Characterization of nucleoside transport in the intestinal protozoan parasite Giardia intestinalis.

T-

The Characteñzation of nucleoside transport in the

intestinal pr otozoan p arasi te G i ar di a

ⁱ

nt

e

s

t i

nal i s

.

Robert Andrew Davey

Department of Microbiology and Immunology The University of Adelaide,

Adelaide, 5005 Australia

A thesis submitted for the Degree of Doctor of Philosophy.

August, 1993.

fi;'.,c,

r.\,-, \ \'

^{ti'ì i i}

Abstract

A rapid sampling technique has been

adapted

and used to

measure nucleoside transport

in a humanderived

isolate

of

the protozoan parasite

Giardia intestinalís

^(syn. G.

lamblía). The study was based on published

findings thatGiardia

cannot produce nucleotides

by

de

novo

synthesis; that labelled adenine, guanine and

uracil

are incorporated

directly into

the ribonucleotide

pool

by the action

of

specific phosphoribosyltransferases; that exogenously acquired ribonucleosides are degnded

rapidly within

the cells

by

specific hydrolases

to yield the respective

bases;

and that the

^parasite

lacks ribonucleotide

reductase

and is able

to produce 2'-deoxynucleotides

only

by direct phosphorylation

of

acquired 2'-deoxynucleosides.

As cell membranes are effective barriers to the

diffusion of

nucleosides,

it

was considered that transporters must exist

in Giardia

to mediate their uptake.

Transport assays were conducted at OoC to prevent loss

of

trophozoites

by

adherence,

to minimize intracellular

metabolism

of

transported nucleosides, and

to slow

transport

for

more accurate measurements.

Initially,

the transport

of [3H]thymidine

was measured and this was shown

to follow

simple Michaelis-Menten kinetics,

with

an apparent

K,n of

50

pM

and

V-"* of

70 pmol.min-1.1106

cells¡-I.

Thymine and

uracil

were the most effective inhibitors

of thymidine

^transport

(KÍ 's of 30 pM

and

45 pM rcspectively), followed by

thymidine, deoxyuridine and

uridine (Ki

's between

&-96 pM). In

contrast, cytosine,

its

ribonucleoside and 2'-deoxyribonucleoside derivatives, and adenosine, were not

effective inhibitors,

even at high concentrations. These results indicated that the transporter responsible

for

a major part

of thymidine influx recognized the nucleoside base moiety. Recognition may require

the presence

of

an oxygen attached to the carbon at position 4

of

the

pyrimidine ring,

as occurs

in thymine

and

uracil, to allow

hydrogen bonding.

The 5-methyl

group

of

thymine apparently does not interfere

with

the recognition process.

Although

the furanose

ring

appeared not to be

involved in

recognition and binding to this transporter

(uracil,

uridine and deoxyuridine were

all efficient inhibitors of

thymidine transport),

it

may be required

for

the translocation step, as bases enter the cells via a separate carrier ^(see below).

al_

A

detailed analysis

of inhibition of thymidine influx by

adenosine and deoxycytidine,

which exhibited a maximum þlateau) level of inhibition only

^20-30Vo

of that

effected by

either uracil or uridine,

suggested

the

existence

of a

second nucleoside transporter

with

a lower

affinity for

thymidine. The presence

of

a second pennease was confirmed by measuring

influx of ¡3Hldeoxycytidine,

¡3Hladenosine

and

¡3Hlguanosine.

The

transport

of

each

of

these nucleosides

was inhibited strongly by all naturally-occurring

nucleosides

but only poorly or not at all by

nucleobases. These

fîndings

were consistent

with a

Eansporter that

recognized structural features on the furanosyl moiety of ribonucleosides and

^2'-

deoxyribonucleosides. Both 2'- and S'deoxyadenosine were potent

inhibitors of influx

^(>95Vo

inhibition at 2 mM),

3'-deoxyadenosine was less

effective

^(=70vo

inhibition)'

^whereas

2"3'- dideoxycytidine

and cytosine arabinoside

were virtually inactive

^(O-207o

inhibition).

^These

observations suggest that the

2'and5'hydroxyl

^groups

ofthe

^furanosyl

ring

are not necessary

for

recognition

of

nucleosides

by

the second transporter,

whilst

the 3'

hydroxyl

appears to be

important. Michaelis-Menten

^constants

(Kr) were calculated for the influx at

^OoC

of

deoxycytidine

^(2291.116

pM)

and adenosine

(45!24 pM), with

respective

V*u*

values

of

1314

and

1112

pmol.min-1.(106 ce[s)-l. Thymidine

exhibiæd

a Ki of

205190

pM

against

¡3uldeoxycytidine influx.

The second transporter, which has a broad specificity,

would

appear to be sufficient

to

mediate the uptake

of all

nucleosides and 2'-deoxynucleosides required

by

the parasite. The

tfrymine/uracil-specific thymidine

rransporrer,

which has high affinity for uridine

and thymidine, may be important when these compounds are scarce.

No

evidence could be found

for

the existence

of

any other nucleoside transporter under the ^assay conditions used.

In work

extending these studies, the presence

of

a

distinct

base transporter

which

has

specificity for

purines and

pyrimidines

was demonstrated.

In

order to clone the genes encoding the

Giardia

nucleoside transporters, a nucleoside rransport

deficient strain of E. coli (5Õ1660) was obtained. This

enabled

a

^strategy

of

complementarion

to

be employed,

by

selection

for

transformants able

to utilize pyrimidine

nucleosides

(uridine or cytidine)

^as

the

source

of

carbon.

Two

genes, designated

bc2

and

l1ca,were

cloned

from

a

Giardia

^genomic

DNA library by

this technique. Sequence analysis

of

the cloned fragments that had been isolated

by

complementation revealed an open reading

iii

frame (ORF)

of

1,134 bp and 984 bp, encoding a putative 43-kDa polypeptide and a 29-kDa

polypeptide for bc2

and 10ca respectively. Transcripts

of bc2

and

l0ca

werc detected as a

1.6-kb and

1.1

kb RNA

species respectively

on

Norttrern

blots using total RNA

extracted from G iar díø trophozoites.

The

likely

secondary structure

of

the BC2 polypeptide determined

from

a consensus

of the

structures predicted

by eight

algorithms

is

consistent

with a known

class

of

transporter,

i.e. the porins, which have been

characterized

from mitochondria and bacærial

outer membranes.

The

structural

model

contains

12

hydrophobic

or amphiphilic

B-strands, each

sufficiently long to

span

a lipid bilayer.

These are connected

by hydrophilic

loops, which

contain the 7 predicted ø-helices, all comprised predominantly of hydrophilic

residues.

Southern hybridizations confirmed that bc2 was

of

giardial and not bacterial

origin,

and that

it was

present

in

trophozoites

as a single

gene.

This

class

of

transporter

may provide

the structural

stability

required

for

proteins that are exposed

to

the lumenal environment

of

the gut.

In contrast, the 10CA polypeptide

shares

significant

sequence

homology to DNA- binding

^proteins.

This

indicates that although complementation

in E. coli

strain 5<Þ1660 has

enabled the cloning of a putative transporter

^gene

(bc2), other

^genes

may be able

to complement the bacterial mutation.

It will

therefore be important to

further

analyse the ^genes (and

their

encoded polypeptide products) isolated

by this

technique,

to confirm their role

as

fansporter ^s

in

G íar dia.

In

summary,

this

study has

led to

the discovery and

kinetic

characterization

of

two distinct nucleoside transporters

in Giardia

intestinalis and to the isolation and characterization

of a

gene encoding

a porin-like

transporter.

The

data

provide

an

insight into

the pathways

involved in

nucleoside and nucleobase transport

in

this organism, and

of

the features

of

the substrate molecules

that

are necessary

for recognition in this

parasite.

Further

study

of

the putative nucleoside transporter gene and its polypeptide product(s)

will

be needed to confirm

its role

as a nucleoside transporter

in Giardia

and may

provide

an

insight into

the structure and

function of the

transporters.

The

data have

important implications for future

design

of

chemotherapeutic drugs that may be used in the treatment

of

giardiasis.

vl_ l_ l_

Contents

Chapter

^L

1.1 1.2 L.3

t.4

t.4.1

t.4.2

1.5 1.6

r.7

1.8 1.9

1.9.1

t.9.2

1.9.3

1.9.3.1

t.9.3.2

r.9.3.3 1.10 1.11

1.11.1

t.lt.2

1.11.3

l.L2

1.13

Chapter

2

2.1

2.r.1

Introduction

Historical

background Nomenclature

Evolutionary history

Morphology

The trophozoite The cyst

Life

cycle Attachment

Determination of Gíardía species Prevalence and epidemiology Biochemistry

of

trophozoites Growth in culture

Energy metabolism

in Gíardia

Nucleic acid metabolism

Purine metabolism

of

G. intestinalis

Pyrimidine

metabolism

Deoxynucleotide biosynthesis Nucleoside Eansport

Treatment

of

giardiasis by chemotherapy

Action of

standard drugs used to combat giardiasis Development of drug resistant strains

of

G. íntestínalis Design

of

novel drugs

for

use

in

treatment

Infection,

immunity

and vaccine development Project

definition

Materials

and

Methods

Transport Assays

Chemicals and reagents

1

1

2 2 3 3 4 5 5 6 9 10 10 10

t2

T3

t7

2l

22 24 25 26 27 z8 30 32 32 32

2.L.2 2.1.3

2.t.4 2.r.5

2.1.6

2.t.7

2.1.7.1

2.t.7.2 2.r.8

2.r.8.1 2.1,.8.2 2.2

2.2.t.

2.2.2 2.2.3 2.2.4

2.2.4.L 2.2.5 2.2.6 2.2.7

2.2.7.1 2.2.7.2 2.2.8

2.2.8.t 2.2.8.2 2.2.8.3

ix

Giardia

cultures

Harvesting trophozoites

[3H] Nucleoside stock solutions

Sampling assay

Liquid

scintillation counting and data analysis Calculation

of

^3H and 14C content

Calculation of extracellular

fluid

contamination Analysis of transport kinetics

Calculation of IC5g values Calculation of

K.

^and

K¡

^values

Molecular biology and recombinant

DNA

techniques Chemicals and reagents

Enzymes Growth media

Bacterial strains and plasmids Maintenance

of

bacterial strains Preparation of competent bacteria

Transformation of Es c he rí c hia c o I í K- 12 ^s trains

DNA

extraction procedure ^s

PlasmidDNA

isolation Preparation

of

genomic DNA, Analysis and manipulation of

DNA

Nucleic acid quantitation

Restriction endonuclease digestion

of DNA Analytical

and preparative separation

of

restriction fragments

Calculation of restriction fragment size Dephosphorylation of

DNA

End-filling with Klenow

fragment

32 33 33 34 34 35 3s 35 36 36 36 36 36 37 37 38 38 38 39 39 39 4L

4t

41 42

42 42 42 43 2.2.8.4

2.2.8.5 2.2.8.6

Manufacture

of

l4C-acetylated bovine serum albumin

I

2.2.8.7 2.2.8.8 2.2.8.9 2.2.8.rO

2.2.8.tr

2.2.9 2.2.9.r 2.2.9.2 2.2.10 2.2.11

2.2.t2

2.2.13 2.2.13.1 2.2.13.2

Chapter

3

3.1 3.2

3.2.t

3.2.2 3.2.3 3.2.4 3.2.5

3.2.5.1 3.2.5.2 3.2.6

x

Ligation

of DNA

In-gel ligation of

DNA

fragments Preparation of

DNA

and

RNA

_Probes Southern transfer and hybridization Colony Blots

Construction

of

^genomic

DNA

libraries

Selection

of

^genes encoding putative nucleoside transporters Detection

of

genomic fragments containing

full

length ^genes

Sequencing using dye labelled primers

DNA

sequencing gels

Analysis of

DNA

^sequences

Analysis and maniPulation

of RNA

Preparation

of RNA

Northern Transfer

Development of an ^assay

for measuring transport kinetics in Giørdia trophozoites

Introduction

Materials and methods

Composition

of

the dense

oil

^laYer Density of

oil

_Phase components Recovery of cells

Preparation

o¡

1251-66elled bovine serum albumin

(BSA)

Extracellular drag volume

Use

of

1251-1u6e11ed BSA to determine extracellular volume Use

of l4c-lub"ll"d

^BSA to determine extracellular volume Assessment

of

non-specific binding

of l4c-labelled

BSA

to trophozoites Quench monitoring

Quenching characteristics

of

solutions Counting efficiency versus'H' number

43 43

M

45 47 47 47 48 48 49 49 50 50 51

52 52 54 54 54 54 55 55 55 56

3.2.7 3.2.7.1 3.2.7.2

57 57 57 57

-T---

3.2.7.3 3.2.7.4 3.2.8 3.3

3.3.1 3.3.2 3.3.3

3.3.3.r 3.3.3.2 3.3.4 3.3.s

3.3.5.1 3.3.5.2 3.3.6 3.4

3.4.r

3.4.2 3.4.3

xl-

Determination

of

the energies

of

B-particle emissions Spreadsheet calculations

Osmolarity of assay medium Results

Density

of

the

oil

^phase

Sedimentation of cells through

oil

Radioactive labelling

of

bovine serum albumin Iodination of

BSA

l4c-acetylation of BSA

Extracellular

fluid

volume

Quenching of radioactive emis sions

Dibutylphthalate reduces counting

efficiency

Quench correction

Osmolarity

of

assay medium Discussion

Assay design

Extracellular drag volume

58 58 61 61 61 64 65 65 65 68 68 68 72 75 75 75 76

78 78 79 79 80

8l

3.4.3.1 3.4.3.2 3.4.3.3 3.4.4 3.5

Chapter

4 83

4.1 4.2

4.2.r

83 85 Determining the amount

of

l3H]substrate transported

in

cells

Measurement of the 3H _and

14c

_content

of

samples Measuring quench

Spreadsheet software design Assay medium

Summary

Detection

and

characterization of thymidine transport

Introduction

Materials and Methods

Temperature dependence of

efflux from

cells pre-loaded

wittr l3H]ttrymidine

⁸⁵

4.2.2 4.2.3

4.2.3.1 4.2.3.2 4.3

4.3.1 4.3.2

Intracellular metabolism

of

transported ¡3Hl thymidine

Efflux

of preloaded

¡3Hlthymidine

xl- l-

Measurement of

efflux

from pre-loaded cells at OoC

Phosphorylation Hydrolysis Results

The effect on

tansport

capability of incubating trophozoites

in

assay medium

Uptake

of

thymidine at OoC

Choice

of

uptake period for

kinetic

studies Saturation

of

uptake

Effect of

glucose on thymidine uptake Metabolism of intracellular thymidine

Specificity of

the thymidine transporter

Kinetic

analyses

IC56 curves Discussion

Measurement of thymidine transport

Efflux of

thymidine

Transport capability after prolonged incubation

of

cells

in

medium

Influx

and

efflux of

thymidine

Uptake, metabolism and

influx of

thymidine

Specificity of

thymidine

influx

Summary

The

detection and

characterization

of a second nucleoside

transporter having specificity for

ribonucleosides and

2'-deoxyri

bonucleosides Introduction

Materials and Methods

85 86 86 86 87 87

89 92 92 95 95 98 100 100

r02

109 109 109 4.3.3

4.3.3.r 4.3.3.2 4.3.3.3 4.3.4 4.3.5 4.3.6

4.3.6.r 4.4

4.4.r

4.4.r.1 4.4..1.2

4.4.1.3

4.4.t.4

4.4.2 4.5

Chapter

5

5.1 5.2

109 110 111 111 119

120

r20

Lzl

5.2.2 5.3

5.3.1 5.3.2 5.3.3

5.3.3.1 5.3.4

5.3.4.r 5.3.4.2

5.2.r

5.4 5.4.1 5.4.2 5.4.3 5.4.4

5.4.4.1 5.4.4.2 5.4.4.3

xl- l- 1

Intracellular metabolism of transported I 3H12'-deoxycytidine and [3H]adenosine

Inhibition

of thymidine transpofi by

uracil

and deoxycytidine Results

Measurement of 2'-deoxycytidine

influx

Metabolism of deoxycytidine

Specificity

of

the deoxycytidine transporter

Inhibition

of deoxycytidine

influx

by thymidine Transport

of

ottrer nucleosides

Specificity

of

adenosine and guanosine uptake

Relative involvement

of

the broad-specificity (deoxycytidine) transporter and the thymine/uracil-specific transporter

in

thymidine transport

Discussion

Deoxycytidine uptake is mediated by a speciñc permease Calculation of intracellular aqueous volume

Intracellular metabolism of deoxycytidine

Inhibition of

nucleoside

influx

Specificity

of

the deoxynucleoside transporter Transport

of

other nucleosides

Thymidine transport via the broad-specificity nucleoside transporter

Recognition

of

subsnate Summary

Ctoning of

^{genes encoding}

putative

nucleoside

transporters

Introduction

Materials and Methods Results

Construction

of

a Sau3A genomic

library

Isolation of clones

t27

tzt

122 122

t22

126

IN

130 130

t32

134 135 136 136

t37

r37 t37

5.4.5 5.5

Chapter

6

6.1 6.2 6.3

6.3.1 6.3.2

138

r39 r42

148 148 151 151 151

t54

6.3.3 6.3.4

6.3.4.1 6.3.5 6.3.6 6.3.7

6.3.7.r 6.3.7.2 6.4

6.4.1 6.4.2 6.4.3

6.5

Chapter

7

7.1 7.2

7.2.1 7.3

7.3.1

7.3.2

7.4 7.4.1

X]-V

Resriction

analysis of

plOCA

and p10UD

Nucleotide sequence determination

of

the p10CA and

plOUD

genomic inserts

Detection

of

open reading frames Southern analysis

Northern analysis

Efficiency

of re-transformation and complementation

for

nucleoside uptake

with

^p10CA and pBC2

Transformation and complementation

with

pBC2 constructs Transformation and complementation

with

^p 10CA

Discussion

Detection

of

the

l)ca

and bc2 sequences

in

trophozoite

DNA

Analysis

of

the bc2 and

l0ca

nucleotide ^sequences

Potential recognition sites

for

polyadenylation

of

primary

RNA

transcripts

Summary

Structural prediction of the

bc2-encoded

polypeptide

by analysis

of primary structure

Introduction Methods

Prediction of polypeptide secondary structwe Results

Searches of

DNA

and protein sequence databases

for

sequences homologou s to bc2 or its deduced polypeptide products

Secondary structure prediction

of

^the BC2

poþeptide

sequence Discussion

BC2

forms ^a

porin-like

transport protein

t57

r57 160 170 175

t82

r82

183 189 190 191

195 195 r97 197 198 192 194

198

t99

207 zo7

7.4.2

7.5.

Chapter I

8.1 8.2 8.3

8.3.1

8.3.2

8.3.3 8.4 8.5

Chapter

9

9.1 9.2

Chapter

¹⁰

Appendix

XV

Reliability of

the secondary structure predicted

for

BC2 Summary

Predictions of

the

structure

and

function of the

polypeptide

encoded by the 70cø geneof G.

intestínalis

Introduction

Methods Results

Searches

for

sequences homologous to 10ca

andits

polypeptide product(s)

Analysis

of

hydrophobic and charged amino acids

in

the deduced amino acid sequence

of l)CA

Secondary structure prediction for 10CA Discussion

Summary

General

Discussion

Detection and characterization

of

nucleoside transport Isolation of genes encoding potential nucleoside transporters

Bibliography

Published work arisirig from this study

208

2r2

zr4

214 215 216

2t6

2r9

222 225 230

239 266

23t

231 236