The related yeasts Saccharyomyces cervisiae and Candida albicans can grow by producing a bud which is identical to the mother cell. What other forms of growth and development do these species undergo? Is there a connection between different growth forms and infection of humans by Candida?
C. albicans and S. cervisiae are both able to grow as pseudohyphae and yeast. In C. albicans there is a third growth form, the hyphal growth form.
In the presence of varying concentrations of nutrients, S. Cervisiae cells may adopt a number of different fates. In the presence of favourable environmental conditions, i.e. abundant nitrogen supply and abundant fermentable carbon source, S. Cervisiae will adopt the yeast form, characterized by asymmetric budding, and proliferate. If nutrients are limited but not exhausted, e.g. a limiting nitrogen source and an abundant fermentable carbon source, S. Cervisiae will switch from the yeast form to a filamentous form. This is known as pseudohyphal differentiation, and growth in this form produces pseudohyphae. When nutrients are exhausted, i.e. limiting nitrogen source, limiting fermentable carbon source, S. Cervisiae will undergo sporulation producing spores. (1, lecture notes).
C. albicans cells assume different growth forms and morhologies depending on environmental conditions. In cultures grown at low temperature and/or pH, e.g below 30 C or pH 4.0 the yeast form is prevalent. Hyphae develop from yeast cells in a response to a number of growth conditions including; temperatures above 34 C in the presence of Serum, in Lees medium at 37 C, in cultures at 37 C and neutral pH, and in the presence of N-acetylglucosamine. Given any of these environmental conditions the majority of growth is hyphal. Cultures grown in intermediate temperatures and pH are seen to contain pseudohyphae. Pseudohyphae can be reliably induced in cultures at pH 6.0 and 35 C, in Nitrogen limited growth on a solid medium or in the presence of high concentrations of phosphate. It is worth noting that pseudohyphal cultures in C. albicans always contain some yeast and/or hyphae and the proportions at which the various morphological forms are present depends on the environmental conditions. There are also growth conditions which have not been well characterised which induce filamentous growth, these include; engulfment by macrophages, growth in mouse kidneys and Iron deprivation (5, 3). In early study’s on C. albicans there was often little attempt to distinguish between hyphae and pseudohyphae with both forms being placed under the umbrella of filamentous growth. This is misleading as the two forms, despite superficial similarities, are morphologically distinct. In fact, despite their filamentous form, pseudohyphae may be more accurately described as yeast cells altered by polarized growth that don’t completely separate after cytokenesis (2, 3).
S. Cervisiae and C. albicans, in common with all yeasts, are able to grow by budding which produces a daughter cell identical to the mother. This type of growth is termed the yeast mode (2). The yeasts of the two species are similar in terms of shape, size and the order of cell cycle progression, although there are significant differences on a molecular level (5). Different stages in the yeast cell cycle are defined by the movements of actins and septins which make up the cytoskeleton and, along with the polarisome, regulate polarised growth. They also dictate the site of bud emergence, mitosis and cytokenisis. In the the first growth phase, G1, actin patches congregate at one pole of the cell and a septin ring appears in the same location, this is known as the pre-bud site. It is at this point that the new bud will envaginate. The location of this site is determined by the previous site of cytokenesis as well as cell type and environmental factors such as temperature (5, 6, 7). This leads to a regulatory point of the cell cycle known as START, located at the transition between G1 and S phase, at which point the cell is committed to reproduce. Passage through START is nessecary for the initiation of bud formation, DNA replication and spindle pole body duplication. After the passage through START a new bud emerges. The septin ring remains at the budding site, defining the junction between mother and bud known as the mother-bud neck. The actin patches and the polarisome remain at the tip of the cell resulting in polarised growth. As the cell cycle continues into the second growth phase, G2, growth in the bud switches from being polarised to isotropic (5). This switch occurs when the cell is about two thirds of its final size and results in even growth across the surface of the bud. This is reflected in the movement of the actin patches from localisation at the tip of the bud to even distribution throughout (3). At the same time, the nucleus, aided by microtubules, moves to the mother-bud neck in preparation for mitosis. In budding yeast cells, mitosis occurs across the mother-bud neck at the septin ring. The spindle pole bodies separate, dividing the nucleus, and cytokenesis takes place. This pulls one chromosomal complement into the mother cell and one into the daughter cell and separates the cytoplasm between mother and bud. After the completion of mitosis the septin ring splits into two, a true septum is formed, and mother and daughter cells separate. At the point of separation the daughter cell is slightly smaller than the mother cell. This means that the daughter cell begins the next cell cycle at a slightly later point resulting in an asymmetric growth pattern. Proliferation in the yeast mode produces colonys which are smooth and round (2, 3, 5, 6, 7).
A second mode common to both yeasts is pseudohyphal growth. When S. Cervisiae or C. albicans cells undergo pseudohyphal differentiation, and switch from producing single yeast cells, to producing pseudohyphae, a number of changes occur. Cells become elongated in relation to yeast cells, remain physically attached, and divide in a uni-polar budding pattern (2). These changes correspond to changes in the cell cycle which is in turn regulated by signalling pathways responding to environmental stimulus. The pseudohyphal cell cycle is extremely similar to the cell cycle in budding yeast and as such there is little need for a full description here. There are however, two main differences. Firstly, growth in the daughter cell is more polarised in pseudohyphae than in yeast, and cells remain in the second growth phase G2, for longer, this results in an elongated cell shape (5). Secondly, the mother and daughter cell do not fully separate after cytokenesis. The end result of the first cell cycle, starting with an unbudded yeast cell, is two attached but fully differentiated cells: a mother cell, which retains the same form as when the cycle started, and a daughter cell which is elongated. Following the initial switch of form, pseudohyphae continue to divide in the same manner, producing elongated daughter cells which generally remain attached to the mother cell at the site of the nucleur division. Unlike yeast which bud in a bi-polar pattern, budding in pseudohyphae is unipolar. This means that growth is apical, resulting in long filaments, constricted at the septum, which are able to invade a growth medium. The budding cycle is more synchronised in pseudohyphae than in yeast. This is a result of the longer period spent in G2 which means that mother and daughter cells reach START at roughly the same time (5). In S. cervisiae, pseudohyphae are induced by the presence of limiting nutrients. Given this, it has been postulated that the role of pseudohyphae in S. Cervisiae is to forage for nutrients in poor environmental conditions enabling this non-motile species to increase its chances of survival (1) In C. albicans, pseudohyphae have often been seen as an intermediate state between the yeast and hyphal forms with no unique function. This may seem intuitive as they occur in conditions intermeidiate to those which induce yeast or hyphae, and this remains a possibility. However, given that pseudohyphae are morphologically distinct to both yeast and true hyphae, it is equally likely that pseudohyphae may have unique biological properties and play a distinct role in the life cycle of C. albicans, perhaps in infection (2, 3). Consistent with this pseudohyphae rarely produce hyphae and visa versa. In both S. cervisiae and C. albicans pseudohyphae form invasive colonys which are fibrous and rough (5).
C. albicans has a third distinct growth mode: hyphal growth. In terms of cell cycle and morphology, hyphae in C. albicans are extremely different to both yeast and pseudohyphae. In fact, they bear more similarity to the hyphae of filamentous fungi. Hyphal growth results in narrow multicellular filaments know as germ tubes with parallel walls seperated by septai without constriction.( ) Rather than appearing at the mother-bud neck, the septum is formed in the elongating germ tube and mitosis occurs at this point. These differences reflect differences in cell cycle as well as the presense of the spitzenkorper, an organelle specific to hyphae, which along with the majority of cortical actin patches, remains at the tip of the growing germ tube. The spitzenkorper along with the polarisome is resposiblele for the continuously polarised growth seen in hyphae. In the hyphal cell cycle, germ tubes, unlike buds, appear before START during G1 and grow continuously throughout the cell cycle, even after cytokenesis has taken place. In mitosis, the nucleus migrates from the mother cell into the germ tube and undergos nucleur division there. One nucleus then migrates back into the mother cell while the other moves towards the tip of the growing germ tube. The movement of the nuclei is orchestrated by mictrotubules. Another distinguishing feature of hyphae is continuous linear growth. Linear growth and hyphal branching are regulated by cytoplasm and vacuole inheritance. In the first cell cycle a large vacoule appears in the mother cell. After cytokenesis the germ tube inherits mainly cytoplasm while the mother cell inherits mainly vacuole. This causes the mother cell to remain in the G1 phase for several generations, accumulating cytoplasmic mass until a certain threshold of cytoplasm is reached in relation to the vacuole. At this point the mother cell re-enters the cell cylce, elaborating another germ tube. While the mother cell remains in G1, the germ tube continues to grow and divide. This pattern is consistent in all subsequent cell divisions with the apical cell inheriting the majorityy of cytoplasm and the sub-apical cell inheriting the majority of vacuole. Sub-apical cells are only able to produce a new branch when a certain ratio of vacuole to cytoplasm is reached. As a result hyphal growth is linear and hyphae are less branched than pseudohyphae with a less regular branching pattern. (2, ,3 ,5).
C. albicans and S. cervisae also have other distinct morpholgical forms which relate to specific functions and environmental conditions. The haploid cells of S. Cervisiae are able to grow invasively if left on a rich growth medium for long periods of time. The main aim of S. Cervisiae haploid cells is to locate sexual partners and reproduce. Low concentrations of mating pheremones greatly stimulate haploid invasive growth and yeast cells are known to respond to gradients of mating pheremones. These facts suggest that haploid invasive growth in S. Cervisiae may be a mechanism by which haploid cells are able to find mating partners (1). C. albicans has several other distinct morphologys including chlamydospores and opaque cells. Chlamydospores are round projections, formed at the end of suspendor cells, with large thick walls and a high lipid and carbohydrate content. They may form in environments low in oxygen, light, temparature or nutrients.. Although the function of chlamydospores is unknown, there formation is associated with the pathogenic form of C. albicans and it is therefore thought that they may play some role in the pathogenic lifestyle (2). The opaque form is the best studyed example of ‘phenotype switching’ in C albicans and reprasents the state in which C. albicans, which was until recently considered to be an asexual orgainsm, is able to reproduce sexualy. Opaque cells are elongated and assymetrical with surface pimples. This compares with the usual form of C. albicans cell, White, which are relatively round and smooth. The white-opaque switch is refected in a change in the appearance of colonies from round and white to flat and grey (8).
Changes in the morpholgy of cells reflect an underlying change in cell cycle which is in turn regulated by signal transduction pathways responding to extrenal signals. There are two main signal transduction pathways which control morhogenisis and growth in C. albicans and S. Cervisiae. These are the MAP Kinase or mitogen-activated protein kinase cascades, and the nutrient-sensing cAMP or cyclic AMP pathway. The target of cAMP is the cAMP dependant protein PKA; protein Kinase A. These pathways consist of complex signalling cascades involvong posistive and negative regulation between receptors, proteins and transcription factors, ultimately regulating the expression of genes. These pathways, under various conditions, are able to give rise to different developmental fates (1). Although there are fundamental differences between the pathways in C.albicans and S.cervisae the basic features are conserved. MAP Kinase cascades contain three protein kinases which act in a series: a MAP kinase kinase kinase, a MAP kinase kinase, and a MAP kinase, with each activating the next protein in the cascade by phosphorilation. (9). The cAMP-PKA pathway involves cAMP which is produced in response to external factors, targeting PKA. PKA’s are made up of a regulatory sub-unit and catalytic sub-units. These pathways are not isolated and it has been shown that there is cross talk between them. In S.cervisiae the G protein Ras2 activates both the MAP Kinase and the cAMP-PKA pathways and both pathways together regulate the promoter of the FLO11 gene which is nessecary for pseudohyphal growth (2).
The roles played by signalling pathways can be investigated by studying mutant strains for genes involved in the pathways or disabling genes directly. By examining the phenotype of a mutant strain it is possible to deduce the role played by the gene in which it is deficient. One example of this can be seen in the transcription factor Flo8, which is part of the cAMP signalling pathway and plays a role in pseudohyphal differentiation in S. cervisae. The S. cervisiae strain S288C has a naturally occurring Flo8 mutation which means it is unbale to produce Flo8. This strain is unable to produce pseudohyphae (4).
The MAP kinase and cAMP-PKA pathways play a major role in regulating yeast, psedohyphae and the transition between them in S. cervisiae, and yeast, pseudohyphae, hyphae and the transition between them in C. albicans.
It has long been assumed that, rather than one single growth form being resposible for the pathogenic properties of C, albicans, it is the ability to switch morphologies, specifically between the yeast and hyphal modes of proliferation, that is required for pathogenesis (10) Although there is still no direct, unambiguous evidence for this, there is a wealth of experimental data which implies that this may be the case. Mutants for Efg1p and Cph1g which are locked into the yeast form are avirulent. Mutants for Tup1p and Nrg1p which are locked into the hyphal form are also avirulent.
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