The Lyme disease spirochete feasts on tick antifreeze

In the northeastern United States the Lyme disease spirochete Borrelia burgdorferi spreads from one white-footed mouse to another by hitching a ride in the tick Ixodes scapularis. Transmission between tick and mouse occurs during the tick's rare blood meals.  The larval tick acquires B. burgdorferi from an infected mouse during a blood meal late in the summer, and the spirochetes take up shelter in the tick's midgut.  Later the larva molts into a nymph, which then completes the transmission cycle by feeding on an uninfected mouse during the next spring or early summer.

Although blood is potentially a rich source of nutrients for both tick and spirochete, the cells lining the tick's gut rapidly engulf the nutrients, including glucose, an energy-rich sugar favored by B. burgdorferi.  The spirochetes must therefore rely on other energy sources if it is to survive the many months between tick feedings.  How does B. burgdorferi fuel its survival during this period?

A study in the July issue of PLoS Pathogens has shown that B. burgdorferi metabolizes the tick's antifreeze while living in its midgut.¹  Many arthropods and insects produce large amounts of antifreeze to protect themselves from freezing temperatures.  The Ixodes tick's antifreeze is glycerol, the same stuff that's often added to enzymes to keep them from freezing in laboratory freezers.  The amount of glycerol found in other organisms is too low to serve as antifreeze.  Instead glycerol is metabolized to extract the chemical energy stored in its bonds and to make cell membrane components.  I describe below how B. burgdorferi handles glycerol, but the same enzymes are found in most organisms that metabolize glycerol, including humans.

The B. burgdorferi genome encodes homologs of a glycerol transporter (GlpF), glycerol kinase (GlpK), and glycerol-3-phosphate dehydrogenase (GlpD), which are used by the bacteria to take up and metabolize glycerol (see figure below).  The figure also shows that B. burgdorferi can break down glucose by the glycolytic pathway (glycolysis) to supply its carbon and energy needs.

Modified from Figure 1 of Pappas et al., 2011.  The BB numbers are the gene ID numbers assigned when the B. burgdorferi genome was sequenced.  The individual steps of glycolysis are not shown.  Source

After the glycerol transporter brings glycerol into the cytoplasm, glycerol kinase (GlpK) quickly phosphorylates glycerol at the expense of ATP to generate glycerol-3-phosphate.

Glycerol-3-phosphate is located at a branch point in glycerol metabolism.  This key metabolite can be shunted to one of two pathways.  One pathway leads to assembly of lipids, and the other leads to the glycolytic pathway, which generates ATP for B. burgdorferi.

To make more lipids, additional molecules are attached to glycerol-3-phosphate by other enzymes to generate phospholipids, glycolipids, and lipoproteins.  For example, one of the two major phospholipids in B. burgdorferi membranes is phosphotidylcholine (the other is phosphotidylglycerol).  Note in the figure below that glycerol-3-phosphate (in black and blue) makes up the core of phosphotidylcholine.  (R₁ and R₂ denote fatty acid chains.)  The glycerol or glycerol-3-phosphate base also forms the core of other phospholipids, glycolipids, and lipoproteins needed to assemble the bacterial cell membrane.

ATP provides the energy to build lipids and other components of B. burgdorferi.  To generate ATP, glycerol-3-phosphate is converted by glycerol-3-phophate dehydrogenase (GlpD or G3PDH) into dihydroxyacetone phosphate, which feeds into the middle of the glycolytic pathway.

Glycerol is not a great energy source.  For each molecule of glycerol, one ATP is consumed to make glycerol-3-phosphate, and two molecules of ATP are made via glycolysis, netting B. burgdorferi one molecule of ATP.  On the other hand, each molecule of glucose, which is plentiful in blood, nets two molecule of ATP, twice the amount of energy extracted from glycerol.

To enlarge the glycolytic pathway, click on the image above

B. burgdorferi lacks the TCA cycle enzymes and electron transport chain, which could unleash the chemical energy stored in the bonds of pyruvate, the end product of glycolysis, to generate even more ATP.  Instead pyruvate is converted into the fermentation end product lactate by lactate dehydrogenase.

Modified from Figure 2c of Harper and Harris 2005

For their study the investigators knocked out the B. burgdorferi glpD gene encoding glycerol-3-phosphate dehydrogenase so that the spirochete couldn't use glycerol as an energy source to make ATP.  As expected, the glpD mutant was unable to grow to a high cell density when glycerol was the major carbon and energy source in the culture medium.  Nevertheless the mutant was still able to infect laboratory mice and spread throughout their bodies almost as well as the wild-type (unmutated) strain.  This makes sense since energy sources other than glycerol (such as glucose) are readily available in mammals.

To see how well the glpD mutant survived in ticks, larval Ixodes scapularis ticks were allowed to feed to satiation on groups of mice infected with the mutant and wild-type strains.  Similar numbers of the mutant (632 ± 343 spirochetes/tick) and wildtype (737 ± 369 spirochetes/tick) ended up in the larva (P = 0.5646).  The infected larva were maintained in the lab and allowed to molt into nymphs.  7-8 weeks after larval feeding, the number of mutant spirochetes in the nymphs (254 ± 137 spirochetes/tick) was much lower than the number of wildtype (1173 ± 637 spirochetes/tick; P = 2.76 x 10^-8).  This result suggests that to thrive in the tick's midgut, B. burgdorferi has to break down glycerol, the tick's antifreeze, to generate ATP.

The glpD mutation also slowed the rapid increase in spirochete numbers seen when the infected nymph starts to feed on a mouse (see below).  It's unclear how much of the blood nutrients are available to B. burgdorferi early during feeding.  Blood consumption by the tick is slow initially, and a membrane called a peritrophic matrix forms in the tick midgut to encase the blood.  The spirochetes in the midgut may therefore rely primarily on glycerol to power its rapid multiplication even as the nymph is feeding.  Within a few days a small number of spirochetes eventually break through the midgut lining and make their way to the salivary glands, where they end up as passengers in the saliva flowing into the mouse's skin.

Figure 11 from Pappas et al., 2011.  Infected nymphs were placed on mice at time zero.  Filled circles, wild-type B. burgdorferi; open squares, glpD mutant.  Source

The impaired growth of the glpD mutant in the feeding nymph also delayed their transmission into the mice.  The nymphs fed for 62 hours before the wild-type strain was transmitted to the mice, whereas 72 hours elapsed before transmission of the glpD mutant was detected.

Why does the glpD mutant survive at all in the ticks?  The answer is that there are probably other energy sources available to B. burgdorferi.  The authors proposed that the sugar chitobiose, a component of the tick's cuticle and peritrophic membrane, can be consumed by B. burgdorferi living in the midgut.  The transporter encoded by chbC brings chitobiose into the spirochete, where it is processed by several enzymes before being fed into the glycolytic pathway.  In fact the authors found that B. burgdorferi expressed larger amounts of the chbC mRNA when in the unfed nymph than it did when in the mouse host.  This result would be expected if B. burgdorferi was trying to metabolize the tick's chitobiose, which is not found in the mouse.

So to sum things up, B. burgdorferi appears to use different organic carbon sources to fulfill its energy needs depending on where it's living.  In the mouse host B. burgdorferi most likely breaks down glucose, a sugar rich with potential chemical energy.  Since glucose isn't available in the tick, the spirochete consumes glycerol and possibly chitobiose while living in the tick's midgut.

Reference

Pappas, C.J., Iyer, R., Petzke, M.M., Caimano, M.J., Radolf, J.D., & Schwartz, I. (2011). Borrelia burgdorferi requires glycerol for maximum fitness during the tick phase of the enzootic cycle PLoS Pathogens, 7 (7) DOI: 10.1371/journal.ppat.1002102

Image sources

Unless otherwise stated in the figure legends, the chemical reactions were taken from Biochemistry (5th Edition) by Berg, Tymoczko, and Stryer.

Harper ET and Harris RA (2005).  Glycolytic Pathway, from eLS.  DOI: 10.1038/npg.els.0003883


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