQ intensity of expression of the corresponding protein, and each bar is related to the protein’s UniProt ID. Protein categories in the right panel are listed from the histogram clockwise.

Figure 4

Bubble chart showing the differentially expressed proteins in R. prolixus hemolymph at 6 h and 24 h post-T. cruzi infection. Each bubble corresponds to a differentially expressed protein. x axis represents the fold change of protein expression, which is proportional to the bubbles size. y axis represents the log values of the intensity of protein expression.

Figure 5

Western blot validation of defensins’ temporal expression profile in the hemolymph at 6 h and 24 h post-blood feeding and T. cruzi infection. The relative expression of defensins was calculated by normalizing the band intensity of defensins to the intensity of the total proteins signal. The results are expressed as the mean ± SEM (n = 3). Statistical significance is shown by * (* p ≤ 0.05 and ** p ≤ 0.01), calculated by unpaired t-test.

Figure 6

Effect of blood ingestion and T. cruzi development on PO and PPO expression and activity in R. prolixus hemolymph at 6 h and 24 h post-challenge. (A) Profile plot representing the LFQ expression intensity of POs/PPOs isoforms under blood-fed and T. cruzi ingestion showing insignificant variation of the protein expression (n = 4); (B) Evaluation of PO and PPO activity in R. prolixus hemolymph from starved, blood-fed and infected insects. The results are expressed as the mean ± SEM (n = 3), and statistical significance is shown by * (* p ≤ 0.05, ** p ≤ 0.01) calculated by unpaired t-test.