Mammalian African trypanosome VSG coat enhances tsetse's vector competence

Abstract

Tsetse flies are biological vectors of African trypanosomes, the protozoan parasites responsible for causing human and animal trypanosomiases across sub-Saharan Africa. Currently, no vaccines are available for disease prevention due to antigenic variation of the Variant Surface Glycoproteins (VSG) that coat parasites while they reside within mammalian hosts. As a result, interference with parasite development in the tsetse vector is being explored to reduce disease transmission. A major bottleneck to infection occurs as parasites attempt to colonize tsetse's midgut. One critical factor influencing this bottleneck is the fly's peritrophic matrix (PM), a semipermeable, chitinous barrier that lines the midgut. The mechanisms that enable trypanosomes to cross this barrier are currently unknown. Here, we determined that as parasites enter the tsetse's gut, VSG molecules released from trypanosomes are internalized by cells of the cardia-the tissue responsible for producing the PM. VSG internalization results in decreased expression of a tsetse microRNA (mir-275) and interferes with the Wnt-signaling pathway and the Iroquois/IRX transcription factor family. This interference reduces the function of the PM barrier and promotes parasite colonization of the gut early in the infection process. Manipulation of the insect midgut homeostasis by the mammalian parasite coat proteins is a novel function and indicates that VSG serves a dual role in trypanosome biology-that of facilitating transmission through its mammalian host and insect vector. We detail critical steps in the course of trypanosome infection establishment that can serve as novel targets to reduce the tsetse's vector competence and disease transmission.

Keywords: VSG; peritrophic matrix; trypanosome; tsetse; vector competence.

Fig. 1.

Tsetse gut responses to trypanosome challenge. Transcriptome analysis of guts from 8-d-old adults after exposure to T. b. rhodesiense bloodstream parasites. (A and B) Heat maps denoting relative transcript abundance of immune-related genes and genes associated with digestion and PM structural integrity. Tsetse gene IDs are indicated in parentheses. Heat maps denoting relative transcript abundance of immune-related genes and genes associated with digestion and PM structural integrity at 48 and 72 hpa. Fold-change values are represented as a fraction of the average normalized gene expression levels from age-matched trypanosome-challenged versus -unchallenged flies. A number 1 by the gene ID indicates differential expression (FDR value < 0.05) in the 72-hpa dataset by EdgeR. n = 2 or 3. In A and B, # or * indicates preferential expression in the midgut or cardia organ, respectively. “Peritrophic Matrix Components” designations are based on proteomic analysis (15).

Fig. 2.

Mir-275 affects the Wnt-signaling pathway. (A) Normalized read counts for mir-275 expression from 72-hpb and 72-hpa individuals. (B) Relative expression of mir-275 between cardia and midgut at 24 hpb. (C) Relative mir-275 expression in ant-275– and ant-ms–treated tsetse (control). Ant-ms encodes a scrambled derivative of mir-275. (D) Relative midgut weight of tsetse (n = 13) treated with either ant-275 or ant-ms measured 24 h post per os antagomir inoculation. (E) Relative gene expression of PM-associated peritrophins (pro1–3), Iroquois/IRX family transcription factors (caupolican, araucan, mirror), hedgehog (hh) and wingless (wg) in cardia 24 h post ant-275, or ant-ms per os dietary provisioning. (F) Relative gene expression of peritrophins, Iroquois transcription factors, and wg in cardia 24 h post siGFP, siWingless, or siMirror per os dietary provisioning. Biological replicates n = 5–6. Midgut weight experiments were performed in duplicate. Results are shown as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.0001. Student’s t-test (midgut weight comparison) and REST software Pair Wise Fixed Reallocation Randomization Test (relative gene expression) were used for statistical analyses. All primers used in the study are detailed in SI Appendix, Table S1.

Fig. 3.

Components of BSF trypanosome modulate the functional integrity of tsetse’s PM. Tsetse survival curves after sequential per os provisioning with heat-killed bloodstream (BSF) or procyclic (PCF) form T. b. rhodesiense (Tbr), T. b. brucei 427 (Tbb), and T. congolense Trans Mara (Tc) extracts followed by live Serratia. (A) Fly diets supplemented with heat-killed BSF Tbr, heat-killed PCF Tbr, PSG (Phosphate Saline Glucose) buffer with BSA, or PCF culture media (Becks) with BSA before receiving Serratia. (B) Fly diets supplemented with heat-killed BSF Tbb, heat-killed PCF Tbb, and BSF culture media (HM19) with BSA or Becks with BSA. (C) Fly diets supplemented with heat-killed BSF Tc or PSG with BSA. ***P < 0.0001, log-rank (Mantel–Cox) test. Statistical comparisons are between the respective control and experimental group (PSG/HM19 BSA vs. BSF and Becks BSA vs. PCF). The n corresponds to the number of flies used per group across two independent biological experiments (displayed in SI Appendix, Fig. S8).

Fig. 4.

Trypanosome VSG modulates tsetse gut physiology. (A) Western blot analysis of purified sVSG (VSG117 MITat1.4) using anti-VSG polyclonal antibodies (M, Marker; 1 VSG). (B) Midgut weights measured 24 hpb from flies that received 1 µg/mL BSA or 1 µg/mL sVSG supplemented blood meals as 8-d-old adults, respectively. (BSA average = 6.89 mg and VSG average = 10.0 mg.) (C) Tsetse survival curve after sequential per os provisioning with either BSA (1 μg/mL) or sVSG (1 μg/mL) followed by live Serratia in the next blood meal. (D) Serratia densities determined from tsetse midgut previously exposed to either BSA or sVSG. Each point indicates one fly midgut, and the bar represents the group average. Experiment was performed only once. (E) Immunohistochemistry of 5-µm-thick sectioned cardia 24 h after provisioning in the blood meal BSA (Left) and live T. b. brucei (strain Lister 427) purified from rat blood (Middle) and purified sVSG (Right). Full images are displayed in SI Appendix, Fig. S7. (F and G) Relative expression of mir-275, peritrophins, Iroquois/IRX family transcription factors, iNOS, at 72 hpb of either BSA or sVSG. Data for midgut weight, Serratia survival assay, and gene expression were pooled from two independent biological experiments. Experiments are displayed as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.0001 between the two independent experiments. (H) Infection prevalence in flies that were provisioned with either 10 µg/mL BSA (1.31%) or 10 µg/mL VSG (6.69%) as 8-d-old adults before receiving BSF Tbr parasites in the next blood meal, respectively. Results are displayed as the mean ± SEM between three independent experiments. (I) Infection prevalence in teneral flies co-fed PCF Tbr parasites with either 1 µg/mL BSA (26.33%) or 1 µg/mL VSG (48.67%), respectively. Results are displayed as the mean ± SEM between three independent experiments. The n corresponds to the total number of flies used in the experiment. Independent biological experiments for survival, gene expression, and individual infection experiments are displayed SI Appendix, Fig. S8.