Spiroplasma endosymbiont reduction of host lipid synthesis and Stomoxyn-like peptide contribute to trypanosome resistance in the tsetse fly Glossina fuscipes

Abstract

Tsetse flies (Glossina spp.) vector African trypanosomes that cause devastating diseases in humans and domestic animals. Within the Glossina genus, species in the Palpalis subgroup exhibit greater resistance to trypanosome infections compared to those in the Morsitans subgroup. Varying microbiota composition and species-specific genetic traits can significantly influence the efficiency of parasite transmission. Notably, infections with the endosymbiotic bacterium Spiroplasma have been documented in several Palpalis subgroup species, including Glossina fuscipes fuscipes (Gff). While Spiroplasma infections in Gff are known to hinder trypanosome transmission, the underlying mechanisms remain unknown. To investigate Spiroplasma-mediated factors affecting Gff vector competence, we conducted high-throughput RNA sequencing of the gut tissue along with functional assays. Our findings reveal elevated oxidative stress in the gut environment in the presence of Spiroplasma, evidenced by increased expression of nitric oxide synthase, which catalyzes the production of trypanocidal nitric oxide. Additionally, we observed impaired lipid biosynthesis leading to a reduction of this important class of nutrients essential for parasite and host physiologies. In contrast, trypanosome infections in Gff's midgut significantly upregulated various immunity-related genes, including a small peptide, Stomoxyn-like, homologous to Stomoxyn first discovered in the stable fly, Stomoxys calcitrans. We observed that the Stomoxyn-like locus is exclusive to the genomes of Palpalis subgroup tsetse species. GffStomoxyn is constitutively expressed in the cardia (proventriculus) and synthetic GffStomoxyn exhibits potent activity against Escherichia coli and bloodstream form of Trypanosoma brucei parasites, while showing no effect against insect stage procyclic forms or tsetse's commensal endosymbiont Sodalis in vitro. Reducing GffStomoxyn levels significantly increased trypanosome infection prevalence, indicating its potential trypanocidal role in vivo. Collectively, our results suggest that the enhanced resistance to trypanosomes observed in Spiroplasma-infected Gff may be due to the reduced lipid availability necessary for parasite metabolic maintenance. Furthermore, GffStomoxyn could play a crucial role in the initial immune response(s) against mammalian parasites early in the infection process in the gut and prevent gut colonization. We discuss the molecular characteristics of GffStomoxyn, its spatial and temporal expression regulation and its microbicidal activity against Trypanosome parasites. Our findings reinforce the nutritional influences of microbiota on host physiology and host-pathogen dynamics.

Fig 1. Tsetse Gut Tissue Responses to Spiroplasma and Trypanosome Infection.

A. Differentially expressed (DE) genes identified in the Spi⁺ and Tbb⁺ groups relative to uninfected Ctrl are presented as follows: The orange and red areas represent up-regulated genes in the Spi⁺ and Tbb⁺ state respectively, while light blue and dark blue indicate down-regulated genes in Spi⁺ and Tbb+ respectively, at a significance level of 10% adjusted p-value and log₂ fold change (log₂FC) ≥ 1. B. Venn diagram illustrates the number of DE genes that are unique to or shared between the Spi⁺ and Tbb⁺ states relative to uninfected controls (Ctrl). C. Heat maps display the DE genes with putative PM-associated and immunity-related functions that are shared between the Spi⁺ and Tbb⁺ datasets. Fold-change values are represented as a fraction of the average normalized gene expression levels from age-matched Spi⁺ or Tbb⁺ versus uninfected control flies (Ctrl). The heat maps (dendrograms) were clustered using Euclidean distance calculation methods in R-package software. The clusters were manually separated into various categories.

Fig 2. Functional classification of DE-pathways and abundant DE genes unique in Spi⁺ or Tbb⁺ state.

A. Gene ontology (GO) enrichment analysis for biological processes was conducted with unique DE genes. B. Functional annotations are provided for some of the most abundant and highly DE genes unique to each microbidal infection state, defined by normalized counts ≥ 700 and (log₂FC) ≥ 2. In this represenetation, red dots indicate genes that are DE in the Tbb⁺dataset, while blue dots indicate genes that are DE in the Spi⁺ dataset.

Fig 3. Impact of Spiroplasma and/or Trypanosome Infection on Host Lipid Level.

A. Levels of triacylglyceride (TAG) circulating in the hemolymph of virgin female Gff were measure for Control (Ctrl), Spi⁺, Tbb⁺ and Spi⁺Tbb⁺ groups. Letters A, B and C indicate significant differences (p<0.05) in TAG levels, while the same letter indicates no significant difference (p>0.05). B. The amount of TAG in the hemolymph of Spi⁺ and Control (Ctrl) male Gff flies. Statistical significance was determined using ANOVA for virgin female flies and unpaired t-test for male flies.

Fig 4. Molecular characterization of GffStomoxyn.

A. Sequence alignment of putative Stomoxyn peptides was performed, including sequences from S. calcitrans (ScalStomoxyn; SCAU016937 and ScalStomoxyn 2; SCAU016907), Gff (GffStomoxyn-like; GFUI18_001176), Gpp (GppStomoxyn; GPPI027903), M. domestica (MdomStomoxyn-like; MDOA008330), L. cuprina (LcupStomoxyn; KAI8119624.1 and LcupStomoxyn-like; XP_023308701.2), S. bullata (SbulStomoxyn; DOY81_004902) and L. sericata (LserStomoxyn-like; XP_037825072.1). The proteolytic cleavage site is shown in bold letters. The alignment indicates the pre-pro-mature domains of the full predicted peptides, with identical residues indicated by and asterisk (*). Conservative substitutions are marked with a colon (:) and semi-conservative substitutions are marked by a period (.). B. Distance matrix analysis was conducted with the mature peptide domains of Stomoxyns from different Diptera.

Fig 5. Spatial and Immune Responsive Expression of GffStomoxyn.

A. The qRT-PCR expression profile of stomoxyn was analyzed from the cardia, midgut, fat body, female ovary, and male testes tissues. Eight biological replicates were used, each consisting of three individual fly tissues collected from 10-day old adults. Results are presented relative to gapdh expression. Letters A, B and C above each bar represent significant differences in stomoxyn expression (p<0.05), while the same letter indicates no significant difference (p>0.05). B. Temporal expression of stomoxyn was measured from late pupa and whole gut tissue at multiple time points: 24 and 72 h post-eclosion, 72 h after the first bloodmeal (designated as 72hr*), and 15 day-old adult flies analyzed 72 h after last bloodmeal. Six to ten biological replicates, each comprised of individual midguts, were used in the experiment. Results are presented relative to gapdh expression. Letters A, B and C on top of each bar indicate significant differences (p<0.05) in stomoxyn expression, while the same letter indicates no significant difference (p>0.05).

Fig 6. In vitro and in vivo Bioactivity of Stomoxyn.

A. In vitro antibacterial activity: The antibacterial activity of mature ScalStomoxyn, ScalStomoxyn 2 and GffStomoxyn peptides was assessed against E. coli and Sodalis bacteria. B. In vitro bioactivity against trypanosomes: The trypanocidal activity of mature ScalStomoxyn, ScalStomoxyn 2 and GffStomoxyn peptides was assessed against mammalian bloodstream forms (BSF) and insect stage procyclic form (PCF) trypanosomes. The blue lines indicate BSF parasite inhibition and the red lines show PCF parasite inhibion over the range of peptide concentrations tested. Different recPeptides are shown by varying symbols. C. Gene-silencing validation: The relative expression of stomoxyn was measured from dsGFP and dsStomoxyn-treated Gff flies, showing a significant decrease in expression (p<0.0001) in the case of dsStomoxyn-treated group. Ten biological replicates, each comprising individual flies, were used to validate silencing efficacy. D. Prevalence of trypanosome infections: The prevalence of midgut trypanosome infections in dsRNA-treated Gff flies was microscopically analyzed 15 days post-parasite acquisition, with significant difference observed (p<0.0011). The total number of flies (N) used in each experimental condition is shown. The experiment included a mixture of male and female flies across two biological replicates, with no significant difference in infection rates between the dsRNA-treated males and females.