Partial Decay of Thiamine Signal Transduction Pathway Alters Growth Properties of Candida glabrata

Abstract

The phosphorylated form of thiamine (Vitamin B1), thiamine pyrophosphate (TPP) is essential for the metabolism of amino acids and carbohydrates in all organisms. Plants and microorganisms, such as yeast, synthesize thiamine de novo whereas animals do not. The thiamine signal transduction (THI) pathway in Saccharomyces cerevisiae is well characterized. The ~10 genes required for thiamine biosynthesis and uptake are transcriptionally upregulated during thiamine starvation by THI2, THI3, and PDC2. Candida glabrata, a human commensal and opportunistic pathogen, is closely related to S. cerevisiae but is missing half of the biosynthetic pathway, which limits its ability to make thiamine. We investigated the changes to the THI pathway in C. glabrata, confirming orthologous functions. We found that C. glabrata is unable to synthesize the pyrimidine subunit of thiamine as well as the thiamine precursor vitamin B6. In addition, THI2 (the gene encoding a transcription factor) is not present in C. glabrata, indicating a difference in the transcriptional regulation of the pathway. Although the pathway is upregulated by thiamine starvation in both species, C. glabrata appears to upregulate genes involved in thiamine uptake to a greater extent than S. cerevisiae. However, the altered regulation of the THI pathway does not alter the concentration of thiamine and its vitamers in the two species as measured by HPLC. Finally, we demonstrate potential consequences to having a partial decay of the THI biosynthetic and regulatory pathway. When the two species are co-cultured, the presence of thiamine allows C. glabrata to rapidly outcompete S. cerevisiae, while absence of thiamine allows S. cerevisiae to outcompete C. glabrata. This simplification of the THI pathway in C. glabrata suggests its environment provides thiamine and/or its precursors to cells, whereas S. cerevisiae is not as reliant on environmental sources of thiamine.

A NovelcisElement Achieves the Same Solution as an AncestralcisElement During Thiamine Starvation inCandida glabrata

“…(Nosaka et al 2012). Additionally, while deletion of THI2 removes the majority of expression of THI promoters, there is still some induction in the absence of Thi2 ( Figure S6), and overexpression of THI3 can compensate for the loss of THI2 presumably by making Pdc2 fully active while in a complex with Thi3 (Iosue et al 2016). However, loss of PDC2 removes all induction in response to thiamine starvation, suggesting Pdc2 is core to the transcriptional response.…”

“…Thi2 dependence -CgPMU3 UAS with ScThi2 is different from the THI UAS with ScThi2 The data in Figure 6 with the switching of the UAS elements suggest that the CgPMU3 promoter fundamentally behaves differently from the other THI promoters, but the THI and CgPMU3 promoters are still dependent on the two known transcriptional regulators CgPdc2 and CgThi3 (Iosue et al 2016). Because CgPMU3 evolved recently, and likely did not experience selective pressures from the ancestral Thi2, we hypothesized that it may behave differently in a setting where Thi2 is important.…”

“…Most of the experiments were performed in C. glabrata wild-type (Cormack and Falkow 1999) and S. cerevisiae wild-type (Wykoff and O'Shea 2001) strains. Additional strains used in this study were deletions of the thiamine pathway regulators: Cgthi3Δ (DG141), and Cgpdc2Δ (DG271), Scthi2Δ (DC126), Scthi3Δ (DC143) (Iosue et al 2016) and Scpdc2Δ, which was generated in this this study. Because PDC2 is essential in glucose-containing medium in S. cerevisiae, NATMX6 was amplified using PCR (primers in Supplemental Material, Table S1) and transformed into a diploid strain to delete ScPDC2.…”

“…We previously identified that C. glabrata, unlike most yeast species, is auxotrophic for thiamine because of a partial loss of the biosynthetic pathway, but it still upregulates 5 genes involved in biosynthesis and scavenging (CgTHI4,CgTHI20,CgTHI10,CgPET18,and CgPMU3) .50 fold in response to thiamine starvation (Iosue et al 2016;Nahas et al 2018). Similar to S. cerevisiae, this upregulation is dependent on the DNA binding protein, Pdc2 and its regulator, Thi3 (Iosue et al 2016;Nahas et al 2018).…”

“…We previously identified that C. glabrata, unlike most yeast species, is auxotrophic for thiamine because of a partial loss of the biosynthetic pathway, but it still upregulates 5 genes involved in biosynthesis and scavenging (CgTHI4,CgTHI20,CgTHI10,CgPET18,and CgPMU3) .50 fold in response to thiamine starvation (Iosue et al 2016;Nahas et al 2018). Similar to S. cerevisiae, this upregulation is dependent on the DNA binding protein, Pdc2 and its regulator, Thi3 (Iosue et al 2016;Nahas et al 2018). However, C. glabrata lost the transcription factor Thi2, which is necessary for the thiamine starvation response in S. cerevisiae and in the ancestor of these yeast species, suggesting that there is some rewiring of how thiamine responsive genes are regulated (Gabaldón Estevan et al 2013;Huerta-Cepas et al 2014) (Figure 1).…”