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1. Pyruvate decarboxylase and thiamine biosynthetic genes are regulated differently by Pdc2 in S. cerevisiae and C. glabrata

   https://doi.org/10.1371/journal.pone.0286744  

   Iosue, Christine L. ; Ugras, Julia M. ; Bajgain, Yakendra ; Dottor, Cory A. ; Stauffer, Peyton L. ; Hopkins, Rachael A. ; Lang, Emma C. ; Wykoff, Dennis D. ( June 2023 , PLOS ONE)

   Misra, Hari S. (Ed.)

   Understanding metabolism in the pathogen Candida glabrata is key to identifying new targets for antifungals. The thiamine biosynthetic (THI) pathway is partially defective in C . glabrata , but the transcription factor Cg Pdc2 upregulates some thiamine biosynthetic and transport genes. One of these genes encodes a recently evolved thiamine pyrophosphatase ( CgPMU3 ) that is critical for accessing external thiamine. Here, we demonstrate that Cg Pdc2 primarily regulates THI genes. In Saccharomyces cerevisiae , Pdc2 regulates both THI and pyruvate decarboxylase (PDC) genes, with PDC proteins being a major thiamine sink. Deletion of PDC2 is lethal in S . cerevisiae in standard growth conditions, but not in C . glabrata . We uncover cryptic cis elements in C . glabrata PDC promoters that still allow for regulation by Sc Pdc2, even when that regulation is not apparent in C . glabrata . C . glabrata lacks Thi2, and it is likely that inclusion of Thi2 into transcriptional regulation in S . cerevisiae allows for a more complex regulation pattern and regulation of THI and PDC genes. We present evidence that Pdc2 functions independent of Thi2 and Thi3 in both species. The C-terminal activation domain of Pdc2 is intrinsically disordered and critical for species differences. Truncation of the disordered domains leads to a gradual loss of activity. Through a series of cross species complementation assays of transcription, we suggest that there are multiple Pdc2-containing complexes, and C . glabrata appears to have the simplest requirement set for THI genes, except for CgPMU3 . CgPMU3 has different cis requirements, but still requires Pdc2 and Thi3 to be upregulated by thiamine starvation. We identify the minimal region sufficient for thiamine regulation in CgTHI20 , CgPMU3 , and ScPDC5 promoters. Defining the cis and trans requirements for THI promoters should lead to an understanding of how to interrupt their upregulation and provide targets in metabolism for antifungals. 

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2. Independent Mechanisms for Acquired Salt Tolerance versus Growth Resumption Induced by Mild Ethanol Pretreatment in Saccharomyces cerevisiae

   https://doi.org/10.1128/mSphere.00574-18  

   McDaniel, Elizabeth A. ; Stuecker, Tara N. ; Veluvolu, Manasa ; Gasch, Audrey P. ; Lewis, Jeffrey A. ; Mitchell, Aaron P. ( December 2018 , mSphere)

   ABSTRACT All living organisms must recognize and respond to various environmental stresses throughout their lifetime. In natural environments, cells frequently encounter fluctuating concentrations of different stressors that can occur in combination or sequentially. Thus, the ability to anticipate an impending stress is likely ecologically relevant. One possible mechanism for anticipating future stress is acquired stress resistance, where cells preexposed to a mild sublethal dose of stress gain the ability to survive an otherwise lethal dose of stress. We have been leveraging wild strains of Saccharomyces cerevisiae to investigate natural variation in the yeast ethanol stress response and its role in acquired stress resistance. Here, we report that a wild vineyard isolate possesses ethanol-induced cross protection against severe concentrations of salt. Because this phenotype correlates with ethanol-dependent induction of the ENA genes, which encode sodium efflux pumps already associated with salt resistance, we hypothesized that variation in ENA expression was responsible for differences in acquired salt tolerance across strains. Surprisingly, we found that the ENA genes were completely dispensable for ethanol-induced survival of high salt concentrations in the wild vineyard strain. Instead, the ENA genes were necessary for the ability to resume growth on high concentrations of salt following a mild ethanol pretreatment. Surprisingly, this growth acclimation phenotype was also shared by the lab yeast strain despite lack of ENA induction under this condition. This study underscores that cross protection can affect both viability and growth through distinct mechanisms, both of which likely confer fitness effects that are ecologically relevant. IMPORTANCE Microbes in nature frequently experience “boom or bust” cycles of environmental stress. Thus, microbes that can anticipate the onset of stress would have an advantage. One way that microbes anticipate future stress is through acquired stress resistance, where cells exposed to a mild dose of one stress gain the ability to survive an otherwise lethal dose of a subsequent stress. In the budding yeast Saccharomyces cerevisiae , certain stressors can cross protect against high salt concentrations, though the mechanisms governing this acquired stress resistance are not well understood. In this study, we took advantage of wild yeast strains to understand the mechanism underlying ethanol-induced cross protection against high salt concentrations. We found that mild ethanol stress allows cells to resume growth on high salt, which involves a novel role for a well-studied salt transporter. Overall, this discovery highlights how leveraging natural variation can provide new insights into well-studied stress defense mechanisms. 

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3. Tup1 Paralog CgTUP11 Is a Stronger Repressor of Transcription than CgTUP1 in Candida glabrata

   https://doi.org/10.1128/msphere.00765-21  

   Bui, Lilian N. ; Iosue, Christine L. ; Wykoff, Dennis D. ( April 2022 , mSphere)

   Mitchell, Aaron P. (Ed.)

   ABSTRACT TUP1 is a well-characterized repressor of transcription in Saccharomyces cerevisiae and Candida albicans and is observed as a single-copy gene. We observe that most species that experienced a whole-genome duplication outside of the Saccharomyces genus have two copies of TUP1 in the Saccharomycotina yeast clade. We focused on Candida glabrata and demonstrated that the uncharacterized TUP1 homolog, C. glabrata TUP11 ( CgTUP11 ), is most like the S. cerevisiae TUP1 ( ScTUP1 ) gene through phenotypic assays and transcriptome sequencing (RNA-seq). Whereas CgTUP1 plays a role in gene repression, it is much less repressive in standard growth media. Through RNA-seq and reverse transcription-quantitative PCR (RT-qPCR), we observed that genes associated with pathogenicity ( YPS2 , YPS4 , and HBN1 ) are upregulated upon deletion of either paralog, and loss of both paralogs is synergistic. Loss of the corepressor CgCYC8 mimics the loss of both paralogs, but not to the same extent as the Cgtup1 Δ Cgtup11 Δ mutant for these pathogenesis-related genes. In contrast, genes involved in energy metabolism ( CgHXT2 , CgADY2 , and CgFBP1 ) exhibit similar behavior (dependence on both paralogs), but deletion of CgCYC8 is very similar to the Cgtup1 Δ Cgtup11 Δ mutant. Finally, some genes ( CgMFG1 and CgRIE1 ) appear to only be dependent on CgTUP11 and CgCYC8 and not CgTUP1 . These data indicate separable and overlapping roles for the two TUP1 paralogs and that other genes may function as the Cg Cyc8 corepressor. Through a comparison by RNA-seq of Sctup1 Δ, it was found that TUP1 homologs regulate similar genes in the two species. This work highlights that studies focused only on Saccharomyces may miss important biological processes because of paralog loss after genome duplication. IMPORTANCE Due to a whole-genome duplication, many yeast species related to C. glabrata have two copies of the well-characterized TUP1 gene, unlike most Saccharomyces species. This work identifies roles for the paralogs in C. glabrata , highlights the importance of the uncharacterized paralog, called TUP11 , and suggests that the two paralogs have both overlapping and unique functions. The TUP1 paralogs likely influence pathogenicity based on tup mutants upregulating genes that are associated with pathogenicity. 

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4. An expanded toolkit of drug resistance cassettes for Candida glabrata , Candida auris , and Candida albicans leads to new insights into the ergosterol pathway

   https://doi.org/10.1128/msphere.00311-23  

   Gregor, Justin B. ; Gutierrez-Schultz, Victor A. ; Hoda, Smriti ; Baker, Kortany M. ; Saha, Debasmita ; Burghaze, Madeline G. ; Vazquez, Cynthia ; Burgei, Kendra E. ; Briggs, Scott D. ( December 2023 , mSphere)

   Mitchell, Aaron P. (Ed.)

   The World Health Organization recently published the first list of priority fungal pathogens highlighting multipleCandidaspecies, includingCandida glabrata,Candida albicans, andCandida auris. However, prior studies in these pathogens have been mainly limited to the use of two drug resistance cassettes,NatMXandHphMX, limiting genetic manipulation capabilities in prototrophic laboratory strains and clinical isolates. In this study, we expanded the toolkit forC. glabrata,C. auris, andC. albicansto includeKanMXandBleMXwhen coupled with anin vitroassembled CRISPR-Cas9 ribonucleoprotein (RNP)-based system. Repurposing these drug resistance cassettes forCandida, we were able to make single gene deletions, sequential and simultaneous double gene deletions, epitope tags, and rescue constructs. We applied these drug resistance cassettes to interrogate the ergosterol pathway, a critical pathway for both the azole and polyene antifungal drug classes. Using our approach, we determined for the first time that the deletion ofERG3inC. glabrata,C. auris,andC. albicansprototrophic strains results in azole drug resistance, which further supports the conservation of the Erg3-dependent toxic sterol model. Furthermore, we show that anERG5deletion inC. glabratais azole susceptible at subinhibitory concentrations, suggesting that Erg5 could act as an azole buffer for Erg11. Finally, we identified a synthetic growth defect when bothERG3andERG5are deleted inC. glabrata,which suggests the possibility of another toxic sterol impacting growth. Overall, we have expanded the genetic tools available to interrogate complex pathways in prototrophic strains and clinical isolates.

   The increasing problem of drug resistance and emerging pathogens is an urgent global health problem that necessitates the development and expansion of tools for studying fungal drug resistance and pathogenesis. Prior studies inCandida glabrata,Candida auris, andCandida albicanshave been mainly limited to the use ofNatMX/SAT1andHphMX/CaHygfor genetic manipulation in prototrophic strains and clinical isolates. In this study, we demonstrated thatNatMX/SAT1, HphMX, KanMX,and/orBleMXdrug resistance cassettes when coupled with a CRISPR-ribonucleoprotein (RNP)-based system can be efficiently utilized for deleting or modifying genes in the ergosterol pathway ofC. glabrata,C. auris, andC. albicans. Moreover, the utility of these tools has provided new insights intoERGgenes and their relationship to azole resistance inCandida. Overall, we have expanded the toolkit forCandidapathogens to increase the versatility of genetically modifying complex pathways involved in drug resistance and pathogenesis.

    

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5. Herbivores disrupt clinal variation in plant responses to water limitation

   https://doi.org/10.1111/1365-2745.14237  

   Diethelm, Aramee C. ; Reichelt, Michael ; Pringle, Elizabeth G. ( December 2023 , Journal of Ecology)

   Plasticity in plant traits, including secondary metabolites, is critical to plant survival and competitiveness under stressful conditions. The ability of a plant to respond effectively to combined stressors can be impacted by crosstalk in biochemical pathways, resource availability and evolutionary history, but such responses remain underexplored. In particular, we know little about intraspecific variation in response to combined stressors or whether such variation is associated with the stress history of a given population.

   Here, we investigated the consequences of combined water and herbivory stress for plant traits, including relative growth rate, leaf morphology and various measures of phytochemistry, using a common garden ofAsclepias fascicularismilkweeds. To examine how plant trait means and plasticities depend on the history of environmental stress, seeds for the experiment were collected from across a gradient of aridity in the Great Basin, United States. We then conducted a factorial experiment crossing water limitation with herbivory.

   Plants responded to water limitation alone by increasing the evenness of UV‐absorbent secondary metabolites and to herbivory alone by increasing the richness of metabolites. However, plants that experienced combined water and herbivory stress exhibited similar phytochemical diversity to well‐watered control plants. This lack of plasticity in phytochemical diversity in plants experiencing combined stressors was associated with a reduction in relative growth rates.

   Leaf chemistry means and plasticities exhibited clinal variation corresponding to seed source water deficits. The total concentration of UV‐absorbent metabolites decreased with increasing water availability among seed sources, driven by higher concentrations of flavonol glycosides, which are hypothesized to act as antioxidants, among plants from drier sites. Plants sourced from drier sites exhibited higher plasticity in flavonol glycoside concentrations in response to water limitation, which increased phytochemical evenness, but simultaneous herbivory dampened plant responses to water limitation irrespective of seed source.

   Synthesis. These results suggest that climatic history can affect intraspecific phytochemical plasticity, which may confer tolerance to water limitation, but that co‐occurring herbivory disrupts such patterns. Global change is increasing the frequency and intensity of stress combinations, such that understanding intraspecific responses to combined stressors is critical for predicting the persistence of plant populations.

    

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