Chewing the fat - Long chain fatty acid uptake and assimilation in Mycobacterium tuberculosis.
Lead Research Organisation:
University of Leicester
Department Name: Infection Immunity and Inflammation
Abstract
With one third of humanity infected, roughly 8 million new cases every year and over 2 million deaths, tuberculosis remains a major global health problem; it is also a significant problem in many urban centres in the UK such as Leicester. We urgently need better diagnostics, better treatments and better vaccines.
Most of what we know about the bacterium responsible for tuberculosis, Mycobacterium tuberculosis (Mtb), derives from growing the bug in laboratory cultures. However, there is always a danger that the organism?s behaviour may be different in test tubes compared to its behaviour in humans.
We have observed that Mtb cells frequently contain fat droplets in sputum samples from tuberculosis patients but that these droplets are difficult to demonstrate in laboratory cultures. We have been studying the metabolic process responsible for droplet formation in Mtb and its relatives for several years. These organisms have highest demand to make fats and related compounds to manufacture their cell coating. It is widely believed that this coating is critical to the way Mtb survives against the attack of the human immune system, causes disease and resists many antibiotics. We believe that the droplets reflect the importance to Mtb of processing fats and that the related metabolic processes are central to its growth and survival during infection.
In this study we aim find out how Mtb processes external resources into and out of fat droplets. Because many Mtb cells grow inside the phagocytic human immune cells called macrophages much of our work will be based on growing the organism in a laboratory model macrophage system. We will use a combination of genetic, biochemical and microscope-based techniques. The last of these will be particularly advantageous when, probably in a subsequent project, we investigate whether the metabolic processes we elucidate are relevant to how Mtb behaves in samples from human infections.
A significant part of our study will be concerned with finding out how interfering with the fat droplet system affects Mtb growth and survival inside macrophages. We argue that careful regulation of the fat droplet system may be very important to Mtb and that this project will provide us not only with a better understanding of tuberculosis but also with important opportunities to develop new treatments.
Most of what we know about the bacterium responsible for tuberculosis, Mycobacterium tuberculosis (Mtb), derives from growing the bug in laboratory cultures. However, there is always a danger that the organism?s behaviour may be different in test tubes compared to its behaviour in humans.
We have observed that Mtb cells frequently contain fat droplets in sputum samples from tuberculosis patients but that these droplets are difficult to demonstrate in laboratory cultures. We have been studying the metabolic process responsible for droplet formation in Mtb and its relatives for several years. These organisms have highest demand to make fats and related compounds to manufacture their cell coating. It is widely believed that this coating is critical to the way Mtb survives against the attack of the human immune system, causes disease and resists many antibiotics. We believe that the droplets reflect the importance to Mtb of processing fats and that the related metabolic processes are central to its growth and survival during infection.
In this study we aim find out how Mtb processes external resources into and out of fat droplets. Because many Mtb cells grow inside the phagocytic human immune cells called macrophages much of our work will be based on growing the organism in a laboratory model macrophage system. We will use a combination of genetic, biochemical and microscope-based techniques. The last of these will be particularly advantageous when, probably in a subsequent project, we investigate whether the metabolic processes we elucidate are relevant to how Mtb behaves in samples from human infections.
A significant part of our study will be concerned with finding out how interfering with the fat droplet system affects Mtb growth and survival inside macrophages. We argue that careful regulation of the fat droplet system may be very important to Mtb and that this project will provide us not only with a better understanding of tuberculosis but also with important opportunities to develop new treatments.
Technical Summary
In spite of over 100 years research on Mycobacterium tuberculosis (Mtb) we still do not know how this scourge of humanity feeds itself during infection. Here, by extension of our established studies on the mycobacterial triacylglycerol?lipid body (TAG-LB) system, we aim to elucidate the metabolic processing of long chain fatty acids by Mtb in axenic and macrophage cultures. We have recently shown that, while TAG-LBs are scarce in Mtb cells grown in vitro, they are abundant in human sputum samples and that a gene (Rv3130c/tgs1) encoding an enzyme, which we have demonstrated contributes directly to LB formation, is strongly expressed in sputum compared to lab cultures.
Our hypothesis is that regulation of the TAG-LB pool is critical to Mtb in vivo. TAG provides a means of rapidly storing LCFAs whenever they are available and providing a consistent flow of resources for mycolic acid, other complex lipid and phospholipid biosynthesis. As lipids comprise ~60% of its dry weight (~15% in E. coli), Mtb faces a major challenge to obtain and process the resources to meet its demand for lipid biosynthesis. LCFAs are potentially available to Mtb in various forms during infection and they constitute the most energetically favourable source from which to manufacture mycolic acids. However, LCFAs are intrinsically toxic, indeed macrophages release them as one of their bactericidal mechanisms. We propose that the TAG-LB system is central to how Mtb reconciles its demand for LCFAs against their toxicity.
Our objectives are: 1) To define the mechanisms and identify the genes involved in LCFA uptake by Mtb (a single putative fatty acid transporter provides an attractive target); 2) To determine major pathways of LCFA acquisition by Mtb in macrophages; 3) To determine the effects of suppressing LCFA uptake, incorporation into TAG and downstream utilisation on growth and survival in macrophages. These will be met by a combination of genetic, radio-isotopic labelling and fluorescence-based cytological methodologies. Our work will be especially assisted by the availability of fadD mutants from Professor WR Jacobs and by collaboration with Dr Brian Robertson through which we will deploy antisense RNA technology to determine the roles of key genes within and outside macrophages. In view of the prominence of LBs in sputum relative to lab cultures our work focuses on Mtb in macrophages where we believe investigation of the TAG-LB system could reveal important insights and potential new therapeutic targets relevant to the control of tuberculosis.
Our hypothesis is that regulation of the TAG-LB pool is critical to Mtb in vivo. TAG provides a means of rapidly storing LCFAs whenever they are available and providing a consistent flow of resources for mycolic acid, other complex lipid and phospholipid biosynthesis. As lipids comprise ~60% of its dry weight (~15% in E. coli), Mtb faces a major challenge to obtain and process the resources to meet its demand for lipid biosynthesis. LCFAs are potentially available to Mtb in various forms during infection and they constitute the most energetically favourable source from which to manufacture mycolic acids. However, LCFAs are intrinsically toxic, indeed macrophages release them as one of their bactericidal mechanisms. We propose that the TAG-LB system is central to how Mtb reconciles its demand for LCFAs against their toxicity.
Our objectives are: 1) To define the mechanisms and identify the genes involved in LCFA uptake by Mtb (a single putative fatty acid transporter provides an attractive target); 2) To determine major pathways of LCFA acquisition by Mtb in macrophages; 3) To determine the effects of suppressing LCFA uptake, incorporation into TAG and downstream utilisation on growth and survival in macrophages. These will be met by a combination of genetic, radio-isotopic labelling and fluorescence-based cytological methodologies. Our work will be especially assisted by the availability of fadD mutants from Professor WR Jacobs and by collaboration with Dr Brian Robertson through which we will deploy antisense RNA technology to determine the roles of key genes within and outside macrophages. In view of the prominence of LBs in sputum relative to lab cultures our work focuses on Mtb in macrophages where we believe investigation of the TAG-LB system could reveal important insights and potential new therapeutic targets relevant to the control of tuberculosis.
