Amino Acid Transporters & Amino Acid Metabolism in Trypanosoma brucei brucei Charles E ...

249 Pages·2008·4.29 MB·English

Amino Acid Transporters & Amino Acid Metabolism
in
Trypanosoma brucei brucei

Charles E. Ebikeme

Division of Infection & Immunity
Faculty of Biomedical & Life Sciences

Thesis submitted for the degree of Doctor of Philosophy
University of Glasgow
October 2007
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1 Author’s Declaration
The results presented in this thesis are my own work, except where stated otherwise.

Charles Ebikeme.
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2 Abstract
The development of new drugs against Human African Trypanosomiasis is much needed
due to toxicity, efficacy and availability problems with current drug treatments for this
resurgent parasitic disease. Delivery of drugs into cells is an important determinant of
therapeutic efficacy of drugs. An effective means of selective drug delivery is to use
plasma membrane transport systems to mediate the entry of drugs into the cell.
Some amino acid transporters fulfil the criteria needed for successful exploitation of
nutrient transport systems for drug delivery. The Trypanosoma brucei genomic database
was screened to identify the full gene repertoire of amino acid transporters. From this,
candidate genes were selected and functional genetic approaches were employed to
characterise candidate amino acid transporter genes. Further characterisation of TbAATP1,
a RNAi cell line shown to be a transporter of small neutral amino acids (serine, glycine,
cysteine, asparagine and alanine), showed a role in threonine uptake.
Amino acid analogues were tested for trypanocidal activity. Of the 96 tested, two
(Azaserine and Levodopa) were investigated in more detail, paying special attention to the
nature of their trypanocidal action and possible route of entry through an amino acid
transporter. Azaserine showed a trypanostatic action as well multiple routes of entry into
the protozoan interior (as shown by inhibition of glutamine, phenylalanine and tyrosine
uptake). The trypanocidal Levodopa showed entry through a tyrosine specific transporter.
However, it is possible that Levodopa’s trypanocidal activity may not be as a result of the
analogue itself, but secondary products of the analogue.
Amino acids are important for protozoa as energy sources as well as forming pools of
soluble osmolites. Amino acid usage in trypanosomes was investigated. Upregulation of
proline transport and catabolism in response to reduced glucose availability was exhibited
by the genome strain of T. brucei. Moreover, this metabolic shift could be mimicked by
addition of GlcNAc to the medium, which blocks the hexose transporter limiting glucose
entry to the cell. Systems biology approaches were initiated to investigate the undergoing
metabolic changes. More specifically, mass spectrometry methodologies were employed to
investigate underlying metabolite changes in procyclic form trypanosomes grown in
differing medium.
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3 Table of Contents
1 Author’s Declaration......................................................................................................2
2 Abstract..........................................................................................................................3
3 Table of Contents...........................................................................................................4
4 List of Figures................................................................................................................7
5 List of Tables................................................................................................................10
6 Definitions....................................................................................................................11
7 Acknowledgements......................................................................................................13
1 Introduction....................................................................................................................1
1.1 The Disease............................................................................................................2
1.2 Vector Biology & Control......................................................................................4
1.3 Life Cycle...............................................................................................................6
1.4 Control Strategies...................................................................................................7
1.5 Chemotherapy......................................................................................................10
1.6 The Genome & Genomics....................................................................................12
1.7 Metabolomics.......................................................................................................14
1.8 Trypanosome Molecular Biology........................................................................17
1.9 RNA Interference & Gene Knockout...................................................................19
1.10 Perspectives for Drug Targets..............................................................................22
1.11 Drug Uptake via Nutrient Transporters................................................................24
1.12 Transport Phenomena across Membranes............................................................25
1.13 Amino Acid Transporters.....................................................................................27
1.14 Human Amino Acid Transporters........................................................................30
1.15 Amino Acids & Trypanosomes............................................................................32
1.16 Amino Acid Transporters in Trypanosomes........................................................36
1.17 Genes of the Amino Acid Transporter Family.....................................................38
1.18 Drug Uptake via Amino Acid Transporters.........................................................43
1.19 Trypanosomes & the Blood-Brain Barrier...........................................................44
2 Materials & Methods....................................................................................................48
2.1 Cell Culture..........................................................................................................49
2.1.1 Trypanosomes..............................................................................................49
2.1.2 Mammalian cells..........................................................................................49
2.2 Alamar Blue Assay..............................................................................................50
2.3 Isolation of genomic DNA...................................................................................50
2.4 Total RNA Isolation.............................................................................................51
2.5 Molecular Cloning Techniques............................................................................52
2.5.1 Polymerase Chain Reaction (PCR)..............................................................52
2.5.2 Plasmid Vectors...........................................................................................52
2.5.3 E.coli Transformations.................................................................................53
2.6 Northern Blot Analysis........................................................................................54
2.7 Probe Labelling....................................................................................................55
2.8 RNA Interference in T. brucei.............................................................................55
2.9 Plasmid Construction...........................................................................................56
2.10 Transfection of Trypanosomes.............................................................................57
2.11 Analysis of Growth Rates....................................................................................57
2.12 Radiolabeled Uptake Assays................................................................................58
2.13 Metabolomic Profiling.........................................................................................59
2.14 Bioinformatic Analyses........................................................................................60
3 RNAi Knock-down Approaches to Characterising Candidate Amino Acid Transporter
Genes in Trypanosoma brucei.............................................................................................61
3.1 Introduction..........................................................................................................62
3.1.1 Amino Acid Usage in Trypanosomes..........................................................62
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3.1.2 Genes of the Amino Acid Transporter Family.............................................63
3.2 Results..................................................................................................................67
3.2.1 Identification of Genes.................................................................................67
3.2.2 TbAATP1 & Growth of PCF trypanosomes................................................69
3.2.3 L-threonine Transport in T. brucei...............................................................73
3.2.4 Osmoregulation............................................................................................78
3.2.5 Cloning of TbAATP6 & TbAATP12 RNAi Constructs..............................79
3.2.6 Northern Blot Analysis................................................................................82
3.2.7 Tetracycline Induction and Growth Curves.................................................83
3.2.8 TbAATP6 Transporter Phenotype...............................................................85
3.3 Discussion............................................................................................................86
3.3.1 TbAATP1.....................................................................................................86
3.3.2 TbAATP6 & TbAATP12.............................................................................91
4 Proline Transport Regulation as a Result of a Metabolic Shift Induced by N-Acetyl D-
Glucosamine.........................................................................................................................93
4.1 Introduction..........................................................................................................94
4.2 Results..................................................................................................................97
4.2.1 GlcNAc Stimulates Growth of PCF Trypanosomes....................................97
4.2.2 GlcNAc is not Internalised by PCF Trypanosomes...................................100
4.2.3 Presence of GlcNAc Causes a Switch to Proline Metabolism...................102
4.2.4 Oligomycin Sensitivity..............................................................................104
4.2.5 GlcNAc Protects Against Death by 2-Deoxy D-glucose...........................105
4.2.6 Proline Regulation as a Result of the Overall Metabolic Status of the Cell
106
4.3 Discussion..........................................................................................................108
5 Metabolomics.............................................................................................................111
5.1 Introduction........................................................................................................112
5.2 Results................................................................................................................116
5.2.1 Global View of the Metabolome................................................................116
5.2.2 Metabolic Fingerprint.................................................................................123
5.2.3 Metabolic Footprint....................................................................................128
5.2.4 Metabolic Profile........................................................................................130
5.3 Discussion..........................................................................................................133
6 Uptake and Effects of Trypanotoxic Amino Acid Analogues...................................136
6.1 Introduction........................................................................................................137
6.1.1 Azaserine....................................................................................................137
6.1.2 Levodopa....................................................................................................140
6.1.3 Cell Penetrating Peptides...........................................................................143
6.1.4 Eflornithine................................................................................................144
6.2 Results................................................................................................................148
6.2.1 Amino Acid Analogue Screens..................................................................148
6.2.2 Trypanocidal Activities..............................................................................150
6.2.3 Mode of action...........................................................................................151
6.2.4 Azaserine & the L-glutamine Transport System........................................154
6.2.5 Levodopa & the Aromatic Transport System............................................159
6.2.6 Cell Penetrating Peptides...........................................................................163
6.2.7 DFMO Mode of Uptake.............................................................................165
6.3 Discussion..........................................................................................................171
6.3.1 Azaserine & Glutamine Transport.............................................................172
6.3.2 Levodopa & Aromatic Amino Acid Transport..........................................175
6.3.3 Cell Penetrating Peptides...........................................................................177
6.3.4 Eflornithine................................................................................................178
7 Closing Discussion.....................................................................................................182
8 Appendix....................................................................................................................186
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8.1 Amino Acids......................................................................................................186
8.2 SDM80...............................................................................................................187
8.3 CBSS Buffer......................................................................................................187
8.4 Cunningham’s Medium......................................................................................188
8.5 TELT Buffer.......................................................................................................188
8.6 TE Buffer...........................................................................................................189
8.7 1X TAE Buffer...................................................................................................189
8.8 LB Medium........................................................................................................189
8.9 20X SSC.............................................................................................................189
8.10 10X MOPS.........................................................................................................189
8.11 ZPFM Buffer......................................................................................................190
8.12 PBS.....................................................................................................................190
8.13 100X Denhardt’s Solution.................................................................................190
8.14 Amino Acid Analogues......................................................................................190
8.15 List of Metabolites.............................................................................................192
8.16 Biochemical Transformations............................................................................195
9 References..................................................................................................................199
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4 List of Figures
Figure 1-1: Human African trypanosomiasis endemicity in the WHO African Region........2
Figure 1-2: Life cycle of the African trypanosome in the human and tsetse fly....................7
Figure 1-3: General RNAi mechanism (taken from Balaña-Fouce & Reguera, 2007)........20
Figure 1-4: Schematic representation of carbon source metabolism in the procyclic form of
T. brucei (taken from Lamour et al., 2005)..........................................................................34
Figure 1-5: Amino acid permeases from L. major, T. brucei and T. cruzi, respectively
(taken from Berriman et al., 2005).......................................................................................38
Figure 1-6: Trypanosoma brucei AAT loci (taken from Jackson, 2007).............................41
Figure 2-1: RNAi vector p2T7Ti.........................................................................................56
Figure 3-1: Amino Acid Transporter location (TbAATP6 and TbAATP1)........................64
Figure 3-2: Amino Acid Transporter location (TbAATP12)...............................................65
Figure 3-3: Dendogram of aligned AATP genes.................................................................67
Figure 3-4: Growth of TbAATP1 4E8 RNAi cell lines.......................................................70
Figure 3-5: Growth of PCF trypanosomes (strain 427) in threonine deficient medium......70
Figure 3-6: Growth counts of TbAATP1 4E8 RNAi cell lines............................................71
Figure 3-7: Growth and measure of threonine uptake.........................................................72
Figure 3-8: Threonine uptake...............................................................................................73
Figure 3-9: Threonine inhibition..........................................................................................74
Figure 3-10: Serine inhibition..............................................................................................75
Figure 3-11: Threonine inhibition by AATP1 specific amino acids....................................76
Figure 3-12: Kinetics of threonine uptake in uninduced TbAATP1 RNAi cell lines..........77
Figure 3-13: Kinetics of threonine uptake in tetracycline induced TbAATP1 RNAi cell
lines......................................................................................................................................78
Figure 3-14: Ethidium bromide stained gel showing PCR amplification of a segment of
TbAATP6.............................................................................................................................80
Figure 3-15: Ethidium bromide stained gel showing excision of p2T7Ti ligated TbAATP6
insert.....................................................................................................................................80
Figure 3-16: Ethidium bromide stained gel showing both fragments of TbAATP12
amplified by PCR.................................................................................................................81
Figure 3-17: Ethidium bromide stained gel showing excision of p2T7Ti ligated
TbAATP12a insert...............................................................................................................81
Figure 3-18: Northern blot analysis of RNA from TbAATP6 RNAi cell lines...................82
Figure 3-19: Northern blot analysis of RNA from TbAATP12 RNAi cell lines.................83
Figure 3-20: Growth of TbAATP6 RNAi cell lines.............................................................84
Figure 3-21: Growth of TbAATP12 RNAi cell lines...........................................................84
Figure 3-22: Proline uptake in TbAATP6 RNAi cell lines..................................................86
Figure 3-23: Volume regulation in trypanosomatid parasites under hypotonic shock (taken
from Viera et al., 1998)........................................................................................................90
Figure 4-1: Cell counts of PCF trypanosomes (strain TREU 927)......................................97
Figure 4-2: Cell counts of PCF trypanosomes (strain TREU 927) grown in SDM80
medium.................................................................................................................................98
Figure 4-3: Alamar Blue assay using PCF trypanosomes grown in SDM80.......................99
Figure 4-4: Cell counts of PCF trypanosomes (strain TREU 927) grown in SDM80.......100
Figure 4-5: 2-Deoxy-D-glucose inhibition.........................................................................101
Figure 4-6: Uptake of Glucose analogues..........................................................................102
Figure 4-7: Glucose utilisation in presence of GlcNAc.....................................................103
Figure 4-8: L-proline uptake..............................................................................................104
Figure 4-9: Oligomycin sensitivity....................................................................................105
Figure 4-10: 2-Deoxy-D-glucose sensitivity......................................................................106
Figure 4-11: Proline uptake in presence of D-glucose.......................................................107
Figure 4-12: Proline uptake after long term D-glucose exposure......................................108
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Figure 5-1: Nodes and edges..............................................................................................115
Figure 5-2: KEGG Network of T. brucei metabolites.......................................................116
Figure 5-3: CM state network............................................................................................118
Figure 5-4: GlcNAc state network.....................................................................................119
Figure 5-5: Glucose state network.....................................................................................120
Figure 5-6: Proline state network.......................................................................................121
Figure 5-7: Principal Component Analysis........................................................................122
Figure 5-8: Metabolites unique to proline metabolism (comparing GL and PR states)....124
Figure 5-9: Metabolites unique to GlcNAc supplementation (comparing CM and NA
states)..................................................................................................................................125
Figure 5-10: Glucose specific metabolites (comparing PR and GL states).......................126
Figure 5-11: Glucose specific metabolites (comparing NA and CM states).....................127
Figure 5-12: Pyruvate abundance within the cell pellet and supernatant...........................130
Figure 5-13: Targeted amino acid analysis........................................................................132
Figure 6-1: Schematic representation of DNA synthesis showing L-glutamine usage in T.
brucei (A) and the specific chemical reaction (B).............................................................138
Figure 6-2: Schematic representation of CTP synthesis showing L-glutamine usage in T.
brucei (A) and the specific chemical reaction (B).............................................................139
Figure 6-3: Tyrosine pathway............................................................................................141
Figure 6-4: Schematic representation of L-tryptophan metabolism in mammalian cells..142
Figure 6-5: General scheme for polyamine pathway in trypanosomes..............................147
Figure 6-6: Structures of some of the relevant amino acid analogues used.......................148
Figure 6-7: Trypanocidal cell counts.................................................................................151
Figure 6-8: Amino acid recovery of azaserine action........................................................152
Figure 6-9: Amino acid recovery of L-Dopa action...........................................................152
Figure 6-10: Glutamine uptake in PCF trypanosomes in the presence and absence of
azaserine.............................................................................................................................154
Figure 6-11: Phenylalanine uptake in PCF trypanosome in presence and absence of
azaserine.............................................................................................................................155
Figure 6-12: Glutamine uptake in BSF trypanosomes in presence and absence of azaserine.
............................................................................................................................................156
Figure 6-13: Tyrosine uptake in BSF trypanosomes in presence and absence of azaserine.
............................................................................................................................................156
Figure 6-14: Glutamine inhibition by 20 amino acids.......................................................157
Figure 6-15: Glutamine inhibition.....................................................................................158
Figure 6-16: Phenylalanine inhibition by amino acid analogues.......................................159
Figure 6-17: Tyrosine uptake in PCF trypanosomes in the presence and absence of L-Dopa.
............................................................................................................................................160
Figure 6-18: Tyrosine uptake in BSF trypanosomes in the presence and absence of L-Dopa.
............................................................................................................................................160
Figure 6-19: Tyrosine inhibition by 20 amino acids..........................................................161
Figure 6-20: Tyrosine inhibition........................................................................................162
Figure 6-21: Phenylalanine inhibition................................................................................163
Figure 6-22: CPP activity against BSF trypanosomes.......................................................164
Figure 6-23: DFMO uptake................................................................................................166
Figure 6-24: DFMO self-inhibition....................................................................................166
Figure 6-25: DFMO inhibition by 20 amino acids. ...........................................................167
Figure 6-26: DFMO inhibition by basic and aromatic amino acids. .................................168
Figure 6-27: Lysine inhibition by DFMO and lysine.........................................................169
Figure 6-28: Tyrosine uptake in BSF trypanosomes in presence and absence of DFMO.170
Figure 6-29: Tyrosine inhibition by DFMO.......................................................................171
Figure 6-30: Schematic representation of a possible L-glutamine transport system in PCF
trypanosomes. ....................................................................................................................173
Figure 6-31: Synthesis of CdS-tiopronin-Tat (taken from la Fuente et al., 2005).............178
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Figure 6-32: Structure of L-lysine and DFMO..................................................................179
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5 List of Tables
Table 1: Amino acid transporter genes from T. brucei........................................................40
Table 2: Amino acid transporter genes from T. brucei........................................................68
Table 3: Calculated IC for the TbAATP1 specific amino acids........................................76
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Table 4: Calculated IC values for analogue inhibitors....................................................101
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Table 5: Total nodes and edges across the metabolic states..............................................121
Table 6: Carbon source specific metabolites (pellet).........................................................127
Table 7: Putative formula generated from pellet metabolites............................................128
Table 8: Carbon source specific metabolites (supernatant)...............................................129
Table 9: Putative formula generated from supernatant metabolites...................................129
Table 10: Targeted amino acid analysis.............................................................................131
Table 11: Trypanocidal activities of lead candidate amino acid analogue........................149
Table 12: Trypanocidal activities of metabolic downstream analogues............................153
Table 13: Calculated IC values for inhibitors of L-glutamine uptake.............................158
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Table 14: CPP amino acid sequence..................................................................................163