Disruption of the Candida albicans TPS1 gene encoding trehalose-6-phosphate synthase impairs formation of hyphae and decreases infectivity

Abstract

The TPS1 gene from Candida albicans, which encodes trehalose-6-phosphate synthase, has been cloned by functional complementation of a tps1 mutant from Saccharomyces cerevisiae. In contrast with the wild-type strain, the double tps1/tps1 disruptant did not accumulate trehalose at stationary phase or after heat shock. Growth of the tps1/tps1 disruptant at 30 degreesC was indistinguishable from that of the wild type. However, at 42 degreesC it did not grow on glucose or fructose but grew normally on galactose or glycerol. At 37 degreesC, the yeast-hypha transition in the mutant in glucose-calf serum medium did not occur. During growth at 42 degreesC, the mutant did not form hyphae in galactose or in glycerol. Some of the growth defects observed may be traced to an unbalanced sugar metabolism that reduces the cellular content of ATP. Mice inoculated with 10(6) CFU of the tps1/tps1 mutant did not show visible symptoms of infection 16 days after inoculation, while those similarly inoculated with wild-type cells were dead 12 days after inoculation.

FIG. 1

Structure of the TPS1 region from C. albicans and Southern blot analysis of tps1 disruptants. (A) Restriction maps of the C. albicans DNA inserts in pOZ11 and pOZ31. Dotted lines indicate yeast DNA. The region corresponding to the CaTPS1 gene and its direction of transcription are indicated. (B) Disruption of CaTPS1 with the hisG-URA3-hisG cassette (see Materials and Methods for details of the construction). (C) Southern analysis of the chromosomal disruption of CaTPS1. The probe used was the 0.3-kb ScaI-EcoRV fragment and is indicated in panel A as PROBE. Genomic DNA was digested with ScaI and HindIII. The relevant genotypes of the strains used for DNA analysis are indicated under the lanes. Sizes of the fragments are indicated at the left.

FIG. 2

Phylogenetic tree of trehalose-6-P synthase from different organisms and regions of putative regulatory importance in the CaTPS1 promoter. (A) Amino acid sequences of the various proteins were aligned with the CLUSTAL V program (22). A maximum parsimony consensus tree (100 bootstrap resamplings) was obtained with the PHYLIP 3.5 package (14). The sequences were retrieved from the SWISSPROT database. Numbers at the intersections indicate the bootstrap value as a percentage. The percentage of identity with respect to the C. albicans amino acid sequence is shown at the right. Figures in brackets were obtained by comparing only the 500 initial amino acids in the case of Arabidopsis thaliana or using only amino acids 290 to 415 from C. albicans in the case of Methanobacterium thermoautotrophicum. (B) Part of the 5′ noncoding region of the CaTPS1 gene. Putative regulatory sequences are underlined.

FIG. 3

Influence of disruption of the CaTPS1 gene on formation of hyphae. (A) Yeasts were grown in YP-glucose until stationary phase and suspended in the same medium with 10% newborn calf serum. The pictures were taken at the time of serum addition (0 h) and 4 h later. (B) Morphologies of cultures of wild-type and tps1/tps1 disruptant on YP-glycerol during the exponential phase of growth at 42°C.

FIG. 4

Infectivity of C. albicans tps1/tps1 mutants. Mice were inoculated with different cell suspensions of the tps1/tps1 mutant (squares), the TPS1/tps1 single disruptant (triangles), and the wild type (circles), and survival was scored over time. Panels A, B, and C correspond to inocula of 10⁶, 10⁷, and 10⁸ CFU. All strains were prototrophic for uracil.