GENETIC PROFILING OF ABC (CDR1) TRANSPORTER GENES OF AZOLE RESISTANT STRAINS OF CANDIDA SPECIES ISOLATED FROM WOMEN OF REPRODUCTIVE AGE

ABSTRACT

Vulvovaginal candidiasis (VVC) caused by excessive growth of yeasts in the vaginal mucosa is estimated to be the second most common vaginal infection affecting women of reproductive age. The azole antifungal agents are the most frequent class of antifungals used to treat Candida infections, however; there is an extensive documentation of intrinsic and developed resistance to azole antifungals among several Candida species. Some transport proteins such as ATP- Binding Cassette transporter proteins (cdr1p) have been implicated to play a vital role in antifungal resistance and these cdr1p transporter proteins are encoded by CDR1 gene. The purpose of this study was to investigate the genetic profile of ABC (CDR1) transporter genes of azole resistant strains of Candida species isolated from women of reproductive age (20-39) years. In this study, a total of 127 HVS samples from women of reproductive age (20-39 years) were collected. The results showed 64 (50.4%) of the sample to be positive (+ve) to candida screening and 63 (49.6%) samples were negative. Further screening of the positive ones showed that, 56 (87.5%) had single species isolates, 7 (10.9%) had two (mixed) species isolate and 1 (1.6%) had three (mixed) species isolate; totaling 73 isolates, of which there were 43 (58.9%) C. albicans, 12 (16.4%) C. glabrata, 11 (15.1%) C. tropicalis, and 7 (9.6%) C. krusei. The in-vitro susceptibility was performed by the disk diffusion method and the drug susceptibility pattern of Candia species against the two azole (fluconazole and voriconazole) antifungal drugs tested showed that 11 (15.1%) of the isolated Candida species were susceptible, 2 (2.7%) were susceptible dose dependent (SDD), 17 (23.3%) were resistant to 25 µg fluconazole (FLU) and 26 (35.6%) were susceptible, 4 (5.5%) were susceptible dose dependent (SDD) and 7 (9.6%) were resistant to 25 µg voriconazole (VOR). In addition, 36 (49.3%) and 7 (9.6%) of the isolates were susceptible and resistant to both fluconazole and voriconazole respectively. Three (3) out of the seven (7) that were resistant to both fluconazole and voriconazole showed no zone inhibition. There was no significant difference in the value of zone of inhibition of the Candida isolates to 25 μg fluconazole at 24 hours (22.82±10.13) and at 48hours (22.89±10.16) conditions; t (72) = 19.243, p <0.001 and there was also no significant difference in the value of zone of inhibition of the

Candida isolates to 25 μg voriconazole at 24 hours (27.38±10.11) and at 48 hours (27.45±10.12) conditions; t (72) = 23.183, p < 0.001. Three isolates with no zone of inhibition were selected for molecular analysis. A segment of their CDR1 genes were sequenced (Candida Iso-28: 56bases, Candida  Iso-49:  161bases  and  Candida  Iso-64:  73bases).  The  expression  fold  of  the  three isolates relative to their reference gene is Candida Iso-28CDR1:18SrRNA = 0.68±0.60, Candida Iso-49CDR1:18SrRNA = 1.64±0.43 and Candida Iso-64CDR1:18SrRNA = 2.34±0.27 while their expression  fold  relative  to  one  another  is  Candida  Iso-28CDR1:Candida  Iso-49CDR1  = 0.44±0.14,    Candida    Iso-28CDR1:Candida    Iso-64CDR1    =    0.29±0.06,    Candida    Iso- 49CDR1:Candida  Iso-28CDR1  =  2.42±0.76,  Candida  Iso-49CDR1:Candida  Iso-64CDR1  = 0.69±0.14, Candida Iso-64CDR1:Candida Iso-28CDR1 = 3.46±0.70 and Candida Iso-64CDR1 :Candida Iso-49CDR1 = 1.48±0.34; the relative fold of the isolates to the control (C. albican Strain ATCC 14053) is Iso-28CDR1:Control = 0.85±0.07, Iso-49CDR1:Control = 2.04±0.53 and Iso-64CDR1:Control = 2.74±0.55. The phylogenetic experiment showed that the three isolates share the same ancestral origin but Candida Iso-28 and Candida Iso-49 are more related than Candida Iso-64.

CHAPTER ONE

INTRODUCTION

Over the years, microbes have developed resistance to antimicrobial agents and the phenomenon of fungal resistance to antifungal agents has significant clinical implications (Sanglard and Odds,

2002; David, 2009; Patrick et al., 2012). Antifungal drug resistance has impacted negatively on the clinical outcome for patients with serious mycoses. Candida species are of clinical relevance because of the rate at which they colonize, infect humans and cause diseases ranging from lesions in the mucous membrane and skin to infections in body organs (Colombo and Guimarães,

2003). Their resulting spread through the bloodstream usually characterizes invasive candidemia. The most common pathogenic Candida spp. are Candida albicans, Candida tropicalis, Candida parapsilosis and Candida krusei (Stock, 2010). Candida spp. are commensal microorganisms that inhabit various sites in the human body such as the gastrointestinal and respiratory tracts. They comprise of the vaginal and urethral microbes (Alonso-Valle et al., 2003).

Yeast of the genus Candida account for 70% of every hospital-environment fungal infections (Azie et al., 2012). Arunaloke (2011) asserted that candidemia is the fourth leading cause of blood-stream infections with 35-55% mortality rate. In spite of considerable research to develop new therapeutic strategies there are limited number of drugs available to fight invasive fungal infections (David, 2009; Patrick et al., 2012). Drugs such as polyenes, azoles, allylamines, echinocandins, and 5-flucytosine are the widely used antifungal agents (Zuza-Alves et al., 2017). Azoles are the mostly used antifungals in clinical practice, and they are also the mostly studied antifungal agents regarding their mode of action, pharmacological properties, and the resistance mechanisms developed by Candida spp. Candida spp develop mechanisms to counteract the fungicidal  effects  of  antifungals  by  reducing  the  drug  accumulation  within  fungal  cells, decreasing the affinity of antifungal drugs for its target and modification of metabolism (Perea et al., 2001; Sanglard and Odds, 2002).

1.2.0    ATP- Binding Cassette Transporters

ATP-binding cassette (ABC) transporters are protein structures associated with cell membrane. They are actively involved in the influx or efflux of a wide range of substrates such as micronutrients, sugars, amino acids, peptides, antibiotics, and antimicrobial peptides (Durmort

and Brown, 2015). ABC transporters utilize energy of ATP hydrolysis to import or export substrate across a membrane against a concentration gradient. Each ABC transporter is relatively specific for a given substrate (Higgins, 1992). ABC transporters are integral membrane proteins are also involved in diverse cellular processes such as maintenance of osmotic homeostasis, nutrient uptake, resistance to xenotoxins, antigen processing, cell division, bacterial immunity, pathogenesis and sporulation, cholesterol and lipid trafficking, and developmental stem cell biology (Jones and George, 2004). These transporters were termed ABC transporters in recognition of the “cassette-like” nature of the ATP-binding subunit (Wilkens, 2015). The ATP- binding cassette (ABC) superfamily is regarded as one of the largest super-families of trans- membrane transporters found in nature given that between 1 and 3% of bacterial and archaeal genomes  code  for subunits  of ABC  transporters  and  over 50  ABC  transporters are known (Higgins, 1992; Wilkens, 2015). Eukaryotes contain an additional large group of ABC proteins (non-transporters) located in the cytosol and mostly used for DNA maintenance and repair and for gene regulation. These two major families of ABC proteins (transporters and non- transporters) are known collectively as the ABC-ATPase superfamily. ATP-binding cassette (ABC) superfamily consist of both importers and exporters (Jones and George, 2004; Wang et al., 2009). In most eukaryotes, have exporters and their ABC transporters are composed of four parts: two membrane-integral domains and two ATP-hydrolyzing domains (Cervelatti et al.,

2006; Wilkens, 2015). The membrane integral domains span the membrane six times while the domains  are  characterized  by  two  short  ATP-hydrolysis  sequence  motifs  in  their  primary structure (Wilkens, 2015). Eukaryotic ABC transporter domains can be organized either as full- transporters combining all four domains (two trans-membrane domains; TMDs and two nucleotide-binding domains; NBDs) in a single polypeptide, or as half-transporters, comprising of two domains (1 TMD and 1 NBD) which can be arranged either as TMD-NBD or NBD-TMD as shown in Figure 1 and 2. The NBDs of all ABC transporters contain three highly conserved motifs: Walker A, Walker B and an ABC “signature sequence” (C motif) located in between (Broehan et al., 2013).

Fig. 1: The structure of ABC transporter (Hollenstein et al., 2007)

The catalytic cycle of ABC transporters starts from the ground state which comprises of the binding of substrate-binding proteins to the TMDs, binding of two Mg ATP molecules to the NBDs, dimerization of the NBDs, switching of the TMDs between the in- and outward or out- and inward-facing conformations, ATP hydrolysis, phosphate, ADP and transport substrate release concomitant with NBD dissociation to reset the transporter to the ground state for the next cycle (Wilkens, 2015).

Fig. 2: Scheme of the mechanism of ABC exporters and importers (Wilkens, 2015)

Most prokaryotic ABC proteins are encoded as separate TMD and NBD subunits or as one-half- size (one TMD and one NBD) transporters (Jasinski et al., 2003). Wang et al. (2009) using five distinct computer programs provided convincing statistical data suggesting that the trans- membrane domains of ABC exporters are polyphyletic. ABC transporters have been implicated in multidrug resistance of microbes against antimicrobial drugs because of their ability to transport drugs. The multidrug resistance proteins (MDRs) or P-glycoproteins (P-gps) belonging to the ATP binding cassette subfamily B (ABCB) subfamily. The P-gp ABCB was the first ABC transporter identified to be overexpressed in multidrug resistant tumor cell lines (Dermauw and Van-Leeuwen, 2014). According to Klein, et al (2011) ABC proteins of most yeast on phylogenetic relationships basis, have been grouped into five sub-families referred to as the MDR (multidrug resistance protein), CFTR (cystic fibrosis trans-membrane conductance regulator), ALDP (adrenoleukodystrophy protein), YEF3 (yeast elongation factor 3) RLI (RNase L inhibitor 1) and PDR (pleiotropic drug resistance) subfamilies.

The PDR family is typified by a configuration in which the ABC module is closer to the N- terminal end of the protein than the trans-membrane segments domain (ABC–TMS) which is the reverse form of the configuration observed in multidrug resistance-associated protein (MDR, ABCA and MRP) in which the TMS module is towards the N terminus of the protein (TMS– ABC). The PDR family of transporters is characterized by the specific modular configuration of its members and by their association with the transport of antifungal agents in yeasts. The

expression  of PDR  genes  is  associated with  antifungal  drug resistance (Van-den-Brule and Smart, 2002). ABC proteins in some yeast have tremendously broad substrate specificity (e.g. Pdr5 or Snq2) while others are known to transport only a limited quantity of substrates (e.g. Ste6) and in some instance, transport substrates have not even been discovered (e.g. Pdr18). The mechanism of ABC transporters on the basis of variable but restricted substrate specificity as well as the precise translocation mechanism remains enigmatic despite numerous efforts and studies (Puri et al, 1999; Klein, et al, 2011). PDR is the largest of the subfamilies of ABC transporters, with nine members of which four members have been characterized; Cdr1p, Cdr2p, Cdr3p and Cdr4p (Smriti et al., 2002). Cdr1p and Cdr2p are involved in drug transport and phospholipid translocation while Cdr3p and Cdr4p translocate phosphoglycerides between the two monolayers of the lipid bilayer of the plasma membrane (Sanglard et al., 1999; Paumi, et al.,

2009). Cdr1p has a vast spectrum of structurally unrelated molecules as its substrates. Cdr1p substrates possess high hydrophobicity index, high aromaticity, molecular branching and the presence of an atom-centered fragment (R-CH-R) (Prasad et al., 2015).

1.2.1    CDR1 ATP- Binding Cassette Transporter Gene

CDR1 gene belongs to the CDR gene family of candida species which codes for proteins that belong to the superfamily of ATP-binding cassette (ABC) transporter (Balan et al., 1997). It is implicated as a specific multidrug efflux transporter (Ribeiro et al., 2005). CDR1 encodes for protein 169.9 kDa whose structural organization is characterized with homologue halves. They each possess a hydrophobic region each and a set of six trans-membrane stretches preceded by a hydrophilic binding fold (Prasad et al., 1995; Franz et al., 1998).

1.3.0    Vulvovaginal Candidiasis

Vulvovaginal Candidiasis (VVC) is a fungal or yeast infection. It is a common worldwide female medical problem, which occurs mostly in women of childbearing age caused by inflammatory changes    in    the    vaginal    and    vulvar    epithelium.    VVC    is    secondary    to    infection with Candida species characterized by irritation, itching and dysuria (Das-Neves et al., 2008). Candida blastospores migrate from the lower gastrointestinal tract to the adjacent vestibule and vagina and the colonization of the vagina follows usually in low numbers after adherence of Candida to vaginal epithelial cells (Sobel, 2016). Candida adheres to the amino-terminal and

cell-binding domains of brinogen and vitronectin and can promote persistence of the organism in the vagina (Mardh et al., 2002). It is estimated that 75% of all women experiences VVC at least once during their lives, and about half of them have at least one recurrence (Nedovic et al.,

2014). VVC can be classified as either uncomplicated or complicated and about 10%–20% of women that experience VVC will have complicated VVC (Dovnik et al., 2015). The most common cause of acute vulvovaginal candidosis is Candida albicans, followed by Candida glabrata though in chronic recurrent vulvovaginal candidosis, Candida albicans and Candida glabrata are often equally distributed (Stock, 2010). Studies have shown that patients with vulvovaginal candidiasis usually have vaginal pH of less than 5, and at least three proteinases are associated with the intracellular compartments of Candida (Mardh et al., 2002). It has been shown from studies some exogenous factors such as changes or imbalances in reproductive hormones, as a result of oral contraception, pregnancy, hormone replacement therapy, antibiotic usage and diabetes mellitus can be linked to VVC (Sobel et al., 2007; Ringdahl, 2000).

1.3.1    Vulvovaginal Candidiasis Causing Species

Candida species such as Candida albicans, Candida glabrata, Candida kursei, Candida parapsilosis and Candida tropicalis are mostly implicated in VCC and Candida albicans is the most common fungal pathogen isolated from clinical samples of patients with vulvovaginal candidiasis (Owen and Clenney, 2004; Shan et al., 2014).

1.2.1.1 Candida albicans

Candida  albicans is  an opportunistic fungal  pathogen that  is  responsible for candidiasis  in human hosts. They can grow in several different morphological forms, ranging from unicellular budding yeast to true hyphae with parallel-side wall (Sudbery et al., 2004). Candida albicans usually live as harmless commensals in the gastrointestinal and genitourinary tract and are found in over 70% of the population. The overgrowth of these organisms lead to disease and it usually occurs in immunocompromised individuals, such as HIV-infected victims, transplant recipients, chemotherapy patients, and low birth-weight babies (Kabir et al., 2012). Candida albicans is highly polymorphic and its phenotypic flexibility is an important virulence factor that aids its invasion of epithelia, dissemination throughout the host, survival in different host niches and modulation of the host immune response (Gow and Yadav, 2017). Candida albicans was one of

the first eukaryotic pathogens with a sequenced genome (Jones et al., 2004). Its genome is highly plastic  and  high  levels  of  heterozygosity,  intra-chromosome  recombination  and  aneuploidy (Jones et al., 2004). It has been shown from studies that the high level of heterozygosity in Candida albicans might play an important role in achieving diversity within the species which might be required for its survival in different adverse conditions. C. albicans has been of great interest to the scientific community for its pathogenic nature. In the pathogenicity of C. albicans the immune status of the host and the virulence factors of this pathogen are the major factors (Kabir et al., 2012). C. albicans possess an array of secreted hydrolytic enzymes of which SAPs (secreted aspartyl proteinases) which are considered to be virulence factors has been implicated to contribute to their pathogenesis (Naglik et al., 2003). C. albicans can also secrete phospholipases A, B, and C, which are considered to be putative virulence factors; they are associated with the function related to host cell damage, adherence, and penetration (Ghannoum,

2000). The ability of C. albicans to form biofilm on biotic and abiotic surfaces is thought to play a major role in its pathogenicity (Ganguly and Mitchell, 2011; Ramage et al., 2009).

1.2.1.2 Candida glabrata

Candida glabrata is non-dimorphic yeast that exists as small blastoconidia under all environmental conditions as a pathogen. It is closely related to the non-pathogenic yeast Saccharomyces cerevisiae than most of the other Candida species associated with human disease phenotypically; C. glabrata lacks some of the virulence factors associated with Candida pathogens, such as the secretion of hydrolases and hyphal growth but despite this, C. glabrata is a growing challenge in clinical settings (Fidel et al., 1999; Kaur et al., 2005). Candida glabrata, like Candida albicans, is an endosaprophytic yeast pathogen which has adapted to colonize most segments of the human gastrointestinal tract and vagina (Jawhara et al., 2012). Systemic C. glabrata  infections  are  associated  with  higher  mortality  than  C.  albicans  infections  as  C. glabrata is mostly resistant to some antifungal drugs, particularly azoles, making it clinically difficult for management (Lee et al., 2010; Edlind et al., 2010). C. glabrata has the ability to synthesize β-mannosides (β-Mans) which has been shown that to display a wide range of specific characteristics related to pathogenesis especially by modulating the cell wall hydrophobicity and acting as specific adhesins through interaction with galectin-3. This interaction with galectin-3 promotes adhesion and gut colonization, thereby stimulating the host cells to produce cytokines

(Kohatsu et al., 2006). C. glabrata has a high survival rate (≥ 5 months) due to its ability to respond to stress, nutrient limitation, competition with other microorganisms and the lack of sporulation (Rodrigues et al., 2014). The central core structure of C. glabrata cell wall is a branched β-(1, 3)-, β-(1,6)-glucan linked to chitin by a β-(1,4)-glucan linkage in which a number of them (chitin and glucan chains) extend out through the full depth of the cell wall structure and the outermost glycoprotein layer of C. glabrata which is comparable to that of S. cerevisiae is considered to play a key role in host cell recognition and adherence (West et al., 2013). Many glycosylation enzymes are found to be conserved in C. glabrata and their deletion is said to reduce virulence (West et al., 2013). This change in virulence is usually attributed to the reduced adherence to host cells. The ability of C. glabrata to adhere to the epithelial tissue of hosts is mediated by the expression of glycosylphosphatidylinositol (GPI) linked adhesin genes and these genes encode for the GPI-anchored cell wall proteins which can bind host cell carbohydrates (Riera et al., 2012; Rodrigues et al., 2014). From experiments, the deletion of two cell wall GPI- anchored  proteins;  Pwp7p  and  Aed1p  found  in  C.  glabrata  are  crucial  to  C.  glabrata’s adherence. This suggests that C. glabrata has unique mechanisms for specialized interaction with host cells (Desai et al., 2011). When compared with C. albicans, C. glabrata displays better efficiency at invasion of host cells (Tam et al., 2015).   The ability of C. glabrata to produce biofilms is one of its major virulence mechanisms. Biofilm formation involves cell-substrate and cell-cell interactions, the production of highly specific adhesins are crucial to successful biofilm development (Berila et al., 2011; Riera et al., 2012). The biomass of C. glabrata biofilms is generally less, yet they contain high quantities of proteins and carbohydrates when compared with other Candida species. The main adhesin involved in biofilm production is encoded by EPA6 (Riera et al., 2012; Rodrigues et al., 2014). C. glabrata has the ability to produces and release extracellular phospholipases, a heterogeneous enzyme which can promote the interaction and destruction of host mucus. Phospholipases facilitates the infiltration of the epithelial cell phospholipid barrier by hydrolysing ester linkages in glycerophospholipids, a molecule common in human cell membranes (Ghannoum, 2000; Bialkova and Šubík, 2006).   Phospholipase B (PLB) and lyso-phospholipase are two major phospholipases secreted by C. glabrata and the association of these phospholipase activity and persistent candidemia infections have been established;  however  the  exact  mechanisms  of  the  secretion  of  these  phospholipase  in  C. glabrata is still remains unclear (Ghannoum, 2000). C. glabrata is also known to produce

haemolysins, which is capable of breaking down blood cells to obtain elemental iron for its metabolic processes. HLP gene has been implicated to be associated with haemolysis in C. glabrata clinical isolates (Bialkova and Šubík, 2006).

1.2.1.3 Candida tropicalis

Candida tropicalis which has been identified as a prevalent pathogenic yeast species of the Candida-non-albicans group. It is a diploid dimorphic yeast which exists as either ellipsoidal budding cells or as pseudomycelium and possesses the ability to produce true hyphae unlike C. glabrata which cannot produce hyphae (Kothavade et al., 2010; Zuza-Alves et al., 2017). C. tropicalis is associated with superficial and systemic infections; and produces more persistent systemic infections than C. albicans (Kontoyiannis et al., 2001; Colombo et al., 2006). The cell wall structure of C. tropicalis is composed of hydrophobic proteins embedded in a cellular matrix which favors the initial interaction (Tronchin et al., 2008). The adhesins of C. tropicalis are involved in the formation of biofilm and are regulated by the BCR1 gene which is considered a cell wall regulator (Punithavathy and Menon, 2012). From episodes of fungemia, C. tropicalis isolates are said to be the strongest biofilm producers and the most efficient biofilm producers among   bloodstream   isolates   when   compared   to   other   Candida-non-albicans   species (Pannanusorn  et  al.,  2013;  Marcos-Zambrano  et  al.  2014).    Candida  biofilm  formation  is initiated when they adhere to a surface with cells attached to each other and they begin to proliferate, ultimately resulting in the formation of highly structured mature biofilm which is comprised of complex intertwining layers of yeast and pseudohyphae mostly embedded in the extracellular matrix (Kumamoto, 2002).    The secretion of enzymes which hydrolyze phospholipids into fatty acids by C. tropicalis are implicated to be involved in its pathogenicity just as most other candida species and are often considered to contribute to host cell membrane damage which could also expose receptors to facilitate adherence (Punithavathy and Menon, 2012).

1.2.1.4 Candida parapsilosis

Candida parapsilosis is now the second or third most common cause of candidiasis (Ashford

1928). The early reports of C. parapsilosis described the organism as a relatively non-pathogenic yeast in the class of normal flora of healthy individuals (Weems 1992). Recently C. parapsilosis

is of clinical importance as an opportunistic pathogen which can cause infections ranging from thrush to invasive diseases such as fungemia, endophthalmitis, arthritis, endocarditis and peritonitis (Van-Asbeck et al., 2009). C. parapsilosis invasion can lead to severe disease especially in hosts that are immunosuppressed. C. parapsilosi is mostly acquired from exogenous sources and it adheres to indwelling devices, thereby invades the host. C. albicans and other non- albicans Candida species have the ability to adhere to a great extent to the mucosal tissue than C. parapsilosis (Kuhn et al. 2004; Van-Asbeck et al., 2009). The ability of C. parapsilosis to produce slime has been implicated as a key virulence factor as it is very important for its adhesion to foreign body material and also in biofilms formation (Nett et al., 2007). Phospholipase plays a major role in the pathogenesis of C. parapsilosis by enhancing their adhesion and penetration of host cells. Lipase seems to be another enzyme that is a key virulence factor in C. parapsilosis; these enzymes affect adhesion and may deny the host nutrients (Gacser et al. 2007). C. parapsilosis is mostly associated with vaginal or gastrointestinal colonization and disease and much less frequently with oral mucosal disease (Weems, 1992).

1.2.1.5 Candida krusei

Candida  krusei  is  an  elongated  budding yeast  and  an  emerging  fungal nosocomial pathogen (Samaranayake and Samaranayake, 1994). C. krusei is an uncommon cause of blood stream infections and like every other candida spp. C. krusei adheres to host cells and can produce phospholipase and proteinase. The formation of hyphae has been implicated as a key factor of its virulence (Duggal, 2015).

1.3.2    Vulvovaginal Candidiasis (VCC) as a Health Challenge

Vulvovaginal candidiasis is estimated to be the second most common cause of vaginitis after bacterial vaginosis with Candida albicans accounting for over 85% of the cases (Anderson,

2004). It is estimated that approximately 10–15% of asymptomatic women are colonized with Candida, 70–75% of women will experience an episode of VVC in their lifetime, 50% of initially infected women will suffer a second VVC event and 5–10% of all women will develop recurrent vulvovaginal candidiasis (RVVC) (Sobel, 2007). Signs and symptoms of VVC are not specific to the disease and the presence of Candida in the vagina is not necessarily an indication of  VVC  since  asymptomatic  women  can  be  colonized  hence,  VVC  diagnosis  require  the

correlation of clinical findings and laboratory confirmation of Candida (Anderson, 2004; Goncalves et al., 2016).

1.4.0    Types of Antifungal Drugs

Antifungal drugs are substances which  can destroy  or prevent the growth of fungi. Unlike bacteria, fungi and humans are eukaryotes, thus there is a bit similarity between fungi and humans at the molecular level, making it more difficult to find a target for an antifungal drug to attack (Scorzoni et al., 2017). Antifungal drugs are of clinical importance as they are used in the treatment infections such as athlete’s foot, ringworm and candidiasis. Antifungal drugs can be classified into azoles, amphotericin, nystatin and echinocandin (Dismukes, 2000).

1.4.1    Azoles

Azoles are a group of antifungal drugs characterized by the presence of an azole ring structure that   includes   imidazoles   and   triazoles.   Triazoles   (e.g.   ketoconazole   and   miconazole, clotrimazole) have three nitrogen atoms in the azole ring while Imidazoles (e.g., itraconazole and fluconazole) have only two nitrogen atoms (Sheehan et al., 1999). The azoles exert a fungistatic effect by dose-dependent inhibition of CYP-dependent 14-α-demethylase, which converts lanosterol to ergosterol (Niwa et al., 2005). Ergosterol plays a key role in the stability of the fungal cell membrane, and the inhibition of its synthesis alters the integrity of the cell membrane (Gallis et al, 1990; Scorzoni et al., 2017). Triazoles can also target other steps in the ergosterol biosynthesis pathway, for intance fluconazole inhibits ergosterol partially and completely blocks obtusifoliol synthesis, whereas voriconazole inhibits both ergosterol and obtusifoliol synthesis completely (Sanati et al., 1997). Itraconazole and fluconazole may also inhibit 3-keto reductase, which catalyzes the reduction of the 3-ketosteroid obtusifolione to obtusifoliol in C. neoformans (Ghannoum and Rice, 1999). Azoles are usually eliminated from the body system through an extensive oxidative (CYP) metabolism (Isoherranen et al., 2004; Hyland et al., 2003), except for posaconazole would usually undergo minimal (2%) CYP metabolism and most of its metabolites are glucuronide conjugates formed by uridine diphosphate glucuronosyltransferase (UGT) pathways (Ghosal et al., 2004; Krieter et al., 2004). When compared to other Azoles, fluconazole is hydrophilic and is highly soluble in water; hence it requires less biotransformation to be eliminated from the body (Spellberg et al., 2006). Itraconazole, voriconazole, and posaconazole are highly lipophilic and have limited aqueous solubility. Therefore, these azoles must undergo

extensive enzymatic conversion to more polar metabolites to ensure their adequate elimination from the body (Gavarkar et al., 2013). Azole drug disposition are facilitated by a variety of transport proteins which are expressed in tissues throughout the body in humans. Azoles interact with P-glycoprotein, especially the efflux transport protein (Wang et al., 2002). The primary toxicity associated with azoles involves the liver; ranging from the common transient elevations in serum transaminases to the less common fulminant hepatoxicity and liver failure (Lutsar et al.,

2003; Tan et al., 2006).

1.4.2    Amphotericin

Amphotericin (AmB) is an amphipathic fermentation product of the Gram-positive bacterium- Streptomyces nodosus. It is a polyene antifungal antibiotic that causes damage to fungal and host cells by altering the permeability of their cell membrane (Sau et al., 2003). AmB has effects on the permeability of the fungal cell membrane at two different levels; it binds to the fungal membrane sterol, ergosterol and induces pore formation which results in a lethal leakiness and induction of oxidative damage (Sau et al., 2003; Palacios et al., 2011). Reports from early studies suggest that AmB inserts into the fungal lipid bilayer through the hydrophobic domains that bind to ergosterol resulting in the formation of multimeric pores with the lipophilic polyene chains of Amphotericin in contact with their membrane lipids (Finkelstein and Holz, 1973; Brajtburg et al., 1990). Liu et al. (2005) in their genome-wide expression analysis confirmed that AmB, not only has an effect on the expression of genes involved in ergosterol synthesis pathway, but also induces the expression of stress genes and this indicates that AmB has pleiotropic effects in the fungal cells. The expression of these stress genes has implicated oxidative damage as a way in which AmB destroys the integrity fungal cell membrane (Mesa-Arango et al., 2012). AmB has an affinity to cholesterol; it binds the mammalian membrane and results in a conformational change which activates NADPH oxidase. AmB can bind TLR, triggering the polymerization of receptors which results in the recruitment of adaptor protein, MyD88. This signaling produces the nuclear translocation of NF-kB, which induces the expression of stress genes (Sau et al., 2003; Mesa-Arango et al., 2012).

1.4.3    Nystatin

Nystatin is a polyene macrolide antibiotic antifungal agent with activity against many species of yeast and which is used largely to treat skin and or pharyngeal candidiasis (Lyu et al., 2016). Nystatin acts by binding sterols in the plasma membranes of fungi creating pores or leakages within the cell membranes and eventually resulting in fungal cell death (Groll et al., 1998). Nystatin is usually biosynthesized by a bacterial strain, Streptomyces noursei and the structure of this   is   characterized   as   a   polyene   macrolide   with   a   deoxysugar   D-mycosamine,   an aminoglycoside (Fjaervik and Zotchev, 2005).

1.3.4    Echinocandins

Echinocandins are large lipopeptide antifungal molecules which targets the synthetic cell wall enzyme complex -1,3-D-glucan synthase of the fungi cell walls and its spectrum is restricted to Candida spp and Aspergillus spp (Denning, 2003). When Candida species are exposed to echinocandins, the cells become greatly enlarged and distorted, with aberrant daughter cell, chlamydospore-like formation and ultimately, lysis of the cell membrane resulting in fungicidal activity against Candida species (Estes et al., 2009). Echinocandins are administered only via the intravenous route because of their poor oral bioavailability and are mainly degraded in the liver by hydrolysis and N-acetylation (Grover, 2010). Echinocandins are well distributed into tissues, including lung, liver, and spleen, but with minimal penetration into  central nervous system (CNS) tissues, including the eye, due to their high protein binding and large molecular weight (Aguilar-Zapata et al., 2015).

1.5.0    Antifungal Resistance

The clinical resistance of antifungal drugs has become a global problem. Reports of non-Candida species resistant to azoles and of multidrug-resistant Candida species, such as Candida glabrata are increasing with  an  alarming frequency  (Kontoyiannis, 2017). Fungi can be intrinsically resistant to antifungal drugs or can develop resistance in response to exposure to the drug during treatment; fungi have tremendous genomic plasticity which enable them become tolerant or fully resistant to antifungal drugs (Perea and Patterson, 2002; Healey et al., 2016). The alteration of the target enzyme, cytochrome P-450 lanosterol 14a-demethylase, either by overexpression or by point mutations in its encoding gene (ERG11) has been implicated as a possible way for the

development of antifungal resistance (Perea and Patterson, 2002). The second major mechanism is a consequence of enhanced drug efflux which is mediated by 2 types of multidrug efflux transporters, the major facilitators (encoded by MDR genes) and those belonging to the ATP- binding cassette superfamily (ABC transporters, encoded by CDR genes) resulting to the failure of azole antifungal agents to accumulate inside the yeast cell. The upregulation of the CDR (CDR1and CDR2) genes seems to confer resistance to multiple azoles, whereas upregulation of the MDR1 gene alone result in fluconazole resistance (Nolte et al., 1997; Perea et al., 2001). Upregulation of CDR1 has been implicated in the development of fluconazole resistance in C. albicans that caused disseminated infection in an immunocompromised patient (Marr et al.,

1998). The primary resistances of C. krusei to azole antifungals (fluconazole and itraconazole) appear to be mediated through reduced susceptibility of the target enzyme, lanosterol 14- ademethylase to inhibition by this drug (Orozco et al. 1998).

1.5.0    Role  of  CDR1  ATP-  Binding  Cassette  Transporter  in  Antifungal  Resistance  in

Vulvovaginal Candidiasis

CDR1 gene of C. albicans is the first ABC efflux pump characterized in any known pathogenic yeast. This gene codes for ABC Transporter protein-Cdr1p. This protein is promiscuous in nature has a vast spectrum of structurally unrelated molecules as its substrates (Prasad et al., 2011; Prasad et al., 2015). Multiple Drug Resistance (MDR) phenomenon have been demanding a better understanding of the basis of the promiscuity of Cdr1p. The structure-activity relationship (SAR) analysis has been extensively employed to evaluate Cdr1p and other polyspecific proteins alike and it has been proposed that Cdr1p substrates generally possess a high hydrophobicity index, a molecular branching, high aromaticity, and the presence of an atom-centered fragment (R-CH-R) (Puri et al., 2010). Remarkably, Cdr1p has a large number of aromatic residues in the binding pocket with a significant clustering of aromatic residues near the ectodomains; hence the substrates with high aromaticity could easily be involved in stacking interactions with such residues. The binding cavity of Cdr1p is anticipated to be large and numerous residues are involved in its interaction with the substrates, branching is likely to increase the reactive surface area for substrate molecules, which results in better passage through the channel (Tyndall et al.,

2013; Prasad et al., 2015). Cdr1p expels steroid hormones such as estradiol and corticosteroids and the transport of steroid could be vied by an excess of drug substrates which suggests that

Cdr1p has binding sites in common with the drugs (Krishnamurthy et al., 1998a). Steroids have some implications in Candida infections as high level of steroid hormones such as estrogen and glycogen content in vaginal secretions during pregnancy can predispose women to vulvovaginal candidiasis (Soong and Einarson, 2009). It has been observed that a brief exposure of Candida cells to steroid hormones results in an upregulation of the CDR1 gene, therefore it could be possible that a link exist between MDR and the steroid response pathway. Cdr1p which are largely localized within microdomains and are enriched with sphingolipids and ergosterol. It is reported to be responsible for the asymmetric distribution of phosphoglycerides in C. albicans plasma membranes (Krishnamurthy et al., 1998b; Dogra et al., 1999; Pasrija et al., 2008). The translocation of phosphoglyceride which is similar to the transport of steroid could be outcompeted by certain drugs, which not only points to the polyspecificity of the Cdr1p but highlights the overlapping of its binding sites within the large, flexible drug binding cavity. These findings imply that in the course of evolution, C. albicans and other fungi could have learned how to utilize their available collection of transporters to bring about the efflux of xenobiotics, though they have their own set of physiological substrates (Prasad et al., 2015).

Studies have shown that CDR1 is a well-regulated gene and also a major multidrug transporter in clinical antifungal resistance and its transcription is controlled by several well-characterized trans factors that interact with a host of cis-elements interspersed in its promoter. Tac1 is a prominent  transcription  regulator  of  CDR1  and  is  often  associated  with  gain-of-function mutations which results in hyperresistance in clinical Candida isolates (Manoharlal et al., 2008). The genome-wide transcription profiling and its comparison in matched pairs of AR and azole- susceptible (AS) clinical isolates showed the hyperexpression of CDR1.  Genome-wide location analysis of Upc2, a zinc cluster family transcription factor that is involved in sterol biosynthesis implicated CDR1 as one of its target genes, hence showing its importance in MDR (Znaidi et al.,

2008). The Genome-wide occupancy and expression profiling study of CAP1 has shown that CAP1 binds to the promoters of a host of genes involved in the oxidative stress response and drug resistance, including CDR1 and phospholipid transport. Recently, the functional analysis of transcription factors has helped in the characterization of the role of Mrr2 in fluconazole resistance  mediated  through  overexpression  of  CDR1  (Schillig  and  Morschhauser,  2013). Elevated CDR1 transcription and mRNA stability are two major factors implicated in the development  of  azole  resistance  in  antibiotic  resistance  (AR)  isolates  in  comparison  with

matched azole-susceptible (AS) isolates study (Manoharlal et al., 2008). The transcriptional regulation of CDR1 by Ncb2, the β subunit of the NC2 complex, a heterodimeric regulator of transcription has also been reported (Shukla et al., 2011).

1.6.0    Rationale for the study

The rationale for this study was to investigate the distribution, azole susceptibility, ABC (CDR1) transporter gene profile and phylogenetic relationship of the resistant Candida species isolated from vagina of women of reproductive age (20-39 years).

1.6.1    Aim of Study

The purpose of this study was to investigate the genetic profile of ABC (CDR1) transporter genes of azole resistant strains of Candida species isolated from women of reproductive age (20-

39 years).

1.6.2    Specific Objectives of the Study

The specific objectives of this study were to;

•   Screen woman of reproductive age (20-39 years) for Candida species using conventional screening methods.

•  Screen Candida species isolates using differential/selective media (chromogenic agar).

•  Carry out antifungal (fluconazole, voriconazole) susceptibility studies on the Candida

species isolated

•  Check for the expression of the CDR1 ATP- Binding Cassette transporter using its cDNA

•  Sequence and compare the CDR1 gene segments of the isolates of interest.

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