Evolutionary unique N-glycan-dependent protein quality control system plays pivotal roles in cellular fitness and extracellular vesicle transport in Cryptococcus neoformans
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eLife Assessment
This important study confirms the molecular function of putative components of the N-glycan-dependent endoplasmic reticulum protein quality control (ERQC) system in the pathogen Cryptococcus neoformans. The study demonstrates an involvement in fitness, virulence, and the secretion and composition of extracellular vesicles, albeit in ways that are not yet fully understood. The evidence provided is largely convincing, with rigorous well-controlled assays and the use of complemented strains.
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Abstract
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 lacks the glucose addition step to the core N -glycan precursors in the ER but includes additional basidiomycetes-specific mannosidases. 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 α1,2-mannose trimming enzymes ( MNS1 , MNS101 , MNL1 , and MNL2 ). The ugg1 Δ, mns1 Δ, mns101 Δ, and mns1 Δ 101 Δ 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 Δ 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, which is indicative of induced ER stress in ugg1 Δ. 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 Δ. These findings highlight the essential role of the functional ERQC system for cellular fitness under adverse conditions and proper EV-mediated transport of virulence bags, which are crucial for the full fungal pathogenicity of C. neoformans .
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eLife Assessment
This important study confirms the molecular function of putative components of the N-glycan-dependent endoplasmic reticulum protein quality control (ERQC) system in the pathogen Cryptococcus neoformans. The study demonstrates an involvement in fitness, virulence, and the secretion and composition of extracellular vesicles, albeit in ways that are not yet fully understood. The evidence provided is largely convincing, with rigorous well-controlled assays and the use of complemented strains.
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Reviewer #1 (Public review):
Summary:
Using gene deletion analysis, the authors confirm the molecular function of putative components of an N-glycan-dependent endoplasmic reticulum protein quality control (ERQC) system (UGG1, MNS1, MNS101, MNL1, and MNL2), in the basidiomycetous fungal pathogen Cryptococcus neoformans. Specifically, they confirm the essential role of these components in the ERQC system and their role in ER stress which contributes to cellular fitness and pathogenicity.
The second part of the study links the components to secretion, mainly EV biogenesis and composition. However, this part of the study is less convincing.
Strengths:
Although it is unclear why ER stress in the mutants would not manifest into a classical secretion defect, this is a rigorous, well-controlled study, with the use of complemented strains that …
Reviewer #1 (Public review):
Summary:
Using gene deletion analysis, the authors confirm the molecular function of putative components of an N-glycan-dependent endoplasmic reticulum protein quality control (ERQC) system (UGG1, MNS1, MNS101, MNL1, and MNL2), in the basidiomycetous fungal pathogen Cryptococcus neoformans. Specifically, they confirm the essential role of these components in the ERQC system and their role in ER stress which contributes to cellular fitness and pathogenicity.
The second part of the study links the components to secretion, mainly EV biogenesis and composition. However, this part of the study is less convincing.
Strengths:
Although it is unclear why ER stress in the mutants would not manifest into a classical secretion defect, this is a rigorous, well-controlled study, with the use of complemented strains that demonstrate phenotypic restoration. The diagram in Figure 1 is very useful in orientating the reader to a complex subject matter, although the legend could be more descriptive.
Weaknesses:
A major weakness is the sheer volume of data presented (in the main text and supplement), which makes the results difficult to follow and retain: the work could essentially be two separate studies.
Another major weakness is the lack of mechanistic insight into the role of the ERQC system in EV secretion and its disconnection to "classical" secretion, which is difficult to reconcile. Some insight into why EV secretion is decreased, and classical secretion is unaffected, would strengthen the significance of the findings. No mechanism is provided to explain why the ERQC mutants (Ugg1 mutant in particular) would have reduced and heterogeneously sized EVs. Furthermore, it is not convincing that the EV content changes would greatly impact fitness and virulence. The proteomics data showing reduced cargo in the Ugg1 mutant is not convincing and difficult to follow. -
Reviewer #2 (Public review):
Summary:
This study investigates the molecular function of the N-glycan-dependent endoplasmic reticulum protein quality control system (ERQC) in Cryptococcus neoformans and correlates this pathway with key features of C. neoformans virulence, especially those mediated by extracellular vesicle transport. The findings provide valuable insights into the connection between this pathway and the biogenesis of C. neoformans extracellular vesicles.
Strengths:
The strength of this study lies primarily in the careful selection of appropriate and current methodologies, which provide a solid foundation for the authors' results and conclusions across all presented data. All experiments are supported by well-designed and established controls in the study of C. neoformans, further strengthening the validity of the results …
Reviewer #2 (Public review):
Summary:
This study investigates the molecular function of the N-glycan-dependent endoplasmic reticulum protein quality control system (ERQC) in Cryptococcus neoformans and correlates this pathway with key features of C. neoformans virulence, especially those mediated by extracellular vesicle transport. The findings provide valuable insights into the connection between this pathway and the biogenesis of C. neoformans extracellular vesicles.
Strengths:
The strength of this study lies primarily in the careful selection of appropriate and current methodologies, which provide a solid foundation for the authors' results and conclusions across all presented data. All experiments are supported by well-designed and established controls in the study of C. neoformans, further strengthening the validity of the results and conclusions drawn from them. The study presents novel data on this important pathway in C. neoformans, establishing its connection with C. neoformans virulence. Interestingly, the findings led the authors to understand the relationship between this pathway and the transport of key fungal virulence factors via extracellular vesicles. This was demonstrated in the study, paving the way for a deeper understanding of extracellular vesicle biogenesis-a field still filled with gaps but one to which this study contributes solid data, helping to clarify aspects of this process.
Weaknesses:
I do not see significant weaknesses in this study. The experiments are well-grounded, and the results are clearly presented. I believe the only weakness is that the paper could be condensed. Sections like the discussion, for instance, are extremely lengthy, which may make reading and, consequently, understanding more challenging for many readers. Regarding the presentation of the results, while clear, the figures contain a lot of information, and I believe that some of this content could be moved to supplementary figures.
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Reviewer #3 (Public review):
Summary:
Cryptococcus neoformans is a global critical threat pathogen and the manuscript by Mota et al demonstrates that the pathogen's N-glycan-dependent protein quality control system regulates the capacity of the fungus to cause disease. The system makes sure that glycoproteins are folded correctly. The system is involved in the fitness and virulence of the fungus by regulating aspects of cellular robustness and the trafficking of virulence-associated compounds outside of the cell via transport in extracellular vesicles.
Strengths:
The investigators use multiple modalities to demonstrate that the system is involved in cryptococcal pathogenesis. The investigators generated mutant C. neoformans to explore the role of genes involved in the protein folding system. Basic microbiology, genetic analyses, …
Reviewer #3 (Public review):
Summary:
Cryptococcus neoformans is a global critical threat pathogen and the manuscript by Mota et al demonstrates that the pathogen's N-glycan-dependent protein quality control system regulates the capacity of the fungus to cause disease. The system makes sure that glycoproteins are folded correctly. The system is involved in the fitness and virulence of the fungus by regulating aspects of cellular robustness and the trafficking of virulence-associated compounds outside of the cell via transport in extracellular vesicles.
Strengths:
The investigators use multiple modalities to demonstrate that the system is involved in cryptococcal pathogenesis. The investigators generated mutant C. neoformans to explore the role of genes involved in the protein folding system. Basic microbiology, genetic analyses, proteomics, fluorescence and transmission microscopy, nanotracking analyses, and murine studies were performed. The validity of the findings are thus very high. Hypotheses are robustly demonstrated.
Weaknesses:
Aspects of the results should be better explained. Some results are extrapolated in their meaning beyond the extent of the data.
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Author response:
Reviewer #1:
We thank the reviewer for recognizing the impact of our work on the pivotal roles of N-glycan-dependent ERQC in cellular fitness and pathogenicity and providing valuable comments to be considered to improve the manuscript. As suggested, we will rearrange data, reduce text volume, and discuss the possibility of how ERQC mutation decreases EV secretion without significant defect in conventional secretion. Regarding the proteomics data, we have already initiated a comparative analysis of total intracellular and EV-associated proteins to determine whether the reduced cargo loading in the Ugg1 mutant is specific to EV-associated proteins. Additionally, we may extend the analysis to include total secretion, enabling a clearer comparison between classical secretion and EV-mediated secretion to better evaluate the …
Author response:
Reviewer #1:
We thank the reviewer for recognizing the impact of our work on the pivotal roles of N-glycan-dependent ERQC in cellular fitness and pathogenicity and providing valuable comments to be considered to improve the manuscript. As suggested, we will rearrange data, reduce text volume, and discuss the possibility of how ERQC mutation decreases EV secretion without significant defect in conventional secretion. Regarding the proteomics data, we have already initiated a comparative analysis of total intracellular and EV-associated proteins to determine whether the reduced cargo loading in the Ugg1 mutant is specific to EV-associated proteins. Additionally, we may extend the analysis to include total secretion, enabling a clearer comparison between classical secretion and EV-mediated secretion to better evaluate the extent of classical secretion defects in the Ugg1 mutant.
Reviewer #2:
We sincerely thank the reviewer for the positive evaluation of our work. As recommended, we will reduce the text and reorganize the data to enhance the manuscript's readability.
Reviewer #3:
We sincerely thank the reviewer for the high appreciation of our work. As recommended, we will provide a more detailed explanation of the results with improved interpretation, strongly grounded on the obtained data.
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