Metabolic disruption impacts tick fitness and microbial relationships

Abstract

Arthropod-borne microbes rely on the metabolic state of a host to cycle between evolutionarily distant species. For instance, arthropod tolerance to infection may be due to redistribution of metabolic resources, often leading to microbial transmission to mammals. Conversely, metabolic alterations aids in pathogen elimination in humans, who do not ordinarily harbor arthropod-borne microbes. To ascertain the effect of metabolism on interspecies relationships, we engineered a system to evaluate glycolysis and oxidative phosphorylation in the tick Ixodes scapularis. Using a metabolic flux assay, we determined that the rickettsial bacterium Anaplasma phagocytophilum and the Lyme disease spirochete Borrelia burgdorferi, which are transstadially transmitted in nature, induced glycolysis in ticks. On the other hand, the endosymbiont Rickettsia buchneri, which is transovarially maintained, had a minimal effect on I. scapularis bioenergetics. Importantly, the metabolite β-aminoisobutyric acid (BAIBA) was elevated during A. phagocytophilum infection of tick cells following an unbiased metabolomics approach. Thus, we manipulated the expression of genes associated with the catabolism and anabolism of BAIBA in I. scapularis and detected impaired feeding on mammals, reduced bacterial acquisition, and decreased tick survival. Collectively, we reveal the importance of metabolism for tick-microbe relationships and unveil a valuable metabolite for I. scapularis fitness.

Keywords: Borrelia burgdorferi; Lyme Disease; Metabolism; Rickettsial Infection; Ticks; Vector-Borne Diseases.

Figure 1:. Glycolysis and OxPhos are critical metabolic pathways for fitness programs in I. scapularis.

A. Abbreviated representation of glycolysis (green), TCA (pink) and OxPhos (blue) with inhibitors that block enzymatic function (red). (B-F). Viability measurement of 1× 10⁶ ISE6 cells. Cells were treated with the corresponding inhibitors at indicated concentrations for 48 hours prior to analysis. Data are representative of at least two independent experiments N=4–8. Concentrations that caused a significant decrease in viability are shaded red and non-significant effects are highlighted in blue. G. Extracellular acidification rate (ECAR) of 1.2× 10⁵ ISE6 cells treated with chemical inhibitors. Glucose (Glu) and 2-Deoxy-D-Glucose (2-DG) were administered at 25 mM and 50 mM, respectively. Data are representative of at least three independent experiments N=6. H. Oxygen consumption rate (OCR) of 1.2× 10⁵ ISE6 cells treated with chemical inhibitors. 2,4-Dinitrophenol (DNP), rotenone and antimycin were administered at 20 µM, 0.1 µM and 0.5 µM, respectively. Data are representative of at least two independent experiments N=6. I. Ticks were injected with the respective amounts of 2-DG and placed on C57BL/6 mice overnight (grey). Percentage of ticks that successfully attached on C57BL/6 mice are displayed in blue. Data are representative of at least two independent experiments. Number of ticks used ranged from 25–100 per treatment. J. Molting length of ticks following microinjection with oligomycin. Ticks were injected with a sublethal amount of oligomycin (0.8 pmol) prior to feeding. K. Percentage of ticks that molted following feeding. Number of ticks used in J and K ranged from 10–11 per treatment. (B-F) One-way ANOVA followed by Dunnett’s test. (I) Chi-square test. (J) Log rank (Mantel-Cox) test. (K) Fisher’s exact test. *, p<0.05. Anti = Antimycin A, Rot=rotenone. NS – not significant.

Figure 2:. A. phagocytophilum and B. burgdorferi induce glycolysis upon infection of tick cells.

A. Schematics of infection assay. I. scapularis ISE6 cells were cultured in L15C300 medium. At day 0, the L15C300 medium was replaced with the mL15C medium for cell culture. Tick cells were infected with distinct microbial agents at indicated MOI and the Seahorse analysis was done at day 2 post-infection. B-D. Extracellular acidification rate (ECAR) of 1.2 ×10⁵ ISE6 cells stimulated with (B) A. phagocytophilum, (C) B. burgdorferi, or (D) R. buchneri. Data normalized to unstimulated cells. Data are representative of three independent experiments N=6. E-G. Oxygen consumption rate (OCR) of 1.2 ×10⁵ ISE6 cells stimulated with (E) A. phagocytophilum, (F) B. burgdorferi, or (G) R. buchneri. Data normalized to unstimulated cells. Data are representative of at least three independent experiments N=6. MOI=multiplicity of infection. Glu=Glucose, 2-DG=2-deoxy-glucose, DNP=2,4-dinitrophenol, Rot=rotenone, Anti=antimycin. DNP, Rot and Anti were administered at 20 µM, 0.1 µM and 0.5 µM, respectively. Glu and 2-DG were administered at 25 mM and 50 mM, respectively.

Figure 3:. A. phagocytophilum and B. burgdorferi enhance the glycolytic flux from glucose to lactate in tick cells.

A. Schematic representation of glycolysis (green), TCA (pink) and OxPhos (blue). Readouts for the glycolytic flux to lactate in tick ISE6 cells are highlighted in red. (B-M) 1 ×10⁶ ISE6 cells were stimulated with A. phagocytophilum at a multiplicity of infection (MOI 50) (blue), B. burgdorferi (MOI 50) (red), R. buchneri (MOI 50) (orange) or left unstimulated (grey) for 1 or 24 hours. (B-D) Phosphoglucoisomerase (PGI) and (E-G) lactate dehydrogenase (LDH) activity, (H-J) lactate and (K-M) nicotinamide adenine dinucleotide (NADH) measurements through colorimetric assays. Data represent at least two independent experiments N=5. Statistical significance was evaluated by the unpaired t test with Welch’s correction. *, p<0.05. NS – not significant.

Figure 4:. A. phagocytophilum depends on tick cell metabolites for I. scapularis infection.

A. Schematics of microbial infection upon inhibitor treatment. B-C 1 ×10⁶ ISE6 cells were treated with oligomycin – 0.5 µM; 2,4-DNP - 20 µM; rotenone – 0.1 µM; antimycin A – 0.5 µM or 2-DG – 50 mM 1 hour prior to infection. Cells were then incubated with (B) A. phagocytophilum (MOI 50) or (C) R. buchneri (MOI 50) for 48 hours. Data were normalized to untreated but infected control cells (−) to calculate fold changes in bacterial load. Data are representative of at least three independent experiments N=4–6. D. Schematics of BAIBA metabolism. Enzymes involved in catabolism and anabolism of BAIBA are highlighted in red. E. BAIBA levels measured in uninfected, A. phagocytophilum-infected, or R. buchneri-infected 5 ×10⁷ ISE6 cells at MOI 50 24 hours post-infection N=4–6. F-H. Gene expression of ticks fed on uninfected mice or mice infected with A. phagocytophilum. Relative expression of (F) upb1, (G) agxt2, or (H) abat normalized to tick actin. Data are representative of two independent experiments N=10. I-L. Nymphs were injected with siRNA or scrambled control before feeding on A. phagocytophilum-infected mice for three days. Silencing efficiency in (I) upb1 or (K) agxt2 ticks. (J and L) A. phagocytophilum burden in silenced and control ticks. Bacterial burden was calculated by using the A. phagocytophilum specific 16S rDNA gene and its relative expression normalized to actin. Data are representative of at least three independent experiments N=22–33. M. A. phagocytophilum burden in BAIBA-treated ticks. Nymphs were injected with 40 pmol of BAIBA, the α-aminoisobutyric acid (isomer) or phosphate-buffered saline (PBS) (−) and ticks were placed on A. phagocytophilum-infected mice for three days. Bacterial burden was calculated by using the A. phagocytophilum specific 16S rDNA gene and its relative expression normalized to actin. Data are representative of two independent experiments N=24–28. Statistical significance was evaluated by one-way ANOVA followed by Dunnett’s post-hoc test (B, C, E and M) or unpaired t test with Welch’s correction (F-L). *, p<0.05. NS=not significant.

Figure 5:. BAIBA regulates tick feeding and survival.

A. Silencing efficiency of upb1 in ticks. Nymphs were injected with the upb1 siRNA (siupb1) or scrambled control (scupb1) before ticks were placed on uninfected mice to feed for three days. N=21–26. B. Weight of ticks post-feeding N=21–26. C. Silencing efficiency of agxt2 in ticks. Nymphs were injected with the agxt2 siRNA (siagxt2) or scrambled control sequence (scagxt2) before ticks were placed on uninfected mice to feed for three days. N=16–20. D. Weight of ticks post-feeding N=16–20. E. Survival of siupb1- or scupb1-injected ticks recorded 18 days post-blood meal N=23–26. F. Survival of siagxt2- or scagxt2-injected ticks recorded 18 days post-blood meal. N=17–19. Data are representative of at two independent experiments. G. Nymphs were injected with corresponding amounts of BAIBA before feeding on mice. Survival was recorded for 18 days. Data are representative of two independent experiments N=9–14. Statistical significance was evaluated by (A-D) unpaired t test with Welch’s correction or (E-G) Log rank (Mantel-Cox) test. *, p<0.05. NS=not significant.