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.

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.

Publications

10 25 50
 
Description Clinical Research Training Fellowship
Amount £296,000 (GBP)
Funding ID 107302/Z/15/Z 
Organisation Wellcome Trust 
Department Wellcome Trust Bloomsbury Centre
Sector Academic/University
Country United Kingdom
Start 01/2015 
End 09/2018
 
Description IMI Model-based preclinical development of anti-tuberculosis drug combinations
Amount € 675,904 (EUR)
Funding ID 115337 
Organisation European Commission 
Sector Public
Country European Union (EU)
Start 05/2012 
End 04/2017
 
Description Project Grant
Amount £578,141 (GBP)
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 12/2016 
End 11/2019
 
Description Studies on Resuscitation Promoting Factor Dependent (RPF-D) M. tuberculosis cells in sputum 
Organisation University of the Witwatersrand
Country South Africa 
Sector Academic/University 
PI Contribution In collaboration with Dr Bavesh Kana (U Witwatersrand) We are supporting studies aimed at replicating /validating our observations on RPF-D Mtb cells in sputum. We are making RPF preparations and studying changes in the Rpf-D population during chemotherapy. The work is in conjunction with Richard Haffner (NAID) and Bill Mac Kenzie (CDC)
Collaborator Contribution Dr Kana is replicating our methods (Mukamolova et al, 2010) and we are exchanging reagents as necessary.
Impact None as yet
Start Year 2012
 
Description Studies on the transmission phenotype of M. tuberculosis 
Organisation Harvard University
Country United States 
Sector Academic/University 
PI Contribution This is a Gates funded pilot study aimed at determining characteristics of patients and aerosolised bacilli that map to onward transmission. The work has been provoke by our sudies on the sputum phenotype of Mtb (Garton et al 2008, Mukamolova et al 2010) and by the work of Kevin Fennelly at the University of Florida (Fennelly AJRCM papers 2004, 12 & 13) using a Cough Aerosol Sampling System (CASS) which detects the number of colony forming cells aerosolised over a period of 5-10min coughing. The work will be done in the South African MRC AIR facility which involves a collaboration between Harvard and the University of Pretoria and in which the project lead, Ed Nardell (Harvard) has established a unit in which all the air from TB patient rooms passes to Guinea Pigs who act as sentinel detectors for infectious aerosolised Mtb. Our Role is to study aerosolised material captured in the CASS for Resuscitation Promoting Factor dependent bacilli (we expect to find a lage excess ove the cfu counts) and to attempt to obtain Mtb transcriptional signalls from paralell aerosol samples. We have used the sampling system described above to obtain transcriptomic profiles from aerosolised M. tuberculosis cells from patients before treatment was initiated. RNA-Seq yielded coherent profiles that were compared to contemporaneous sputum samples. The comparison revealed over 100 differentially expressed transcripts between the two samples. We were also able to compare the 16S profiles reflecting the other bacteria present in sputum and aerosol. These reveal that reads in aerosol were more than 95% Mtb while they comprised less than 70% in sputum. Further, the ranking of other bacteria present was quite different. We interpret these results as indicating that aerosol is not a simple sample of sputum and a selection process is likely to be involved in aerosolisation. We have explored this possibility with colleagues from Birmingham and Leeds in a recently rejected MRC application and are now taking this further in a Wellcome trust application.
Collaborator Contribution The Harvard groupd manage the facility with colleagues fron the SAMRC and U Pretoria. They recruit and admit patients and run the CASS. Kevin Fennelly is supervising CFU counting in the CASS and Ed Nardell is supervising the Guinea pig work which is embedded in a a larger GP vaccine study. We have done all the RNA work in Leicester helped by Prof Jay Hinton in Liverpool
Impact some of the work will be presented in a poster at The Microbiology Society meeting in March and further at the American Thoracic Society in May. We expect to submit a manuscript this year.
Start Year 2012
 
Description Studies on the transmission phenotype of M. tuberculosis 
Organisation University of Florida
Country United States 
Sector Academic/University 
PI Contribution This is a Gates funded pilot study aimed at determining characteristics of patients and aerosolised bacilli that map to onward transmission. The work has been provoke by our sudies on the sputum phenotype of Mtb (Garton et al 2008, Mukamolova et al 2010) and by the work of Kevin Fennelly at the University of Florida (Fennelly AJRCM papers 2004, 12 & 13) using a Cough Aerosol Sampling System (CASS) which detects the number of colony forming cells aerosolised over a period of 5-10min coughing. The work will be done in the South African MRC AIR facility which involves a collaboration between Harvard and the University of Pretoria and in which the project lead, Ed Nardell (Harvard) has established a unit in which all the air from TB patient rooms passes to Guinea Pigs who act as sentinel detectors for infectious aerosolised Mtb. Our Role is to study aerosolised material captured in the CASS for Resuscitation Promoting Factor dependent bacilli (we expect to find a lage excess ove the cfu counts) and to attempt to obtain Mtb transcriptional signalls from paralell aerosol samples. We have used the sampling system described above to obtain transcriptomic profiles from aerosolised M. tuberculosis cells from patients before treatment was initiated. RNA-Seq yielded coherent profiles that were compared to contemporaneous sputum samples. The comparison revealed over 100 differentially expressed transcripts between the two samples. We were also able to compare the 16S profiles reflecting the other bacteria present in sputum and aerosol. These reveal that reads in aerosol were more than 95% Mtb while they comprised less than 70% in sputum. Further, the ranking of other bacteria present was quite different. We interpret these results as indicating that aerosol is not a simple sample of sputum and a selection process is likely to be involved in aerosolisation. We have explored this possibility with colleagues from Birmingham and Leeds in a recently rejected MRC application and are now taking this further in a Wellcome trust application.
Collaborator Contribution The Harvard groupd manage the facility with colleagues fron the SAMRC and U Pretoria. They recruit and admit patients and run the CASS. Kevin Fennelly is supervising CFU counting in the CASS and Ed Nardell is supervising the Guinea pig work which is embedded in a a larger GP vaccine study. We have done all the RNA work in Leicester helped by Prof Jay Hinton in Liverpool
Impact some of the work will be presented in a poster at The Microbiology Society meeting in March and further at the American Thoracic Society in May. We expect to submit a manuscript this year.
Start Year 2012
 
Description Transcriptome analyses of M. tuberculosis in sputum 
Organisation St George's University of London
Department Department of Cellular and Molecular Medicine
Country United Kingdom 
Sector Academic/University 
PI Contribution We obtained the patient samples, extracted and stabilized the RNA and conducted confirmatory qRTPCR analyses . We also conducted parallel analyses relating the transcriptome to lipid body content of tubercle bacilli and to antibiotic tolerance developing in response to NO exposure.
Collaborator Contribution We conducted microarray-based transcriptome analyses with Prof Butcher's group who deployed an RNA amplification method to enable us to obtain results from small amounts. Initial direct analyses were confirmed to show poorly replicating mycobacteria in sputum and other features consistent with our cytological analyses.
Impact The work was published in in PLoSMedicine (PMID: 18384229).
 
Description Radio interviews 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Primary Audience Public/other audiences
Results and Impact Our paper in PLoSMedicine (PMID: 18384229) attracted significant media attention with several newspaper reports. The PI was interviewed on local radio, the BBC World service and BBC Radio 4's science programme, Leading Edge.

None beyond dissemination of knowledge and understanding
Year(s) Of Engagement Activity 2008