Fluctuations in glycolytic mRNA levels during morphogenesis in Candida albicans reflect underlying changes in growth and are not a response to cellular dimorphism

Abstract

The levels of pyruvate kinase (PYK1), alcohol dehydrogenase (ADH1), phosphoglycerate kinase (PGK1) and phosphoglycerate mutase (GPM1) mRNAs were measured during batch growth and during the yeast-to-hyphal transition in Candida albicans. The four mRNAs behaved in a similar fashion. PYK1, ADH1, PGK1 and GPM1 mRNA levels were shown to increase dramatically during the exponential growth phase of the yeast form, and then to decrease to relatively low levels in the stationary phase. The dimorphic transition was induced using two sets of conditions: (i) an increase in temperature (from 25 degrees C to 37 degrees C) combined with the addition of serum to the medium; and (ii) an increase in temperature (from 25 degrees C to 37 degrees C) and an increase in pH of the growth medium (from pH 4.5 to pH 6.5). Additional cultures were analysed to control for the addition of serum, and for changes in temperature or pH. Immediately following dilution of late-exponential cells into fresh media the levels of all four glycolytic mRNAs decreased rapidly in contrast to the ACT1 mRNA control, the level of which increased under most conditions. The recovery of glycolytic mRNA levels depended on the culture conditions, but there was no direct correlation with the formation of germ tubes, with the addition of serum to the medium, the increase in culture temperature, the medium pH, or the glucose concentration. This indicates that the changes in glycolytic gene expression that accompany the dimorphic transition in C. albicans reflect the underlying physiological status of the cells during morphogenesis and not alterations to cell shape.

Structure and regulation of a Candida albicans RP10 gene which encodes an immunogenic protein homologous to Saccharomyces cerevisiae ribosomal protein 10

“…Reprobing of the same filters revealed that the PYK1 mRNA also was repressed under identical conditions (Fig. 6) but the ADH1 mRNA was not repressed by heat shock (55). Therefore, the decrease in RP10 mRNA levels observed at 37ЊC during the dimorphism experiments (Fig.…”

“…Like RP10, the mRNAs encoding actin (ACT1) and the essential fungal translation elongation factor 3 (TEF3) increased when late-exponential-phase cells were inoculated into fresh medium (54). In contrast, the levels of the glycolytic mRNAs GPM1, PGK1, PYK1, and ADH1 decreased transiently under identical conditions (55).…”

“…7). The levels of numerous housekeeping mRNAs are known to change during the transition: for example, those of the ACT1, ADH1, GPM1, PGK1, PYK1, and TEF3 mRNAs (11,54,55). Therefore, it seems more reasonable to suggest that the changes in RP10 mRNA levels observed during dimorphism (Fig.…”

“…For example, the identification of S. cerevisiae genes that influence pseudohyphal growth in this yeast (17,30) may lead ultimately to the isolation of homologs that are important for dimorphism in C. albicans. Also, differential hybridization strategies are proving successful in the identification of genes which are regulated during dimorphism despite the fact that the expression of many genes fluctuates during the yeast-to-hyphal form transition (54,55). Such strategies have led to the isolation of the C. albicans ECE1 and PHR1 genes, which are expressed specifically in the hyphal growth form (4,14), but these genes are not required for the transition under all conditions.…”

“…Hybridizations were performed with excess probe, and signals were quantified directly by two-dimensional radioimaging with an AMBIS Radioanalytic System (LabLogic, Sheffield, United Kingdom). To our knowledge, there is no report of a C. albicans mRNA that remains at sufficiently constant levels during the dimorphic transition to use as an internal loading control on Northern blots (11,54,55). Therefore, mRNA levels were measured relative to those of the rRNAs by loading of approximately equal amounts of total RNA in each lane of the Northern gels.…”