Microbes get fat when they’re unhappy, too…*

We’ve talked before about microbes playing dead to avoid the effects of antibiotics.   A recent paper (Baek et al. 2011.  Metabolic regulation of mycobacterial growth and antibiotic sensitivity, PLoS Biol. 9 e1001065) identifies a new mechanism that Mycobacterium tuberculosis uses for switching into a low-metabolism, drug-tolerant state.

M. tuberculosis, as you undoubtedly know, is the bacterium that causes tuberculosis (TB).  It’s a nasty pathogen, made worse by the fact that it’s really hard to kill.  Treating tuberculosis involves a 6-month-long course of antibiotics — anything shorter, and not only does the infection come back, it’s now drug-resistant.  Multi-drug resistant TB (MDR-TB) is increasingly a nasty public health problem.  People just aren’t very good at taking pills for 6 months, without fail, even after they’ve started feeling better.

Why does M. tuberculosis take so long to kill?  The way these bacteria survive is rather bold: they live inside macrophages, the cells that normally help get rid of bacteria, and indeed inside the vesicles (phagosomes) that are intended to chew them up.  Here they grow, but very slowly: they divide maybe once every 100 hours.  Many studies have shown that antibiotics generally do better at killing bacteria that are growing rapidly.  Maybe this slow growth has something to do with the poor killing.

It’s a stressful environment inside a phagosome.  If you’re a bacterium, this is an environment that’s designed to kill you.  There’s not much oxygen, the pH is low, and important nutrients, including iron, are lacking.   Simply restricting the oxygen supply in vitro causes the bacterium to become slow-growing and antibiotic-tolerant.  Baek et al. used these hypoxic bacteria in a transposon-based genetic screen for mutants that don’t slow down their growth when oxygen is limited, to look for genes involved in the pathway that controls the growth shutdown.  The mutants they find are… in genes to do with the production of fat.  Triacylglyceride, to be precise.  Huh?

What seems to be happening here is that when the bacterium is in a stressful environment, it shifts its metabolism away from growth and towards making storage molecules.  Up-regulation of the pathway that makes triacylglyceride appears to be a general stress response: it comes up not only in oxygen stress, but also in the very different situation of low iron availability. If you have a given amount of energy available, the more of it you put into making stores the less you will have available for growth and division; so the authors wondered whether this fat-producing pathway is competing with growth-enabling pathways.  One bit of metabolism that you might want to shut down if you’re not planning to grow much is the citric acid cycle.  And the key metabolite for both fat production and the citric acid cycle is acetyl-coA.

You’ve all learned how acetyl-coA gets into the citric acid cycle, and perhaps you even remember.  Citric acid synthase condenses acetyl-coA with oxaloacetate, producing citrate.  If activating triglyceride synthesis blocks growth by removing acetyl-coA from the citric acid cycle, then making more citric acid should redress the balance and prevent the growth slowdown.  And it does: the authors show that adding more oxaloacetate makes the cells grow better in low oxygen conditions.  So does overexpressing the citrate synthetase gene.  Quantitative measurements of carbon flux, using 14C-labeled acetate, also show a big shift away from the citric acid cycle and towards fat synthesis in a low-oxygen environment.

But does this have anything to do with the antibiotic resistance the authors set out to understand?  Yes, luckily: the bacteria with mutations in the triglyceride pathway, which grow better in reduced oxygen, are also less resistant to antibiotics when oxygen or iron is low.   And bacteria with increased expression of citric acid synthase — which no longer show the growth shutdown response — are more antibiotic-sensitive too.  So, growth inhibition via increased fatty acid synthesis seems to protect M. tuberculosis not only against stress, but also against antibiotics.

This means that if you want M. tuberculosis to be more sensitive to antibiotics, you should increase its growth rate.  That sounds like a strategy that the FDA will be really excited about, right?  I’m not sure I would sign up for that clinical trial.  But wait: it’s true that blocking the triglyceride pathway allows the bacteria to continue to divide in hypoxic conditions, but this also causes problems for them.  Using an assay based on the loss of an unstable plasmid, Baek et al. were able to show that these cells continued to divide even after the density of the cultures stabilized: in other words, the death rate increased as well.  So the fitness advantage of the triglyceride pathway mutants over wild-type is only temporary, at least in vitro.  What about in vivo?  Baek et al. infected mice with triglyceride pathway mutants and wild-type M. tuberculosis, and treated the mice with various antibiotics targeting different cellular functions.  In all cases the triglyceride pathway mutants were easier to kill than the wild type.  Perhaps I might sign up for the growth-enhancing clinical trial after all.

The authors emphasize that although they’ve shown that diversion of acetyl-coA to the triglyceride pathway is important, this is undoubtedly not the only mechanism regulating the rate of metabolism in response to stress.  Also, although replication rate was correlated with antibiotic sensitivity in many cases, the correlation wasn’t perfect.  Baek et al. suggest that the important factor may not be so much whether the cell is growing as whether the citric acid cycle is operating, as this cycle produces reactive oxygen species that may be essential for the bacteriocidal activity of antibiotics.  But in any case, this study suggests that finding a way to manipulate the carbon flux in clinical M. tuberculosis infections might, eventually, lead to better combination therapies.

Baek SH, Li AH, & Sassetti CM (2011). Metabolic regulation of mycobacterial growth and antibiotic sensitivity. PLoS biology, 9 (5) PMID: 21629732

*If you’re one of those people who get thin, not fat, when you’re unhappy, just don’t tell me about it, OK?