PubMed · 2679922
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1989. Yeast genetic engineering.. https://pubmed.ncbi.nlm.nih.gov/2679922/
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BACKGROUND: During host colonization, fungal plant pathogens secrete effector-like proteins that alter host cell physiology and target plant-associated microbes. However, rapid evolution and low sequence conservation hinder the study and characterization of these proteins. The fungus Zymoseptoria passerinii infects Hordeum spp. and includes lineages adapted to wild and domesticated barley. To date, the evolution of effector-like proteins in this species has not been addressed. RESULTS: We combined multiple structure-based and network analyses to unravel the secretome of Z. passerinii. We first compared AlphaFold2 and ESMFold predictions to establish the baseline for structural analyses. We identified 72 structural clusters in the secretome, revealing fold-level relationships across divergent sequences. We showed that effector-like proteins with predicted host immune-interfering functions evolved from a limited group of protein folds, whereas proteins with predicted antimicrobial properties were distributed across fold groups. Physicochemical comparisons indicate that putative antimicrobial effectors predominantly emerged through amino acid replacements on common effector-enriched scaffolds in Z. passerinii, reconfiguring surface charge and electrostatics. We analyzed intra- and interspecific variation in selected effector-enriched families by comparing Z. passerinii proteins and homologs across the genus Zymoseptoria. We describe constrained core folds, with local variation in loop and surface-exposed regions, consistent with fold stability while still enabling protein diversification. We further report that putative antimicrobial effector homologs are broadly distributed across the genus despite sequence divergence. CONCLUSIONS: The secretome of Z. passerinii is organized around common structural folds that support diverse biological roles, including host manipulation and host-associated microbial interactions. Conserved scaffolds combined with surface and physicochemical variation likely contribute to rapid adaptive evolution of effector-like proteins in Z. passerinii.
The ability to transition between yeast and filamentous growth states is critical for virulence of the leading human fungal pathogen Candida albicans. Large-scale genetic screens have identified hundreds of genes required for this morphological switch, but the mechanisms by which many of these genes orchestrate this developmental transition remain largely elusive. In this study, we characterized the role of Ent2 in governing morphogenesis in C. albicans. We showed that Ent2 is required for filamentous growth under a wide range of inducing conditions and is also required for virulence in a mouse model of systemic candidiasis. We found that the epsin N-terminal homology (ENTH) domain of Ent2 enables morphogenesis and virulence and does so via a physical interaction with the Cdc42 GTPase-activating protein (GAP) Rga2 and regulation of its localization. Further analyses revealed that overexpression of the Cdc42 effector protein Cla4 can overcome the requirement for the ENTH-Rga2 physical interaction, indicating that Ent2 functions, at least in part, to enable proper activation of the Cdc42-Cla4 signaling pathway in the presence of a filament-inducing cue. Overall, this work characterizes the mechanism by which Ent2 regulates hyphal morphogenesis in C. albicans, unveils the importance of this factor in enabling virulence in an in vivo model of systemic candidiasis and adds to the growing understanding of the genetic control of a key virulence trait. IMPORTANCE Candida albicans is a leading human fungal pathogen that can cause life-threatening infections in immunocompromised individuals, with mortality rates of ~40%. The ability of this organism to grow in both yeast and filamentous forms is critical for the establishment of systemic infection. Genomic screens have identified many genes required for this morphological transition, yet our understanding of the mechanisms that regulate this key virulence trait remains incomplete. In this study, we characterized Ent2 as a core regulator of C. albicans morphogenesis. We show that Ent2 regulates hyphal morphogenesis through an interaction between its ENTH domain and the Cdc42 GAP, Rga2, which signals through the Cdc42-Cla4 signaling pathway. Finally, we show that the Ent2 protein, and specifically its ENTH domain, is required for virulence in a mouse model of systemic candidiasis. Overall, this work identifies Ent2 as a key regulator of filamentation and virulence in C. albicans.
In wild-type Hansenula polymorpha the proliferation of peroxisomes in induced by various unconventional carbon- and nitrogen sources. Highest induction levels, up to 80% of the cytoplasmic volume, are observed in cells grown in methanol-limited chemostat cultures. Based on our accumulated experience, we are now able to precisely adjust both the level of the peroxisome induction as well as their protein composition by specific adaptations in growth conditions. During the last few years a series of "peroxisome-deficient (per) mutants of H. polymorpha have been isolated and characterized. Phenotypically these mutants are characterized by the fact that they are not able to grow on methanol. Three mutant phenotypes were defined on the basis of morphological criteria, namely: (a) mutants completely lacking peroxisomes (Per-;13 complementation groups); (b) mutants containing few small peroxisomes which are partly impaired in the peroxisomal import of matrix proteins (Pim-; five complementation groups); and (c) mutants with aberrations in the peroxisomal substructure (Pss-; two complementation groups). In addition, several conditional Per-, Pim- and Pss- mutants have been obtained. In all cases the mutant phenotype was shown to be caused by a recessive mutation in one gene. However, we observed that different mutations in one gene may cause different morphological mutant phenotypes. A detailed genetic analysis revealed that several PER genes, essential for peroxisome biogenesis, are tightly linked and organized in a hierarchical fashion. The use of both constitual and conditional per mutants in current and future studies of the molecular mechanisms controlling peroxisome biogenesis and function is discussed.