Trypanosome infection establishment in the tsetse fly gut is influenced by microbiome-regulated host immune barriers

Abstract

Tsetse flies (Glossina spp.) vector pathogenic African trypanosomes, which cause sleeping sickness in humans and nagana in domesticated animals. Additionally, tsetse harbors 3 maternally transmitted endosymbiotic bacteria that modulate their host's physiology. Tsetse is highly resistant to infection with trypanosomes, and this phenotype depends on multiple physiological factors at the time of challenge. These factors include host age, density of maternally-derived trypanolytic effector molecules present in the gut, and symbiont status during development. In this study, we investigated the molecular mechanisms that result in tsetse's resistance to trypanosomes. We found that following parasite challenge, young susceptible tsetse present a highly attenuated immune response. In contrast, mature refractory flies express higher levels of genes associated with humoral (attacin and pgrp-lb) and epithelial (inducible nitric oxide synthase and dual oxidase) immunity. Additionally, we discovered that tsetse must harbor its endogenous microbiome during intrauterine larval development in order to present a parasite refractory phenotype during adulthood. Interestingly, mature aposymbiotic flies (Gmm(Apo)) present a strong immune response earlier in the infection process than do WT flies that harbor symbiotic bacteria throughout their entire lifecycle. However, this early response fails to confer significant resistance to trypanosomes. Gmm(Apo) adults present a structurally compromised peritrophic matrix (PM), which lines the fly midgut and serves as a physical barrier that separates luminal contents from immune responsive epithelial cells. We propose that the early immune response we observe in Gmm(Apo) flies following parasite challenge results from the premature exposure of gut epithelia to parasite-derived immunogens in the absence of a robust PM. Thus, tsetse's PM appears to regulate the timing of host immune induction following parasite challenge. Our results document a novel finding, which is the existence of a positive correlation between tsetse's larval microbiome and the integrity of the emerging adult PM gut immune barrier.

Figure 1. Immunity-related gene expression in teneral Gmm ^WT flies following per os challenge with infectious trypanosomes.

Log₂ fold-change in the expression of immunity-related genes in teneral Gmm ^WT individuals 24 hpc (A) and 3 dpc (B) with T. b. rhodesiense parasites. Gene expression in challenged and unchallenged teneral Gmm ^WT individuals is normalized relative to constitutively-expressed tsetse β-tubulin. All log₂ fold-change values are represented as a fraction of average normalized gene expression levels in trypanosome-challenged vs. unchallenged flies. Samples sizes are represented by individual dots, and the red bars indicate the median log₂ fold-change for each gene assayed. All quantitative measurements were performed in duplicate. No significant difference in the expression of immunity-related genes was observed between challenged and unchallenged teneral Gmm ^WT individuals at either of the monitored time points (Student's t-test).

Figure 2. Immunity-related gene expression in mature Gmm ^WT flies following per os challenge with infectious trypanosomes.

Log₂ fold-change in the expression of immunity-related genes in mature Gmm ^WT individuals 24 hpc (A) and 3 dpc (B) with T. b. rhodesiense parasites. Gene expression in challenged and unchallenged mature Gmm ^WT individuals is normalized relative to constitutively-expressed tsetse β-tubulin. All log₂ fold-change values are represented as a fraction of average normalized gene expression levels in trypanosome-challenged vs. unchallenged flies. Samples sizes are represented by individual dots, and the red bars indicate the median log₂ fold-change for each gene assayed. All quantitative measurements were performed in duplicate. Genes that presented a significant change in expression in parasite challenged versus unchallenged mature Gmm ^WT flies are represented by red dots (p≤0.05; Student's t-test).

Figure 3. Immunity-related gene expression in mature Gmm ^Apo flies following per os challenge with infectious trypanosomes.

Log₂ fold-change in the expression of immunity-related genes in mature Gmm ^Apo individuals 24 hpc (A) and 3 dpc (B) with T. b. rhodesiense parasites. Gene expression in challenged and unchallenged mature Gmm ^Apo individuals is normalized relative to constitutively-expressed tsetse β-tubulin. All log₂ fold-change values are represented as a fraction of average normalized gene expression levels in trypanosome-challenged vs. unchallenged flies. Samples sizes are represented by individual dots, and the red bars indicate the median log₂ fold-change for each gene assayed. All quantitative measurements were performed in duplicate. Genes that presented a significant change in expression in parasite challenged versus unchallenged mature Gmm ^Apo flies are represented by red dots (p≤0.05; Student's t-test).

Figure 4. Tsetse symbiont status correlates with structural integrity of the fly's peritrophic matrix.

(A) Midguts from 10 day old flies (3 days after consuming their last blood meal; n = 3) of each treatment group were microscopically dissected, fixed, sectioned and stained. Prepared sections were observed in an effort to compare PM structural integrity between tsetse treatment (Gmm ^Apo, Gmm ^WT/Apo and Gmm ^WT/Sgm−) and control (Gmm ^WT) groups. Tsetse flies that underwent intrauterine larval developed in the presence of their endogenous microbiome (Gmm ^WT, Gmm ^WT/Apo and Gmm ^WT/Sgm−) appear to have a structurally robust PM, while those that matured in the absence of their symbionts (Gmm ^Apo) do not. Red arrows identify the PM in gut sections where the structure was visible. 100× scale bars = 100 µm and 400× scale bars = 25 µm. (B) Dextran feeding assay of teneral Gmm ^WT adults, and mature Gmm ^WT and Gmm ^Apo adults (n = 10 per group). Flies were administered modified blood meals (see Materials and Methods, sub-section ‘Dextran feeding assay’, for details) supplemented with 500 kDa FITC-labeled dextran molecules. Six hours post-inoculation, midguts were dissected and examined under a fluorescence-emitting dissecting microscope. Scale bar (which is the same for all 3 panels) = 500 µm.

Figure 5. Age and symbiont status modulate trypanosome infection outcomes in the tsetse fly.

Approximately 50% of teneral WT tsetse flies become infected when challenged with trypanosomes. Flies at this stage of development exhibit an immature PM, and present a weak and innocuous innate immune response following parasite challenge. Some teneral tsetse flies are refractory to parasite infections, likely because they acquire more maternally-transmitted PGRP-LB than their susceptible counterparts. Mature Gmm ^WT flies present a vigorous immune response following challenge with trypanosomes and are thus highly resistant to parasite infection. In contrast, age-matched Gmm ^Apo flies, which undergo their entire lifecycle (including intrauterine larval development) in the absence of endogenous microbes, are relatively susceptible to trypanosome infection. Although mature Gmm ^Apo flies also up-regulate the expression of several immunity-related genes following trypanosome challenge, notably absent from this list is trypanolytic pgrp-lb. Interestingly, the timing of this response also occurs earlier in the infection process in Gmm ^Apo compared to Gmm ^WT individuals. We propose that this premature immune response results from the fact that aposymbiotic tsetse house a structurally compromised PM that allows these flies to detect parasites immediately upon entry into the fly's midgut. Our model suggests that symbiotic microbes present in larval tsetse modulate the ability of subsequent adults to produce an intact PM. In turn, this structure regulates trypanosome infection outcomes by controlling the timing of tsetse's immune response following parasite challenge. Other invertebrates, including D. melanogaster and Hirudo verbana (the medicinal leech), house phagocytic cells in their alimentary canal that engulf pathogenic organisms , . We speculate that WT tsetse may house similar cells in it's digestive tract that assist in the fly's immune response against trypanosome challenge. PV, proventriculus; BT, bacteriome; GL, gut lumen; EPS, ectoperitrophic space; CP, crop; PM; peritrophic matrix; AMP, antimicrobial peptide; ROS, reactive oxygen species; PGRP-LB, peptidoglycan recognition protein LB.