Microbes Manipulate Tick Metabolism Impacting Vector Fitness and Infection

Ticks are blood-feeding arthropods responsible for transmitting many human and other animal pathogens in the United States and parts of Europe. Microbes, including bacteria, viruses, and parasites, are acquired from, or transferred to a vertebrate host while a tick feeds on blood. Interestingly, ticks can carry these microbes without facing significant disruptions in their fitness, or ability to survive and reproduce. Given that metabolism provides energy and the building blocks for sustaining all biological processes, we posited that ticks balanced fitness and microbial infection by reprogramming their metabolism and reallocating energy resources. Specifically, we investigated how tick-borne microbes altered tick metabolism and aimed to understand how energy resources were redistributed during an infection. 
While it has been shown that microbial infection can alter tick metabolism, a systematic approach to measuring these metabolic changes was lacking, and no key metabolites involved in tick-microbe interactions had been mechanistically identified. Therefore, we began measuring metabolism in tick cell culture systems following microbial challenges. We first tested the survival of cell lines derived from different tick species using a range of drugs that affect energy metabolism through targeting glycolysis and oxidative phosphorylation. Among all cell types tested, the blacklegged tick Ixodes scapularis ISE6 line exhibited permissiveness to biochemical manipulation. By carefully determining the drug doses and culture conditions, we developed a functional system capable of quantitatively measuring glycolytic and oxidative phosphorylation changes in ISE6 cells through the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), respectively.    

Measuring glycolysis and oxidative phosphorylation in ISE6 cells through extracellular acidification rate and oxygen consumption rate. (a) Extracellular acidification rate (ECAR) and (b) oxygen consumption rate (OCR) was measured in cells treated with glucose (Glu),  2-deoxy-D-glucose (2-DG),  2,4-dinitrophenol (DNP), rotenone (Rot) and antimycin a (Anti) at different timepoints (indicated by dashed lines and arrows) by the Seahorse analyzer compared to untreated cells (-). 

To assess whether altered metabolic responses were elicited after infection, we challenged ISE6 cells with three evolutionarily diverse bacteria. Pathogens that could infect humans and are maintained between life stages (Anaplasma phagocytophilum or Borrelia burgdorferi) were compared to an endosymbiont bacterium from the tick I. scapularis (Rickettsia buchneri). We showed that stimulation of ISE6 tick cells with human pathogens redirected metabolism towards glycolysis, while infection with R. buchneri led to minimal bioenergetic changes. These results demonstrate contrasting alterations in energy metabolism during infection with distinct tick-borne microbes.  

Glycolytic alterations elicited after infection with tick-borne microbes. Seahorse analysis 2 days post-infection at indicated multiplicity of infection (MOI). Extracellular acidification rate (ECAR) of ISE6 cells stimulated with (a) A. phagocytophilum (blue), (b) B. burgdorferi (red), or (c) R. buchneri (orange). Tick cells were treated with glucose (Glu) and 2-deoxy-D-glucose (2-DG) at indicated timepoints (dashed lines and arrows) and compared to untreated cells (-). 

Notably, disruption of either glycolysis or oxidative phosphorylation not only influenced bacterial infection, but also affected various aspects of tick physiology. Altering glycolysis prevented ticks from attaching to hosts, while inhibiting oxidative phosphorylation hindered the molting process from nymphs to adults. Collectively, our results indicated that alterations in energy metabolism redirects programs associated with tick fitness and infection. 

 Disruption of glycolysis or oxidative phosphorylation alters tick physiology. (a) Percentage of ticks that attached to a host (blue) 24 hours post-injection with increasing concentrations of 2-deoxy-D-glucose (2-DG). (b) Percentage of ticks that successfully molted (orange) to the adult life stage after injection with oligomycin. 

 Intrigued by metabolic changes on tick fitness and infection, we focused on identifying individual metabolites that play a role in these processes. Using metabolomics, we identified and quantified cellular metabolites, comparing the abundance in uninfected to infected cells. We found that A. phagocytophilum significantly modified the metabolism of ISE6 cells compared to R. buchneri, involving alterations in carbohydrate, lipid, nucleotide, and protein pathways. Among the metabolites noted, we identified a pleiotropic metabolite involved in these pathways that showed elevated levels after infection: β-aminoisobutyric acid (BAIBA). 

 Involvement of the pleiotropic metabolite, BAIBA, in nucleotide, protein, carbohydrate, and lipid metabolism. Schematic of BAIBA metabolism, including its two enantiomers: D-BAIBA and L-BAIBA (red), and its connection with metabolic pathways upregulated during A. phagocytophilum infection of tick cells.  

Finally, we evaluated the effect of BAIBA on tick fitness and infection. Administering BAIBA to ticks affected their ability to feed and subsequently reduced their survival. Moreover, BAIBA injection reduced A. phagocytophilum infection after a blood meal. Conversely, if ticks were already infected with A. phagocytophilum when BAIBA was administrated, their survival decreased compared to the control group. Thus, we provided evidence that a metabolic circuit, here represented by the BAIBA metabolite, affected interspecies relationships and fitness parameters in the tick I. scapularis.  

The effect of BAIBA on tick fitness and A. phagocytophilum infection. (a) Survival of ticks injected with increasing concentrations of BAIBA. (b) Detection of A. phagocytophilum by RT-qPCR in individual ticks after injection with BAIBA and feeding on an infected blood meal. (c) Survival of ticks already infected with A. phagocytophilum and injected with BAIBA (BAIBA_In) compared to those injected with the isomer control (Isomer_In).  

Overall, we developed a structured approach to understand metabolism in ticks that has broad applications in arthropod vector biology. We demonstrated the interdependence of infection and metabolism in tick fitness. The changes in tick metabolism depended on the type of microbial association. A reductionist view of the system enabled to pinpoint a metabolite that influenced the balance between fitness and infection.  
https://www.nature.com/articles/s41564-024-01756-0