Triglyceride Accumulation Decreases Growth Rate and Virulence of Mycobacterium Tuberculosis Th

175 Pages·2015·12.38 MB·English

Mycobacterial Metabolic Syndrome: Triglyceride
Accumulation Decreases Growth Rate and
Virulence of Mycobacterium Tuberculosis
Citation
Martinot, Amanda Jezek. 2015. Mycobacterial Metabolic Syndrome: Triglyceride Accumulation
Decreases Growth Rate and Virulence of Mycobacterium Tuberculosis. Doctoral dissertation,
Harvard University, Graduate School of Arts & Sciences.
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http://nrs.harvard.edu/urn-3:HUL.InstRepos:14226055
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Mycobacterial Metabolic Syndrome: Triglyceride accumulation decreases growth
rate and virulence in Mycobacterium tuberculosis
A dissertation presented
By
Amanda Jezek Martinot
to
The Committe on Higher Degrees in
Biological Sciences In Public Health
in partial fulfillment of the requirements
for the degree of
Doctor of Philosophy
in the subject of
Biological Sciences in Public Health
Harvard University
Cambridge,
Massachusetts
September 2014
© 2015 by Amanda Jezek Martinot
All rights reserved.
Advisor: Eric J Rubin Author: Amanda Jezek Martinot
Mycobacterial Metabolic Syndrome: Triglyceride accumulation decreases growth
rate and virulence in Mycobacterium tuberculosis
Abstract
Mycobacterium tuberculosis (Mtb) mutants lacking the operon Rv1411c-1410c encoding
a lipoprotein, Rv1411c (LprG) and a putative transporter, Rv1410c (Rv1410) are
dramatically attenuated for growth in mice. Previous work in our lab, using the model
organism Mycobacterium smegmatis, suggested that this operon regulated the lipid
content of the cell wall. Work in other laboratories characterized LprG as a lipid-binding
lipoprotein leading us to hypothesize that these bacteria grew poorly due to loss of a key
lipid important in the host-pathogen interaction. Based on structural and biochemical
studies we hypothesized that this attenuation was due to a lipid transport defect. Using
whole cell lipidomic analysis, we found changes in LprG-1410 mutants including
accumulation of triacylglyceride (TAG) species in the absence of the transport system.
We have identified TAG in outer membrane fractions and supernatants of Mtb, have
demonstrated the ability of LprG to transport TAG in an in vitro vesicle transfer assay,
and have co-crystallized LprG with TAG. Moreover, accumulation of intracellular TAG
substantially decreases growth under carbon stress in vitro and in vivo in the mouse
model. Our results suggest a far different model – that TAG is ordinarily transported out
of the cell and, in the absence of a transporter, limits cell proliferation independent of the
host immune response. This suggests that TAG is a key metabolic regulator of cellular
growth within the host.
iii
Table of Contents
Abstract……………………………………………………………………………….….iii
Table of Contents…………………………………………………………………...…….iv
List of abbreviations……………………………………………………………………...ix
List of figures……………………………………………………………………………..xi
Acknowledgements…………………………………………………….…………...…...xiv
Dedication………………………………………………………………………………..xv
Chapter 1. Introduction………………………………………………..………………….1
Section 1.1 TB continues to be a major global health threat.……..………..……..2
Section 1.2 Synopsis…………………………………………………….….……..3
Section 1.3 Mycobacterial Lipoproteins…………………………………………..7
Section 1.4 Hypothesis………………………………………………….………..10
Section 1.5. Triacylglycerides in Prokaryotes…………………………...………11
Section 1.6 Triacylglyceride Metabolism in Actinomycetes………….......….….13
Section 1.7. Hypoxia Model for Growth Arrest—Implications for the TB
transition to Latency……………………………………….….....…19
Section 1.8. Triacylglyceride Accumulation is a feature of Mtb growth
in vivo ……………………………………………................……25
Section 1.9 References…………….…………..………………………………....27
Chapter 2. LprG-1410 function regulates mycobacterial growth rate in vivo
independent of immune function…………...………….….………………….40
Section 2.1. Attributions……………………………………………………...………….41
iv
Section 2.2. Preface………………………………...………………….…………41
Section 2.3. LprG-1410 function does not affect in vitro growth rate in
rich media or macrophages………………………….……..……….43
Section 2.4. LprG-1410 function is necessary for survival of Mycobacterium
tuberculosis in mice…………………………….……………….….46
Section 2.5. Growth of LprG-1410 mutants cannot be rescued in mice with
deficiencies in key components of innate immunity important for
Mtb control……………..……………………………………...……47
Section 2.6. Rv1410c is required for M. tuberculosis virulence in the absence
of an adaptive immune response……………………………...….…51
Section 2.7. Mutations in RvLprG-1410 result in attenuation due to decreased
growth rate……………………….…………………………..……..52
Section 2.8. Discussion….…………………………………….…...…………….59
Section 2.9 Methods…………………………………………………….………..65
2.9.1 Culture of Mycobacteria…………………………..…….…………65
2.9.2 Cell Culture and Macrophage Infections……………..........………66
2.9.3 Mouse strains………………………………………………………66
2.9.4 Mouse infections…………………………………………….……..67
2.9.5 Histopathological analysis of Mouse Lungs……………….………68
Section 2.10. References………………...…………………...……………….….69
Chapter 3. LprG-1410 function is an important mediator of global lipid
flux in Mycobacterium tuberculosis…………….……...……………………74
Section 3.1. Attributions………………………………...…………...…………..75
Section 3.2. Preface……………………………………………...………………75
Section 3.3. Loss of LprG-1410 function results in global increase in
lipids compared to WT Mycobacterium tuberculosis……………... 76
Section 3.4 Overexpression of LprG1410 decreases triglyceride
v
levels in Mtb during log phase growth………………..…………….81
Section 3.5 Disruption of the LprG1410 operon results in triglyceride
accumulation during stationary phase growth………….……….…..82
Section 3.6 Disruption of Rv1410 is sufficient for TAG accumulation……....…85
Section 3.7 LprG1410 function affects intracellular levels of
triacylglycerides…………………………………………….….……91
Section 3.8 Triacylglycerides are a feature of the Mycobacterium
tuberculosis outer membrane………………………….……….....…96
Section 3.9 Overexpression of LprG1410 increases presence of triglycerides
in supernatants of Mycobacterium tuberculosis………………….…97
Section 3.10. LprG transfers triacylglyceride across
vesicles…………..………………………………………...…........98
Section 3.11. LprG co-crystallizes with triacylglyceride………………….....…..99
Section 3.12. Discussion………………………………………..………..……..100
Section 3.13. Methods………………………………………………........……..106
3.13.1 Culture of mycobacteria…………………………………..……..106
3.13.2 Construction of the ΔLprG1410 mutant…………...…...……….106
3.13.3. Construction of Complementation Constructs……..………..….112
3.13.4. Lipid extraction, normalization, and HPLC/MS…………….….112
3.13.5 Reverse micellar extraction of outer membrane lipids……….....113
3.13.6. Collisional Mass spectrometry………………………….........…114
3.13.7 Extraction of supernatant derived lipids…………………...……114
3.13.8 R, Gene Pattern, Statistical Analysis……………………..…….114
3.13.9. Cloning of lipoprotein expression vectors…………...........……115
3.13.10 Overexpression of His-tagged recombinant proteins…………..115
3.13.11 Purification of His-tagged proteins……………….……...…….115
vi
3.13.12 Triglyceride transfer assays………………................................116
3.13.13 Co-crystallization with triglyceride………………….….……..117
3.14 References………………………………………………………….……...118
Chapter 4. Triacylglyceride Levels Regulate Growth Rate in Mtb in vitro during
carbon source restriction………………………………………….…….…...….121
Section 4.1. Preface…………………………………………………........……..121
Section 4.2. Carbon source restriction limits growth of LprG-1410
mutants………………………………………………………….…123
Section 4.3. LprG-1410 mutants are hypersusceptible to propionate
as sole carbon source compared to WT………………………...…126
Section 4.4. LprG-1410 mutants have a generalized growth defect
on fatty acids………………………………………...………...…..129
Section 4.5. Acetate and Glycerol Rescue LprG-1410 mutant growth defect
on propionate…………………………………...……...…………129
Section 4.6. Blocking lipase activity exacerbates LprG-1410 growth
defect on cholesterol………………………………………………132
Section 4.7 Suppressor mutation links LprG-1410 function to
regulation of Fas1 via FasR………………………………………..136
Section 4.8 Rv3208 F123C exacerbates growth defect of ΔLprG-1410
in propionate and cholesterol………………………………………139
Section 4.9 Fatty acids do not rescue growth of LprG-1410 mutant with
Rv3208 F123C in propionate………………………………………140
Section 4.10 Discussion………………………………………..……….………143
Section 4.11. Methods……………………………………………….………….147
4.11.1 Carbon restriction in Mtb…………………….…….…………....147
4.11.2 Genetic manipulation of Mtb………………………..……..……148
4.11.3 Drug testing……………………………………......…………….149
vii
4.11.4 Malachite green susceptibility assay…………………………….149
4.11.5 Sequencing…………………………………….......…………….149
Section 4.12. References………………………………………………....……..150
Chapter 5. Summary and Future Directions……………………………………………153
Section 5.1. Summary …………………………………………….…..………..153
Section 5.2. Future Directions………………………………….......…………..157
Section 5.3. References…………………………………………..……………..159
viii
List of Abbreviations for Lipid Species
Ac PIM triacylated phosphoinositol mannoside
2 3
Ac SGL diacylsulfolipid
2
Ac SGL triacylated sulfolipid
3
Ac SGL tetraacylated sulfolipid (a.k.a SL-1)
4
C16 PGL monoglycosyl palmityl phenolphthiotriol dimycocerosates
CL Cardiolipin
CM carboxymycobactin
DAG diacylglycerol
DAT diacyltrehalose
DDCM dideoxycarboxymycobactin
DDMB dideoxymycobactin
FA fatty acid
GMM glucose monomycolate
GPL Glycopeptidolipid
GroMM Glycerolmonomycolate
LPI lysophophatidylinositol
LPG lysophosphatidylglycerol
LPE lysophosphatidylethanolamine
MA mycolic acid
MAG monoacylglycerol
MB mycobactin
MDCM monodeoxycarboxymycobactin
ix