Expression of Growth Form Factors during Morphogenesis in Candida albicans Amna Shafiq1,Aziz Fatima1, Qudsia Hussain1, Shazia Tabassum Hakim1, Sayyada Ghufrana Nadeem1* 1Department of Microbiology, Jinnah University for Women, Karachi -74600, Pakistan ABSTRACT The transition of Candida albicans from unicellular yeast form to filamentous form i.e, pseudohyphae and hyphae is referred to as morphogenesis. C.albicans has the ability to respond to environmental conditions and accordingly changing its cell morphology. Three main morphological forms of C.albicans are unicellular yeast, pseudohyphae and hyphae. Environmental factors are important in selectively favouring yeast or hyphal form, most important being the growth medium, incubation temperature and external pH value. Cell morphology in C.albicans is associated with changes in the expression of specific factors that are expressed exclusively in particular growth form. These growth form factors are an important target in the investigation of morphogenesis in C. albicans and in the discovery of antifungal drugs that targets the specific growth form of C. albicans. Keywords: Candida albicans, Environmental factors, Growth specific form, Morphogenesis, INTRODUCTION Morphogenesis is an essential trait in the pathogenic fungus C. albicans and it is clearly required for virulence (Lo et al., 1997; Braun et al., 2000; Braun et al., 2001, Murad et al., 2001; Saville et al., 2003). Morphogenesis in C. albicans is defined as transition from unicellular yeast form to filamentous form (pseudohyphae or hyphae) (Khan et al., 2010). It can grow in a variety of morphological forms such as yeast, pseudohyphae and hyphae (Sudbery et al., 2004; Merson-Davies et al., 1989). C. albicans also can form chlamydospores that is to say; thick walled spherical cells or asexual spores which develop over pseudohyphal support cells and appear under unfavourable environmental conditions (Sudbery et al., 2004). In pseudohyphae daughter bud elongates and, after septum formation, the daughter cell remains attached to the mother cell. The elongation of buds in pseudohyphae can be so extreme that these filaments can superficially resemble hyphae. Because of this, it is often useful to be able to refer to pseudohyphae and hyphae collectively and we will use the term ‘filamentous’ for this purpose. (Sudbery et al., 2004). However, hyphae are narrower than pseudohyphal cells ("2 mm) and have parallel walls with no obvious constriction at the site of septation (Sudbery et al., 2004). Germ tube are the initial projections observed when C. albicans switches from yeast form to hyphal growth (Yang, 2003). Growth is polarized in C. albicans hypha, with continuous apical growth throughout the cell cycle and parallel cell walls at the septal junctions. In contrast, the growth of pseudohypha and blastophores is only limited to the apical tip during the initial part of the cell cycle (Court et al., 2007). Pseudohypha can also be distinguished from true hypha on the basis of their morphological index which quantifies the dimensions of cell compartments. Alternatively they can be distinguished on the basis of the positions of their septal junctions. These lie at the bud neck for pseudohypha, and within the germ tube for emerging hypha (Sudbery et al., 2004). Hyphae, pseudohyphae and yeast differ from each other in the rate and order of cell cycle events (Berman, 2006). *Corresponding author: huma_45@hotmail.com 75 Vol 4 (2), July 2013; 75-79 Morphogenesis is interrelated to the pathogenesis of C. albicans. Adherence has been shown to play critical role in the pathogenesis of infections. Germ tubes, that are short hyphal elements, are important in the adherence of organism to the host epithelium. (Kimura et al., 1978, Lee et al., 1938, Sobel el al., 1984). Hypha of C. albicans are also important for tissue destruction and host invasion (Berman and Sudbery, 2002). The yeast form of C. albicans also have virulence attributes and is thought to promote dissemination within the blood stream and establishment of infection at distant sites (Braun et al., 2000; Gow, 2002; Sudbery et al., 2004; Sundstrom, 2006). Several researchers have investigated that hyphal form is more virulent than yeast form. The principal determinant in the development of disease is the ability of C. albicans to switch between yeast and hyphal forms rather than the individual morphologies (Saville et al., 2003). Morphological changes between the yeast and the various filamentous forms occur in response to alterations in the growth conditions. Parameters that promote hyphal development in vitro include a growth temperature above 35 °C, a pH greater than 6 . 5 , n i t r o g e n a n d / o r c a r b o n s t a r v a t i o n , nonfermentable carbon sources, low oxygen concentrations, and a wide range of chemicals including N-acetylglucosamine, proline (and other amino acids) and alcohols. Serum is one of the most potent inducer of hyphal development (Brown, 2002). The effect of serum is complex but it is proposed to act, in part, by conferring amino acid starvation. Two known inducers of hyphal formation, N- acetylglucosamine (GlcNAc) and proline, may contribute to the serum effect since they are generated by degradation of serum (glyco-) proteins. It is well established that a pH around neutrality favours hyphal development of C. albicans in vitro, while a low pH (pH <6.5) blocks hyphal formation and stimulates growth of the yeast form. Growth of cells in the yeast form is promoted by an inoculums above 106 cells/ml, a growth temperature below 35°C, a pH of less than 6.5, glucose and ammonium salts (Odds, 1988). Compared to liquid medium, induction on solid media appears to represent a weaker hypha- inducing condition, because minor defects in filamentation show a defective phenotype on solid but not in liquid media. So the mycelial tendency is stronger in liquid than on solid media (McClary, 1952) and stationary phase cells are most responsive to hyphal and pseudohyphal induction signals (Berman, 2006). It is generally assumed that changes in cell morphology in C. albicans are associated with changes in the expression of specific factors that are expressed exclusively in particular growth form. A total of 5 true hyphae specific genes have been identified. All appear to encode structural proteins, as opposed to regulatory proteins, and four of the five appear to encode cell surface proteins. (Brown, 2002). ECE1 (extent of cell elongation 1) was the first hyphae specific gene to be identified. ECE1 is probably an intracellular protein; having a predicted molecular size of 28,886 Da. This protein consists of eight degenerate repeats, 34 amino acids in length. ECEI was highly expressed when hyphae were formed, regardless of the induction signal, and ECEI expression occurred soon after the stimulus to form hyphae was given. (Birse et al., 1993) In previous work (Marot-Leblond et al., 1995), a C. albicans germtube-specific antigen 3D9 antigen was recovered by the use of a MAb. The relative molecular mass ranged from 120 to 220 kDa. Later (Beucher et al., 2009) it was concluded that antigen 3D9 is ALS3 with same molecular mass. ALS3 is a hypha specific protein (Coleman et al., 2009) member of one of the major families of C. albicans adhesins ALS (agglutinin-like sequence) that encode cell-surface glycoproteins.(Hoyer et al., 2008). ALS3 have molecular size of 119,927 Da, it consists of 10 degenerate repeats and 36 amino acids in length (Hoyer et al., 1998). ALS3 has been shown to be required for mature-biofilm formation, binding 76 Vol 4 (2), July 2013; 75-79 extra cellular matrix, adhesion to host cells, and internalization of C. albicans by endothelial cells (Hoyer et al., 2008; Sheppard et al., 2004; Zhao et al., 2006). ALS3 is activated under serum, N- acetylglucosamine and proline-inducing conditions (Hoyer et al., 1998). ALS8 has been mentioned several times in the literature as an ALS3-like gene (Leng et al., 2001; Murad et al., 2001) ALS8 gene was also shown to encode a hypha-specific cell surface agglutinin. More detailed analysis confirmed that ALS8 is expressed under serum, N-acetylglucosamine and pH-inducing conditions and hence that ALS8 is a hypha-specific factor (Leng & Brown, unpublished data). ALS8 have a molecular mass of 111,945 Da - UniProtKB (http://www.uniprot.org/uniparc/UPI000006A36A). HWP1 (hyphal wall protein 1) is expressed during hyphal development (Kim et al., 2007; Staab et al., 1996). The gene was tentatively called ECE2 (Sharkey et al.,1999). It functions as hypha-specific cell surface adhesion (Sharkey et al., 1999; Staab et al., 1999) Molecular mass of HWP1 is 65,342 Da. Peptide mass obtained from Candida DB (http://genolist.pasteur.fr/CandidaDB/). HWP1 (hyphal wall protein) encodes an outer surface mannoprotein on the hyphal wall; the amino terminal sequences of this adhesin are recognized as mammalian transglutaminase substrate (TGase) and form covalent binding with HBEC (Chaffin et al.,1998; Staab et al.,1999) Its mRNA being expressed during pH and cell culture medium- i n d u c i n g c o n d i t i o n s ( S t a a b s e t a l . , 1 9 9 6 ) HYR1 (hyphally regulated gene) is putative hypha- specific cell surface glycoprotein. The HYR1 sequence revealed a 2,810-bp open reading frame capable of encoding a 937-amino-acid protein with a predicted molecular mass of 94,000 Da. HYR1 is induced specifically during hyphal development during,serum,pH, or N-acetylglucosamine-induction (Bailey et al.,1996) ALS3, ALS8 and HYR1 are all members of multigene families, and a second ECE locus appears to exist in C. albicans (CGD). These are genes that are expressed specifically during hyphal development (Swoboda et al., 1994). CONCLUSION Several experimental approaches have been applied to the investigation of morphogenesis in C. albicans. The key purpose of investigation on morphogenesis in C. albicans is to define and analyze the expression of growth-form specific factors that determines the cell shape. The growth specific factors are the putative targets in the discovery of antifungal drugs that are aimed at inhibiting hyphae formation in C. albicans. 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