EPIGENETIC MECHANISMS INVOLVED IN PHASE SWITCHING PROCESS EXHIBITED BY CANDIDA ALBICANS

1. Scientist-III, Central Research laboratory, Meenakshi Ammal Dental College and Hospital, Maduravoyal, Chennai-600095, India 2. Reader, Department of Microbiology, Meenakshi Ammal Dental College and Hospital, MAHER, Meenakshi University, Madhuravoyal, Chennai – 600 095, Tamilnadu, India 3. Post doctoral research associate, Centre for Cellular and Molecular Biology, Uppal road, Hyderabad-500007, Telangana, India.

appreciation of the importance of epigenetic mechanisms in the gene regulation of phenotypic switching has emerged in recent decades (Grant Downton and Dickinson, 2006). Indeed, among the various epigenetic mechanisms like DNA methylation, phosphorylation, ubiquitination, centromeric chromatin alterations, histone acetylation leads its role in phenotypic switching . With this background, this review throws insight on the novel mechanism of epigenetic phase switching prevailing among the commensal C.albicans in mucocutaneous tissues transforming it as an opportunistic pathogen: the "frenemy" within the host.

Major virulence factors:-
The factors responsible for initiation, propagation and sustenance of disease in host need to be clearly defined in the case of an opportunistic pathogen like C.albicans. In this context, an opportunistic pathogen should accomplish the following criteria: (a) component which damages host should be present in the pathogen, (b) the pathogen should exhibit all traits to establish disease,(c) factors present in the pathogen should directly interact with host cells. C.albicans adapts to host niche by inducing transcriptional and translational changes which enhances its survival under host environmental conditions. Several specialized mechanisms have to be activates so as to promote the establishment, dissemination and pathogenesis (Brock, 2009). Thus, specialized adaptations of Candida to changing host microenvironments aid in sustaining infection in the host. The major factors which contribute to the pathogenesis and virulence of Candida are mentioned in Table 1.

Morphogenesis and phase switching:-
The transition of C.albicans from unicellular yeast form to filamentous form (hyphae or pseudohyphae) is termed as morphogenesis. C.albicans and C.dubliniensis are the only species of the genus which can undergo morphogenesis. The process of morphogenesis is enabled by the presence of an array of factors including nutrients, near-neutral pH, a temperature range of 37 -40°C, CO 2 concentration of 5.5%, presence of N-acetyl D-glucosamine, serum, amino acids and biotin. The reverse of this process produces yeast forms from hyphal forms which is accelerated by low temperature, acidic pH, absence of serum and high concentration of glucose (Corner and Magee, 1997;Eckert et al., 2007). The mechanism of transition morphogenesis is vital for pathogenesis as the yeast forms are suited for propagation in tissues, whereas hyphae are required for tissue invasion and damage.

White-Opaque cells (W-O cells):-
C.albicans possess a unique property to switch between two specific types of cells, white and opaque, which is not found even in closely related species. The process of white-opaque switching was discovered by David Soll and Colleagues in 1987 (Slutsky et al., 1987). It was only after a decade or so the principle role of phase switching in the mating cycle was established. Each cell type can propagate for many generations without any change in their genome sequence. About seven percent of the genome, coding for about 400 genes are known to regulate the phase switching process (Magee and Magee, 2004;Bennett and Johnson, 2005). The properties of white and opaque cells have been tabulated in table 3. Table 3: Characteristics of white and opaque cells.
The difference in phenotype and transition from white to opaque forms contributes to the virulence of the organisms in different biological environments. Kvaalet al, in 1997 provided evidence for the niche specific transition of W-O cells. When mice were infected with opaque type of cells, only a few cells could be recovered from their kidney which was the target organ in the infection model, when compared to white cells. The results indicated that most of the cells switched from opaque to white type which conferred a selective advantage under the host conditions. In contrast to the above study, opaque cells colonized the skin of mice more effectively than white cells in a cutaneous infection model. Protease secreted by opaque cells was attributed for this virulence property. Hence, conditions prevailing in the host decide the transition of W-O cells, making it advantageous for colonization or dissemination of infection (Kvaalet al., 1999).

Mechanism of phase switching (PS):-
The process of phase switching is strictly regulated by the transcriptional regulators encoded at the Mating type-like locus (MTL). Two different alleles, MTLa and MTLα exist in majority of C.albicans strains. Cells heterozygous for MTL locus produce a heterodimer a1-α2 which is found to be a major repressor of phase switching. However, in conditions leading to loss of heterozygosity (LOH), either by mitotic recombination or loss of one copy of chromosome 5 containing one allele and duplication of the other homolog, the cells turn out to be either MTLa or MTLα (Wuet al., 2005, 2007). These cells lose their ability to produce the heterodimer and hence become switching competent. The cells thus produced are stable for several generations under normal laboratory conditions. This property is attributed to the transcriptional circuit consisting of several interlocking feedback loops. They tend to regain their potential to synthesize heterodimer after mating of a andα producing a tetraploid cells, which in turn aids the cells to switch phase. The phase switching mechanism does not involve changes in the DNA sequence but is chiefly controlled by epigenetic factors (Bennett and Johnson, 2003) (Figure 1).  Numerous studies have provided evidence on the function of Wor 1 as the key regulatory molecule effecting phase switching process. Artificial or induced expression of Wor 1 from heterologous promoter in a/α cells promotes W-O switching, but the mating process is hindered as the genes essential for mating are repressed by a1/α2 repressor (Huanget al., 2006). Vinceset al., 2007, studied the effect of Cfz 1 overexpression on switching process, which revealed that even high concentration of Czf 1 could not overcome the repression of Wor 1 by the heterodimer a1/α2. Interestingly, the deletion of Efg 1, the repressor of opaque switching, did not show any effect on the phase switching process as the expression of Wor 1 is strongly regulated by the expression of a1/α2 (Zordan etal.,2007).

Epigenetics tags involved in PS process:-
The phase switching in either direction is modulated by external environmental conditions, which substantiates the involvement of epigenetic factors in the process. Chromatin modification by histone deacetylation has been documented as an important epigenetic mark influencing the transition. Inhibition of histone deacetylation by trichostatin A or deletion of HDA1 gene encoding histone deacetylase, are known to stimulate white to opaque switching. In addition, deletion of RPD3 encoding histone acetylase increased frequency of bidirectional switching . Cells lacking HDA1 gene or mutants expressing low levels of Hda1 protein shows increased switching to opaque phase, which is paralleled with the reduced expression of Efg1 . Another interesting finding is that the Efg1∆ mutant opaque cells switch to white phase after the repression of Set3, a histone deacetlyase complex, which provides evidence on the derepression of Wor 1 protein in the absence of Efg1 is driven by histone deacetlyase. Consequently, these experimental evidences prove that Candidal cells sense the biological environment prevailing in the host and adapt themselves to the selective pressure by way of phenotype transition.

Clinical significance:-
The formation of white and opaque cells is an adaptive response exhibited by the yeast cells to escape host defence mechanisms. Although both white and opaque cells are phagocytosed with an equal frequency, white cells exhibit more resistance to killing than opaque cells. Opaque cells are more sensitive to ROS (reactive oxygen species) produced by phagocytes (Kolotila et al., 1990). Interestingly, opaque cells have developed mechanisms to avoid recognition by host defences, while white cells fail to do so (Geiger et al., 2004). A study by Lohse and Johnson, 2008 showed that opaque cells are not effectively phagocytosed when compared to white cells when exposed to two different types of innate immune cells the Drosophila S2 and mouse macrophage derived cell line. Hence, it is vivid that the phase switching mechanism in a way aid C.albicans to oppose or evade host immune components.
In addition to the above views, the transition state also enables the organism to survive at different biological niches, for example, opaque cells are found to stable at lower temperatures like the skin surface, where as white cells have been recovered from internal organs of infected mice (Kvaal et al., 1999). Biofilm formation and adherence is also facilitated by this transition although the underlying molecular mechanism has not been clearly defined.

Conclusion:-
Candida albicans establishes diverse mucosal and systemic diseases in mucocutaneous tissues in immunosuppressed and immuno-compromised patients. This versatility depends on the co-ordination of gene expression, albeit, epigenetic regulation in C.albicans is poorly characterized. In recent decades, among the various virulence factors implicated, phenotypic phase switching seems to be alarming among the treating medical mycologists and geneticists. Transition frequency varies depending on the growth temperature, oxygen levels and adaptive mating mechanisms. Genetic based analysis of phenotypic switching relates this occurrence to DNA methylation and histone deacetylation. More definitive conclusions and understanding of this simple relation between epigenetic mechanisms with phenotypic switching will aid in the achievement of novel therapeutic, preventive and diagnostic targets in C.albicans.