Trehalose pathway regulates lamentation response in Saccharomyces cerevisiae

: In Saccharomyces cerevisae , the diploid cells undergo either pseudohyphal differentiation or sporulation in response to carbon and nitrogen source depletion. Distinct pathways are known to regulate the processes of filamentation and sporulation in response to nutritional stress. Here, we report the novel finding that the trehalose pathway which is essential for sporulation, is involved in pseudohyphae formation both via GPR1 as well as RAS2 mediated signaling. Our observations indicate that GPR1 is epistatic over TPS1 in signaling for filamentation. Further, we have demonstrated that the pseudohyphal defect of the ras2 mutant is overcome upon disruption of TPS2 . Thus, our results indicate that TPS1 and TPS2 may be involved in cell fate decision between meiosis and filamentation response under nutrient depleting conditions. Further, monitoring pseudohyphae formation under limiting glucose condition unravelled the possibility that TPS1 and TPS2 exert opposing effects to trigger filamentation response.


INTRODUCTION
Organisms have evolved a plethora of developmental and differentiation mechanisms to overcome nutritional deprivation. Yeast, Saccharomyces cerevisiae, undergoes both metabolic as well as morphologic differentiation in order to overcome nutrient deprivation (Gagiano et al. 2002, Gancedo 2008. Diploid cells of S. cerevisiae have the potential to either sporulate or achieve pseudohyphal differentiation in response to carbon and/or nitrogen depletion (Gimeno et al. 1992, Iyer et al. 2008. Sporulation occurs when carbon and nitrogen are depleted (Honigberg and Purnapatre 2003). Although pseudohyphal differentiation was originally thought to occur only in response to low ammonium and high glucose (Lengeler et al. 2000, Lorenz 1997), Iyer et al. (2008) demonstrated that in addition to signaling from low ammonium, low glucose signaling was also necessary for filamentation response. Despite intense investigations, it is still unclear as to how these two developmental processes of pseudohyphae formation and sporulation emerge in response to low nitrogen and low glucose (Cullen and Sprague 2012, Iyer et al. 2008). While distinct low glucose and low ammonium signaling pathways implement pseudohyphal differentiation by regulating the levels of cAMP, the nature of the crosstalk between glucose and ammonium signaling has remained largely elusive (Cullen and Sprague 2012, Gagiano et al. 2002) Signaling for pseudohyphal transition in response to low ammonium occurs via MEP2, an ammonium transporter, (Lorenz and Heitman 1998). It was demonstrated that MEP2 is a transceptor i.e. in addition to signaling, the transport function of Mep2 was necessary for filamentation response (Rutherford et al. 2008). NPR1, a TORC1 effector kinase, positively regulates MEP2 under conditions of poor nitrogen availability to trigger pseudohyphae formation (Boeckstaens et al. 2007, Boeckstaens et al. 2014. Although it has been demonstrated that MEP2 signals via cAMP (Lorenz and Heitman 1998), the underlying mechanisms have not yet been fully elucidated. (Rutherford et al. 2008).
In contrast to the above, how the glucose responsive, GPR1-GPA2 signaling impinges on the cAMP-PKA pathway (Cullen and Sprague 2012, Xue and Hirsch 1998) to induce filamentation response is relatively well understood. It was previously reported KRH1/2 interfere with GPR1-GPA2 coupling thereby inhibiting downstream signaling via cAMP (Harashima and Heitman 2005). Although it was tacitly assumed that KRH1 and KRH2 are functionally redundant, Iyer and Bhat demonstrated that they are non-redundant and uncovered distinct roles for these two kelch proteins in inducing pseudohyphae in low glucose (Iyer and Bhat 2017 , invoking the possibility of a role for trehalose metabolism in pseudohyphal differentiation. This idea does not seem to be farfetched given the observation that the components of the trehalose biosynthetic pathway are essential for sporulation (De Silva-Udawatta and Cannon 2001), which also is a response to nutrient depletion. In yeast, trehalose performs a variety of functions ranging from serving as a carbon source to a stress protectant, by conferring resistance to adverse environmental conditions (Bonini et al. 2000, Miao et al. 2017). The trehalose pathway involves two enzymes namely TPS1 (trehalose phosphate synthase) and TPS2 (trehalose phosphate phosphatase). TPS1 catalyzes the formation of T6P from UDP-Glucose and glucose-6-phosphate (Thevelein and Hohmann, 1995). T6P appears to regulate glucose mediated signaling (Deroover et al. 2016).
Interestingly, observations in Candida albicans suggested a possible link between trehalose synthesis and virulence. Evidence in C. albicans indicated that disruption of TPS1 results in a decrease in virulence of the strain (Zaragoza et al. 1998). Disruption of TPS2 caused a reduction in virulence without affecting hyphae formation (Van Dijck et al. 2002). Further, it was demonstrated that TPS2 and GPR1 functioned synergistically in trehalose metabolism as well as virulence (Maidan et al. 2008). In contrast to S. cerevisiae, TPS1 disruption in C. albicans did not affect growth on glucose (Zaragoza et al. 1998, Van Dijck et al. 2002. The TPS genes have also been shown not only to regulate differentiation in C. albicans but also to regulate several processes in plants ranging from cell morphology to architecture of inflorescence and other developmental processes (Chary et al. 2008).
Based on the above and the observation that low glucose is pivotal in pseudohyphae formation as well as in restoring the glycolytic imbalance in a tps1 mutant, we hypothesized that the trehalose biosynthetic pathway may be involved in filamentation response. This hypothesis is further supported by the correlation observed between low trehalose levels and pseudohyphae formation (Iyer et al. 2008). Despite the availability of a vast body of literature on the components of trehalose pathway, it's role in pseudohyphal differentiation has hitherto been unexplored in S. cerevisiae. Here, we show that the tps1 but not tps2 mutant is defective in pseudohyphae formation. This is the first report on the involvement of the trehalose biosynthetic pathway in pseudohyphal differentiation in S. cerevisiae. The use of SLALD (Synthetic low ammonia low dextrose) medium enabled the dissection of the independent roles of TPS1 and TPS2. Our results demonstrate that TPS1 and TPS2 may regulate RAS2 differentially depending upon the availability of nutrients to signal filamentation via cAMP.

Media, Strains and Plasmids:
The strains used in this study are isogenic derivatives of 1278b strain. The strains were constructed using standard methods (Adams et al. 1997, Wach et al. 1994) and are listed in Table 1. Genes were disrupted using either the KanMX or Hygromycin cassette. The double disruptants were generated by mating the individual mutants followed by sporulation and segregation of haploids.
Pseudohyphal growth assay: Synthetic low ammonia dextrose (SLAD) medium with 2% glucose or Synthetic low ammonia low dextrose (SLALD) medium with 0.05% glucose were used to score pseudohyphal growth (Gimeno et al.1992, Lorenz and Heitman 1997, Iyer et al. 2008). Cells were spread for single colonies and incubated for 6 days at 30 o C unless mentioned otherwise. Images of colonies were captured at 10X magnification using a Nikon Coolpix 8400 attached to a Nikon TS 100 microscope. The images are representative of at least three repetitions of each experiment.
Spotting Assay: Cells were grown to 0.5 OD, washed twice and five-fold serial dilutions were spotted on yeast extract peptone dextrose (YPD) or yeast extract peptone galactose (YPGal) agar. Images were captured after 2 days of incubation.
Western Blot Analysis: Crude cell extracts were prepared as described by (Peeters et al. 2017). Briefly, cells at OD of 3-4 in YPD medium were collected and washed with ice-cold water. For analysis in SLALD medium, cells at OD 3-4 were collected by centrifugation, washed twice, transferred to SLALD (one-fifth volume) and incubated for an additional 8 hrs. 200mg cells were lysed with 0.2g of glass beads after adding lysis buffer as described (Peeters et al. 2017). Centrifugation at 8000 rpm for 5 min yielded the crude protein extract used for western blot analysis. The blots were developed with antibodies against yeast Ras2p from Santacruz Biotechnology Inc., according to the manufacturer's recommendations.

RESULTS:
Glucose growth defect of the tps1 mutant is both strain as well as ploidy dependent  (2014), the diploid strain of S288c was used (personal communication). Our analysis showed that although the tps1Δ haploid strain in the ∑1278 background had a growth defect (Fig. 1), the diploid strain was able to grow well on glucose (Fig. 2). Thus, the glucose growth phenotype of the tps1 mutant strains varies with different lineages as well as the ploidy status of the cell.

TPS1 is required for filamentation response
Trehalose synthesis in yeast occurs in response to adverse environmental conditions, including nutritional stress. Since filamentation occurs in response to nutrient limitation, we hypothesized that TPS1 and/or TPS2 may have a role in pseudohyphal differentiation. Therefore, independent mutants of tps1 and tps2 as well as the tps1tps2 double mutant were analyzed on SLAD as well as SLALD media (Fig. 3). As expected, tps1 was defective in pseudohyphae formation. In contrast, however, the tps2 mutant had no filamentation defect. Further, pseudohyphae formation was slightly enhanced in the tps2 mutant as compared to the wild type indicating that TPS2 may be a negative regulator. Surprisingly the tps1tps2 double mutant formed pseudohyphae on SLALD but not SLAD medium. In high glucose the double mutant phenotype was the same as that of the tps1 mutant whereas in low glucose it was the same as that of tps2 mutant, indicating that TPS1 and TPS2 may signal differently depending on the availability of glucose. Further, heterodiploids in the TPS1 as well as the TPS2 loci exhibit different phenotypes as compared to the homodiploids (Fig. 4), indicating that the effective concentrations of intermediates of the trehalose synthesis pathway may have a role in signaling.
The next question was to determine the pathway through which TPS1 acted. Studies in C. albicans have demonstrated that TPS enzymatic activity was higher in the gpr1Δ strain. Further a synergistic action between TPS2 and GPR1 had been proposed in virulence (Maidan et al. 2008). To determine if there was any interaction between GPR1 and TPS1 or TPS2, in S. cerevisiae, pseudohyphal growth of tps1gpr1 and tps2gpr1 mutants was monitored. Both the haploid as well as the diploid strains of the tps1gpr1 double mutant exhibited a growth defect on high glucose (data not shown). However, in SLALD medium, the filamentation defect of tps1 mutant was overcome upon disruption of GPR1 (Fig. 5 and Fig. 6), indicating that GPR1 was epistatic over TPS1 in signaling for pseudohyphae via the cAMP-PKA pathway. This is corroborated by the observation that extraneous addition of cAMP overcomes the filamentation defect of the tps1 mutant (Fig. 7). It was demonstrated earlier that GPR1 suppressed filamentation under low glucose conditions (Iyer et al. 2008). The current observations indicate that GPR1 is epistatic over TPS1 in signaling for filamentation when glucose is limiting. Although, the individual effects of tps2 or gpr1 mutation is enhanced pseudohyphae, the combined effect results in reduction of filamentation (see tps2/tps2 in Fig. 3, gpr1/gpr1 and gpr1/gpr1 tps/tps2 in Fig. 5). This is as opposed to that TPS1 and TPS2 have a differential role in regulating RAS2 depending upon nutrient availability It has been observed that tps1 mutation results in activation of RAS2. However, the effect of tps2 mutation on RAS2 is not known. In order to determine whether the effect on RAS2 is limited only to TPS1 or does TPS2 also have a role, RAS2 was disrupted in the tps1 as well as the tps2 mutant. Interestingly, disruption of tps2 but not tps1 restored pseudohyphae formation in the ras2 mutant (Fig. 8). This phenotype was enhanced in SLALD as compared to SLAD medium (compare left and right panels of Fig. 8). Therefore, expression of Ras2p was monitored under nutrient complete (YPD medium) as well as nutrient depleted (SLALD medium) conditions in the diploid strains of tps1Δ and tps2Δ. Contrary to earlier reports in the haploid tps1 mutant where RAS2 is activated in glucose causing the growth defect (Peeters et al., 2017), we observed that Ras2p expression was suppressed in the diploid tps1 mutant in YPD medium (Fig. 9). This meant that regulation of Ras2p by TPS1 resulted in opposite effects in the haploid versus the diploid cell. Our observations indicated that this mutant was unable to form pseudohyphae. Since, Ras2p is suppressed in high glucose in a diploid tps1 mutant, the defect in pseudohyphae formation is probably due to repression of Ras2p. In SLALD medium, however, Ras2p expression was suppressed in tps2 but not tps1 mutant (Fig. 10). Thus indicating that TPS1 and TPS2 regulate RAS2 differentially under conditions of nutrient abundance or depletion. Given that the ras2tps2 double mutant puts forth pseudohyphae in SLALD medium, it is possible that TPS1 and TPS2 regulate RAS2 to appropriate levels for filamentation by exerting opposing effects.

DISCUSSION
Trehalose has been shown to perform a range of functions in the cell (Perfect et al. 2017), eventually leading to stress protection. Although there is a large body of data available on the deleterious effects of TPS1 or TPS2 mutations in S. cerevisiae, the exact mechanism of action of trehalose is unclear. Gibney et al. (2015) demonstrated that phenotypes of the tps1 mutant could not be reversed by simply increasing intracellular concentration of trehalose. This meant that the phenotypes were not due to the depletion of intracellular trehalose concentration per se. It is possible that the trehalose pathway exerts a more complex metabolic effect on the physiology of the cell.
It has been demonstrated that both TPS1 and TPS2 are essential for sporulation (De Silva-Udawatta and Cannon 2001). It is possible that these enzymes of the trehalose pathway have a signaling function in addition to the metabolic effect. Our observations unravel a hitherto unknown role for the trehalose pathway in regulation of pseudohyphal differentiation in yeast. We hypothesize that TPS1 and TPS2 coordinate to regulate the expression of RAS2 based on glucose availability and thereby affect the downstream concentration of cAMP. It is thus possible that components of the trehalose biosynthetic pathway determine whether the cell goes into pseudohyphal differentiation or sporulation in response to nutritional stress. This is in accordance with an earlier observation that intracellular concentration of trehalose is inversely proportional to pseudohyphae formation (Iyer et al. 2008). It is significant to note that this pathway, which is important for virulence and stress resistance of several pathogenic fungi, does not exist in mammals. Thus, understanding the nuances of the pathway have far reaching consequences in developing novel antifungal targets. Since it has been observed that trehalose increases protein stability under stressful conditions including hypoxia, it also has a potential to be applied in therapeutics for mammalian cell injury due to hypoxia or anoxia.      Glucose growth phenotype of the diploid tps mutants. Three dilutions ( ve-fold) were spotted Pseudohyphal growth phenotype of the tps mutants on SLAD and SLALD media as indicated. Three independent colonies are shown Figure 4 Pseudohyphal growth phenotype of the heterodiploid tps mutants on SLAD as well as SLAD medium as indicated. The three images represent independent colonies Filamentation of the tps mutants in the background of gpr1 disruption. Three independent colonies are shown Pseudohyphae formed by the tps1gpr1 double mutant is enhanced on prolonged incubation of 8 days. Three independent colonies are shown Effect of extraneous addition of cAMP on lamentation of the tps1 mutant. The three images represent independent colonies Figure 8 Pseudohyphal growth phenotype of the tps mutants in the background of ras2 mutation. Three independent colonies are shown Figure 9 Expression of Ras2p in the tps mutants grown in YPD medium Figure 10 Expression of Ras2p in the tps mutants grown in SLALD medium