Biology subjects

Sofras, D.

Publications and source records attributed to Sofras, D..

4 recordsLinked to original sources

bioRxiv2025

Epistasis at the cell surface: what is the role of Erg3 loss-of-function in acquired echinocandin resistance?

Echinocandins, which target the fungal {beta}-1,3-glucan synthase (Fks), are essential for treating invasive fungal infections, yet resistance is increasingly reported. While resistance typically arises through mutations in Fks hotspots, emerging evidence suggests a contributing role of changes in membrane sterol composition due to ERG3 mutations. Here, we present a clinical case of Nakaseomyces glabratus (Candida glabrata) in which combined mutations in ERG3 and FKS2, but not FKS2 alone, appear to confer echinocandin resistance. Integrated analyses reveal a recurrent association between Erg3 loss-of-function and echinocandin resistance mediated by Fks variation across Candida species, but exclude ERG3 loss-of-function as an independent resistance mechanism. Advances in Fks structural biology and insights into echinocandin-Fks interactions support a model of epistatic crosstalk between membrane sterols and Fks function. Understanding this interaction is crucial, as it may underlie not only acquired echinocandin resistance but also the broader development of multidrug resistance across major antifungal drug classes.

molecular biology↗

bioRxiv2025

Survival of the unfittest: clinical emergence of hyper-multidrug-resistant Nakaseomyces glabratus with rare nonfunctional Erg3 and Erg11 and severely impaired fitness.

BackgroundNakaseomyces glabratus (Candida gabrata) poses a significant clinical challenge due to common drug resistance. We report a case of a complicated urinary tract infection (UTI) progressing to prostatitis and urosepsis, with the emergence of a hyper-multidrug-resistant isolate with low stress tolerance, slow growth and a short life span. This study elucidates the genetic mechanisms and phenotypic characteristics underlying antifungal hyper-resistance with strong fitness trade-offs, and explores potential alternative therapies for resistant UTIs. MethodsWhole-genome sequencing was performed to identify resistance-associated mutations and gene knock-out strains were generated to assess the relative impact of putative loss-of-function (LoF) mutations on antifungal resistance, fitness and membrane sterol composition. Drug susceptibility testing of the antibiotic nitroxoline and related compounds was conducted to evaluate it as a therapeutic alternative and study the mechanism of action. FindingsLoss-of-function mutations in ERG3 and ERG11 were identified and linked to the accumulation of 4,14-dimethylzymosterol and lanosterol instead of ergosterol. Engineered ERG3{Delta}+ERG11{Delta} strains recapitulated the clinical isolates hyper-multidrug resistance and associated fitness deficits. While ERG3{Delta} strains showed no resistance but enhanced thermotolerance, ERG11{Delta} and ERG3{Delta}+ERG11{Delta} strains exhibited multidrug resistance with severe fitness trade-offs. Interestingly, ERG3{Delta}+ERG11{Delta} strains showed mild resistance to flucytosine, but an additional FUR1 mutation in the clinical isolate most probably underlies hyper-resistance to flucytosine. The UTI antibiotic nitroxoline demonstrated high antifungal activity against all strains, and the LoF of ERG3 and/or ERG11 induced collateral sensitivity to this drug. Testing of related compounds suggest a mode of action beyond iron chelation. InterpretationThis case demonstrates that hyper-resistant strains of N. glabratus can emerge despite significant fitness costs and persist under prolonged antifungal therapy in specific clinical settings. These findings underscore the importance of vigilant antifungal resistance monitoring and highlight nitroxoline as a promising alternative treatment for complicated fungal UTIs. These results challenge the notion that strains with fitness deficits are clinically irrelevant and emphasize the need for novel therapeutic strategies including repurposed agents.

microbiology↗

bioRxiv2025

A comparative evaluation of CRISPR-Cas9 allele editing systems in Candida auris: challenging research in a challenging bug

Candida auris is an emergent fungal pathogen of significant interest for molecular research because of its unique nosocomial persistence, high stress tolerance and common multidrug resistance. To investigate the molecular mechanisms of these or other phenotypes, a handful of CRISPR-Cas9 based allele editing tools have been optimized for C. auris. Nonetheless, allele editing in this species remains a significant challenge, and different systems have different advantages and disadvantages. In this work, we compare four systems to introduce the genetic elements necessary for the production of Cas9 and the guide RNA molecule in the genome of C. auris, replacing the ENO1, LEU2 and HIS1 loci respectively, while the fourth system makes use of an episomal plasmid. We observed that the editing efficiency of all four systems was significantly different and strain dependent. Alarmingly, we did not detect correct integration of linear CRISPR cassette constructs in integration-based systems, in over 4,900 screened transformants. Still, all transformants, whether correctly edited or not, grew on selective nourseothricin media, suggesting common random ectopic integration of the CRISPR cassette. Although the plasmid-based system showed a low transformation success compared to the other systems, it has the highest editing efficiency with 41.9% correct transformants on average. In an attempt to improve editing efficiencies of integration-based systems by silencing the non-homologous end joining (NHEJ) DNA repair pathway, we deleted two main NHEJ factors, KU70 and LIG4. However, no improved editing or targeting efficiencies were detected in ku7011, lig411, or ku7011/lig411 backgrounds. Our research highlights important challenges in precise genome editing of C. auris and sheds light on the advantages and limitations of several methods with the aim to guide scientists in selecting the most appropriate tool for molecular work in this enigmatic fungal pathogen. Author summaryCandida auris is a rapidly emerging fungal pathogen that poses serious challenges to global healthcare. Understanding the genetic mechanisms that underlie its nosocomial persistence, virulence, multidrug resistance and other traits is essential for developing new treatments and preventing the spread and burden of C. auris infections. However, precise genetic manipulation in C. auris has proven difficult due to inefficient genome editing tools. This study compares four different CRISPR-based allele editing systems in C. auris, identifying their strengths and limitations. The findings provide crucial insights into selecting the best tools for genetic research in C. auris, guiding future efforts to combat this formidable pathogen.

microbiology↗

bioRxiv2024

Functional Redundancy in Candida auris Cell Surface Adhesins Crucial for Cell-Cell Interaction and Aggregation

Candida auris is an emerging nosocomial fungal pathogen associated with life-threatening invasive disease due to its persistent colonization, high level of transmissibility and multi-drug resistance. Aggregative and non-aggregative growth phenotypes for C. auris strains with different biofilm forming abilities, drug susceptibilities and virulence characteristics have been described. Using comprehensive transcriptional analysis we identified key cell surface adhesins that were highly upregulated in the aggregative phenotype during in vitro and in vivo grown biofilms using a mouse model of catheter infection. Phenotypic and functional evaluations of generated null mutants demonstrated crucial roles for the adhesins Als5 and Scf1 in mediating cell-cell adherence, coaggregation and biofilm formation. While individual mutants were largely non-aggregative, in combination cells were able to co-adhere and aggregate, as directly demonstrated by measuring cell adhesion forces using single-cell atomic force spectroscopy. This co-adherence indicates their role as complementary adhesins, which despite their limited similarity, may function redundantly to promote cell-cell interaction and biofilm formation. Functional diversity of cell wall proteins may be a form of regulation that provides the aggregative phenotype of C. auris with flexibility and rapid adaptation to the environment, potentially impacting persistence and virulence.

microbiology↗