Transcriptional regulatory circuitries in the human pathogen Candida albicans involving sense--antisense interactions

Abstract

Candida albicans, a major human fungal pathogen, usually contains a diploid genome, but controls adaptation to a toxic alternative carbon source L-sorbose, by the reversible loss of one chromosome 5 (Ch5). We have previously identified multiple unique regions on Ch5 that repress the growth on sorbose. In one of the regions, the CSU51 gene determining the repressive property of the region was identified. We report here the identification of the CSU53 gene from a different region on Ch5. Most importantly, we find that CSU51 and CSU53 are associated with novel regulatory elements, ASUs, which are embedded within CSUs in an antisense configuration. ASUs act opposite to CSUs by enhancing the growth on sorbose. In respect to the CSU transcripts, the ASU long antisense transcripts are in lesser amounts, are completely overlapped, and are inversely related. ASUs interact with CSUs in natural CSU/ASU cis configurations, as well as when extra copies of ASUs are placed in trans to the CSU/ASU configurations. We suggest that ASU long embedded antisense transcripts modulate CSU sense transcripts.

Figure 1

Schematic presentation of two CSU/ASU sense/antisense configurations in the context of Ch5. CSU51/ASU51 configuration from region A or CSU53/ASU53 configuration from region 135 is located within a 209-kb portion of Ch5, which is critical for growth on sorbose. This portion also carries other regions B, C, and 139, as indicated (Kabir et al. 2005). The size of Ch5 is indicated and the centromere (C) and telomeres (T) of Ch5 are shown. Also indicated are the ORF sizes of CSUs and ASUs.

Figure 2

Diagrams representing various co-overexpression plasmids that were derived from the backbone plasmid pCA88. Shown are the following: (1) an original low copy number replicative plasmid pRC2312; (2) its derivative pCA88 overexpressing the metabolic SOU1 gene; (3–9) various plasmids co-overexpressing SOU1 with a sequence of interest. The sequences are indicated by open, shaded, and solid blocks. These correspond to the Ch5 region, to the portion of region encompassing the CSU gene, which always contains an embedded ASU gene (CSU + ASU), and to the ASU gene, as indicated. Mutations are marked with an X. The phenotype of the recipient Sou⁻ cells of the strain CAF4-2 conferred by each type of plasmid on L-sorbose medium, is indicated by Sou⁻, Sou⁺, Sou⁺⁺, and Sou ^+/−. The Sou⁻ or Sou⁺ phenotypes due to the original pRC2312 or pCA88 (SOU1), respectively, are considered as the general negative or positive controls for growth. Note that in the CSU/ASU configuration, the stronger repression phenotype of CSU, Sou⁻, dominates the weaker phenotype of ASU, Sou⁺⁺, in the CSU/ASU configuration. Also note that the intact CSU always contains the embedded ASU. However ASU can be separated by destroying CSU, while the intact CSU cannot be separated from ASU.

Figure 3

Phenotypes conferred to a recipient Sou⁻ strain CAF4-2 by various replicative plasmids. Two major elements CSU53/ASU53 or ASU53 that derived from region 135 of Ch5 (Figures 1 and 4A), are co-overexpressed with the SOU1 gene in different combinations, as indicated in parentheses. For more information see Figure 2 legend. Note that the names of the plasmids and tested portions that are presented schematically in Figure 4A are the same. Shown are examples of spot assay for growth on medium containing sorbose as a sole carbon source. Approximately 5 × 10⁵ cells per spot were plated in duplicates. (A) Analysis of different portions of region 135 (Figure 4A) resulting in the identification of CSU53 and the CSU53/ASU53 configuration. The Sou phenotypes conferred by plasmids are indicated as follows: the control Sou⁻ lack of growth in the presence of an empty vector pRC2312; the control Sou⁺ growth due to pCA88 (see also Figure 2 legend); the Sou⁻ repression of growth due to pCA135 or pEA144; and the Sou⁻* repression of growth with multiple Sou⁺ colonies due to pEA105 or pEA143. When the CSU53/ASU53 configuration is carried on a shorter portion than an entire region 135, it is designated CSU53**. Note that repression of growth occurs due to the natural CSU53/ASU53 configuration in which CSU53 always dominates ASU53. Also shown is the Sou⁺⁺ enhanced growth due to pEA104 co-overexpressing SOU1 with ASU53. Also shown is the control Sou⁺ growth due to pEA145 and pEA140 that co-overexpress SOU1 with the representative portions of region 135 lacking CSU and/or ASU elements. (B) Mutational analysis of the CSU53 ORF implicates the putative Csu53 protein with the Sou phenotype. Plasmids pEA227, pEA201, and pEA158, each containing a region 135 with a different frameshift mutation of CSU53 ORF (Figure 2, diagram 5; Figure 4A) abolish the Sou⁻ repression phenotype of region 135 in favor of the control growth due to SOU1, Sou⁻ → Sou⁺. (C) Plasmids pEA104 or pEA234 co-overexpressing ASU53 and SOU1, enhance growth on sorbose medium, Sou⁺⁺, independent of the insert orientation on a plasmid, as indicated by an arrow. See above for more explanations. (D) Mutational analysis of the ASU53 ORF does not implicate the putative Asu53 protein with the Sou phenotype. Plasmids pEA232 or pEA205 carrying frameshift or stop codon mutation, respectively, in the ASU53 ORF (Figure 2, diagram 7; Figure 4A), do not abolish the original Sou⁺⁺ phenotype. See above for more explanations. (E) ASU53 and CSU53 interact at the phenotypic level, as shown with the plasmid pEA162 (Figure 2, diagram 8) co-overexpressing three elements: SOU1; CSU53 in a natural configuration CSU53/ASU53, which is designated CSU53**; and ASU53. An extra ASU53 changes the repressive phenotype of CSU53 to the growth, which is almost equal to the control Sou⁺ growth. See above for more explanations.

Figure 4

Schematic presentation of the analyses of CSU/ASU configuration in region 135 or region A of Ch5 (Figure 1). The entire region or a portion of the region was individually co-overexpressed with SOU1 on a plasmid (Figure 2) and assayed on sorbose medium, as exemplified in Figure 3. The co-overexpression phenotypes of portions fell into three categories: repression of growth Sou⁻, enhanced growth Sou⁺⁺, and control growth Sou⁺, as indicated on the right (see Figure 3A legend for more explanations). (A) CSU53/ASU53 configuration in the context of region 135, which is also designated pCA135 by the name of the corresponding plasmid. Also shown are the sense CSU53 and antisense ASU53 transcripts, as determined by RACE in the strains 3153A and CAF4-2 (see Mapping UTRs of sense and antisense transcripts). Also shown are representative portions of region 135 that were co-overexpressed with SOU1 to identify CSU53 and ASU53. Mutations within an entire region 135 in CSU53 ORF or within a portion of the region in ASU53 ORF are indicated by stars. (B) CSU51/ASU51 configuration in the context of region A, which is also designated pAK65 by the name of the corresponding plasmid. Also shown are the sense CSU51 and antisense ASU51 transcripts that were determined as indicated in A. Also shown are representative portions of region A that were co-overexpressed with SOU1 to identify ASU51.

Figure 5

Northern blot analyses reveal the CSU53/ASU53 and CSU51/ASU51 sense/antisense transcripts, as exemplified with the strains 3153A and SC5314. (A) Hybridization signals obtained with total RNA and dsDNA probe for CSU51 transcript, as well as mRNA and riboprobes for CSU53, ASU51, and ASU53 transcripts, as indicated. For quantitative estimate of amounts of the corresponding CSU and ASU transcripts, see Relative amounts of antisense ASU and sense CSU transcripts. (B) Nucleotide sequence of CSU53/ASU53 or CSU51/ASU51 ORF configuration. Start codons of CSU sense elements, as well as sequences comprising ASU antisense elements are in boldfaced type. The sequences that were used to prepare riboprobes hybridizing with mRNA transcribed from either Watson or Crick strand on the Northern blot in A are underlined.

Figure 6

Amount of the SOU1 transcript is inversely related to the amount of CSU transcript. Co-overexpression of SOU1 with CSU51 from a low copy number plasmid (Figure 2, diagram 3) decreases the SOU1 transcript, while increasing, as expected, the CSU51 transcript, as determined with Northern blot analysis. The recipient CAF4-2 cells carry the following plasmids: (1) negative control pRC2312, an empty vector; (2) positive control pCA88 overexpressing SOU1; and (3) pAK65 co-overexpressing SOU1 with region A carrying a natural CSU51/ASU51 configuration in which CSU51 always dominates ASU51. Hybridization signals were obtained with total RNAs and dsDNA probes prepared from SOU1 or CSU51, as indicated. Normalization for the amounts of loaded RNA was done with the same blot using dsDNA probe prepared from 18S rDNA. Note the decrease of the SOU1 signal from pAK65, as compared to pCA88.