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1. 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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2. Cause and Effectors: Whole-Genome Comparisons Reveal Shared but Rapidly Evolving Effector Sets among Host-Specific Plant-Castrating Fungi

   https://doi.org/10.1128/mBio.02391-19  

   Beckerson, William C. ; Rodríguez de la Vega, Ricardo C. ; Hartmann, Fanny E. ; Duhamel, Marine ; Giraud, Tatiana ; Perlin, Michael H. ; Taylor, John W. ( December 2019 , mBio)

   ABSTRACT Plant pathogens utilize a portfolio of secreted effectors to successfully infect and manipulate their hosts. It is, however, still unclear whether changes in secretomes leading to host specialization involve mostly effector gene gains/losses or changes in their sequences. To test these hypotheses, we compared the secretomes of three host-specific castrating anther smut fungi ( Microbotryum ), two being sister species. To address within-species evolution, which might involve coevolution and local adaptation, we compared the secretomes of strains from differentiated populations. We experimentally validated a subset of signal peptides. Secretomes ranged from 321 to 445 predicted secreted proteins (SPs), including a few species-specific proteins (42 to 75), and limited copy number variation, i.e., little gene family expansion or reduction. Between 52% and 68% of the SPs did not match any Pfam domain, a percentage that reached 80% for the small secreted proteins, indicating rapid evolution. In comparison to background genes, we indeed found SPs to be more differentiated among species and strains, more often under positive selection, and highly expressed in planta ; repeat-induced point mutations (RIPs) had no role in effector diversification, as SPs were not closer to transposable elements than background genes and were not more RIP affected. Our study thus identified both conserved core proteins, likely required for the pathogenic life cycle of all Microbotryum species, and proteins that were species specific or evolving under positive selection; these proteins may be involved in host specialization and/or coevolution. Most changes among closely related host-specific pathogens, however, involved rapid changes in sequences rather than gene gains/losses. IMPORTANCE Plant pathogens use molecular weapons to successfully infect their hosts, secreting a large portfolio of various proteins and enzymes. Different plant species are often parasitized by host-specific pathogens; however, it is still unclear whether the molecular basis of such host specialization involves species-specific weapons or different variants of the same weapons. We therefore compared the genes encoding secreted proteins in three plant-castrating pathogens parasitizing different host plants, producing their spores in plant anthers by replacing pollen. We validated our predictions for secretion signals for some genes and checked that our predicted secreted proteins were often highly expressed during plant infection. While we found few species-specific secreted proteins, numerous genes encoding secreted proteins showed signs of rapid evolution and of natural selection. Our study thus found that most changes among closely related host-specific pathogens involved rapid adaptive changes in shared molecular weapons rather than innovations for new weapons. 

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3. Duplications in corneous beta protein genes and the evolution of gecko adhesion

   https://doi.org/doi:10.1093/icb/icz010  

   Gamble, T. ( January 2019 , Integrative and comparative biology)

   Corneous proteins are an important component of the tetrapod integument. Duplication and diversification of keratins and associated proteins are linked with the origin of most novel integumentary structures like mammalian hair, avian feathers, and scutes covering turtle shells. Accordingly, the loss of integumentary structures often coincides with the loss of genes encoding keratin and associated proteins. For example, many hair keratins in dolphins and whales have become pseudogenes. The adhesive setae of geckos and anoles are composed of both intermediate filament keratins (IF-keratins, formerly known as alpha-keratins) and corneous beta-proteins (CBPs, formerly known as beta-keratins) and recent whole genome assemblies of two gecko species and an anole uncovered duplications in seta-specific CBPs in each of these lineages. While anoles evolved adhesive toepads just once, there are two competing hypotheses about the origin(s) of digital adhesion in geckos involving either a single origin or multiple origins. Using data from three published gecko genomes, I examine CBP gene evolution in geckos and find support for a hypothesis where CBP gene duplications are associated with the repeated evolution of digital adhesion. Although these results are preliminary, I discuss how additional gecko genome assemblies, combined with phylogenies of keratin and associated protein genes and gene duplication models, can provide rigorous tests of several hypotheses related to gecko CBP evolution. This includes a taxon sampling strategy for sequencing and assembly of gecko genomes that could help resolve competing hypotheses surrounding the origin(s) of digital adhesion. 

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4. Genetic basis for probiotic yeast phenotypes revealed by nanopore sequencing

   https://doi.org/10.1093/g3journal/jkad093  

   Collins, Joseph H ; Kunyeit, Lohith ; Weintraub, Sarah ; Sharma, Nilesh ; White, Charlotte ; Haq, Nabeeha ; Anu-Appaiah, K A ; Rao, Reeta P ; Young, Eric M ( April 2023 , G3: Genes, Genomes, Genetics)

   Whiteman, N (Ed.)

   Abstract Probiotic yeasts are emerging as preventative and therapeutic solutions for disease. Often ingested via cultured foods and beverages, they can survive the harsh conditions of the gastrointestinal tract and adhere to it, where they provide nutrients and inhibit pathogens like Candida albicans. Yet, little is known of the genomic determinants of these beneficial traits. To this end, we have sequenced 2 food-derived probiotic yeast isolates that mitigate fungal infections. We find that the first strain, KTP, is a strain of Saccharomyces cerevisiae within a small clade that lacks any apparent ancestry from common European/wine S. cerevisiae strains. Significantly, we show that S. cerevisiae KTP genes involved in general stress, pH tolerance, and adherence are markedly different from S. cerevisiae S288C but are similar to the commercial probiotic yeast species S. boulardii. This suggests that even though S. cerevisiae KTP and S. boulardii are from different clades, they may achieve probiotic effect through similar genetic mechanisms. We find that the second strain, ApC, is a strain of Issatchenkia occidentalis, one of the few of this family of yeasts to be sequenced. Because of the dissimilarity of its genome structure and gene organization, we infer that I. occidentalis ApC likely achieves a probiotic effect through a different mechanism than the Saccharomyces strains. Therefore, this work establishes a strong genetic link among probiotic Saccharomycetes, advances the genomics of Issatchenkia yeasts, and indicates that probiotic activity is not monophyletic and complimentary mixtures of probiotics could enhance health benefits beyond a single species. 

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5. Arabidopsis Abscisic Acid Repressor 1 is a susceptibility hub that interacts with multiple Pseudomonas syringae effectors

   https://doi.org/10.1111/tpj.15110  

   Schreiber, Karl J. ; Hassan, Jana A. ; Lewis, Jennifer D. ( January 2021 , The Plant Journal)

   Pathogens secrete effector proteins into host cells to suppress host immunity and promote pathogen virulence, although many features at the molecular interface of host–pathogen interactions remain to be characterized. In a yeast two‐hybrid assay, we found that thePseudomonas syringaeeffector HopZ1a interacts with the Arabidopsis transcriptional regulator Abscisic Acid Repressor 1 (ABR1). Further analysis revealed that ABR1 interacts with multipleP. syringaeeffectors, suggesting that it may be targeted as a susceptibility hub. Indeed, loss‐of‐functionabr1mutants exhibit reduced susceptibility to a number ofP. syringaestrains. The ABR1 protein comprises a conserved APETALA2 (AP2) domain flanked by long regions of predicted structural disorder. We verified the DNA‐binding activity of the AP2 domain and demonstrated that the disordered domains act redundantly to enhance DNA binding and to facilitate transcriptional activation by ABR1. Finally, we compared gene expression profiles from wild‐type andabr1plants following inoculation withP. syringae, which suggested that the reduced susceptibility ofabr1mutants is due to the loss of a virulence target rather than an enhanced immune response. These data highlight ABR1 as a functionally important component at the host–pathogen interface.

    

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