The role and relevance of phospholipase D1 during growth and dimorphism of Candida albicans

Abstract

The phosphatidylcholine-specific phospholipase D1 (PLD1) in Saccharomyces cerevisiae is involved in vesicle transport and is essential for sporulation. The gene encoding the homologous phospholipase D1 from Candida albicans (PLD1) was used to study the role of PLD1 in this pathogenic fungus. In vitro and in vivo expression studies using Northern blots and reverse transcriptase-PCR showed low PLD1 mRNA levels in defined media supporting yeast growth and during experimental infection, while enhanced levels of PLD1 transcripts were detected during the yeast to hyphal transition. To study the relevance of PLD1 during yeast and hyphal growth, an essential part of the gene was deleted in both alleles of two isogenic strains. In vitro PLD1 activity assays showed that pld1 mutants produced no detectable levels of phosphatidic acid, the hydrolytic product of PLD1 activity, and strongly reduced levels of diacylglycerol, the product of lipid phosphate phosphohydrolase, suggesting no or a negligible background PLD1 activity in the pld1 mutants. The pld1 mutants showed no growth differences compared to the parental wild-type in liquid complex and minimal media, independent of the growth temperature. In addition, growth rates of pld1 mutants in media with protein as the sole source of nitrogen were similar to growth rates of the wild-type, indicating that secretion of proteinases was not reduced. Chlamydospore formation was normal in pld1 mutants. When germ tube formation was induced in liquid media, pld1 mutants showed similar rates of yeast to hyphal transition compared to the wild-type. However, no hyphae formation was observed on solid Spider medium, and cell growth on cornmeal/Tween 80 medium indicated aberrant morphogenesis. In addition, pld1 mutants growing on solid media had an attenuated ability to invade the agar. In a model of oral candidosis, pld1 mutants showed no attenuation of virulence. In contrast, the mutant was less virulent in two different mouse models. These data suggest that PLD1 is not essential for growth and oral infections. However, they also suggest that a prominent part of the phosphatidic acid and diacylglycerol pools is produced by PLD1 and that the level of these components is important for morphological transitions under certain conditions in C. albicans.

Phosphatidylinositol-4-phosphate 5-kinase activity is stimulated during temperature-induced morphogenesis in Candida albicans

“…Earlier experiments performed in this laboratory have shown that PLD1 plays a significant role in the morphogenetic process (Hube et al, 2001 ;McLain & Dolan, 1997). Given that the product of PLD1 catalysis is PA At -25n8 11n1 18n9 22n2 19n2 21n5 20n9 17n2 74n8 14n3 17n3 20n9 20n4 Ca -12n3 23n9 22n3 21n5 21n9 22n1 21n1 27n8 10n1 48n5 45n2 24n1 Ce -11n3 10n9 11n0 11n7 11n2 12n1 10n4 10n5 10n7 11n7 12n3 Dm -49n2 41n5 44n2 46n3 38n7 19n6 10n5 15n1 18n7 19n7 Hsα -59n9 60n1 91n4 65n8 23n5 10n5 22n0 24n3 24n1 Hsβ -95n9 56n2 57n6 18n8 10n3 17n3 18n3 23n4 Mmα -56n6 60n3 19n9 9n4 18n6 22n1 23n2 Mmβ -63n4 22n0 10n3 19n8 23n6 23n4 Mmγ -17n7 12n4 14n5 19n2 23n6 Nr -17n3 18n5 20n5 22n4 Os -11n...…”

“…C. albicans strains used were CAF2-1 (ura3 ::imm434\URA3) and JDC12 (strain also designated pld1∆1 in Hube et al, 2001 ;ura3 ::imm434\ ura3::imm434 pld1::hisG::URA3::hisG\pld1 ::hisG), which are isogenic except as indicated (Hube et al, 2001). S. cerevisiae strains used were 23567 (a\α his3∆1\his3∆1 leu2∆0\ leu2∆1 met15∆0\j lys2∆0\j ura3∆0\ura3∆0 mss4 : : Kan R \ j ; Research Genetics), SEY6210.1 and AAY202.1 (SEY6210.1 with mss4∆ ::HIS3MX6 carrying YEplac-mss4-102).…”

“…The small magnitude of stimulation raises the question of physiological impact. By way of comparison, PLD1 activity is stimulated only 60 % during morphogenesis, but the null mutant is unable to form hyphae under specific conditions and is avirulent in a mouse model system (Hube et al, 2001 ;McLain & Dolan, 1997). Thus, there is a solid precedent, supported by both biochemical and genetic analysis, for small changes in activity having relevant and significant physiological impact in the process of morphogenesis in C. albicans.…”

“…To determine whether PLD1 was playing a role in the stimulation of PI4P5K, a homozygous pld1∆ knockout strain, JDC12 (Hube et al, 2001), was assayed for PI4P5K activity during morphogenesis. PI4P5K activity was observed to decline steadily during morphogenesis in the pld1∆ cells (Fig.…”

Functional analysis of the phospholipase C gene CaPLC1 and two unusual phospholipase C genes, CaPLC2 and CaPLC3, of Candida albicans

“…The dimorphic transition is regulated by a number of signal transduction pathways such as the MAPK and cAMP/protein kinase A pathways (reviewed by Sudbery et al, 2004;Whiteway & Oberholzer, 2004). In addition, lipid signalling pathways with second messengers such as DAG may play a role during morphogenesis of C. albicans (Hube et al, 2001).…”