Alspaugh, A. | bioRxiv Science

bioRxiv2024

Evolutionary unique N-glycan-dependent protein quality control system plays pivotal roles in cellular fitness and extracellular vesicle transport in Cryptococcus neoformans

A conserved N-glycan-dependent endoplasmic reticulum protein quality control (ERQC) system has evolved in eukaryotes to ensure accuracy during glycoprotein folding. The human pathogen Cryptococcus neoformans possesses a unique N-glycosylation pathway that affects microbial physiology and interactions with the infected host. To investigate the molecular features and functions of the ERQC system in C. neoformans, we characterized a set of mutants with deletion of genes coding for the ERQC sensor UDP-glucose:glycoprotein glucosyltransferase (UGG1) and putative 1,2-mannose-trimming enzymes (MNS1, MNS101, MNL1, and MNL2). The ugg1{Delta}, mns1{Delta}, mns101{Delta}, and mns1{Delta}101{Delta} mutants showed alterations in N-glycan profiles, defective cell surface organization, decreased survival in host cells, and varying degrees of reduced in vivo virulence. The ugg1{Delta} strain exhibited severely impaired extracellular secretion of capsular polysaccharides and virulence-related enzymes. Comparative transcriptome analysis showed the upregulation of protein folding, proteolysis, and cell wall remodeling genes, indicative of induced ER stress. However, no apparent changes were observed in the expression of genes involved in protein secretion or capsule biosynthesis. Additionally, extracellular vesicle (EV) analysis combined with proteomic analysis showed significant alterations in the number, size distribution, and cargo composition of EVs in ugg1{Delta}. These findings highlight the essential role of the functional ERQC system for cellular fitness under adverse conditions and proper EV-mediated transport of virulence factors, which are crucial for the full fungal pathogenicity of C. neoformans.

microbiology↗

bioRxiv2020

Transposon mobilization during infection promotes drug resistance in the human fungal pathogen Cryptococcus deneoformans

When transitioning from the environment, pathogenic microorganisms must adapt rapidly to survive in hostile host conditions. This is especially true for environmental fungi that cause opportunistic infections in immunocompromised patients since these microbes are not well adapted human pathogens. Cryptococcus species are yeast-like fungi that cause lethal infections, especially in HIV-infected patients. Using Cryptococcus deneoformans in a murine model of infection, we examined contributors to drug resistance and demonstrated that transposon mutagenesis drives the development of 5-fluoroorotic acid (5FOA) resistance. Inactivation of target genes URA3 or URA5 primarily reflected the insertion of two transposable elements (TEs): the T1 DNA transposon and the TCN12 retrotransposon. Consistent with in vivo results, increased rates of mutagenesis and resistance to 5FOA and the antifungal drugs rapamycin/FK506 and 5-fluorocytosine (5FC) were found when Cryptococcus was incubated at 37{degrees} compared to 30{degrees} in vitro, a condition that mimics the temperature shift that occurs during the environment-to-host transition. Inactivation of the RNAi pathway, which suppresses TE movement in many organisms, was not sufficient to elevate TE movement at 30{degrees} to the level observed at 37{degrees}. We propose that temperature-dependent TE mobilization in Cryptococcus is an important mechanism that enhances microbial adaptation and promotes pathogenesis and drug resistance in the human host. SIGNIFICANCE STATEMENTThe incidence of infections due to fungal pathogens has dramatically increased in the past few decades with similar increases in human populations with weakened or suppressed immune systems. Understanding the mechanisms by which organisms rapidly adapt during human infection to enhance virulence and evolve drug resistance is important for developing effective treatments. We find that transposon mobilization in the human pathogen Cryptococcus causes genomic mutations in a murine model of infection and promotes resistance to antifungal drugs in vitro. Thermotolerance is a key virulence determinant for pathogenic fungi during the environment-to-host transition, and we demonstrate that a temperature increase is sufficient to trigger transposon mobilization in vitro. The link between temperature stress and transposon-associated mutations may significantly impact adaptation to the host during infection, including the acquisition of drug resistance.

microbiology↗