Vitamin B6 generated by obligate symbionts is critical for maintaining proline homeostasis and fecundity in tsetse flies

Abstract

The viviparous tsetse fly utilizes proline as a hemolymph-borne energy source. In tsetse, biosynthesis of proline from alanine involves the enzyme alanine-glyoxylate aminotransferase (AGAT), which requires pyridoxal phosphate (vitamin B6) as a cofactor. This vitamin can be synthesized by tsetse's obligate symbiont, Wigglesworthia glossinidia. In this study, we examined the role of Wigglesworthia-produced vitamin B6 for maintenance of proline homeostasis, specifically during the energetically expensive lactation period of the tsetse's reproductive cycle. We found that expression of agat, as well as genes involved in vitamin B6 metabolism in both host and symbiont, increases in lactating flies. Removal of symbionts via antibiotic treatment of flies (aposymbiotic) led to hypoprolinemia, reduced levels of vitamin B6 in lactating females, and decreased fecundity. Proline homeostasis and fecundity recovered partially when aposymbiotic tsetse were fed a diet supplemented with either yeast or Wigglesworthia extracts. RNA interference-mediated knockdown of agat in wild-type flies reduced hemolymph proline levels to that of aposymbiotic females. Aposymbiotic flies treated with agat short interfering RNA (siRNA) remained hypoprolinemic even upon dietary supplementation with microbial extracts or B vitamins. Flies infected with parasitic African trypanosomes display lower hemolymph proline levels, suggesting that the reduced fecundity observed in parasitized flies could result from parasite interference with proline homeostasis. This interference could be manifested by competition between tsetse and trypanosomes for vitamins, proline, or other factors involved in their synthesis. Collectively, these results indicate that the presence of Wigglesworthia in tsetse is critical for the maintenance of proline homeostasis through vitamin B6 production.

FIG 1

Interactions of synthesis of vitamin B₆ by Wigglesworthia with salvage of vitamin B₆ and proline synthesis in tsetse flies. Enzymes in metabolic pathways: pyridoxal phosphatase (PDXP), pyridoxal kinase (PDXK), pyridoxamine 5′-phosphate oxidase 1 (P5P1), pyridoxamine 5′-phosphate oxidase 2 (P5P2), glyceraldehyde 3-P dehydrogenase A (gapA), erythronate-4-phosphate dehydrogenase (pdxB), phosphoserine aminotransferase (pdxF), 4-hydroxythreonine-4-phosphate dehydrogenase (pdxA), pyridoxal phosphate biosynthetic protein (pdxJ), pyridoxine-phosphate oxidase (pdxH), alanine-glyoxylate transaminase (AGAT), pyruvate carboxylase (PC), ATP citrate synthase (ACS), aconitate hydratase (aconitase), isocitrate dehydrogenase (IDH), glutamate synthase (GS), 1-pyrroline-5-carboxylate dehydrogenase (P5CDH), pyrroline-5-carboxylate (PYCR). Pathways were established based on sequences from the Wigglesworthia (2, 32) and Glossina (41) genomes.

FIG 2

Expression of alanine-glyoxylate transaminase 1 to 3 genes (agat1 to agat3). (A) Temporal expression. (B) Tissue localization. Transcript levels were determined by qPCR analysis. The data were analyzed with CFX Manager software, version 3.1 (Bio-Rad). Data represent the means ± SEs for four samples and were normalized to tubulin. Error bars are omitted from agat2 and agat3 data (no significant differences among treatments).

FIG 3

Expression levels of selected genes from Wigglesworthia and Glossina associated with vitamin B₆ metabolism. (A) Transcript levels for Wigglesworthia genes. (B) Transcript levels for Glossina genes. (C) Expression of pdxB within tsetse tissues during lactation. Transcript levels were determined by qPCR analysis. The data were analyzed with CFX Manager software, version 3.1 (Bio-Rad). Data represent the means ± SEs for four samples and were normalized to tubulin. The asterisk indicates that the expression is significantly higher (P < 0.05) than that in newly emerged teneral flies (0 days) or other tissues/samples.

FIG 4

Vitamin B₆ levels in bacteriome and carcass tissues of wild-type and aposymbiotic tsetse. Data represent the means ± SEs of 4 to 5 samples. The asterisks indicate that the expression in WT flies is significantly different from that in Apo flies (P < 0.05). Gmm, G. morsitans morsitans.

FIG 5

Proline levels in hemolymph throughout lactation from wild-type (WT) and aposymbiotic (Apo) flies. Data represent the means ± SEs for five samples. Gmm, G. morsitans morsitans.

FIG 6

Proline levels in flies after knockdown of agat and after supplementation with microbial extracts and B vitamin cocktails. (A) Knockdown of agat1 through utilization of injection of siRNA. (B) Supplementation of aposymbiotic flies with yeast extract, bacteriome (Wigglesworthia) extract, and B vitamins. (C) Knockdown of agat1 in aposymbiotic flies followed by treatment with yeast extract. (D) Knockdown of agat1 in aposymbiotic flies followed by treatment with B vitamins. (E) Fecundity following supplementation with microbial extracts and after knockdown of agat1. Letters indicate no difference in expression between samples (P > 0.05). Gmm, G. morsitans morsitans.

FIG 7

Proline levels in flies infected with trypanosomes. Data represent the means ± SEs for nine samples. “a” indicates that the proline levels in uninfected and infected flies are significantly different (P < 0.05).

FIG 8

Summary of the role of vitamin B₆ in relation to proline homeostasis.