Mutations in a large number of genes affect cell elongation see Table 1. It is important to note that in some cases the cells may become elongated due to a delay in mitosis and a defect in the switch from apical to isotropic growth.
Nevertheless, it is still debated whether this is the mechanism underlying the change in cell shape observed in pseudohyphae. Among the genes related with elongation, those encoding cyclins, or proteins able to regulate cyclins or the cyclin-dependent kinase Cdc28 itself, are prominent. At least one of the G 1 cyclins, Cln1 or Cln2, is required for PH growth and haploid invasive growth [ 67—69 ], while overexpression of the cyclins Clb1 or Clb2 blocks PH growth triggered by low nitrogen, even in the presence of the dominant active mutations RAS2 Val19 or STE [58] , and suppresses the elongation phenotype of a cdc mutant [63].
Since deletion of CLB2 produces an elongation phenotype, even in rich medium [ 63 , 67 ], it appears that the complexes CdcClb and CdcCln play opposite roles in the morphogenic process [57]. The negative role of Clb2 would be dependent on the Cln3 cyclin, since both a cln3 and a clb2 mutation bypass the requirement of Cln1 for elongation [67].
This indicates that the MAP kinase cascade activates at least two parallel pathways with some degree of redundancy. The observation that a grr1 mutation promotes PH growth [70] may in turn be related with the fact that Grr1 participates in the degradation of Cln1 and Cln2 [71]. The crucial role of Cdc28 in controlling S.
ELM1 encodes a serin—threonine protein kinase [ 72 , 73 ] which appears located mainly at the bud neck [74]. It may activate the protein kinase Hsl1 [63] to allow cytokinesis and mitosis to proceed. The elm1 mutation would then cause a delay in cytokinesis which results in elongation of the cells and eventually PH growth. Another elm mutation has been characterized as cdc12 [70] and its phenotype can be related to the fact that CDC12 encodes one of the septin proteins essential for cytokinesis.
The role played by Cdc55, a regulatory subunit of yeast PPase 2A, is less clear. Whereas a diploid with a single functional copy of the CDC55 gene shows an elongated morphology [72] , a diploid with the homozygous cdc mutation is impaired in cell elongation [5].
While Dfg5 and Dfg10 have not been characterized further, Spa2, Pea2, Bni1 and another protein, Bud6, appear to function together in a complex to promote polarized cell growth [75]. These proteins, and also Tpm1, a major form of tropomyosin, could interact with actin and thereby direct the actin cytoskeleton to specified growth sites in the yeast cells.
Some mutations in actin interfere with cell elongation suggesting that the polarization of actin patches at the tip of the cell is important for elongation [66].
At the end of mitosis there is an asymmetric accumulation of the repressor Ash1 in daughter cells due to an asymmetric distribution of ASH1 mRNA which is dependent on proteins such as Act1, Tpm1 or Bni1 which regulate the actin cytoskeleton [77].
Although this mechanism may seem rather unusual, it has recently been reported that the association between the complex She3—Myo4 and the She2 protein is dependent on the presence of Ash1 mRNA [78].
In a general screen to isolate genes involved in filamentous growth, in which different classes of mutants were identified, only one mutant, dfg16 , was obtained which was defective in invasion but not affected in cell polarity or filament formation [5].
DFG16 encodes a protein predicted to contain six membrane-spanning domains, which could play a role in cell wall synthesis [79] , but has not been further characterized. A cdc mutant was impaired both in cell elongation and in invasiveness [5].
In addition, some actin alleles cause a marked defect in agar invasiveness; a possible reason for this would be the participation of actin in the secretion of putative hydrolases able to facilitate growth through the agar [66].
It should be noted that not every gene which could have been identified was revealed in the screening described in [5]. Overexpression of PHD1 induces vigorous PH growth of diploid cells on rich medium [60] and PHD1 expression is strongly induced in a medium with low nitrogen [80]. Nevertheless, deletion of PHD1 does not interfere with the filamentation response to nitrogen starvation, and overexpression of PHD1 does not induce PH growth in wild-type haploid strains, although it does in a bud4 mutant strain, which has the polar budding pattern [60].
Phd1 has been shown to be localized in the nucleus and to contain a region similar to one present in different fungal transcriptional regulators [60]. This region includes a positively charged stretch followed by a helix-turn-helix motif potentially able to bind DNA; there is no information, however, on the possible target s of Phd1.
In any case the enhancement of filamentous growth by Phd1 is independent of Tpk2 [30] , Tec1 [81] , Ste12 or Ash1 [76]. Here again the target s for Sok2 are not known but it should be noted that the positive effect of a SOK2 deletion on filamentation requires the presence of PHD1 , although sok2phd1 diploids still exhibit PH growth when starved for nitrogen.
Pseudohyphae formation may require an enhanced production of chitin, since pseudohyphae have been shown to accumulate chitin on their whole surface and not only at the septa [8]. It can be noted also that in yeasts with an impairment in PH growth caused by a non-functional FLO8 allele, disruption of ACE2 , which encodes a positive regulator of the chitinase gene CTS1 , results in the production of pseudohyphae.
Since disruption of the CTS1 gene itself in a flo8 background also allows PH growth, but at a reduced level [82] , Ace2 may control another gene involved in PH growth. So many genes have been described as involved in the control of PH growth that it becomes difficult to distinguish between direct and indirect effects.
For instance, the impairment of dom34 mutants in forming pseudohyphae appears related to a decrease in bulk protein translation, which, in turn, alters the cell cycle [83]. In the absence of a functional Shr3, the yeast exhibits starvation responses, even when amino acids are present in the medium. MSN5 is one of the genes identified in different screenings, for which no role has been suggested. Overexpression of the gene restores filamentation in a mep1mep2 mutant, and its deletion in a wild-type strain causes a moderate defect in PH growth [33].
Since Msn5 is involved in the nuclear export of phosphorylated proteins [85] it could facilitate the removal from the nucleus of some regulatory protein which represses PH growth. A form of the RNA polymerase II holoenzyme includes a negative regulator formed by Srb10, a cyclin-dependent kinase, and several associated proteins.
This Srb—CDK complex, which would function through phosphorylation of the RNA polymerase carboxy-terminal domain, appears to down-regulate a small fraction of S. As cells enter the diauxic shift Srb10 levels decrease strongly and other control mechanisms should become operative. Recently, targets for the MAP kinase Kss1 have been looked for by performing transcript profiling experiments [ 68 , 87 ]. Although dozens of genes regulated by the Kss1 signaling pathway have been identified, most of them are not required for invasive growth in haploid cells [68].
This could indicate that many of these genes play a redundant role, but also that some of them participate in a process related to filamentous growth but not required for morphogenesis. A clear-cut example is the PGU1 gene, which encodes a secreted enzyme that hydrolyzes polygalacturonic acid, a component of the plant polysaccharide pectin. This enzyme is not required for invasiveness but would facilitate the access of S. Profiling experiments have also confirmed that there is some functional overlap between the filamentous growth and mating responses [87].
PH growth of diploid cells and invasiveness of haploids present very similar requirements. Both depend on the MAPK cascade, starting with activation of Cdc42, including the Ste20, Ste11, Ste7 and Kss1 proteins and their downstream targets, the transcription factors Ste12 and Tec1 [ 4 , 11 , 12 , 17 ]. Finally the PKA pathway plays a role in haploids [12] as it does in diploids. However, the regulation of these pathways is different in the two types of cells.
Expression of a reporter gene depending on the transcription factors Ste12 and Tec1 was fold higher in haploids than in diploids in a complete mineral medium SC. The rate of transcription of the reporter gene was increased in SLAD medium, which has a low level of ammonium, the effect being more marked in the case of diploid cells nine-fold vs.
It has been stated [12] that while in haploids an increase in protein kinase A activity stimulated the expression of a reporter gene controlled by Ste12 and Tec1, in diploids a very high protein kinase A activity inhibited the transcription of this reporter gene.
Looking at the effects of an activation of the PKA pathway on the expression of the reporter gene controlled by Ste12 and Tec1, no clear difference emerges between haploids and diploids. On the other hand, the effects are strongly dependent on the metabolic conditions of the cell.
As discussed in Section 2. There are, however, some differences in the behavior of haploid and diploid cells. The most striking difference between the two types of cells appeared to be that haploids are able to invade agar in the presence of a good nitrogen source, while diploids are not [4].
However, in a diploid growing on a rich medium YP , filamentation and invasiveness may be induced by cAMP together with a stressful condition such as a poor carbon source or the presence of aliphatic alcohols [8].
Although TEC1 transcription is higher in haploid than in diploid cells, there is little correlation between TEC1 transcription and haploid invasive growth, an observation which suggests that a low level of TEC1 transcription is sufficient for invasive growth [88].
It has been shown that invasive growth in haploids does not take place in rim mutants, while in homozygous diploid rim mutants there is no defect in PH growth [89]. The exact role played by the Rim proteins is not yet known, but from the characteristics of these proteins yeast protein databases several points emerge.
RIM8 has not been cloned, Rim9 may acetylate Rim13 and Rim13, similar to PalB, a calpain-like protein from Aspergillus nidulans , may be the protease which cleaves Rim Although dimorphism has been observed in a large number of fungi, most studies on this process have been carried out in two main species, S.
Pseudohyphae formation is induced by growth on a variety of solid media Spider, milk-Tween, etc. The role of the MAP kinase pathway in the transition from yeast to pseudohypha in C.
Functional homologues of the S. In contrast another Stelike protein, Cla4, appears to be absolutely required for hyphal growth; homozygous cla4 mutants show an aberrant morphology which may indicate a role for Cla4 in cytokinesis [95]. In this respect it can be noted that there is evidence that Cla4 is important for the formation of the septin ring [96] , although a requirement for Cla4 for pseudohyphal growth in S.
The downstream target of the MAP kinase pathway in C. Although an homologue of the S. On the other hand, while PH growth is constitutive in C. Targets for Cph1 have not been identified but INT1 , a gene encoding a surface protein with a cytoplasmic tail, may be a candidate. Besides, overexpression of INT1 led to the production of germ tubes in a haploid S. Morphogenesis in C. Reduced expression or deletion of EFG1 caused an inability to form germ tubes and true hyphae in the presence of serum, but elongated pseudohyphal cells could be observed in certain solid media [ , ].
It has been postulated that Efg1 lies in a Ras-activated, cAMP-dependent pathway [90] , and recent results indicate that mutation of the single possible PKA phosphorylation site in Efg1 to alanine does not allow filamentation, whereas a change to glutamate results in hyperfilamentation D.
Ernst, personal communication. Evidence is also accumulating that a cAMP-dependent protein kinase is involved in the dimorphic transition in C. Dibutyryl-cAMP enhanced germ-tube formation in exponentially growing cells [] , inhibitors of protein kinase A blocked the germ-tube formation induced by N -acetyl glucosamine, although not that induced by serum [] , and Catpk2 mutants are impaired in hyphal development unless EFG1 is overexpressed [].
One of the targets of the cAMP-activated protein kinase may be the glucosaminephosphate synthase required to provide the substrate for chitin synthesis, which is more abundant in mycelium than in yeast cells []. Very recently the RAS1 gene from C. Hyphal development in C. Homozygous tup1 mutants display filamentation in a great variety of media but no germ tube formation progressing to true hyphae in the presence of serum.
A double mutant tup1cph1 behaves as a tup1 mutant, and it has been suggested that repression by Tup1 is relieved via Cph1 []. However, as discussed below, Tup1 and Cph1 are probably acting in different pathways. It can be noted that in S. A further gene involved in C. In hwp1 mutants the ability to form hyphae on solid medium is lost and in the presence of serum there are reduced levels of peripheral hyphae []. The ability to invade the agar beneath the colony is maintained as well as the capacity to form germ tubes in liquid media.
HWP1 is expressed at a reduced level in a strain with a deletion in RBF1 and expression can take place in non-inducing conditions in a tup1 null mutant []. Since the expression of HWP1 is repressed by Tup1 but does not require Cph1, the suggestion that Cph1 acts by counteracting the action of Tup1 remains unproven. Although HWP1 can be situated downstream of EFG1 , the inability of constitutively expressed HWP1 to suppress an efg1 null mutation indicates that Efg1 regulates additional genes required for hyphal development [].
Recent evidence also suggests that Cph1, Efg1 and Tup1 control different pathways which make additive contributions to filamentous growth and that a fourth pathway may still operate when the others are blocked [].
A tentative model integrating the data available is shown in Fig. It should also be noted that, as reported for S. In a serum-containing medium, on the other hand, the cln1 mutation has only a slight effect on the induction of HWP1 and does not interfere with germ tube formation or hyphal growth [].
Signaling pathways triggering hyphal and pseudohyphal growth in C. Arrows indicate activation, lines with bars indicate inhibition. Finally, the interruption in C. This is in contrast with the situation in S. As happens with S.
For instance, disruption of the C. In the first case the impairment could be due to alterations in the metabolism of glucose, whereas in the second it may reflect a need for trehalose for the maintenance of the hyphae at a high temperature. In Yarrowia lipolytica the protein Mhy1, which is able to bind to a STRE sequence, is required for the yeast-to-hypha transition []. During the yeast-to-hypha transition the transcription of MHY1 is strongly increased and Mhy1 is concentrated in the nuclei of actively growing cells found at the hyphal tip.
Mhy1 transcription is unaffected by thermal stress and decreases upon carbon source starvation and osmotic or oxidative shock []. It is interesting to note that while C. A further peculiarity of Y. XPR2 which encodes an alkaline extracellular proteinase from Y. Rim [ , ], while the processing of the native Xpr2 is carried out by a dibasic endoprotease Xpr6 []. This regulatory pathway would be equivalent to the pathway controlled by the RIM genes in S. The product of the homeogene HOY1 , which shows some regions of homology with the S.
The expression of HOY1 increases when the yeast cells are induced to form hyphae but there is not yet information on the genes which could be controlled by Hoy1. Even the fission yeast Schizosaccharomyces pombe may undergo pseudohyphal growth in certain stress conditions. The absence of a coiled-coil protein, Alm1, with similarity to myosin and other fibrous proteins, results in the production of a high proportion of elongated mononucleate cells [].
Since Alm1 is associated with the medial region of the cells during nuclei separation at anaphase, it has been suggested that Alm1 plays a role in initiating cell division. The fungal pathogens Cryptococcus neoformans and Ustilago maydis show some characteristics which set them apart from the fungi we have considered until now. Under suitable environmental conditions haploid, budding cells mate and generate a dikaryotic filamentous cell type [ 9 , ].
However, a true dimorphic transition from a haploid yeast phase to a hyphal phase has also been observed for both species [ , ]. In the case of C. For U. These observations support the idea that morphogenesis in U. However, while in the well studied cases of S. It may be noted, too, that for Mucor rouxii , cAMP levels were found to decrease before the yeast-to-hypha transition induced by exposure to air and that this transition was blocked by addition of dibutyryl-cAMP [].
The MAP kinase cascade is required for cell elongation while the cAMP-dependent protein kinase Tpk2 controls the switch to unipolar budding [30].
For invasiveness both pathways appear redundant to some extent since a defect in one of the pathways may be compensated by an increased activity in the other [40]. However, large gaps in knowledge remain, which concern different aspects of the morphogenetic process. Without being exhaustive, the following points can be listed:. The receptor Gpr1 and its coupled G protein, Gpa2, appear to be important in the response to the nutritional situation, and a role has also been suggested for the membrane protein Sho1.
However, due to the fact that Ras2 is situated upstream of the two well-known regulatory pathways, it should be expected that some signal is transmitted to the Ras2 protein itself and this point has not yet been addressed. Actin appears to play a very important role in determining the budding pattern and the morphology of the cells round vs. It is also clear that the formation of chains of cells as well as invasiveness would require specific cell-surface proteins.
What are the roles for a number of elements involved in PH growth but which have not been yet connected with the main regulatory pathways? One of these elements is the cyclin-dependent kinase Cdc However, the evidence for this is still rather indirect and further research will be necessary to test these suggestions.
Another protein which stimulates PH growth, but for which no mechanism of action has been described is Phd1. It has been shown that Phd1 acts independently of Tpk2 [30] , but Phd1 could be a substrate for Tpk1 or Tpk3. In this context, it can be noted that the Phd1 homologue in C. An intriguing point which has not received much attention is the fact that invasiveness of haploid strains grown in liquid medium and spotted in test plates is strongly dependent on the growth phase at which the cells were sampled from the liquid cultures.
Late-exponential phase yeast cells showed a stronger invasive phenotype. It has been suggested that the phase of growth could even affect the results of epistasis analysis [42]. Differences due to the growth state of the cells used in different laboratories may explain some of the contradictions between reports from different groups.
When the factors which regulate morphogenesis in different yeasts are compared, it is plain that no unified picture emerges. Nitrogen starvation stimulates filamentous growth in both S. With respect to cAMP, it has a positive effect on filamentation in S. There are also marked differences between S. On the other hand some signaling pathways, such as the MAP kinase cascade leading to phosphorylation of Ste12, the cAMP-dependent pathway, or the pathway involving Phd1 in S.
Therefore, even in the absence of extensive studies in species other than S. Depending on the ecological niche where each particular species has evolved, diversity has been generated, through selection of the mechanisms which allowed a better adaptation to the features of each particular environment.
I am grateful to all the colleagues who communicated to me unpublished results. Zaragoza for the pictures shown in Fig. Guilliermond, A. John Wiley and Sons, New York. Kron S. Trends Microbiol.
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Furthermore, the availability of virulent and avirulent isolates is very useful for future works aimed to explore the mechanisms involved in S. Example of colony phenotype switching. We want to acknowledge to I. Noguera from the School of Pharmacy, University of Valencia Spain for its help during the mice assays.
We also want to acknowledge to L. Madrid, Spain , F. Valencia, Spain and J. We would like to thank C. Gil, G. Molero and E.
Reviewed and critically revised the manuscript: LM AQ. Browse Subject Areas? Click through the PLOS taxonomy to find articles in your field. Abstract Saccharomyces cerevisiae plays a beneficial role in health because of its intrinsic nutritional value and bio-functional properties, which is why it is also used as a dietary supplement. Materials and Methods Strains Yeast strains used for the elaboration of commercial dietary products were isolated from 22 products D1—D4 and D6—D23 , listed in Table 1.
Download: PPT. Table 2. Growth Conditions For dietetic strains isolation, a sample from each commercial product 1 pill, 1 ml or a teaspoonful depending on whether the product was in pill, liquid of flake form was grown in 4. Secretion of hydrolytic enzymes protease and phospholipase. Pseudohyphal and Invasive growth. Colony phenotype switching. Western blotting assays. Adherence to polystyrene. Adherence to polyurethane intravenous catheters. Experimental infections.
Animal studies were carried out at the University of Valencia mentioned above. Experimental infection. Evaluation of yeast translocation and dissemination. Determination of Burdens in Representative Organs and Stools When required, stools were obtained by manually pressing the lower abdomen of mice, weighed and placed in 5 ml volumes of sterile saline solution.
Statistical Analysis To compare among strains based on the study of virulence-associated phenotypes and to analyze the association between these phenotypic traits and in vivo virulence by intravenous route, a presence and absence matrix was prepared from which the Spearman's Rho correlation coefficient was calculated both for strains and traits.
Results 1. Identification of Yeasts Isolated from Commercial Products A total of 22 commercial products including enriched beverages and dietary products, as well as the bio-therapeutic agent sold as Ultra-Levura were microbiologically studied see Table 1 for origin and presentation of the products.
Molecular Characterization of Yeasts Isolated from Commercial Products We studied the purity of the nine commercial products containing live yeasts by the molecular technique based on mtDNA restriction analysis with the endonuclease Hin fI. Figure 1. Molecular characterization of yeast strains analyzed.
Table 3. Distribution of DNA types of yeasts isolated from commercial products. Growth at high temperatures. Figure 2. Growth at different temperatures of commercial and control S. Figure 3. Generation time of commercial and control S. Extracellular secretion of degradative enzymes. Figure 4. Extracellular secretion of phospholipase A and protease B of commercial and control S.
Pseudohyphal formation. Figure 5. Differences in pseudohyphal growth of commercial and control S. Invasive growth. Figure 6. Examples of invasive growth in commercial and control S. Table 4. Colony phenotype switching frequencies of commercial Saccharomyces strains. Activation of MAPK signaling pathways. Figure 7. Adherence to polystyrene plastic. Figure 8. Adherence of commercial and control yeast strains to plastic and catheters.
In vivo Virulence by Intravenous Inoculation in Mice The results of the analysis of virulence-associated phenotypes summarized in Table 5 show that isolates D2, D4 and D14 displayed positivity for Table 5. Virulence-associated phenotypes observed in strains isolated from commercial products.
Figure 9. Figure Evaluation of the presence of yeasts in mouse feces during the assay of gastrointestinal infection. Translocation and dissemination from the gut of strains D14 and D23 after oral inoculation. Table 6. Intestinal translocation and dissemination of isolates D14 and D23 based on the number of mice that showed yeast burdens in target organs.
Identity of the Yeast Cells Recovered from Infected Mice In order to confirm that the colonies recovered from the infected mice really belonged to the yeast strain inoculated, mtDNA restriction analysis was performed both in the intravenous and oral models of infection. Discussion In recent years, the perception that S. Table 7. Statistical analysis of association between virulence-associated phenotypic traits and in vivo virulence.
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