Fig 1.
Dynamics of parasite infection in tsetse flies.
(A and B) Midgut (MG) and salivary gland (SG) organs were dissected from 40-day old female flies subjected to a single parasite challenge as newly eclosed adults. (A) Percentage of flies harboring MG infections, and (B) percentage of MG infected flies that also presented SG infections. The numbers in each circle represent the three independent biological replicates. The black bars represent the mean of the three biological replicates (detailed numbers of flies used for the three biological replicates in S1 Dataset). (C) Schematic depiction of trypanosome localization in tsetse fly tissues. In the non-permissive flies (inf+/- state, shown in the upper scheme) only the midgut, including the cardia, is colonized by parasites, which reside in the ES (purple). In permissive flies (inf+/+ state, shown the lower scheme), parasites infect the fly’s and SGs (violet). (D) Percentage of permissive infections (inf+/+) following challenge at teneral stage with parasites obtained from the midgut of either inf+/- or inf+/+ individuals. Four independent experiments were performed for each treatment with 64 infected flies observed in total for each treatment. The black bar represents the mean of the four experiments. SG infection is independent of the initial inf+/- or inf+/+ status of the parasite used for fly infection (GLM, p = 1, detailed model in S1 Dataset). (E) Expression of PM-associated genes proventriculin-1, 2 and 3 (pro1, 2, 3) relative to the housekeeping gene gapdh, three and six days after flies received a blood meal supplemented with 1μg/mL of either BSA (control, light gray) or trypanosome-derived soluble variant surface glycoprotein (sVSG; dark gray). Each bar represents the average (± SEM) of five biological replicates. For each time point of each gene, a Student t-test was used to determine significant differences (* p<0.05; ** p<0.01; *** p<0.001).
Fig 2.
Trypanosome infection establishment process.
(A) Percentage of RNAseq reads, relative to the total, that mapped to the parasite reference geneset from the three independent biological replicates. Inf+/- cardia is depicted in red circles and inf+/+ in blue circles. The numbers in circles represent the three biological replicates. The black bars indicate the mean of the three replicates (detailed numbers of flies used for the three biological replicates in S1 Dataset). (B) Abundance of trypanosome gapdh relative to tsetse gapdh determined from inf+/- cardia (shown by red circles) and inf+/+ cardia (shown by blue circles). The black bars indicate the mean of the replicates. The increase in relative abundance indicates an increase in parasite numbers in the inf+/+ cardia (Student t-test, p = 0.0028). (C) Schematic representation of the cardia organ based on microscopic observations. The cardia is composed of cells originating from the foregut (light orange) and midgut (light blue) at the junction of the foregut and midgut. Specialized midgut cells organized as an annular pad around the invaginated foregut secretes the PM (green) in the annular cleft formed between the foregut and midgut. Sphincter muscles that form a ring above the PM secreting cells, as well as the thin layer of muscle that surrounds large lipid-containing cells (shown in yellow), are indicated. The schematic indicates the upper and lower points where cardia were dissected for all experiments. In this schematic, the crop duct connecting the foregut prior to its invagination in the cardia is not presented. (D-G) Representative TEM micrographs showing cardia from inf+/- (D-E) and inf+/+ (F-G) individuals. (E) and (G) are magnified micrographs of the black and white boxes in (D) and (F), respectively. Cardia from six and five individuals from inf+/+ and inf+/- flies were imaged, respectively. MG: midgut; FG: foregut; ES: ectoperitrophic space; TR: trypanosomes; PM: peritrophic matrix; LU: lumen; mv: microvilli; sm: subcellular microtubules.
Fig 3.
Parasite infection effects on PM synthesis in cardia inf+/+ and inf+/-.
(A) Percentage of transcripts that encode PM-associated proteins, relative to the total number of transcripts in each transcriptome. (B) Differential expression of PM-associated transcripts in cardia of flies that house inf+/+ (blue circles) and inf+/- (red circles) infections. Each dot represents the fold-change in expression of one transcript relative to the non-infected control. The gray area delineates fold-changes that are <1.5, and thus not statistically different from the control cardia (p> 0.05 after FDR correction). For each data point, the Glossina gene ID and function, based on BlastX annotation, is depicted on the x-axis. (C) SG infection prevalence in control (dsRNA-gfp, dsGFP) and treatment (PM compromised; dsRNA-chitin synthase, dsCS) flies. The circles depict the percentage of flies that harbor both gut and SG infections. A total of four independent experiments were set-up for each group. The black bars indicate the mean of the four experiments. A total number of 66 and 63 infected flies were observed in the dsGFP and dsCS treatments, respectively. The dsCS treatment significantly increases trypanosome infection prevelance in tsetse’s salivary glands (GLM, Wald-test p = 0.01408). Detailed counts and a complete statistical model are indicated in S1 Dataset. (D) Effect of cardia inf+/- and inf+/+ extracts on PM integrity. Survival of flies was monitored daily after per os treatment of 8 day-old flies with cardia extracts followed by per os treatment with Serratia marcescens 72 hours later. The Kaplan-Meyer curves show fly survival over time: cardia inf+/- (red), cardia inf+/+ (blue), or cardia from flies that recovered from infection (rec-/-, green) and cardia non-inf (black). Statistical analysis was performed using a full regression model followed by a pairwise test (details in S1 Dataset). Different letters next to fly group designations in the figure legend represent significantly different curves (p< 0.05).
Fig 4.
Trypanosome-PM interactions in cardia from inf+/+ tsetse.
(A-C) Ultrastructure of PM secreting cells in the cardia from inf+/+ flies. (A) Trypanosomes are observed in mass in the lumen. (B-C) Magnified micrographs of the black boxes shown in (A). (B) Trypanosomes are observed embedded in the secreted matrix (blue arrowheads). (C) In this niche parasites are observed in cyst-like bodies (purple arrowhead), and can also be observed out of the PM secretions (yellow arrowheads). Parasite secreted extracellular vesicles are observed (pink arrowheads). Micrographs in this image represent one of six biological replicates analyzed. MG: midgut; FG: foregut; TR: trypanosomes; MS: muscles; PM: peritrophic matrix; PM sc: PM secreting cells; mv: microvilli.
Fig 5.
Trypanosome-PM interactions in cardia from inf+/- tsetse.
(A-D) Ultrastructure of cardia inf+/- near the PM secreting cells. (B) is a magnification of the black frame in (A) showing the PM (blue arrowhead). (C) and (D) two independent cardia organs showing the same region near PM secreting cells. Trypanosomes are observed packed within the ES near the location of PM secretion. At this point, several trypanosomes observed present vacuolation and nuclear condensation (orange arrowheads) indicative of cell death. Micrographs in this image represent three of five biological replicates analyzed. MG: midgut; FG: foregut; TR: trypanosomes; PM: peritrophic matrix; PM sc: PM secreting cells; mv: microvilli.
Fig 6.
Mitochondrial integrity in cardia from inf+/+ and inf+/- tsetse.
(A) Effect of infection on mitochondria related gene expression. Heatmap generated from the fold-changes between control and either inf+/- or inf+/+ cardia. The * denote the level of significance associated with the DE of specific transcripts (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001). (B-D) Ultrastructure of the sphincter myofibrils in control non-inf (B), inf+/- (C) and inf+/+ (D) cardia. White arrows show the mitochondria, red arrowheads show patterns of mitochondria degradation, and yellow arrows show dilatation of sarcoplasmic reticulum. (E-G) Ultrastructure of giant lipid-containing cells in control non-inf (E), inf+/- (F) and inf+/+ (G) cardia. In both infection phenotypes, mitochondria cristae appear disogarnized compared to control. Micrographs in this image represent one of three, five and six of biological replicates from cardia non-inf, inf+/- and inf+/+, respectively. White arrows show the mitochondria. Ld, lipid droplets; Nu, nucleus.
Fig 7.
Muscle structural integrity in cardia from inf+/+ and inf+/-.
(A) Effect of infection on cardia muscle related gene expression. Heatmap generated from the fold-changes between control and either inf+/- or inf+/+ cardia. The * denote the level of significance associated with the DE of specific transcripts (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001). (B-D) Ultrastructure of a sarcomere from muscles surrounding non-inf (B), inf+/- (C) and inf+/+ (D) cardia. The red arrowhead indicates the Z band structure associated with sarcomeres. Micrographs in this image represent one of three, five and six of biological replicates from cardia non-inf, inf+/- and inf+/+, respectively.
Fig 8.
Influence of oxidative stress on infection status.
(A-B) Electron microscopy observations of trypanosomes presenting cell-death swelling within the ES of cardia from inf+/- flies. Micrographs in this image represent two of five of biological replicates. FG: foregut; TR: trypanosomes; PM: peritrophic matrix; LU: lumen. (C) Comparison of peroxide levels in cardia from non-infected (white circles), inf+/- (red circles) and inf+/+ (blue circles) flies 72 hours after a blood meal. Each dot represents an independent quantification of 10 pooled cardia. The black bars indicate the mean of the 3 replicates. Statistical analysis was conducted using a one-way ANOVA followed by a TukeyHSD posthoc test for pairwise comparisons. Statistical significance is represented by letters above each condition, with different letters indicating distinct statistical groups (p< 0.05). (D) SG infection prevalence in normal and anti-oxidant (cysteine) treated flies. The circles depict the percentage of flies that harbor both gut and SG infections in the cysteine treated (10μM) and control groups. A total of 4 independent groups were set-up for each treatment (91 and 89 infected flies were observed in the control and cysteine treatment groups, respectively). The black bars indicate the mean of the 4 experiments. Cysteine treatment significantly increases trypanosome infection prevelance in tsetse’s salivary glands (GLM, Wald-test p<0.001). Detailed counting data and complete statistical model are indicated in S1 Dataset.
Fig 9.
Model illustrating permissive (inf+/+) and non-permissive (inf+/-) infection phenotypes in tsetse’s cardia.
African trypanosomes must pass through the tsetse vector in order to complete their lifecycle and infect a new vertebrate host. Following successful infection of tsetse’s midgut, Trypanosoma brucei parasites either remain trapped indefinitely in this environment (non-permissive infection, inf +/-) or migrate to the fly’s SG where they subsequently transmit to a vertebrate host (permissive infection, inf +/+). For a inf+/+ infection to occur, trypanosomes must successfully circumvent several immunological barriers, including the cardia-synthesized PM. In this situation, parasites that have accumulated in the ES (1) of the cardia traverse the structurally compromised PM at its site of synthesis (where the matrix is most diffuse and fragile) (2) (Figs 4 and S5). The cyst-like bodies of parasites are observed within layers of the PM (S6 Fig), at which point they may force their way out by breaking through the structure’s electron-dense layer (S7 Fig). Otherwise they may stay enclosed within and move along the gut with the continuously generated PM (3) [33]. Parasites that have successfully translocated to the cardia lumen then migrate to the foregut and salivary glands (4). In an effort to facilitate their passage of the PM, trypanosomes may interfere with PM synthesis by secreting modulatory molecules (Figs 3D and 4B) that are taken up by PM synthesizing cells (5). These molecules may subsequently inhibit expression of genes that encode proteinaceous components (Pro1, Pro3, etc) of the matrix (Fig 3B) and trypanocidal reactive oxygen intermediates (ROIs). In non-permissive infections, a relatively small number of parasites reach tsetse’s cardia (Figs 2A, 2B and S2), but they appear damaged or dead (6) (Fig 8A and 8B). The inability for the parasite to sustain in the inf +/- cardia environment is likely caused by a relatively high concentration of ROIs in this environment (Fig 8C). ROI-mediated regulation of the parasite population comes with collateral damage to cardia tissues (Fig 6), especially the muscles lining the outer border of the organ, which present sarcoplasmic dilatation and mitochondrial vacuolation and swelling (7) (Figs 7 and S9). In the inf +/- cardia, PM synthesis is not affected (Fig 3B), probably due to the absence of trypanosome-derived molecules interfering with the PM production (Fig 3D). Ability to restrict parasites in the cardia prohibits the trypanosomes from translocating to the cardia lumen for subsequent transmission (8).