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Disruption in Candida albicans of the TPS2 gene encoding trehalose-6-phosphate phosphatase affects cell integrity and
Oscar Zaragoza1, Claudio de Virgilio, José Pontón
1Instituto de Investigaciones Biomédicas Alberto Sols CSIC-UAM, Unidad de Bioquímica y Genética de Levaduras, 28029 Madrid, Spain.
Abstract:
The gene CaTPS2 encoding trehalose-6-phosphate (T6P) phosphatase from Candida albicans has been cloned and disrupted in this organism. The Catps2/Catps2 mutant did not accumulate trehalose but accumulated high levels of T6P. Disruption of the two copies of the CaTPS2 gene did not abolish growth even at 42 degrees C, but decreased the growth rate. In the stationary phase, the Catps2/Catps2 mutant aggregated, more than 50% of its cells became permeable to propidium iodide and a large amount of protein was found in the culture medium. Aggregation occurred only at pH values higher than 7 and was avoided by osmoprotectants; it was never observed during the exponential phase of growth. The mutant formed colonies with a smooth border on Spider medium. Mice inoculated with 1.5 x 10(6) c.f.u. of wild-type cells died after 8 days, while 80% of those inoculated with the same number of c.f.u. of the Catps2/Catps2 mutant survived for at least 1 month. Reintroduction of the wild-type CaTPS2 gene in the Catps2/Catps2 mutant abolished the phenotypes described. It is hypothesized that the accumulation of T6P interferes with the assembly of a normal cell wall.
Insights
Candida albicans lacking trehalose-6-phosphate (T6P) phosphatase (CaTPS2) accumulated T6P, leading to cell aggregation and reduced virulence. This suggests T6P accumulation impacts cell wall integrity and fungal pathogenicity.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Trehalose is a vital disaccharide in many organisms, playing roles in stress tolerance and metabolism.
- Trehalose-6-phosphate (T6P) is a key intermediate in trehalose biosynthesis, regulated by T6P synthase and phosphatase enzymes.
- Candida albicans, an opportunistic fungal pathogen, possesses genes for trehalose biosynthesis, but their precise roles in virulence remain incompletely understood.
Purpose of the Study:
- To investigate the function of the CaTPS2 gene encoding trehalose-6-phosphate (T6P) phosphatase in Candida albicans.
- To determine the impact of CaTPS2 disruption on trehalose and T6P metabolism, cell physiology, and virulence.
- To explore the relationship between T6P accumulation and cell wall integrity in C. albicans.
Main Methods:
- Gene cloning and disruption of CaTPS2 in Candida albicans.
- Analysis of trehalose and T6P levels in wild-type and mutant strains.
- Assessment of growth characteristics, cell permeability (propidium iodide staining), and protein secretion.
- Evaluation of fungal aggregation under various pH and osmoprotectant conditions.
- In vivo virulence studies using a mouse infection model.
- Complementation of the mutant with the wild-type CaTPS2 gene.
Main Results:
- The Catps2/Catps2 mutant failed to accumulate trehalose but showed significantly elevated T6P levels.
- Disruption of CaTPS2 decreased the growth rate, particularly at 42°C, and led to cell aggregation and increased cell permeability in stationary phase.
- The mutant exhibited reduced virulence in a mouse model, with significantly higher survival rates compared to the wild-type.
- Phenotypes were reversed upon reintroduction of the functional CaTPS2 gene.
Conclusions:
- The CaTPS2 gene is essential for normal trehalose synthesis and T6P homeostasis in Candida albicans.
- Accumulation of T6P due to CaTPS2 disruption negatively affects growth, cell integrity, and promotes aggregation.
- Elevated T6P levels are linked to decreased virulence, suggesting a role in host-pathogen interactions.
- The findings support the hypothesis that T6P accumulation interferes with proper cell wall assembly in C. albicans.
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