Introduction

INTRODUCTION

xfungal kingdom comprises over 100,000 species throughout the world, of

which up to 150 have been documented as causes of infections in humans (Marichal, 1999).

The severity of fungal diseases varies from superficial infections to life-threatening systemic

infections. Most of the infections are opportunistic because the infecting agents are found as

commensal or are ubiquitous in the environment (e.g. Aspergillus species) and can easily

gain access to debilitated patients. In addition to opportunistic pathogenic fungi, a limited

number of fungi exist with a true pathogenic potential for healthy hosts and cause life­

threatening infections restricted 'to their endemic region. These pathogens e;g., Histoplasma

capsulatum, Paracoccidioides brasiliensis, Penicillium marneffei and Coccidioides immitis

are therefore classified in biohazard class (Vanden Bossche et al., 1994a; White et al., 1998).

The incidence of systemic fungal infections is high and has increased over the past

few decades. The nature and the extent of the impairment of normal host defense influence

the manifestation and severity of infection. Several factors contribute ·to the increased

incidence of fungal infections. One is the increase in the size of susceptible population as a

result of increased use of chemotherapy, and long term use of broad-spectrum antibacterial

agents and immunosuppressive drugs. The prevalence of AIDS, malignancies, rising

numbers of elderly individuals, invasive surgical interventions and perhaps paradoxically.the

progress and sophistication of medical treatments have all contributed to the rising toll of

morbidity and mortality caused by fungi. Another factor is the greater clinical awareness of

the importance of fungal infections reflected by an increased motivation to search for the

presence of fungal pathogens (White et al., 1998; Marichal, 1999).

1.1. CANDIDA AND CANDIDIASIS

Candida albicans is the species most frequently associated with fungal infections in humans

(Kluytmans et al., 1996; Vanden Bossche et a!., 1998). C. albicans is a serious agent of

infection, particularly in immunocompromised patients. The delicate balance between the

host and Candida which is commensal may tum into a parasitic relationship, resulting in an

infection called Candidiasis (Odds, 1988). C. albicans attributes include the production of

4

Introduction

secreted hydrolytic enzymes, dimorphic transition (morphogenetic conversion from budding

yeastto the filamentous growth form or hypha), antigenic variability, the ability to switch

between different cell phenotypes, adhesion to inert and biological substrates and

immunomodulation of host defense mechanisms (Odds, 1988). Humans carry yeast fungi,.

including Candida, throughout the gastrointestinal tract {mouth through anus) as part of

normal commensal flora. Removai of bacteria from skin, mouth and gastrointestinal tract

with the use of antibiotics leads to inhibition of endogenous flora, providing reduced

environmental and nutritional competition that favors the growth of Candida! organisms. The

commensal oral isolation of Candida species ranges between 30% and 60% of healthy

patients. It is important to note that Candida species are not part of the normal flora of the

skin. They may, however, transiently colonize fingers or body folds. The ability of yeast

forms to adhere to the underlying epithelium is an important step in the production of hyphae

and tissue penetration. Candida can infect mouth, throat, skin, scalp, vagina, fingers, nails,

bronchi, lungs or gastro-intestinal tract or may become syst~mic as in case of septicemia,

endocarditis and meningitis (Odds, 1988; Prasad, 1991; Vanden Bossche et al., 1993; Khan

and Gyanchandani, 1998). Candidiasis is a primary or secondary infection ranging from

acute, sub~acute and chronic to fatal ~ases. The history of candidiasis dates back to the fourth

century BC when Hippocrates described oral aphthae in two patients with severe underlying

disease in his book, 'Epidemics' (Marichal, 1999). In general, superficial mucocutaneous

candidiasis is frequent in-patients with T- cell deficiencies, such as AIDS patients. The more

serious, life threatening deep seated or disseminated candidiasis is normally found in a

spectrum of severely immunocomprornised patients (Hitchcock, 1993). Candida

vulvovaginitis is found in 10% of women of childbearing age: its prevalence increases up to

30% during pregnancy (Marichal, 1999). Pfaller et a/., reported that Candida species have

become the fourth leading causes of nosocomical blood stream infections in the USA (Pfaller

et al., 1998). About 50% are due to C. albicans. Superficial infections of skin and mucous

membranes are the. most common. Esophagitis, peritonotis or urinary tract infections are less

frequent sites of candidiasis. Although C. albicans is the most common cause of human

infection, the genus Candida includes about 200 species. C. albicans is now listed as one of

the top 5 most frequently isolated organisms from blood infections, as is recognized as a

major contributor to morbidity and mortality worldwide (Frosco and Barrett, 1998). Fischer-

5

Introduction

Hoch et a!., reported on a ·nearly 5-fold increase in the incidence of oropharyngeal

candidiasis in hospital admissions over a ten year period from 1980-1989 (Frosco and

Barrett, 1998). Orpharyngeal ,candidiasis has presented nearly a 15% incidence in all AIDS

patients (Frosco and Barrett, 1998). Likewise, the incidence of disseminated candidiasis

jumped more than 10-fold in the past decade (Frosco and Barrett, 1998). Of all the cases of ·

AIDS reported by WHO, Candidiasis due to C. albicans (though other species of Candida

have also been demonstrated to be responsible) is the predominant "AIDS-indicative" ' .

disease. Different types of Candidiasis their locations, symptoms and predisposing factors are

described in Table-!.

C. albicans, the principal infectious agent of human infection, is an oval yeast of 2-6

)..Lm in diameter. It exists as diploid, asexual, polymorphic yeast with various biochemical

abilities, both assimilative and fermentive, but lack any proper sexual stage and carotenoid

pigments. This medically significant fungi has the ability to undergo phenotypic switching

and has 8 chromosomes (Odds, 1988; Prasad, 1991). It exists in both hypha! and yeast forms.

If pinched cells do not separate; a chain of cells is produced called pseudo hyphae. Different

factors are known to affect the morphogenesis of Candida. Although both bud and hyphae

are found in infected tissues, it seems likely that the elongated hyphae penetrate tissues,

leaving in its path lateral colonies of budding cells that in turn give rise to a new penetrating

hyphae (Shepherd, 1985; Odds, 1985). Based upon this basic phenomenon of morphological I

transition and the capacity to switch heritably but reversibly at relatively high frequency

between a number of general phenotypes distinguishable by colony morphology is shown in

Figure 1 (Soli eta!., l988;Scherer and Magee, 1990;Berger et a!., 1990). The etiological,

biochemical and morphological attributes of Candida makes it a unique eukaryote and its

pathological potential makes it an important microorganism (Khan and Gyanchandani, 1998).

1.2. DRUGS USED AGAINST CANDIDA AND THEIR MECHANISM OF ACTION

Antifungals or drugs used against Candida can be broadly classified into different categories,

allylamines, thiocarbamates, azoles, mbrpholines, pyridines and pyrimidines. Figure 2 show

the site of action of some of the antifungal drugs. With the exception of flucytosine most of

the antifungals interact with or illhibit ergosterol, the major sterol of the fungal plasma

membrane which contributes to many cellular functions.

6

Introduction

TABLE-I

Incidences of Candidiasis, Locations, Symptoms and Predisposing Factors

Type I

Location l Symptoms I Predisposing Factors

SUPERFICIAL

Oral thrush Oropharynx White lesions Old age, infancy resembling milk curd on the surface of throat, tounge,gum linings.

Denature Stomatitis Palate Erythema, oedema of Old age palate

Leukoplakia Inner cheek surface Chronic raised lesions Tobacco smoking, denature wearing

Vulvo-vaginal Vagina, perinal area White discharge, Pregnancy, diabetes Candidiasis intense erythema Candida onychia Brown, greenish Swollen, painful Occupational hazards

discoloration of nails inflammation of nail (fruit canners) and nail folds

SYSTEMIC

Oesophageal Oesophagus Dysphagia, Fatally ill patients, Candidiasis reterosternal pain AIDS patients Gastric Candidiasis Stomach Lesions, vomitting Pre-existing lesions

Parental nutrition, Candidiasis of lower Bronchia, lungs Lesions Aspiration respiratory tract Candiduria Kidney, urinary tract Micro abscesses, Diabetes, old age,

dissemination of the drug abuse tract

Candida endocarditis Heart, aortic and Intracardiac Cardio pulmonary mitral valve vegetation blocking bypass, open heart

blood vessels surgery Candida menengitis Granulomas of Microabscess, No particular factor

ventricle, lesions headaches Lesions

Candida arthritis Necrosis of cartilages, Swelling, painful Intra-articular steroid abscess formation ·joints injection, trauma

Cancer patients DISSEMINATED

Eye, skin, blood Leukaemic patients Bum victims Iatrogenic factors

Compiled from Odds (1988)

7

Figure 1. The various colony phenotypes in the switching system of C. albicans (A) "0-smooth", (B) "Star", (C) " Ring", (D) " Irregular wrinkle", (E) " Stipple", (F) " Hat", (G) " Fuzzy", (H) " r-smmoth"(Adapted from Soli , 1990).

t

t ~·

HMG-CoA reductase

l l Squalene synthase

l

G t + ll"-reductase

Figure 2. The site of action of various antifungals at different steps of ergosterol biosynthetic pathway.

Introduction

Several drugs have been developed which acts and inhibit enzymes which are involved in

different steps of ergosterol biosynthetic pathway. The site of action of these drugs in

ergosterol biosynthetic pathway is described in Table-II (White et al., 1998).

1.3. DRUG RESISTANCE

One of the major problems raised out of the irrational use of above mentioned antifungals, is

multidrug resistance (MDR), which is threatening the pharmaceutical industry. MDR is

defined as resistance against a spectrum of drugs that share neither a comnion target nor a

common enzyme (Vander Bleik and Borst, 1989; Fine, 1989; Prasad et al., 1996; Gottesman

et al., 1996). It was described more than 20 years ago when Kessel et al., and Bielder and

Riehm (Kessel et al., 1968; Bielder and Riehm, 1970) noted that cells lines become

resistance to actinomycin D or vinca alkaloids displayed cross resistance to a wide range of

other components (Kessel et al., 1968; Bielder and Riehm, 1970; Gottesman et al., 1996).

Multidrug resistance, which is a major problem in medical and agricultural development, is

an emerging phenomenon observed in various organisms throughout the evolutionary scale

(Skovsgaard et al., 1994; Ruetz and Gros, 1994a; Gottesman et al., 1996; Kane, 1996;

Lautier et al., 1996; Zamble and Lippard, 1996).

The problem of non-existent chemotherapeutic ability to combat the disease has been

compounded by the emergence of MDR in this clinically important fungi, C. albicans

(Georgopapadakou and Walsh, 1994; Hazen, 1995; Odds, 1996). One of the major problems

with MDR is that clinical isolates of Candida show cross-resistance to many unrelated drugs

apart from showing resistance to one particular drug. In addition, the incidence of C. albicans

cells acquiring resistant to azoles and polyenes has increased considerably in recent years,

which has posed serious problems towards its successful chemotherapy. Superimposed with

this, C. albicans is naturally more resistant to several drugs e.g. cycloheximide, benomyl,

methotrexate than other yeasts' which further compounds the overall problem (Prasad, 1991;

Ben-Yaacov et al., 1994; Gow et al., 1994). In order to combat exodus of antifungals

Candida cells have developed several drug-evading mechanisms, which are discussed below.

8

Introduction

TABLE-II

Drugs used against Candida and their mechanism of action

Drugs

INHffiiTORS OF ERGOSTEROL BIOSYNTHESIS

ALLYLAMINES e.g. Naftitine and terbinafine THIOCARBAMATES e.g. Tolnaftate and tolcitate

AZOLES e.g. Imidazole (ketoconazole and miconazole) e.g. Triazoles (fluconazole, itraconazole and voriconazole)

PYRIDINES e.g. Buthiobate and pyrifenox PYRIMIDINES e.g. Triarimol and fenarimol

MORPHOLINES e.g. Fenpropimorph and amorolfine

OTHER ANTIFUNGAL DRUGS

POLYENES e.g. Nystatin, amphotericinB (AmB) and pimaricin.

5-FLUCYTOSINE (5-FC)

Mechanism of Action

Both allylamines and thiocarbamates inhibit the conversion of · squalene to 2,3-oxidosqualene by the enzyme squalene epoxidase (which is a product of ERGJ gene) leading to accumulation of squalene in the cells.

AZoles inhibit cytochrome P450 dependent 14a-lanosterol demethylase (encoded by ERG II) resulting in the accumulation of 14-methylated sterols, which are toxic to the cells.

Pyridines and Pyrimidines also inhibit lanosterol demethylase and are used in agriculture but not in medicine.

Inhibit 014-reductase and 08-07 isomers.

These polyenes intercalate into plasma membranes containing ergosterol and forms the channel through which cellular components like potassium ions, leak out and destroy the proton gradient within the membrane.

Inside the cell, flucytosine is dearninated to 5-fluorouracil (5-FU). 5-FU is converted into 5-fluorouridine monophosphate (5-FUMP), and 5-fluorodeoxyuridine monophosphate (5-FdUMP). 5-FdUMP, a specific inhibitor of thymidylate synthetase, an essential enzyme for DNA synthesis. 5-FUMP is incorp~rated in RNA, thus disrupting protein synthesis of fungus. ·

Comptled from White et al., (1998) and White eta/., (1997)

9

Introduction

1.3.1. Mechanisms of Drug Resistance in Yeast

Many different types of mechanisms are known to contribute to drug resistance in the cell

(Table-III and Figure 3). The most frequent mechanisms include reduction in the import of

the drug in the cell; modification or degradation of the drug once it is inside the cell; changes

in the interaction of the target enzyme (binding activity); changes in other enzymes in the

same enzymatic pathway and an increased efflux ofdrug from the cell (White, 1997a; Lopez- -

Ribot et al., 1998; Franz et al., 1998a).

1.3.2. Effiux Pumps in Drug Resistance

Efflux pumps, which are generally present in the plasma membranes, have now emerged as

one of the important components of multi drug resistance. Outward efflux by transporters is a

strategy to prevent the access of the toxic compounds from their intracellular targets (Ames,

1986; Higgins, 1992; Fath and Kolter, 1993). These evolutionary conserved drug transporters

are found within four major superfamily of transporters namely, the "ATP Binding Cassette

transporters (ABC)"; a proton motive force driven group: the "Major Facilitators (MFS)"

(Marger and Saier, 1993; Michaelis and Berkower, 1995); the "Small Multidrug Resistance

family (SMR)"; and the "Resistance/Nodulation/cell Division (RND) family". ABC

transporters are frequently associated with active efflux of molecules, which are toxic to cells

and are relatively hydrophobic like azoles. MFS have not been studied extensively but are

also associated with efflux of hydrophobic molecules. While ABC family of transporters are

in general multicomponent active transporters, capable only of transporting small solutes in

response to ATP hydrolysis, the MFS transporters are single polypeptide secondary carriers

capable only of transporting small solutes in response to chemiosmotic ion gradients. ABC.

and MFS superfamilies account for nearly half of the solute transporters encoded in the

genome of microorganisms (Higgins, 1992; Fath and Kolter, 1993; Paulsen and Saier, 1997;

Pao eta/., 1998; van Veen and Konings, 1998).

1.3.2.1. Major Facilitators (MFS)

The MFS has originally been defined as a superfamily of perm eases that are characterized by

two structural units of six transmembrane spanning a- helical segments, connected by a cyto-

10

Introduction

Table-III

Different Mechanisms of Antifungal Resistance

Different Mechanisms of Antifungal Resistance

CELLULAR MECHANISMS

1. Intrinsic resistance of endogenous strains Replacement with more resistant Candida species Replacement with more resistant strain of C. albicans

2. Genetic alterations that results in more resistant strain 3. Transient gene expression that renders a cell temporarily

resistant

4. Alteration in cell type

5. Alteration in the fungal population

MOLECULAR MECHANISMS

1. Drug import 2. Alterations in intracellular drug processing (a) Modification (b) Degradation 3. Modification of ergosterol biosynthetic pathway 4. Alterations of ERGJJ gene (a) Point mutations (b) Overexpression (c) Gene amplification (d) Gene conversion or mitotic recombination

5. Alteration in other Erg genes

6. Decreased accumulation of drug

7. Efflux pumps or transporters

(a) ABC Transporters

(b) MFS Transporters

Examples

* C. krosei is intrinsically· more resistant that C. albicans. C. norvegensis & C. inconspicua are also found to be more intrinsically resistant.

* C. albicans can be divided into two serotypes A, and B, based on carbohydrate surface markers. B serotype are more sensitive to azoles but more resistant to 5-FC than A serotypes.

* Mutations in ERGJJ changing R467K alorie is sufficient to cause azole resistance. Mutation in T315 A causes a reduction in enzymatic activity and reduction in azole binding to active site, resulting in fluconazole resistance.

+ Deletion of ERG3 from C. glabrata causes cell sensitive to fluconazole. * Deletion of ERG/I causes susceptibility to amphotericin B.

Reduced drug accumulation in intrinsically azole resistant strains e.g. C. krosei. Similar thing is observed for C. albicans and C. glabrata for Rh 123, fluconazole etc.

*CDR!, CDR2 of C. albicans. * CaMDRI,FLUI of C. albicans

Compiled from White eta/., ( 1998); White eta/., ( 1997) and Mancha} eta/., ( 1999)

11

~·=:· r:::::\ '- '""~ ~

rod"'"t>'3-.. ~ -·. 8 • ·V 4J Degradation

Drug r; ~ / Substrate target If{

ABC transporter

product

0

Figure 3. Mechanisms of azoic resistance in fungi. I. Changes in the cell wall/ plasma membrane leading to impaired azole uptake. 2. Efflux of drugs mediated by the ABC or the MFS class of efflux pwnps 3. Over-expression of the drug target or of the efflux pumps. 4. Mutation in the drug target (P45014DM) does not allow the drug to bind or allows drug to bind with low a!Tmity. 5. Activation of alternate pathways such as L'l5•6-

desaturase. 6. Sequestration of the drug into an organelle , like vacuole by organellar ptunp. 7. Chromosome alterations or changes in chromosome number as a mean to maintain more copies of the required gene . 8. Modification of azoles (L'l22 -

desaturase and CYP52) to an inactive form or degradation of drug .

Introduction

plasmic loop. The MFS consists of over 50 transporters membrane proteins from bacteria to

higher eukaryotes involved in symport, antiport, or uniport of various substrates (Marger and

Saier, 1993; Pao et al., 1998) which have been classified into five distinct clusters or families

of membrane transport proteins. MFS proteins are shown to be involved in (i) drug

. resistance, (ii) sugar uptake, (iii) uptake of Krebs cycle intermediates, (iv) phosphate

ester/phosphate antiport, and (v) oligosaccharide uptake (Marger and Saier, 1993; Paulsen et

al., 1996a). The list ofMFS family of proteins from microbes to humans classified according

to sequence similarities, their substrates and their functions are listed in Table-IV. The drug

resistance proteins are PMF-dependent antiporters which efllux out drugs exchanging one or

more W ions with a substrate molecule (Paulsen and Skurray, 1993). On the basis of

hydropathy and phylogenetic analyses, drug efllux proteins can be divided into two distinct

types with 12 and 14 transmembrane segments (TMS), which contain more than 100

members (Paulsen et al., 1996b ). The first report of a MDR gene, which acts as a pump in

bacteria came from the studies of Qac locus in Staphylococcus for resistance to quaternary

ammonium compounds (Rouch et al., 1990). qacA has characteristically been found on

multi-resistance plasmids from clinical strains of S. aureus (Gillespie eta/., 1986). Multidrug

efflux protein Bmr from Bacillus subtilis mediates resistance to structurally diverse

compounds, including rhodamine G and acridine dyes, ethidium bromide, TPP, puromycin,

chloramphenicol, doxorubicin and tluoroquinolones. Drug transport mediated by Bmr are

sensitive to inhibitors of the mammalian P-glycoprotein (Pgp) pump, such as reserpine and

verapamil (Neyfakh et al., 1991).

S. aureus protein NorA was identified as a chromosomal tluoroquinolone resistance

gene but was subsequently shown to confer resistance to similar range of substrates as that of

Bmr. InS. cerevisiae, FLRI gene product, a MFS, has been shown to cause resistance to

cycloheximide, fluconazole, cadmium and H20 2 (Alarco et al., 1997). Another MFS, HXTJJ

gene product, which is a sugar transporter of S. cerevisiae, is involved in pleiotropic drug

resistance. Loss of HXTJ 1 and or HXT9 confers cycloheximide, sulfomethuron methyl and 4-

NQO resistance.

1.3.2.2. ABC (ATP-Binding Cassette) Transporters

ABC transporters, also referred as "Traffic-ATPases" constitutes one of the largest and most

12

Introduction

TABLE-IV

List of MFS family of proteins from microbes to humans depicting their substrates and functions

FAMILY FAMILY NAME ORIGIN FUNCTIONS SUBSTRATES NUMBER

1. Sugar Bacteria, Archea, Sugar uniport, Monosaccharides transporter eukaryotic Protists, Sugar:proton symport (hexoses,pentoses ),

Yeasts, Animals, Sugar:sugar antiport diasaccharides, Plants organocations, inositols

Drugs:H+ Bacteria, Yeasts Drug: H+ anti port Multiple or single drugs 2. anti porter

(14TMS)

3. Drugs:W Bacteria, Archea, Drug:W antiport Multiple of single drugs anti porter Yeasts, Animals (12TMS)

4. Organophosphat Bacteria, Animals Organophosphate: Pi Sugarphosphates, e: Pi antiporter antiport glycerolphosphate,

phosphoglycerates 5. Oligosaccharide: Bacteria Sugar:W symport Di and trisaccharides

w Sugar:sugar antiport Syrnporter

6. Metabolite: H+ Bacteria Solute: H+ antiport Dicarboxylases, citrate, symporter betane etc.

7. Fucose- Bacteria Hexose uniport or L-fucose, glucose, galactose- hexose:W symport ·galactose

glucose: H+

8. Nitrate: nitrite Bacteria, Yeasts, nitrite uniport? Nitrite, nitrate porter Plants Nitrate: W symport

9. Phosphate:H+ Yeasts, Piants Pi:H+ symport Inorganic phosphate syrnporter

10. Nucleoside: H+ Gram negative- Nucleoside: H+ Nucleosides syrnporter Bacteria syrnport

ll. Oxalate: formate Bacteria, Anion:anion antiport Oxalate, formate anti porter Archeabacteria,

Animals 12. Sialate: H+ Gram negative- Substrate:H+ symport Sialete

symporter Bacteria

13

introduction

13. Monocarboxylat Yeasts, Animals Substrate:H+ symport Pyruvate, lactate, eporter mevalonate

14. Anion:cation Bacteria, Yeasts, Substrate:H+ or Na Hexauronate, tartarate, symporter Animals symport acetate

15. Aromatic acid: Bacteria Substrate:H+ symport Muconate, benzoate, H+ protocatachurate

16. Unknown major Yeasts Unknown Unknown facilitator

17. Cynate Bacteria Substrate:W symport Nco· permease

H+:

18. Oligopeptide Bacteria, Yeasts, Substrate:W symport Peptides, amino acids, transporter Animals, Plants nitrate

Compiled from Pao eta/., (1998)

highly conserved superfamilies and is found in large numbers in all organisms. ABC protein

family with currently more than 1000 members represents the largest protein family known

to date (Higgins, 1992; Higgins, 1993). ABC transporters have been identified till now in

species including bacteria, yeast's, insects, protozoa, plants and humans(Higgins, 1992; Fath

and Kolter, 1993; van Veen and Konings, 1998; Bauer et a/., 1999a). They fulfill a

remarkable variety of cellular functions. While most ABC proteins are purely ATP-driven

membrane transporters, some of them act as ion channels, channel regulators, receptors,

proteases and even sensing proteins (Higgins, 1995). As far as the transport of substrates is

concerned, ABC transporters mediate translocation of ions, heavy metals, carbohydrates,

anticancer drugs, amino acids, phospholipids, steroids, glucocorticoids, bile acids,

mycotoxins, antibiotics, pigments, peptides through membranes and in . some cases even

whole proteins (Figure 4) (Higgins, 1992; Dean and Allikmets, 1995; Mahe eta/., 1996;

Ambudkar and Gottesman, 1998; Bevers eta/., 1999; Bauer eta/., 1999a). However, each

ABC transporter transport large variety and size of substrates, but at the same time maintains

selectivity for its particular substrate, this represents an intriguing and yet unsolved mystery.

Several mammalian ABC proteins are medically important, because mutations in

14

pheromone

•• Drug

+t

ATP Drug

~ A~ <l I>

p ~ JJ.

<lU~> Pheromones Pv~

Steroid

Figure 4. Schematic representation of ABC transporter spanning the membrane bilayer and transporting various substrates. Six transmembrane segments of ABC transporter are shown in yellow whereas its two nucleotide binding domains are shown in cream colour. Its involvement in transport of various types of substrates is depicted for e.g. drugs may be extruded directly from the membrane into the extracellular space or flipped from inner to outer leaflet and then to extracellular space, efflux of steroids from cells and transport of pheromones. [Adapted and modified from Kolaczkowski and Goffeau ( 1997)].

Introduction

corresponding genes cause severe genetic diseases such as cystic fibrosis (Harris and Argent,

1993), ademoleukodystrophy (Mosser et a!., 1993) and zeweller syndrome (Gartner and

Valle, 1993), dubin-johnson syndrome (Paulusma et a!., 1996), familial hyperinsulincmic

hypoglycemia of infancy, hepatic choleostasis and stargardt's macular dystrophy of the eye

(Klein eta!., 1999).

1.3.2.2.1. Domain structure and organization

All ABC proteins share a similar molecular architecture with a hall mark domain

organization that includes the presence of at least one evolutionary conserved A TP-binding

cassette (ABC), also known as NBD (for nucleotide binding domain), as well as several

predicted a-helical membrane-spanning segments (TMS) (Tusnady eta!., 1997; Klein eta!.,

1999). The TMS and NBDs are normally arranged in duplicated forward (TMS6-NBD)2 or

reverse (NBD-TMS6)2 configuration, but numerous half-size transporters with various

topologies are also known as shown in Figure 5 (Higgins, 1992; Dean and Allikmets, 1995).

Full size ABC proteins usually have six predicted TMS in each halve, connected by a

charged linker region (Figure 5). Additional TMS proximal to N-terrninus also exist in some

ABC proteins (Cole and Deeley, 1998). Finally the hydrophilic NBD domains encompass

approximately 250 residues with five conserved protein motifs. The Walker A and Walker B

motifs, found in all other nucleotide binding proteins (Walker et a!., 1982), and the ABC

signature or C motif with the consensus sequence LSGGQ are diagnostic hallmarks for all

ABC proteins. In addition, there are two less conserved regions, the so-called center region

between Walker A and Walker B and another one downstream of Walker B motif (Michaelis

and Berkower, 1995). Many ABC proteins are located in plasma membrane (Higgins, 1992;

Decottignies and Goffeau, 1997; Kolaczkowski and Goffeau, 1997). In bacteria, where there

are no intracellular membranes, all ABC transporters are situated on the cytoplasmic

membrane mediating the movement of solutes in and out of the cells (George, 1996;

Neyfakh, 1998).

A typical ABC transporter consists of four units, viz. two membrane domains

comprising of six transmembrane segments and two nucleotide binding domains (NBDs),

which bind and hydrolyze A TP. These four modules can be expressed as separate

polypeptides (Higgins, 1992; Ames eta!., 1992; Higgins, 1993). In !:".coli histidine permease,

15

(TM-NBF)2 (TM-NBF) (NBF-NBF)

(NBF-TM)2 NBF-TM NBF

Figure 5. Schematic picture of different ABC type proteins depending on their predicted topology. Nucleotide binding domains are depicted as red circles. Hydrophobic domains are shown in blue lines.Two hydrophobic domains are marked in black and blue lines. [Adapted from Decottignies and Goffeau (1997)]

Introduction

four genes express these polypeptides whereas there are three genes for the ribose

transporter, two encoding membrane bound domains and one encoding the two fused NBDs

(Higgins, 1992; Ames et a!., 1992; Higgins, 1993 ). Among eukaryote members of the ABC

superfamily, the TAPI and TAP2 peptide transporter is encoded by two genes, each

encoding a membrane bound domain fused to an NBD whereas for human MDRJ (Pgp) a

single gene encodes all the four modules (Higgins, 1992; Ames eta!., 1992; Higgins, 1993 ).

1.3.2.2.2. ABC proteins of Saccharomyces cerevisiae

From the genome-sequencing project it has been revealed that there exist atleast 33 distinct

ABC transporters in S. cerevisiae. This budding yeast is a powerful model organism for·

higher eukaryotes as it allows the combination of classical genetics and biochemistry with

recombinant technology Almost each cellular compartment harbors at least one ABC

transporter except endoplasmic reticulum and nuclear membrane. It has been found that 25%

of human genes significantly match yeast proteins. In some cases even human genes are

capable to complement yeast function, facilitating structure/function studies in yeast. Based

on phylogenetic classification tree analysis, yeast ABC proteins are classified into six distinct

subfamilies as shown in Table-V (Taglicht and Michaelis, 1998; Bauer eta/., 1999a). These

families are defined as the MDR, the PDR, the MRPICFTR, the ALDp, the YEFJ and the

RLI. PDR5, SNQ2, YORJ are some of the ABC well known drug transporters.

1.3.2.2.3. ABC proteins in antifungal resistance of C albicans

It has been observed that treatment of fungal infections with triazoles can result in severe

antifungal resistance (Sanglard eta!., 1995; White eta!., 1998; Sanglard eta!., 1998; Ha and

White, 1999). The discovery that Pdr5p mediates pronounced resistance to mycotoxins and

antifungal azoles prompted the hunt for Pdr5p homologues in pathogenic fungi. Intracellular

levels of drug in resistant isolates were increased by the addition of sodium azide suggesting

that an energy dependent efflux pump wasassociated with resistance (Sanglard eta/., 1995;

Albertson eta/., 1996). As a result CDRJ (Candida drug resistance) gene was first cloned by

Prasad et al, by functional complementation of a null mutant of PDR5 of S. cerevisiae

(Prasad et al., l995a). The expression of CDRJ even in low copies inS. cerevisiae conferred

drug resistance, while multiple copies of PDR5 are required to confer resistance.

Introduction

TABLE-V

ABC transporters of S. cerevisiae

ABC TRANSPORTERS SUBSTRATE (S) NUMBER OF LOCALIZATION PHENOTYPIC AND THEIR FAMILIES AMINO

ACIDS :MDR family Ste6p (Sterile 6) a-factor 1290 Plasma membrane, Sterile

Golgi Vesicle, Slow growth Atm I p (A 1P transporter of F e/S proteins 694 Endosome Viable Mitochondria) Not Known 820 Mitochondria Viable Md 12p (Multidrug resistant like) Not Known 696 Not Known Md II p (Multidrug resistant like) Not Known

PDRfamily Pdr5/Stsl/Ydr11Lemlp[(Pleiotropic Drugs, steroids, 1511 Plasma membrane Hypersensitivity to drug resistance) (Sporidesmin toxicity antifungals drugs supperssor) (Yeast drug resistance) (Ligand effect modulator)]

Pdr I Op (Pleiotropic drug resistance) Not Known 1564 Plasma membrane Viable Pdr I 5p (Pleiotropic drug resistance) Not Known 1529 Plasma membrane Viable Snq2p (Saccharomyces Drugs, steroids, 1501 Plasma membrane Hypersensitivity to nitroquilnoline transporter) mutagens drugs

Weak acids 1511 Plasma membrane Hypersensitivity to Pdr I 2p (Pleiotropic drug resistance) weak acid

Not Known 1411 Plasma membrane (?) Viable Pdr II p (Pleiotropic drug resistance) Not Known 1049 Not Known Viable Adp I p (A 1P-<iependent permease)

:MRP/CFfR family Oligomycin, Yorlp (Yeast oligomycin resistance) reveromycin 1477 Plasma membrane Hypersensitivity to

drugs Glutathione 1515

Y cfl p (Yeast cadmium factor) conjugates- Vacuole Hypersensitivity to conjgates/Cd2+ cadmium

Batlp (Bile acid transporter) Bile acids 1661 Vacuole Viable

ALDpfamily Ssh2/Palllpxa l/pat2p (Peroxisomal A1P-binding cassette transporters) Pxa21Patlp (Peroxisomal A1P- Long chain-fatty acids 870 Peroxisome Oleate binding cassette transporters)

Long chain-fatty acids 853 Peroxisome Oleate YEF3family Y ef3p (Yeast translation elongation Drugs 1044 Ribosome Essential factor) Gcn20p (General control of nitrogen Not Known 752 Poly somes Viable metabolism)

Abbreviations used are: ABC, A TP-binding cassette

Compiled from (Bauer et al.. 1999: Dean eta!., 1994; Leighton and Schatz, 1995; Balzi and

Goffeau, 1995; Shani et al., 1995: Shani et a!., 1996: Decottignies and Goffeau, 1997: Ortiz

et al., 1997; Taglicht and Michaelis, 1998).

17

Introduction

The levels of resistance to drugs like cycloheximide was much stronger as compared to

PDR5 which suggested it is intrinsically much more effective against drugs. Reduced

accumulation of radiolabeled fluconazole has been observed in a variety of fluconazole

resistant clinical isolates of C. albicans when compared with fluconazole sensitive isolates

(Sanglard eta/., 1995;Albertson eta/., 1996). Later on another gene from C. a/bicans was

isolated, named as CDR2 which also confer resistance to different drugs. The

characterization of ABC proteins e.g. CDRJ (Krishnamurthy et a/., 1998a; Krishnamurthy et

a/., 1998b; Krishnamurthy eta/., 1998c; Smriti eta/., 1999), CDR2 (Sanglard eta/., 1997)

from C. albicans and their overexpression in certain instances of azole resistant clinical

isolates has confirmed that these transporters are involved in MDR scenario of C. a/bicans

(Sanglard eta/., 1995; Hannun, 1996; Maesaki et al., 1998; Cannon et al., 1998). This led to

the isolation of other ABC traits porters of Candida which are described in Table-VI.

The homologues Cdr3p and Cdr4p show highest homology to Cdr1p and Cdr2p,

however, as compared to Cdr1p and Cdr2p which are more than 90% similar, Cdr3p and

Cdr4p are only 75% similar to Cdrlp and Cdr2p. Interestingly, over expression ofCDR3 and

deletion of CDR3 and CDR4 could not affect drug susceptibilities (Balan et al., 1997; Franz

eta/., 1998b ). It is not yet known why some ABC proteins show drug resistance phenomenon

whereas others do not, inspi~e of their close homology. The ball models of Cdrlp and Cdr3p

drawn from hydropathy plot (Figure 6a & 6b) do not show very striking differences in the

domain organization of the two ABC proteins. Both the proteins have similar topological

arrangements where hydrophilic domain containing nucleotide binding motif precedes

hydrophobic transmembrane stretches. The only apparent difference between the two

proteins appears in C-terminal where Cdr3p has an extended loop connecting TMl I and

TM12. In addition, the last 21 amino acids in the C-terminal of Cdr3p are totally different

from Cdrlp. The molecular basis of drug transport by these transporters will have to await a·

high-resolution 3D protein structure analysis, however analyses of mutations of homologous

and non-homologous regions between different Cdrps can soon provide valuable

information. This situation is similar to the presence of two types of P-glycoproteins in

mammals: the one, which transports hydrophobic drugs while the other, does not transport

18

Introduction

Table-VI

ABC transporters of C alhicans

Transporters Function Number of Domain Localization Amino Acids Arran_gement

HST6 Not Known 1323 (TMS6-ABC)2 Not Known (Homologue of STE6)

CDRJ (Candida drug resistance)

Antifungals 1501 (ABC-TMS6)2 Plasma membrane

CDR2 Anti fun gals 1499 (ABC-TMS6)2 Plasma membrane (Candida drug resistance)

CDR3 Not Known (Candida drug resistance) 1501 (ABC-TMS6)2 Plasma membrane

CDR4 Not Known (Candida drug resistance) 1490 (ABC-TMS6)2 Plasma membrane

ELFJ Not Known 1191 ABC2 Not Known (Elongation like factor)

CaEF3 Not Known 1050 ABC2 Not Known (Candida elongation factor)

Abbreviations used are: ABC, ATP-binding cassette; TMS, transmembrane segment.

Compiled from (Prasad eta!., 1995b; Sanglard eta/., 1997; Balan eta/., 1997; Franz eta/.,

1998b; Raymond eta/., 1998; Bauer eta/., 1999) (http://alces.med.umn.edu/candida.html).

drugs. Mouse mdr1 and mdr3 of, and human MDRJ belong to the type I, which can transport

and confer drug resistance. Mouse mdr2 and human MDR2 (also known as MDR3) belong to

the type II (Ueda et al., 1987; Buschman eta/., 1992; Schinkel eta!., 1994; Smith eta/.,

1994; Ruetz and Gros, 1994b; Leveille-Webster and Arias, 1995). Studies employing MDRJ­

MDR2 chimeric proteins led to the identification of some amino acid residues in the TM6 of

MDRJ which are sufficient to allow an MDR2 backbone in the N-terminal half of P-gp to

transport several MDRJ substrates (Zhou et a/., 1999). These studies indicate a· close

relationship between MDRJ, a multidrug transporter and A1DR2, a phosphatidylcholine

19

Ball model of Cdrlp

Figure 6a. A hyi>Othetical two-dimensional model of Cdrll•· The model is based on the hydrophobicity profiles of amino acid sequences and functional domains. Small circles represent amino acid residues, which are filled with single letter code of amino acid. The numbers indicate the begimting and the end of the transmembrane (1M) domains. The putative A TP-binding sites and signature sequences are shown in yellow and pink respecti\'ely. The first and the last amino acid of each transmembrane segment is colored blue.

Figure 6b. A hy pothetical two-dimensional model of Cdr3p. The model is based on the hydrophobicity profiles of amino acid sequences and functional domains. Small circles represent amino acid residues, which are filled with single letter code of amino acid. The nwnbers indicate the beginning and the end of the transmembrane (TM) domains. The putative ATP-binding sites and signature sequences are shown in red and purple respectively. The first and the last amino acid of each transmembrane segment is colored blue. The extra stretch of amino acids at the C-terminal in Cdr3p are shown in green.

Introduction

flippase. Since Cdrl p, Cdr2p and Cdr3p have similar domain structure, their substrate

preferences are most likely to be determined by some non-identical amino acid residues.

However, this remains to be investigated.

1.3.2.2.3. Other Transporters of Candida which are Involved in Drug Resistance

CaMDRJ and FLUJ are the MFS genes identified in C. albicans to be involved in drug

resistance. CaMDRJ (previously known as BENr) was initially identified as a gene which

conferred resistance to tubulin binding agent benomyl and tetrahydrofolate reductase

inhibitor methotrexate (Fling eta/., 1991; Ben-Yaacov et a/., 1994; Gupta eta/., 1998).

FLUJ is a gene, which confers resistance to fluconazole (Calabrese eta/., 1999). CaMDRJ is

highly homologous to FLRJ of S. cerevisiae, while FLUJ revealed high similarity to putative

MFS transporter YLL028wp (Alarco et a!., 1997; Calabrese et a/., 1999). CaMDRJ

expression in S. cerevisiae confers resistance to several unrelated drugs and its

overexpression has been linked to azole resistance in C. albicans. Recently polymorphic

mutant alleles of CaMDRJ have also been identified by Gupta eta/ (Gupta eta!., 1998). The

complete sequencing of CaMDRJ alleles revealed several in frame point mutations leading to

amino acid residues where insertion/replacement of an aspartate residue in a stretch of serine­

asparagine-aspartate rich domain were noteworthy (Gupta et a/., 1998). Interestingly these

mutant alleles of CaMDRJ showed distinct drug resistant profiles. The relevance of such

alleles of CaMDRJ in azole resistance in C. albicans remains to be ascertained. The

sequencing of PCR amplified serine-aspargine-aspartate rich domain of fluconazole resistant

and laboratory isolates revealed that these isolates harbor different alleles. This would mean

that the polymorphic alleles might exist in nature in response to different environmental

stresses. The expression of CaMDRJ in C. albicans cells was enhanced by benomyl,

methotrexate and several other unrelated drugs and was more pronounced in some of the

azole resistant clinical isolates. This confirms that while CaMDRJ overexpression is linked

to fluconazole resistance, other effiux mechanisms are equally important. Recently,

homologues of CaMDRJ have been identified from C. dubliniensis and C. glabrata which

are termed as CdMDRJ and CgMDRJ, respectively (Sanglard et a!., 1999a; Sanglard et a!.,

1999b ). It appears that increased expression of CdMDRJ is the main mechanism of

fluconazole resistance involved in C. dubliniensis clinical isolates (Moran eta!., 1998). Since

20

S':j-:1 I Introduction

CgMDRI confers specific resistance to fluconazole, its constitutive expression in C. glabrata

may be responsible for the intrinsically low susceptibility of this yeast species to fluconazole

(Sanglard et al., 1999b ).

1.5. IMPORTANCE OF ABC TRANSPORTERS IN HEALTH AND DISEASES

Several genetic disorders are related to mutations or defects in the ABC proteins (Table-VII).

The best examples are cystic fibrosis and peroximal disorders related to CFTR and ALDP

ABC proteins, respectively. The clinical importance of ABC proteins is not restricted to

diseases but evidence accumulated suggests that they have important role in cellular

physiology also. Several homologues of genes causing diseases are also observed in yeasts.

Yeast therefore represents an ideal model organism for the functional dissection of disease

related genes particularly relating to ABC proteins. (Harris and Argent, 1993; Mosser ei al.,

1993; Gartner and Valle, 1993; Paulusma et al., 1996; Decottignies and Goffeau, 1997;

Bauer et al., 1999b; Klein et al., 1999)

1.5.1. PHYSIOLOGICAL RELEVANCE OF ABC PROTEINS

All ABC transporters have distinct ATP binding domain (s), and are

hydrolysis. The precise molecular mechanism, which couples nucleotide hydrolysis to their

physiological functions, is not understood. The involvement of these A TPases in drug

transport and multidrug resistance in various organisms is the most important functions

attributed to members of this superfamily. However, all traffic ATPases are not involved in

drug transport and have been shown to mediate cellular transport, transbilayer movement and

secretion of membrane phospholipids, cholesterol transport (Figure 7) (Higgins, 1992; Dean

and Allikmets, 1995; Mahe eta/., 1996; Ambudkar and Gottesman, 1998; Bevers eta/.,

1999; Bauer et al., 1999a).

In the following sections some of the physiological roles of ABC transporters are discussed

in detaiL

1.5.1.1. Emerging Roles of Drug Transporters

Recently number of roles has been attributed to ABC transporters. In addition to their

involvement in drug transport, such systems can export and import a range of substances and

21

Introduction

TABLE-Vll

Examples of human health diseases related to ABC proteins

ABC Transporters Organism Human Health Closest yeast Impact Homoloeues

CF1R Human Cystic fibrosis YCFJ (Cystic fibrosis (Yeast cadmium factor)

transmembrane conductance regulator protein)

ALD (Adernoleukodystrophy)

Human Adernoleukodystrophy

PMP70 Human Zellweger syndrom PXAJ (Peroxisomal ATP-( Peroxisomal matrix binding cassette transporter)

protein) PXA.J (Peroxisomal ATP-binding cassette transporter)

TAPJITAP2 Human Behcet's disease, multiple MLDJ,MLD2 (Transporter associated with sclerosis, (Multidrug resistant like)

antigen processing) Bare lymphocyte syndrome type 1

MDRJ Human Cancer cells drug STE6 resistance (Sterile)

MRP Human Cancer cells drug YCFJ, Y/101 (Multidrug resistance resistance (Yeast cadmium factor)

protein) SUR Human Hyperinsulinemic YCFJ

(Sulfonylurea receptor) hypoglycemia of infancy (Yeast cadmium factor)

ALDR Human Zellweger syndrome PXAJ (Peroxisomal A TP-binding

cassette transporter) ABC/ Human, Mus musculus Not known No close homologue

(ATP-binding cassette) ABC2 Human, Mus musculus Not known No close homologue

(ATP-binding cassette) ABC7 Human, Mus musculus Not known ATMJ

(ATP-binding cassette) (A TP transporter of Mitochondria)

ABC8 Human, Mus musculus Not known ADPI (ATP-binding cassette) (ATP-dependent permease)

CMOAT Candida albicans Same phenotype as YCFJ (Canalicular multispecific human Dubin-Johnson (Yeast cadmium factor) organic anion transporter) syndrome

CDR/ Cm1dida albicans Drug resistant candidiasis PDR5,PDRJO,PDRJ 5 (Candida drug resistance) (Plcotropic drug resistnacc)

PJMDR2 Plasmodium falciparum ATMJ (Plasmodium falciparum Drug resistance malaria (A TP transporter of

Multidrug resistance) Mitochondria)

EhPgp/ Entamoeba histolytica. Drug resistance ST£6 (Sterile) (Entamoeba histolytica P- amoebiasis glycoprotein)

LdMDR leishmania donovani Drug resistance kala azar ST£6 (Sterile) (leishmania donovani (visceral leishmaniasis) Multi drug resistance)

Compiled from Decottignies and Goffeau (1998).

22

A B c

T R A N s p 0 R T E R

Transport of drugs/xenobiotics

__. Transport/flip-flop oflipids

Cell signalling

Membrane composition

Membrane organization:* asymmetry * lateral domains

Regulation of membrane processes

Transport of pheromones I Translocation of Lipoproteins

I Cell differentiation

j Inhibition of virus production

Figure 7. Schematic representation depicting the physiological functions associated with ABC transporters.

Introduction

provide a wide variety of essential nutrients and protect them from a range of noxious agents.

They help in building of surface layers, regulate development in microorganisms.

1.5.1.2. ABC-Systems can be Exporters or Importers

An outstanding feature of ABC-systems is the extraordinary range of compounds transported

by different members of this family, from ions to large polypeptides and polysaccharides and

it is a major challenge how this is achieved. The range of substrates exported and imported

are described .in Table-VIII. An additional remarkable feature of ABC-systems is that the

movement of the ion or molecule, accompanied by A TP hydrolysis, can be outward (export

or secretion) or inward (import). Import condition is found basically for many prokaryote

systems. They require for the uptake of small solutes e.g. maltose, histidine, peptides or

ribose. Export systems in great majority of cases and is found both in prokaryotes and

eukaryotes(Holland and Blight, 1999).

Some of the other emerged functions of these ABC proteins or traffic A TPases

include behaving as a sex pherome transporter, lipoprotein translocater, phospholipid

translocater, steroid transporter as described in (Table-IX).

* Pheromone transporter: Sexual reproduction in the yeastS. cerevisiae is controlled by two

alternative mating-type alleles, a and a, where haploid cells of opposite mating type

conjugate to give diploid zygotes. The sexual cycle requires the secretion of the mating

factors a and a from MATa and MATa cells respectively (Gooday, 1974; Jackson and

Hartwell, 1990). The pheromone produced by MATa cells, a factor i~ secreted via the ER­

Golgi secretory pathway. Genetic and biochemical evidences suggest that the bioactive a­

factor is translocated from the cytoplasm across the plasma membrane to the extracellular

space by ST£6 (Michaelis, 1993). HST6 of C. albicans, a homologue of STE6 also functions

as pheromone transporter (Raymond eta!., 1998).

* Transport of steroids: CDR/ of Candida albicans specifically transport human steroid

hormones namely 13-estradiol and corticosterone (Krishnamurthy et a!., 1998c). A S.

cerevisiae transformant harboring CDRI gene, accumulated about 3 fold f3-estradiol and

about 2 fold less corticosterone than the non transformed strain. Furthermore the effiux of 13-

estradiol and corticosterone was found to be inhibited by some drugs and thus suggesting

23

Introduction

TABLE-VIII

Range of sustrates transported by ABC systems

Export Import

ABC Organism Substrates ABC Transporters Organism Substrates Transporters

MDRI (Pgp) Human Anti-tumor ALDP Man Fattyacids (Multidrug drugs.hydrphobic (peroxisomes) resistance) analgesics,J3-amyloid

peptides lipids, RbsA E. coli Ribose detergents, hormones, cholesterol AAP E. coli L-amina acids anti-histamine.

PstABC E. coli Phosphate MRP(l-7) Human Organic ions, (Multi drug conjugated and non- CysPTWAM Bacillus subti/is Sulphate-thiosulphate resistance conjugated drugs protein) Dei A Salmonella Dipeptides

Typhimurium MDRJ Human Long chain phosphati

(Multidrug dylcholine, distinct resistance) drugs. OppABCDEF Streptococcus Oligopeptides

pneumoniae SPgp Human Phosphatidy~ne?

(Sister P- Enterobacter glycoprotein) ArniABCDEF Aerogenes Oligopeptides

ABC-I Human Retinol? (AlP-binding E. coli Maltose

casette) MalEFGK ABC-r Human cr CFTR Pseudomonas Copper

(Cystic fibrosis Human Glucocouronate (Na) NosDYF Stutzeri transmembrane

conductance regulator protein)

SUR Human K+ (Sulfonylurea

receptor)

TAP1,2 Plants MHC peptides (Transporter

associated with antigen

processing) ABCX Plants Heme

MRPLike (Multidrug Herbicides, pigments, resistance flavanoids protein) PDRI2 Yeast Weak organic acids

(Pleotropic drug resistance)

STE6 (Sterile) Yeast Pheromone peptide

Compiled from Holland and Blight (1999) and Prasad et al., (1996)

24

Introduction

TABLE-IX

Functions of yeast ABC proteins

ABC Proteins Organism Physiological Function References STE6 Saccharomyces cerevisiae Pheromone transporter (Michaelis, 1993)

HST6 Candida albicans Pheromone transporter (Raymond et a/., CDRJ Candida albicans Phosphatidylethanolamine 1998)

{Ptd.Etn) translocater

PDR5 Saccharomyces cerevisiae Human steroid transporter (Mahe eta!., SNQ2 Saccharomyces cerevisiae Human steroid transporter 1996;Krishnamurth CDR2 Candida albicans Human steroid transporter yet a!.,

Phosphatidylethanolamine 1998c;Dogra eta/., (Ptd.Etn) translocater 1999)

(Dogra eta/., 1999)

LolCDE Gram negative bacteria Transport of Lipoprotein (Y akushi et a!., 2000)

RhT Dictyostel ium Regulator for cell (Good and Kuspa, differentiation 2000)

MDRJ Human Inhibition of HIV virus (Lee eta!., 2000) production, Long chain lipid translocater

MDR3 Human Phosphatidylcholine (Smith eta!., 1994) translocater

commonality among binding site(s). PDR5 and SNQ2 of S. cerevisiae are also known to

efflux human steroids (Mahe eta!., 1996).

* Translocation of Lipoproteins: Recently Yakushi et a!., have found a new complex,

LolCDE, belonging to A TP-binding cassette (ABC) transporter family. Lipoproteins in

Escherichia coli are anchored to periplasmic side of either inner or the outer membrane by a

lipid moiety. Lipoproteins directed to the outer membrane are released from the inner

membrane in an A TP-dependent manner through the formation of a complex with LolA, a

25

Introduction

periplasmic chaperone. Using reconstituted proteoliposomes, they have observed that lolCDE

catalyses the release of lipoproteins in lolA and sorting-signal-dependent manners. The

LolCDE complex differs mechanistically from all other ABC transporters, as it is not

involved in the transmembrane transport of substrates. This new mechanism is evolutionary

conserved in Gram negative bacteria (Figure 8) (Yakushi et al., 2000).

* Cell differentiation: A cellular effiux pump, RhT, has been identified from Dictyostelium,

and is found to be involved in cell differentiation as RhT inhibitors disrupted development

but induced stalk cell formation in submerged cultures (Good and Kuspa, 2000).

* Inhibition of HIV production: Recently MDRI involvement is shown in inhibition of virus

production of HIV when overexpressed in human cell line. Its expression did not

significantly alter the surface expression or distribution of either the HIV -1 receptor CD4 or

the coreceptor CXCR4. Reduction ofHIV-1 infectivity in MDRI expressing cells occurred

both during the fusion of viral and plasma membrane and at the subsequent step(s) in HIV-1

life cycle (Lee eta/., 2000).

1.5.1.3. ABC Multidrug Transporters as Phospholipid Translocators

Another role of ABC transporters is observed in translocation of various classes of lipids

between the two monolayers of plasma membrane (Bevers et al., 1999). It has been observed

by van Helvoort that MDRI gene when overexpressed in epithelial cells, it is able to

translocate various fluorescent phospholipid analogs possessing a short B chain (van

Helvoort et al., 1996). This phenomenon is found to be inhibited by MDRI inhibitors and in

energy depleted cells. Human MDR3 or mouse mdr2 are proteins with high homology with

MDRI. The authors conclude, however that it is unlikely that natural long chain lipids would

be translocated. They speculated that the biological function of translocation of

phospholipids with short chain by MDRI could be to excrete peroxidized lipids or lyso-

. derivatives out of the cell. MDR3 is a phospholipid (phosphatidylcholine (PtdCho )]

translocater (Smith eta/., 1994; van Helvoort et al., 1996) with a highly specific expression

pattern in canalicular membrane ofliver (Smith et al., I 994; van Helvoort eta/., 1996). It has

been observed that knockout mice did not secrete phospholipids into the bile and developed

severe liver disease with symptoms similar to persistent hyperinsulinemic hypoglycemia of

infancy PFIC3 (Klein eta/., 1999). PICF3 is caused by the reduction of biliary phospholipid

26

LOIB

Periplasm

ATP

LOIB

LOICDE complex

Figure 8. Transport of Lipoproteins: Lipoproteins anchored to periplasmic side of the inner membrane are released in an A TP dependent manner from LOICDE complex by their formation of complex with LolA, which acts as chaperone for transporting it to outer membrane.[Adapted from Yakushi et al., 2000].

Introduction

secretion, with normal bile salt concentration. Ruetz and Gros later carried out experiments

with secretory vesicles from yeast in which mdr2 or MDR3 have been expressed (Ruetz and

Gros, 1994b ). They found that mdr2 expression cause slight enhancement of PtdCho

translocation. MRP (multidrug resistance protein) is also shown to be involved in

translocation of sphingolipids and NBD labeled fluorescent phospholipids (Kamp and Haest,

1998; Dekkers et al., 1998a; Raggers et al., 1999a).

In order to appreciate the involvement of these proteins m mediating phospholipid

translocation, it is important to recall the structural organization of membranes.

1.6. MEMBRANES ARE ASYMMETRIC STRUCTURES

Membranes are structurally and functionally asymmetric (Lehninger et al., 1993). The outer

and inner surfaces of all known biological membranes have different components and

different enzymatic activities. Membranes are defined as the external boundary of cells that

regulate the membrane traffic across the boundary; divides the internal space into

compartments; organize complex reaction sequences and are central to biological energy

conservation and cell to cell communication. Each membrane has characteristic protein and

lipid composition, which helps them to mediate different sorts of functions (Lehninger et al.,

1993).

1.6.1. Protein Asymmetry

Membrane proteins have unique orientation because they are synthesized and inserted into a

membrane in an asymmetric manner (Lehninger et al., 1993 ). This absolute asymmetry of

proteins is preserved, as they do not rotate from one side of the membrane to another,

because they are always synthesized from the preexisting membranes. Some proteins appear

one only one face of the membrane, others span the full thickness of the bilayer and protrude

from both inner and outer membrane surfaces, causing an asymmetry of the two membrane

faces that gives them different characteristics. For e.g. the amino terminal of lycophorin

protein of erythrocyte membrane protrudes on outer surface of cell. whereas carboxyl

terminus protrudes on inner side of the cell .. It does not move from one face of bilayer to

other; its disposition iri membrane is asymmetric in nature. The absolute nature of asymmetry

27

Introduction

is observed for glycoproteins, which are mainly present on the outer side of membrane

(Lehninger eta!., 1993).

1.6.2. Lipid Asymmetry

Lipids too are asymmetrically distributed as a consequence of their mode of biosynthesis, but

this asymmetry is not absolute, except in case of glycolipids {Lehninger et a!., 1993). The

percentage of different lipids in inner and outer monolayer of membrane is different. An

almost absolute asymmetry is observed for glycolipids. These are mainly exposed to the

outside of the celL In case of endoplasmic reticulum all glycolipids generally are on lumenal

side. It is not clear how the differences in the lipid composition in the two leaflets arised. One

possibility is that certain lipids bind to certain membrane embedded proteins that occur

preferentially on one leaflet of a membrane, so that these lipids will be present in greater

quantity in that leaflet.

1.6.3. Asymmetry of Phospholipids in Membranes

Proteins and carbohydrates, which are known for their absolute asymmetry, a partial and

dynamic asymmetry has been observed for membrane phospholipids also.

The first contribution was of Bretscher to show that the phospholipids are asymmetrically

distributed in plasma membrane of erythrocytes. He observed that the non-permeant

chemical modification reagent, formyl methionylsulfone methyl phosphate is able to detect

only small fraction of the total accessible PtdEtn (Bretscher, 1972). Several groups later

confirmed his observations. Membrane of human erythrocytes has an unequal distribution of

phospholipids in the two monolayers of plasma membrane. Major portion of

aminophospholipids phosphatidylethanolamine (Ptd.Etn) and phosphatidylserine (PtdSer) is

present in the inner monolayer and phosphatidylcholine (PtdCho) and sphingomylein (SM)

predominate in the outer monolayer (Verkleij et a!., 1973; Zwaal et a!., 1975;Diaz and

Schmit, 1996; Bevers et al., 1999).

Asymmetry of phospholipid arrangement has now been studied in eukaryotic plasma

membrane and various membrane-forms; subcellular organelles, bacteria and viruses (Op den

Kamp, 1979). The general observation is that most biological membranes have different

28

Introduction

phospholipid composition in their inner and outer monolayer. Among all the systems,

erythrocyte membranes are best studied.

1.6.4. FUNCTIONAL SIGNIFICANCE OF PHOSPHOLIPID ASYMMETRY

The asymmetric distribution is very important for cells and the loss of this distribution

function as a cue for several physiological processes is summarized in Table-X

TABLE-X

FUNCTIONS ASSOCIATED WITH REFERENCES ASYMMETRIC DISTRIBUTION OF

PHOSPHOLIPIDS (Fadok eta/., 1992; Bratton eta/.,

1. Procoagulant activity 1992;Martin and Jesty, 1995; Ohga et a!., 1998) (Bevers eta!., 1983; Cerb6n and

1. Membrane enzyme regulation Calderon, 1995)

(Gupta and Mishra, 1981; Kumar and 2. Maintenance of the cell Gupta, 1983; Franck eta!., 1985; Wali

eta/., 1988; Herrmann and Devaux, 1990)

4. Removal of erythrocytes from blood (Fadok, 1992; Connor eta!., 1994)

(Nakao eta/., 1960; Confurius eta/., 5. Maintenance ofthe cell shape I 990; Farge and Devaux, 1992;

Schroit, 1998) (Tanaka and Schroit, -1983; Song et

6. Interactions of membranes a/., 1992; Roelofsen and Kamp, 1998; Bevers eta!., 1999)

7. Modulation of membrane surface potential (Cerb6n and Calderon, 1995)

(Emoto eta/., 1999; Emoto and 8. Role in Cytokinesis Umeda,"2000)

29

Introduction

1.6.4.1. Procoagulant Activity

One important biological function of asymmetry is the modulation of cell-surface properties

by the appearance ofPtdSer on the outer monolayer. For example, blood platelet stimulation

is accompanied by PtdSer exposure on outer monolayer. The PtdSer catalyses the conversion

of prothrombin to thrombin, which is an essential step of coagulation (Fadok et al., 1992;

Martin and Jesty, 1995; Ohga et al., 1998). The main function of the platelet membrane

during the coagulation process is to provide a surface for the assembly of coagulation factors,

leading to an increased local concentration. Furthermore, the cellular internalization of

platelet activation factor (PAF), a potent ether-phospholipid mediator of inflammation, may

be a consequence of enhanced transbilayer movement, accompanying cellular activation.

(Bratton et al., 1992).

1.6.4.2. Membrane Enzyme Regulation

It has been reported that PtdSer regulate the protein kinase activity and autophosphorylation.

Any changes in percentage distribution of PtdSer in cytoplasmic leaflet or inner monolayer

of plasma membrane affect the activity of membrane enzymes, for e.g. by calcium influx

may affect protein kinase activity (Bevers eta/., 1983; Cerb6n and Calderon, 1995).

1.6.4.3. Maintenance of the Cell

It has been observed that the distribution of arninophospholipids gets changed in aged

erythrocytes (Herrmann and Devaux, 1990); erythrocytes from patients with malarial

infections (Gupta and Mishra, 1981); chronic myeloid leukemia and other tumors (Kumar

and Gupta, 1983); diabetes mellitus (Wali et a!., 1988); and hereditary diseases (Franck et

al., 1985).

1.6.4.4. Removal of Erythrocytes from Blood

Presence or exposure of PtdSer on outer membrane of erythrocytes serves as a signal for their

removal from blood circulation. According to Fadok eta/., macrophages recognize PtdSer,

which is present on the outer monolayer of lymphocytes during the process of apoptosis. In a

similar manner activated blood monocytes also recognize exposure of Ptdser on outer

monolayer of cancerous cells (Fadok et al., 1992; Connor et al., 1994).

30

Introduction

1.6.4.5. Maintenance of the Cell Shape

The shape of cell is mainly maintained by: (a) the curvature of the bilayer imposed by the

relative area of the two monolayers; (b) attachment of the plasma membrane to the ·

cytoskeleton, which is present below the cytoplasmic leaflet of plasma membrane; and {c) by

attachment of the plasma membrane to the cell wall. In erythrocytes, changes in shapes have

been observed by the addition of 2%-3% of phospholipids in one layer from discocytes to

echinocytes or to stomatocytes. Further increase causes lysis of the cells. Important changes

in the shape occur like the one in the vesicle formation of Golgi bodies. These shape changes

could be triggered by the transfer of around 1% of phospholipid pool in giant unilamellar

liposomes with a diameter of 20 nrn (Confurius et a/., 1990). It has been observed that if the

diameter of vesicles is 100 run or less, a net transfer of 1% phospholipid pool induces lateral

tension (Farge and Devaux, 1992) rather than bringing a change in shape. The modulation of

lateral tension may be another way of modulating the activity of membrane proteins. In

erythrocytes and blood platelets it has been shown that cell shape not only depends on

addition of exogenous lipids to one layer but also depends on the cytoskeletal constituents.

For example, the addition of amphiphiles to red cells generates echinocytes with many

spicules, whereas in platelets the same addition gives rise to few pseudopodia. It has also

been observed that if red cells are depleted of A TP than there shape changes to echinocytes,

even after repletion with A TP red cells are unable to restore there original shape of

discocytes. Excess of ATP changes their shape to stomatocytes (Nakao et al., 1960).

Membrane budding and exocytosis requires net efflux of phospholipids to the outer

monolayer. On the other hand endocytosis requires net influx of phospholipids to inner

monolayer. Both processes require fusion of membranes. Fusion is transient but drastic

disturbance in lipid distribution takes place as a result again partial redistribution occurs and

various proteins do this. It is interesting that those cells which show high endocytic activity

also possess high translocase activity (Martin and Pagano, 1987a; Schroit, 1998). During all

these process cytoskeleton plays important role, it imposes specified locations for bending.

31

Introduction

1.6.4.6. Interactions of membranes

Interaction of two membranes also depends on the interaction of their phospholipids. In case

of PtdCho interfaces, interaction is unfavorable due to presence of large amount of

interbilayer spacing -30 A .. PtdSer and PtdEtn interfaces on the other hand are separated by

only -15 A, hence they can come closer. It has been observed that presence of acidic lipids

on the outer leaflet of pure lipid vesicles favors calcium induced fusion. The interaction of

PtdSer and PtdEtn with this cation as opposed to PtdCho and Sphingomylein {SM) makes

most cell fusion incompetent, as under normal conditions, relatively higher concentrations of

PtdCho and SM are localized at the outer monolayer. The inner monolayer, by the same

argument can easily fuse with intracellular vesicles, which have a major proportion of

aminophospholipids at their outer surface. Thus the role of aminophospholipids in these

organelles would be to enable fusion with plasma membrane. Intracellular fusion's may be

triggered either by calcium or by specific proteins such as annexins {Song et a/., 1992;

Roelofsen and Kamp, 1998). Phospholipids can influence membrane-membrane interactions

in other ways. Proteins involved in cell-cell recognition may be modified by specific

phospholipid interactions. Very good example . of this is the stimulation of plasma

prothrombinase by exposure of PS dependent recognition of red cells and liposomes by the

reticulo-endothelial system is well-documented (Tanaka and Schroit, 1983). Lipid

scrambling can also be induced by the accumulation of peroxides and malonyl dialdehyde,

which occur in appreciable quantities in aged cells {Bevers eta/., 1999).

1.6.4.7. Modulation of Membrane Surface Potential

According to Cerbon and Calderon {Cerb6n and Calderon, 1995) yeast cells rich in anionic

phospholipids, phosphatidylinosito1 rich cells and PtdSer rich cells show negative surface

potential two times higher than normal cells. This increased surface potential activates the

high affinity proton-linked transport systems and inhibits the transport of anions. from high

external concentrations. The reduction of surface potential by counter ions reverses

completely the above mentioned alterations. Proteins may provide such a localized negative

charge. However, this would mean the expenditure of energy to synthesize the protein,

transport it across the bilayer and retrieve it back when not needed is altogether energy

consuming process. Another alternative is to transiently alter the transbilayer lipid flux so as

32

Introduction

to expose negatively charged lipid molecules at the surface. Such a process would be rapid

and least energy utilizing. Thus the modulation of membrane surface potential, by altering

the surface lipids enables the cell to respond rapidly to environmental conditions.·

1.6.4.8. Role in Cytokinesis

Recently in mammalian cells it has been suggested that PtdEtn is required for completion of

cytokinesis. It has been earlier demonstrated that PtdEtn is exposed on the cell surface of the

cleavage furrow during cytokinesis. Immobilization of the cell surface PtdEtn by a PtdEtn­

binding peptide inhibited disassembly of the contractile ring components, including myosin

II and radixin, resulting in formation of a long cytoplasmic bridge between the daughter cells.

This blockade of contractile ring disassembly was reversed by the removal of surface-bound

peptide, suggesting the role of PtdEtn in cytokinesis. To further examine a role of PtdEtn, a

mutant cell is established with specific decrease in cellular PtdEtn level. On culturing, mutant

ceased cell growth in cytogenesis and the contractile ring remained in the cleavage furrow.

Addition of PtdEtn, a precursor of PtdEtn synthesis, restored the cell surface PtdEtn on

cleavage furrow and normal cytokinesis (Emoto et al., 1999; Emoto and Umeda, 2000).

1.6.5. METHODOLOGIES FOR DETERMINING PHOSPHOLIPID ASYMMETRY

A number of techniques are used to determine the organization of phospholipids in two

monolayers of plasm~ membrane as described in Table-XI. Use of techniques mainly

depends on the type of cells under study and the condition of experiments. The probes to be

used for the study should have following characteristics:

1. The probe must be easily accessible to the lipid under study.

2. The design of the experiment should be in such away so that the probe should not

penetrate inside and label also the lipid under study of inner leaflet of plasma membrane.

3. Labeling should not disturb the original distribution or organization of membrane lipids

of plasma membrane by inducing transbilayer movements.

1. 7. MOVEMENT OF LIPIDS

Although lipid bilayer structure is stable, The individual phospholipid and sterol molecules

move freely within the plane of membrane. They diffuse laterally so fast that an individual

33

Introduction

lipid molecule can circumnavigate an erythrocyte in a few seconds (Lehninger et al., 1993).

Individual hydrocarbon chains of fatty acids are also in constant motion produced by the

rotation about the carbon-carbon bonds of the long acyl side chains. The degree of fluidity

depends on lipid composition and temperature. At low temperature a very less lipid motion

takes place and membrane mainly remains in paracrystalline state (Figure 9). Above a

temperature which is characteristic of each membrane, lipids undergo rapid motion. The

temperature of the transition from a paracrystalline solid to fluid depends upon the lipid

composition of the membrane. The higher the proportion of saturated fatty acids, the higher

is the solid-to-fluid transition temperature of the membrane. Although lateral migration of

membrane components and thermal flexing of the acyl chains clearly occurs, a third kind of

motion takes place: transbilayer or "flip-flop" diffusion, me movement of a lipid from one

face of the bilayer to the other Figure_,;/(Lehninger et al., 1993).

1. 7.1. Proteins Involved in Translocation of Phospholipids

Different classes of proteins that are involved in transmembrane lipid traffic are distributed

within the different membranes of eukaryote cell as shown in Figure 10 (Do lis et al., 1997;

Bevers et al., 1999) . In particular, the enzymes of lipid metabolism are mostly required in

endoplasmic reticulum which also contains nonspecific phospholipid flippase. Whereas,

plasma membrane contains lipid pumps such as aminophospholipid translocase and the

protein responsible for lipid scrambling. Pgp is another group of proteins, which are involved

translocation of phospholipids. A TPases that contain pH gradient and transmembrane

proteins forming as stable electrical dipole as well as cytoskeletal proteins may participate in

the stabilization of lipid asymmetry. It should be noted, however that a pH gradient is not

sufficient to pull a charged lipid from one side of the membrane to the other (Dolis et al.,

1997).

1.7.2. Inward Movement of Lipids: Role of Aminophospholipid Translocase

The first evidence for the involvement of a membrane protein in the maintenance of lipid

asymmetry dates from 1984 when Seigneuret and Devaux (Seigneuret and Devaux, 1984)

demonstrated a specific inward movement of spin-labeled analogs of phosphatidylserine and

phosphatidylethanolamine in human erythrocyte membranes. These findings were supported

34

Introduction

TABLE-XI

Methodologies for determining phospholipid asymmetry

Techniques

CHEMICAL LABELING 1. Trinitrobenzenesulfonic acid (TNBS)

2. Fluorescamine 3. Formyl methionyl (sulfone) methyl phosphate

4. Fluorodinitrobenzenes 5. Diazo sulfaniate ENZYMATIC METHODS I. Phospholipases

Properties

All these probes labels aminophospholipids.

References

(Cerb6n and Calderon, 1995;Dogra eta/., 1999)

(Bretscher, 1972) (Whatmore and Allan, 1994;Calderon and DeVries, 1997)

(Schafer eta/., 1974)

2. Lacteoperoxidase-1251 labeling

This method has been utilized (Mersel et al., 1976) extensively for labeling proteins as well a:s lipids. Generally used to label neutral and charged lipids.

3. Glucose oxidase/Galactose Both these probes are capable oxidase of oxidizing glucose and

galactose residues, linke4 to glycolipids.

4. Protein kinase C (Nishizuka, 1992;Newton, 1993)

INTRODUCTION OF EXOGENOUS LIPID I. Lipid back extraction methods

PHYSICAL METHODS 1. Nuclear Magnetic Resonance (NMR)

2. Electron Spin Resonance (ESR)

3. Electron-electron Double Resonace (ELDOR) 4. Fluorescence Resonance

Radiolabeled, spin labeled or fluorescently labeled lipids are introduced and then their kinetics is followed.

In this shift reagents are used and then their shift is studied.

Spin labeled fatty acyl chains are used and then their reduction is studied.

It employees use of

(Anzai eta/., I 993;Marx eta/., 1999)

(Hutton eta/., 1977) (Gerritsen eta/., 1979)

(Wu and Hubbell, 1993)

(Wolf eta/., 1992)

35

Introduction

Energy Transfer (FRET) NED/rhodamine fluorescence energy ..

5. X-ray diffraction (Casper and Kirshner, 1971) 6. Fluorescence Microscopy 7. Electron Microscopy (Kamp, 1979) AFFINITY METHODS 1. Antibodies Antibodies raised against (Guarnieri, 1975)

various classes of lipids are used.

2. Lectin Binding Lectins binds to different (Kahane and Tully, 1976) carbohydrates.

3. Annexin V binding 4. Exchange proteins (Bloj and Zilversmit, 1981)

by other investigators usmg either fluorescent labeled (Connor and Schroit, 1988), or

radiolabeled (Tilley et al., 1986) phospholipids or short acyl chain phospholipids (Daleke and

Huestis, 1985). Only aminophospholipids are substrates for this transporter, referred to as

aminophospholipid translocase, with a preference to phosphatidylserine over

phosphatidylethanolamine. Transport is sensitive to sulfhydryl reagents (Zachowski et al.,

1986; Connor and Schroit, 1988) and bromophenacylbromide (Zachowski et a/., 1986),

suggesting. the requirement of one or more cysteine and histidine residues respectively.

Aminophospholipid transport is ATP dependent (Seigneuret and Devaux, 1984; Zachowski

et al., 1986) and Belenzay eta/. (Beleznay et al., 1993)have estimated the stoichiometry of

transport. One molecule ATP is consumed for each molecule of phospholipid transport.

Hence inward transport of aminophospholipid was found to be inhibited by vanadate, NEM,

cytosolic calcium and AIF4-, indicating that this transporter belongs to the family of P-type

A TPases. Micromolar levels of intracellular Ca2+ also inhibit aminophospholipid transport

(Bitbol et al., 1987). In summary, it is well established that Mg2+ -A TPase is involved in

translocation of amino lipids from outer to inner leaflet of red cells. Investigation of the lipid

specificity indicated that not only head group is recognized by the aminophospholipid

translocase but also the glycerol backbone and the ester bond on the sn-2 chain (Dolis et al.,

1997). Because the chain length and the degree of unsaturation are not crucial, chemical

modifications of the chains do not impede the rapid translocation of PtdSer analogs. A

significant amount suggests that atleast two different red blood cells proteins play a role in

36

3

TRANSBILAYER DIFFUSION ("FLIP-FLOP")

PARACRYSTALLINE STATE

THERMAL MOTION OF AC r L SIDE

CHAINS (SOLID +fLUID TRANSITION)

Figure 9. Different sorts of lipid movements in biological membranes: I, lateral diffusion oflipids;2,thermal motion of fatty acid chains; 3, transbilayer diffusion.[Adapted from Lehninger, 1996]

Non specific flippase

Lipid • flip-flop ..._

Aminophospholipid

translocase Lipid

~ scrambling ~

Mg-ATP Ca2+

IV V VI

I u

i t Enzyme of H+

lipid ATPase metabolism

P-glycoprotein

/

Mg-ATP

vn

HI

i Electrically polarized protein

Figure 10. Schematic representation of involved in the modulation of lipid distribution. [Adapted from Dolis eta/. , 1997]

various proteins transmembrane

Introduction

aminophospholipid transport. These are the 110-120 kDa Mg2+-ATPase proposed by

Zachowski and Devaux and the 32 kDa Rh-associated proteins as suggested by Connor and

Schroit (Martin and Pagano, 1987b; Diaz and Schroit, 1996; Schroit, 1998) . Based on

labeling experiments with an iodinated photoaffinity PtdSer analog, Schroit (Schmit et a/.,

1990) have identified the aminophospholipid transporter with Rh (D) protein which has a

molecular weight of 32 kDa as mentioned above. However Smith and Daleke (Smith and

Daleke, 1990) found that Rh null cells which are deficient in the expression in Rh proteins

transport PtdSer normally. Experiments carried out by one of France group, on a series of 7

patients with no Rh protein have confirmed Smith and Daleke conclusion. Furthermore

Rh(D) amino acid sequence does not possesses no apparent ATP binding site implying that if

an Rh protein is involved in flippase activity then it must be associated with a separate

ATPase.

Recently Tang and coworker observed a gene DRS2 gene belonging to P-type

A TPases from yeast and a drs2 null mutant was shown to be defective in aminophospholipid

transport in contrast to wild type of this yeast strain (Tang et a/., 1996). Recently, the

potential role of DRS2 protein in aminophospholipid transport was questioned by Siegmund

et a! (Siegmund et a!., 1998)who observed no change in the uptake or distribution of

fluorescent-labeled phosphatidylserine or phosphatidylethanolamine in the mutant strain.

Moreover, the absence of Drs2p did not affect the distribution of endogenous

phosphatidylethanolamine in the plasma membrane.·

The compensatory flux mechanism: Since this translocase has no apparent effect on PtdCho

and SM movement, the translocase action should be expected to cause a progressive

accumulation of aminophospholipids in inner monolayer. Due to the slow diffusion of the

choline phospholipids from in-to-out, an imbalance is created between the lipid population in

the two leaflets, creating curvatures and finally leading to collapse of the membrane. To

account for this, a compensatory flux mechanism was proposed, which takes care of any

imbalance by a process referred to as vesicle shedding. An alternative explanation is that the

role of the putative translocase is only corrective; that is to rectify slow spontaneous lipid

randomization or moderate scrambling induced by external agents.

37

Introduction

1.7.3. Outward Movement of Lipids: Role ofFioppase and P-glycoproteins

Studies with erythrocytes have demonstrated the existence of a 'floppase' which facilitates

lipid migration across the bilayer in a direction opposite to the aminophospholipid

translocase (Bitbol and Devaux, 1988~ Connor et a/., 1990~ Connor et a/., 1992). This

inward-outward movement appeared to be less specific with respect to lipid polar head group

since both choline and aminophospholipids are transported. The rate of outward movement is

rather slow, with an order of 1.5 hr, and much lower than the inward rate by the

aminophospholipid translocase with half- times of approximately 5-10 min. Moreover, it

could be demonstrated that translocase and floppase operate independently, since a rapid

inward movement of aminophospholipid did not affect the rate of outward movement

(Connor eta/., 1992). These observations has led to the suggestion that a concerted action of

both the proteins is responsible for a dynamic asymmetric steady state distribution in which

aminophospholipids occupy the inner leaflet balanced by choline phospholipids in the outer

leaflet (Connor eta/., 1992).

Members of ubiquitous family of membrane transporters characterized by an A TP

binding cassette (ABC-transporters), are the human multidrug resistance MDRJ and MDR3

Pgps (in mice mdr1a and mdr2, respectively). In 1996, van Helvoort demonstrated that

epithelial cells expressing MDRJ non-selectively translocate a broad range of lipid soluble

compounds from inner to outer leaflet. Using stable transfectants of pig kidney epithelial

(LLC-PKl) cells expressing MDRJ Van Helvoort et a/. (1996) demonstrated that newly

synthesized short chain fluorescent-labeled (C6-NBD) analogs of PtdCho, Ptd.Etn,

sphingomyelin and glucosylceramide could be recovered by the albumin-containing medium

from the apical side of these cells. Short chain lipids without a fluorescent tag, such as diC8-

[14C}phosphatidylcholine and diC8-[14C}phosphatidylethanolamine were also found to be

translocated by MDRJ.

Human MDR3, which is abundant in hepatocyte canalicular membranes (Smit eta/.,

1993), with high homology with MDRJ exClusively transports phosphatidylcholine from

inner to outer leaflet, thus playing an important role in the production of bile (Srnit et a/.,

·1993). Nevertheless, outward directed transport of endogenous nidiolabeled

phosphatidylcholine could be observed in fibroblasts of transgenic mice expressing MDR3,

indicating that this protein can translocate naturally occurring phosphatidylcholine (Smith et

38

Introduction

a!., 1994 ). It was found that transgenic mice with an homogenous mdr2 disruption had severe

liver disease with a totally inability to secrete Phosphatidylcholine in bile (Smit et a/., 1993).

It was pointed out, however that it seems unlikely that MDRJ or the mouse mdrla can

translocate long chain membrane phospholipids, since mdr1a cannot compensate for the

function of mdr2 as a phosphatidylcholine transporter in the mdr2 knockout mice (Van

Helvoort eta/., 1996). Recently a French group found indications for a role of MDRJ or Pgp

in the transverse distribution of sphingomyelin (Van Helvoort et a/., 1997). These

investigators have hypothesized that resistance of human leukemia KGla cells to TNF-a

induced apoptosis is related to a limited production of cerarnides due to decreased content of

sphingomyelin in inner leaflet. Incubation ofKGla cells with a specific Pgp blocker restored

apoptosis, a phenomenon which appeared to be related to a shift in the transverse distribution

of sphingomyelin toward the inner leaflet of membrane. Number of lipid probes are used to

address the question whether or not Pgp's are involved in transbi1ayer lipid

organization.Floppase activity could be abolished upon A TP depletion, sulfhydryl oxidation

and histidine modification as was also found for aminophospholipid translocase activity .

. Recently evidence was presented that floppase in red blood cells is identical to multidrug

resistance protein 1 (MRP 1), which seems to recognize the fluorescent-labeled analogs used

in these studies as xenobiotics to be expelled from the cell (Raggers et a/., 1999b ). As a

consequence, a more of extreme accumulation of fluorescent -labeled (NBD)

phosphatidylserine in the red cell's inner leaflet was observed in MRP I Knockout as

compared to wild type mice.

It was shown for MRP I in erythrocytes that may not, under all circumstances, reflect the

behavior of the endogenous lipids. On the basis of a combined kinetic and thermodynamic

model, it was proposed that outward movement of phospholipid analogs in the red cell

membrane is Mg2+ and ATP-dependent, because it merely reflects a flux of lipids to

compensate for aminophospholipid translocase activity, independent of the existence of a

floppase (Frickenhaus et a/., 1998). It has been implied that translocase-driven outward

movement of lipids does not seem to contribute substantially to lipid flop. InS. cerevisiae the

. involvement of two transcriptional regulators Pdrl p and Pdr3p whose target gene is PDR5 is

shown in the maintenance of asymmetry of phosphatidylethanolamine (Kean et al., 1997). In

conclusion, it appears that proteins belonging to MDR family which are members of ABC

39

Introduction

transporters are able to translocate phospholipids from inner to outer membrane of plasma

membrane.

1.7.4. ATP-Independent Flippases

In 1985, Bishop and Bell showed the existence of a rapid ATP-independent translocation of

the water soluble dibutyroyl-PtdCho in microsomal membranes by the action of specific

endoplasmic reticulum (ER) protein, named as phospholipid flippase (Bishop and Bell,

1985). This protein is different from the aminophospholipid translocase, which translocases

aminophospholipids at the expense of the hydrolysis of ATP. Bishop and Bell claimed that

ER flippase is specific for PtdCho, but it has been found that using spin-labeled

phospholipids with one long chain that many different phospholipids transverse the ER

membrane with t112< 20 min (Herrmann and Devaux, 1990). Recent results show that half

time for the translocation of glycerophospholipids could be order of few seconds (Buton et

al., 1996) and about one order of magnitude slower for sphingomylein. This rapid

translocation is bidirectional, saturable and displays a competitive behavior. It does not

involve ATP. It is partially inhibited by N-ethyl maleimide and by trypsin treatment, again

indicating a role of protein.

Rapid transbilayer movement of glycerophospholipids was also found in purified Golgi

fractions from rat liver. Furthermore, protein mediated transbilayer movement of

glucosylceramide was evidenced in rat liver and Golgi apparatus. Finally, protein mediated

transmembrane movement of water soluble analogue of mannosylphosphoryl-dolichol in rat

liver ER has been reported, indicating that this very particular membrane possesses a whole

series of proteins allowing the rapid equilibration of newly synthesized lipids or lipid

precursors (Rush and Waechter, 1995).

1. 7 .5. Bidirectional Movement of Lipids: Function of a Lipid Sera m blase

Although assembly and maintenance of an asymmetric distribution of lipids in membrane is

an energy requiring process, ATP-depletion associated with inhibition of aminophospholipid

translocase as well as floppase and MDR activity, will not readily lead to a significant loss of

lipid asymmetry. A collapse of lipid asymmetry may occur in less than a minute upon

particular conditioned of activation. Scrambling process was first observed in blood platelets ·

40

Introduction

which undergo rapid transverse reorganization of their membrane lipids (Bevers eta!., 1983).

Stimulation with a variety of physiological and non-physiological agonists results in

appreciable randomization of the lipids over both membrane leaflets. This process of lipid

scrambling, is most clearly manifested by surface exposure of phosphatidylserine, the extent

of which is dependent on the type of agonist used. Lipid scrambling has also been

demonstrated in a variety of cells such as lymphocytes (Fadok et a!., 1992; Bratton et a!.,

1997), endothelial cells (Hamilton et a!., 1990; Bombeli et a!., 1997), red blood cells

(Chandra et a!., 1987), smooth muscle cells (Bennett et a!., 1995) and tumorigenic cells

(Utsugi et a!., 1991 ). An increase in intracellular calcium concentration, is an essential

requisite for the onset of scrambling process. However, no direct relationship could be found

between the levels of intracellular calcium and lipid scrambling (Dachary-Prigent et a!.,

1995). Clear evidence has been presented to show that loss of lipid asymmetry does not

result from a mere inhibition of aminophospholipid translocase activity· by an increased

intracellular Ca2+ concentration {Connor et al., 1992; Kuypers .. et a!., 1996). Scrambling

requires the continuous presence of Ca2+; removal of intrace~lular calcium causes an arrest in

scrambling process (Williamson eta!., 1995). The scrambling process is bi-directional and

involves all major phospholipid classes, moving all of them at comparable rates except for

sphingomyelin of which the inward movement tends to be somewhat less in comparison to

the glycerophospholipids(Williamson eta!., 1995). Lipid scrambling is not coupled to ATP

hydrolysis but a gradual loss in scrambling activity in erythrocytes is observed on prolonged

ATP depletion (Martin and Jesty, 1995). Lipid scrambling is sensitive to the sulfhydryl

reactive reagent pyridyldithioethyamine (Williamson et a!., 1995); a synthetic. compound

referred to as R542 1 in micromolar concentrations without affecting aminophospholipid

translocase (Dekkers eta!., 1998b ).

In 1996, two groups reported the successful reconstitution of a protein fraction from

platelet and erythrocyte membranes into proteoliposomes with functional Ca2+ -inducible

scrambling activity (Comfurius et a!., 1996; Basse et a!., 1996). The putative scramblase

from erythrocytes was further purified to homogeneity and found to be a 37-kDa protein. It is

of 318 amino acid residues and is rich in proline (Zhou et a!., 1998). In proximity of the

transmembrane segment, (Coldham et a!., 1997)a putative Ca2+ -binding loop motive of 12

amino acid residues (Asp273-Asp284) was found (Zhou eta!., 1998). A potential protein

41

Introduction

kinase C phosphorylation site was found at Thrl59, which could account for the previously

suggested ATP-dependency observed upon prolonged ATP-depletion (Martin and Jesty,

1995). Lowering pH induces lipid scrambling in absence of calcium as was demonstrated for

inside-out erythrocyte ghost. Light scatter experiments by the same group of investigators

(Stout eta!., 1998) indicated that the scramblase protein may undergo conformational change

including self-association into an oligomer in the presence of Ca2+. MRNA of the

erythrocyte scramblase was detected in a variety of hematologic and non-hematologic cells

(Zhou eta/., 1998) and levels of expression of scramblase were found to correlate with the

Ca2+-induced surface exposure of phosphatidylserine (Zhao et al., 1998). Recently a gene

MmTRAla, which is truncated form ofMmTRAlb is cloned, appeared to be 100% identical

to the human erythrocyte scramblase (Kasukabe et al., 1998).

1.8. AIMS AND OBJECTIVES

As is evident from the foregoing discussion that the pathogenic yeast C. albicans also

harbors several multidrug ABC transporters and only few of them could be attributed to

antifungal resistance. In view of the large family of such proteins the question of their

physiological relevance is of considerable importance. The establishment of physiological

roles of such proteins is also expected to contribute to our understanding of the mechanisms

underlying drug resistance. The work embodied in this thesis was planned to elucidate the

role of ABC transporter proteins of human pathogenic yeast C. afbicans.

Results presented in this thesis clearly show that plasma membrane of C. albicans is

asymmetric in its distribution of phospholipids particularly with respect of Ptd.Etn. (Chapter

1). Furthermore, ABC transporters of C. albicans CDRJ, CDR~ and CDR3 are involved in

translocation of phospholipids between the two monolayers of plasma membrane with

different speceficites. CDRJ and CDR2 functions as floppase i.e. translocating phospholipids

from inner to outer monolayer of plasma membrane where as CDR3 functions as flippase i.e.

translocating phospholipids from outer to inner monolayer of plasma membrane (Chapter 2).

Known functions mediated by CDR/ including maintenance of phospholipid asymmetry are

effected in an altered membrane environment (Chapter 3).

42