The Rab7 C-terminal intrinsically disordered region is a key determinant of development and pathogenicity in Aspergillus flavus.

The Conserved N-Terminal Extension of AtKEA1 Is Largely Dispensable for Plastid Function but Contributes to Potassium Homeostasis

An evolutionarily ancient regulatory module is uncovered that shapes the molecular organization and function of IE KEAs, advancing the understanding of plastid ion and pH homeostasis and plastid ribosome integrity.

Functional dissection of velvet DNA-binding region in Aspergillus nidulans VelB uncovers an essential conserved arginine cluster

Abstract Velvet regulators, characterized by a conserved velvet domain, function as central hubs that coordinately govern fungal development, secondary metabolism, stress adaptation, and pathogenicity. The velvet domain is organized into an N-terminal DNA-binding region of approximately 30 amino acids and a C-terminal dimerization region of approximately 100 amino acids. In this study, Aspergillus nidulans VelB was used as a paradigm to systematically dissect the velvet DNA-binding region. The three arginine residues R71, R80, and R81 in the N-terminal velvet domain that are indispensable for VelB function were identified through alanine-scanning mutagenesis of 15 conserved residues. Alanine substitutions at these positions caused severe defects in long-term spore viability, sexual development, and secondary metabolism. Further comparative characterization of electrostatic-potential dynamics pre- and post-mutation revealed that the three individual substitutions markedly attenuated local electrostatic potential across the DNA-binding interface. Notably, these mutations drove comprehensive remodeling of the protein’s electrostatic properties, whereby electrostatic perturbations propagated across the entire protein exterior. Analysis of 4,999 velvet-domain sequences across the fungal kingdom revealed extraordinary conservation of these positions: arginine was present at position 71 in 85% of sequences, at position 80 in 91%, and at position 81 in 84%. Cross-kingdom complementation experiments further demonstrated that the wild-type velvet DNA-binding region from Capsaspora owczarzaki, a unicellular holozoan lacking the equivalent of R71, failed to rescue the A. nidulans velB deletion phenotype, whereas the introduction of arginine at this position conferred substantial functional restoration. These findings establish that a cluster of conserved arginine residues generates the positive electrostatic surface potential required for velvet–DNA interaction and define the molecular basis of DNA recognition by this ancient family of fungal transcription factors.

A single Omicron mutation reshapes ORF3a-driven host-cell remodelling

Genetic dissection of human ABCE1 in yeast reveals separable requirements for ribosome recycling and suppression of aberrant reinitiation

The ATP‐binding cassette E1 (ABCE1) protein is an essential factor for ribosome recycling, splitting post‐termination ribosomes into subunits for subsequent rounds of translation. Despite high conservation, human ABCE1 (hABCE1) fails to functionally complement the depletion of its essential yeast homolog, Rli1. In this study, we leveraged this species‐specificity to dissect the functional architecture of ABCE1. Through analysis of yeast–human chimeric proteins, we identified the N‐terminal nucleotide‐binding domain (NBD1) as the primary determinant of this incompatibility. To further investigate, we isolated multiple hABCE1 point mutants (revertants) that successfully restored yeast growth. We then developed a novel dual‐luciferase reporter assay to quantify aberrant translation reinitiation in the 3′UTR, an event recognized as a direct consequence of ABCE1 deficiency. Notably, while the revertant mutants rescued yeast viability, they failed to suppress aberrant reinitiation, exhibiting levels equivalent to the nonfunctional hABCE1. This genetic uncoupling of viability from the suppression of reinitiation suggests that the canonical ribosome recycling function required for cell growth and the role in preventing aberrant reinitiation have distinct functional thresholds or are genetically separable aspects of ABCE1 activity.

Distinct STRIPAK subunits drive conserved and subunit-specific signaling programs in Cryptococcus neoformans

ABSTRACT The striatin-interacting phosphatase and kinase (STRIPAK) complex is a conserved protein phosphatase 2A (PP2A)-associated signaling hub that integrates kinase-phosphatase networks, yet its roles in human fungal pathogens remain poorly defined. Here, we dissected STRIPAK functions in the opportunistic pathogen Cryptococcus neoformans by combining genetic, genomic, virulence, and phosphoproteomic analyses across mutants lacking individual STRIPAK subunits. Loss of the core STRIPAK components via PPH22, FAR8, FAR9, or FAR11 mutations caused severe defects in growth, stress adaptation, cell cycle progression, and morphogenesis, accompanied by widespread aneuploidy and genome instability. In murine infection models, far11Δ strains were avirulent, whereas far9Δ mutants caused delayed but ultimately fatal disease and underwent host-associated genome remodeling, with recovered isolates exhibiting chromosome 11 amplification despite no consistent in vitro fitness advantage. In contrast, deletion of MOB3 produced a hypervirulent phenotype. mob3Δ cells exhibited enhanced transmigration across an in vitro blood-brain barrier model, increased survival in macrophages, and generated small-cell morphotypes, features associated with increased dissemination. Phosphoproteomic profiling revealed extensive and overlapping phosphorylation changes among core STRIPAK mutants, affecting pathways involved in signaling, cytoskeletal, cell cycle control, chromatin regulation, RNA metabolism, and stress responses. Conversely, mob3Δ mutants displayed a smaller, largely distinct phosphoproteomic signature. Network and functional enrichment analyses highlighted STRIPAK-dependent regulation of TORC2-associated signaling, MAPK/GTPase signaling, autophagy, nuclear transport, RNA processing, DNA replication, and ribosome biogenesis. Together, these findings establish STRIPAK as a coordinator of genome stability, morphological plasticity, stress adaptation, and virulence in C. neoformans, and demonstrate that individual STRIPAK subunits drive shared yet divergent signaling outputs that shape host-pathogen interactions. IMPORTANCE Fungal pathogens must rapidly adapt their growth, morphology, and stress responses to survive within the host, requiring precise coordination of cellular signaling pathways. The conserved striatin-interacting phosphatase and kinase (STRIPAK) complex controls key developmental programs in eukaryotes, but its roles in fungal pathogenesis are not fully defined. We previously showed that STRIPAK is important for genome stability, development, and virulence in the opportunistic human fungal pathogen Cryptococcus neoformans. Here, we define how individual STRIPAK subunits differentially regulate fungal morphogenesis, genome plasticity, host adaptation, and virulence, revealing both shared and subunit-specific functions within this conserved signaling complex. Core STRIPAK mutants exhibit severe growth and stress-response defects and attenuation of virulence, whereas loss of the Mob3 subunit promotes hypervirulence by enhancing dissemination and persistence within the host. Phosphoproteomic profiling reveals that individual STRIPAK components exert shared yet distinct control over phosphorylation networks that shape host-pathogen interactions, establishing STRIPAK as a central signaling hub and a potential target for antifungal intervention. Fungal pathogens must rapidly adapt their growth, morphology, and stress responses to survive within the host, requiring precise coordination of cellular signaling pathways. The conserved striatin-interacting phosphatase and kinase (STRIPAK) complex controls key developmental programs in eukaryotes, but its roles in fungal pathogenesis are not fully defined. We previously showed that STRIPAK is important for genome stability, development, and virulence in the opportunistic human fungal pathogen Cryptococcus neoformans. Here, we define how individual STRIPAK subunits differentially regulate fungal morphogenesis, genome plasticity, host adaptation, and virulence, revealing both shared and subunit-specific functions within this conserved signaling complex. Core STRIPAK mutants exhibit severe growth and stress-response defects and attenuation of virulence, whereas loss of the Mob3 subunit promotes hypervirulence by enhancing dissemination and persistence within the host. Phosphoproteomic profiling reveals that individual STRIPAK components exert shared yet distinct control over phosphorylation networks that shape host-pathogen interactions, establishing STRIPAK as a central signaling hub and a potential target for antifungal intervention.

The AAA+ chaperone ClpB contributes to stress tolerance and pathogenesis in Mycoplasma bovis.

ClpB, an ATP-dependent molecular chaperone belonging to the Hsp100/Clp subfamily of AAA+ ATPases, plays a crucial role in protein disaggregation, thereby enhancing bacterial survival under stress conditions. Despite its well-conserved function in prokaryotes, the specific contributions of ClpB to the pathogenesis of the ruminant pathogen Mycoplasma bovis remain largely unexplored. In this study, we identified and functionally characterized a ClpB homolog in M. bovis. Biochemical assays confirmed that the recombinant ClpB protein exhibits intrinsic ATPase activity and, in cooperation with the DnaK chaperone system, efficiently mediates protein disaggregation in vitro. Through genome-wide transposon mutagenesis of the M. bovis HB0801 strain, we generated ClpB-deficient mutants that maintained normal growth kinetics and morphology at 37 °C but exhibited significant growth defects under thermal and oxidative stress conditions. Phenotypic analysis demonstrated that ClpB disruption attenuated key virulence traits, including impaired adhesion to host cells, marked reduction in biofilm formation, diminished pro-inflammatory cytokines (IL-1β, IL-6, TNF-α) expression in BoMac cells. Furthermore, the reduced virulence of the ClpB mutant was investigated by DIA proteomic analyses, which revealed that the ClpB mutant strain altered distinct protein expression patterns related to proteostasis, including phosphotransferase system, serine-type peptidase activity, serine hydrolase activity, and chaperone-mediated protein folding that contribute to the stress response and virulence. These findings collectively demonstrate that ClpB serves as a multifunctional virulence determinant in M. bovis, orchestrating stress adaptation, host-pathogen interactions, and pathogenic potential through modulation of both protein quality control systems and virulence-associated pathways.