Fungal Extracellular Vesicles: Isolation, Characterization, and the Immune System Response

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Ikeda and colleagues presented a comprehensive review of the literature on fungal extracellular vesicles, with a major emphasis on isolation protocols and their impact on the immune system response. The manuscript is intriguing and addresses essential findings in this field. However, this reviewer would like to offer some observations that should be taken into consideration by the authors.

 

Section 1.1:

-The paper published by Rodrigues and colleagues represents the first work where fungal extracellular vesicles were successfully isolated. This important information should be added to the text.

 

-There is a third hypothesis for fungal EVs biogenesis that was not included neither in the text not in Figure 1. Please see PMID: 23628115

 

Page 2, lane 67 - Please remove the word “new” from the sentence.

 

Section 1.2:

-Lanes 82-87 – it is crucial to include the filtration step using the Amicon system to the work published by Rodrigues and colleagues. This step appears to be significant in their isolation process.

 

-Lane 122 – Regular Amicon systems supports up to 500 mL.

 

-Lanes 139 – It is important to mention that there are no markers for fungal EVs, except the ones suggested by Dawnson and colleagues (PIMD 32363014) for Candida albicans EVs. This is one of the reasons why it is not feasible to use beads, immunoaffinity-based capture, among other methods used for human EVs.

 

Section 1.3:

- In the reviewer's opinion, this section appears to be quite limited in terms of the information provided. It resembles a brief introduction to section 1.4 rather than a comprehensive exploration of the topic. It would be more engaging to have a more in-depth analysis in this section, comparing conserved and non-conserved proteins, virulence factors, and other compounds.

 

Section 1.4:

-What would be the explanation by which EVs from C. albicans were protective and EVs from S. brasiliensis were not? EVs from P. brasiliensis were also distinct. Could the authors include some discussion on this topic? 

 

-Do the authors think the effect of antibodies in EVs release in H. capsulatum could be a mechanism of action for these protective antibodies? If so, please incorporate the discussion to the manuscript.

 

-Page 7, lanes 267-384. Although both works exploited mice models, the protocols are very distinct. It would be interesting whether the authors add some input in those differences to “explain” these data.

 

-Page 8, lanes 328-330. This paragraph is not clear, please rephrase it. In addition, Oliveira and colleagues (2010) also used EVs from a C. neoformans mutant and their incubation with macrophages induced a distinct response.

 

-Pages 8 and 9, lanes 331-354. It seems that this paragraph should be considered as a distinct topic. The information does not fit as “Fungi EVs and host immune system”. It sounds more are the role of EVs in fungi-fungi communication.

-It would be appreciated whether the authors could consider adding a conclusion and/or perspectives subitem to the manuscript.

 

Table 1:

The table provided is undoubtedly informative. However, to enhance its clarity and conciseness, the authors may consider reducing the size by combining similar data. For instance, if C. albicans EVs have been utilized in various studies to protect Galleria mellonella with similar results, these data could be combined together in the table. This approach would present the information more efficiently and make the table more reader-friendly.

Author Response

Dear,

Thank you for all the suggestions. Follow the responses:

Section 1.1:

-The paper published by Rodrigues and colleagues represents the first work where fungal extracellular vesicles were successfully isolated. This important information should be added to the text.

Thank you for the suggestion. Significant observation and essential to mention Rodrigues's work. We included in the text this information.

Due to their ability to carry bioactive molecules, EVs have gained significant attention in biomedical research. However, it was when in 2007, after more than thirty years of EVs discovery, the first work where fungal EVs were successfully isolated was published by Rodrigues and colleagues describing the EVs from the fungus Cryptococcus neoformans as responsible for transporting glucuronoxylomannan through the cell wall. After Rodrigues's publication, we saw an increase in publications describing new species of fungi that could produce EVs.

-There is a third hypothesis for fungal EVs biogenesis that was not included neither in the text not in Figure 1. Please see PMID: 23628115

Thank you for the suggestion. We changed the text and the figure 1.

Also, Rodrigues and colleagues (2013) propose a third route of release of fungal EVs by cytoplasmic subtraction originated from membrane reshaping with consequent plasma membrane invaginations and formation of an isolated membranous compartment located at the periplasmic space.

Page 2, lane 67 - Please remove the word “new” from the sentence.

 The word new was removed.

Section 1.2:

-Lanes 82-87 – it is crucial to include the filtration step using the Amicon system to the work published by Rodrigues and colleagues. This step appears to be significant in their isolation process.

We included, a suggestion:

2.1. EVs isolation

The first description of fungal EVs being isolated was made by Rodrigues and colleagues in 2007, where EVs were separated by ultracentrifugation based on the different buoyant densities of cells and particles in the solution. From liquid media, a culture of C. neoformans was submitted to two centrifugations in a cold rotor (4^oC) at 4.000g to remove the heavy portion of cells and at 15.000g to remove most of the apoptotic bodies, debris, and molecules with higher density than the EVs. The pellet was discarded and the supernatant was concentrated with an Amicon® ultrafiltration system of 100 kDa cutoff. They were finally ultracentrifuged at 100.000g for 1-2 hours, repeating this step 5 times to wash the pellet. Such protocol granted a reliable and cost-effective method to isolate EVs from many other fungi until today. In a few hours, with only one ultracentrifuge and a flask of TBE or PBS, a pellet of 1x10⁸ to 10¹¹ particles/mL could be easily obtained (Bitencourt et al., 2018; Ikeda et al., 2018; De Paula et al., 2019). However, the main disadvantages regarding centrifugation are working with large volumes and the fact that other molecules, such as proteins, lipoproteins, and nonexosomal particles, will also be isolated given their similar size and density (Gardiner et al., 2016; Mathieu et al., 2019).

-Lane 122 – Regular Amicon systems supports up to 500 mL.

Thank you for your observation. Here we wrote about tubes.

 Similarly, in studies regarding fungal EVs, it is often seen specific ultracentrifugation tubes (Amicon®, Vivapsin®) being employed, which consists of a centrifuge tube varying in size (2 or 15 mL) accoupled with filters with different molecular weight cutoffs (MWCO) ranging from 3 -100 kDa

-Lanes 139 – It is important to mention that there are no markers for fungal EVs, except the ones suggested by Dawnson and colleagues (PIMD 32363014) for Candida albicans EVs. This is one of the reasons why it is not feasible to use beads, immunoaffinity-based capture, among other methods used for human EVs.

 We agree with the observation, and we changed the text.

Although there are many other techniques for EV isolation and purification, such as Polyethylene glycol precipitation (Kim et al., 2015; Deregibus et al., 2016), Magnetic bead separation (Gardiner et al., 2016), Immunoaffinity-based capture (Ingato et al., 2016), Size-exclusion chromatography (SEC), ExoQuick precipitation agent, these approaches are most seen used in the extraction of EV's from human samples (Musante; Tataruch; Holthofer, 2014; Guerreiro et al., 2018; Zhu et al., 2020). Furthermore, these methods are more efficient with the use of EVs protein markers that are only available for mammalian cells. One study by Dawson and colleagues (2020) evaluated two putative targets for C. albicans that may be used as specific markers for this fungi species.

Section 1.3:

- In the reviewer's opinion, this section appears to be quite limited in terms of the information provided. It resembles a brief introduction to section 1.4 rather than a comprehensive exploration of the topic. It would be more engaging to have a more in-depth analysis in this section, comparing conserved and non-conserved proteins, virulence factors, and other compounds.

We included some information:

The characteristics of EVs of small size heterogeneous population, low refractive index, and the possibility of aggregation of proteins and glycoproteins of similar size with EVs do not allow the performance of more conventional analysis techniques. In the study of EVs, the most used single‐particle analysis techniques are nanoparticle tracking analysis (NTA), electron microscopy (EM), atomic force microscopy, high‐resolution microscopy, resistive pulse sensing, high‐resolution flow cytometry, and Raman spectroscopy (Welsh et al., 2023). In mycology, works mainly use NTA and EM techniques for EVs quantification and size measurement. Most studies show that fungal EVs varied in size between 30nm and 600nm (Liebana-Jordan et al., 2021), but there were different fungal growth conditions, EV isolation techniques, and EV analysis techniques, not allowing an accurate comparison between the studies.

Section 1.4:

-What would be the explanation by which EVs from C. albicans were protective and EVs from S. brasiliensis were not? EVs from P. brasiliensis were also distinct. Could the authors include some discussion on this topic? 

Interesting point. We included a paragraph:

S. brasiliensis is notably pathogenic, with strains causing invasive mycosis even in immunocompetent hosts. The immune response to EVs may differ from opportunistic fungi with a different pathogenesis. Furthermore, some proteins present in the EVs, as well as virulence factors, must be different5, depending on the fungus.

-Do the authors think the effect of antibodies in EVs release in H. capsulatum could be a mechanism of action for these protective antibodies? If so, please incorporate the discussion to the manuscript.

 Although it has already been shown in vivo that the treatment with monoclonal antibodies had a protective effect, these antibodies modified the pathobiology of the fungi, and therefore, have an impact on the response of the host immune system.

-Page 7, lanes 267-384. Although both works exploited mice models, the protocols are very distinct. It would be interesting whether the authors add some input in those differences to “explain” these data.

The opposite results may have occurred due to the different routes of inoculation of the fungus. C. neoformans is known to cause infection in the central nervous system, and therefore the hematogenous route may have facilitated the pathogenesis of the fungus.

-Page 8, lanes 328-330. This paragraph is not clear, please rephrase it. In addition, Oliveira and colleagues (2010) also used EVs from a C. neoformans mutant and their incubation with macrophages induced a distinct response.

 We cleared the paragraph and included some information about Oliveira and colleagues.

Genetic modification can result in mutant fungi that release EVs with a different composition that impacts host-cell interaction. Oliveira and colleagues (2010) compared wild-type strains of C. neoformans with an acapsular mutant, and it was observed that macrophages stimulated with EVs obtained from the mutant strain had greater activation of the macrophage response with increased production of nitric oxide, phagocytosis capacity, and fungicidal activity. The same results were obtained in vivo (Rizzo et al., 2021) where immunization with EVs from acapsular mutant prolonged survival in a mice infection model.

Also, Colombo et al. (2019), demonstrated that a mutant strain of C. neoformans that releases EVs enrichened in GXM and sterylglucosides had a protective effect in G. mellonella infection model. In C. albicans (Wolf et al., 2015), EVs obtained from five mutant strains for phospholipid biosynthesis demonstrated different sizes and protein cargo than the wild-type strain, and one mutant had a lower activation of NF-κB signaling pathway in bone marrow-derived macrophage cells. These results show the potential role of using EVs obtained from mutants of important pathways in fungal pathogenesis

-Pages 8 and 9, lanes 331-354. It seems that this paragraph should be considered as a distinct topic. The information does not fit as “Fungi EVs and host immune system”. It sounds more are the role of EVs in fungi-fungi communication.

We agree. We changed the text.

-It would be appreciated whether the authors could consider adding a conclusion and/or perspectives subitem to the manuscript.

 Challenges and perspectives of fungal EVs:

The study of fungal EVs has advanced a lot in recent years, but several challenges are faced in this process. Regarding the isolation of EVs, despite the studies using similar isolation techniques, there is a large variation in the fungal growth conditions, which can cause variation in the size and molecular/biochemical composition of the EVs and consequently have a different activation of the host's immune response.

Concerning the functional capacity of EVs, there are many options for in vitro and in vivo models, which present particular characteristics of each one. Several studies are necessary to determine the real effect of EVs from each fungus on the immune response. With the advancement of laboratory techniques, more and more information can be obtained about the composition and function of fungal EVs. Furthermore, new bioengineering technologies will make it possible to explore the modulation of EVs and obtain new therapeutic targets and diagnostic markers.

Table 1:

The table provided is undoubtedly informative. However, to enhance its clarity and conciseness, the authors may consider reducing the size by combining similar data. For instance, if C. albicans EVs have been utilized in various studies to protect Galleria mellonella with similar results, these data could be combined together in the table. This approach would present the information more efficiently and make the table more reader-friendly.

Thank you for your suggestion. We modified the table 1.

 

 

Reviewer 2 Report

Comments and Suggestions for Authors

In this review paper, Ikeda et al. summarized the current understanding of extracellular vesicles of fungi. The manuscript includes the following sections: 1) the history of discovery of fungal extracellular vesicles. 2) how fungal extracellular vesicles are purified, 3) the components of fungal extracellular vesicles, and 4) how fungal extracellular vesicles affect host immune responses.

Major comments:

1) The first part of the manuscript is "1. Introduction", What are the 2nd, 3rd and 4th part of this manuscript?

2) This manuscript missed an introduction section and a conclusion or summary section at the end of the manuscript.

3) I feel that the authors simply gathered previous findings and observations without digesting previous discoveries and presenting them in a clear way. 

Minor comments:

Ref 57 and 58 are the same ref.

Line 66: "e" should be "and"

Line 209: "...mice bone.." should be "...mouse bone..."

Line 216: "fungi infection" should be "fungal infection"

Line 217: "Fungi EVs" should be "Fungal EVs"

Line: "mice immunization" should be "mouse immunization"

Comments on the Quality of English Language

many errors need to be corrected.

Author Response

Dear,

Thank you for your suggestions. Follow the responses:

Major comments:

1) The first part of the manuscript is "1. Introduction", What are the 2nd, 3rd and 4th part of this manuscript?

We changed the organization:

1. Introduction

1.1 EVs

1.2 Fungal EVs

2. Fungal EVs isolation and analysis

2.1 EVs isolation

2.2 Fungal EVs Characterization

3. Impacts of fungal EVs on Host Immunity

3.1 In vitro assays

3.2 In vivo assays: Galleria mellonella model

3. 3. In vivo assays: animal model

3.4. Fungal EVs as vaccines?

3.5. Modulation of fungal EVs

4. Challenges and perspectives of fungal EVs

2) This manuscript missed an introduction section and a conclusion or summary section at the end of the manuscript.

We agree. We modified the text to clear the sections. We included a conclusion and a  perspective:

Challenges and perspectives of fungal EVs

The study of fungal EVs has advanced a lot in recent years, but several challenges are faced in this process. Regarding the isolation of EVs, despite the studies using similar isolation techniques, there is a large variation in the fungal growth conditions, which can cause variation in the size and molecular/biochemical composition of the EVs and consequently have a different activation of the host's immune response.

Concerning the functional capacity of EVs, there are many options for in vitro and in vivo models, which present particular characteristics of each one. Several studies are necessary to determine the real effect of EVs from each fungus on the immune response. With the advancement of laboratory techniques, more and more information can be obtained about the composition and function of fungal EVs. Furthermore, new bioengineering technologies will make it possible to explore the modulation of EVs and obtain new therapeutic targets and diagnostic markers.

3) I feel that the authors simply gathered previous findings and observations without digesting previous discoveries and presenting them in a clear way. 

Thank you for your comment. We included and discussed more about important files of EVs. We cleared the text.

Minor comments: 

All notes below have been corrected

Ref 57 and 58 are the same ref.

Line 66: "e" should be "and"

Line 209: "...mice bone.." should be "...mouse bone..."

Line 216: "fungi infection" should be "fungal infection"

Line 217: "Fungi EVs" should be "Fungal EVs"

Line: "mice immunization" should be "mouse immunization"

The English was corrected.

 

Reviewer 3 Report

Comments and Suggestions for Authors

In this review, the authors have comprehensively summarized and discussed both historical and current insights into fungal extracellular vesicles (EVs) and their pathobiological impacts. Overall, this review promises to be highly informative to those in the field of medical mycology. To improve this review further, I offer the following comments:

 

[Major comments]

 

1. Improving the Structure for Readability: The authors may want to reconsider the overall structure of this review to enhance its readability. Currently, the review is divided into four sections:

1) General introduction of fungal extracellular vesicles

2) Fungal EV extraction methods

3) Cargo delivered by Fungal EVs

4) Impacts of fungal EVs on host immunity.

 

While the first three sections are well-structured, the last section is somewhat difficult to follow due to its distracted and unfocused presentation. I recommend creating subsections for each category of fungal pathogen, such as 4.1 Candida EVs and host immunity, 4.2 Cryptococcus EVs and host immunity, etc. This restructuring will provide readers with specific, comprehensible information about EVs related to each fungal pathogen, and can replace the current Figure 3 (or Table 1). In addition, the ‘1. Introduction’ section seems to be dispensable for this type of review.

 

2. Adding a Future Perspective Section: The authors might consider adding a section on future perspectives to discuss unknown questions and directions in studying fungal EVs. This would further contribute to the review's depth and relevance.

 

3. Tense Consistency: Please ensure that the present tense is used for published results throughout the manuscript.

Comments on the Quality of English Language

I suggest the following corrections:

Line 2: Replace 'Fungi extracellular vesicles' with 'Fungal extracellular vesicles.'

Line 17: Change ‘fungi EVs’ to 'fungal EVs.'

Line 73: Change to 'sporotrichosis (Ikeda et al., 2018). In the upcoming years,'

Lines 77-79: Ensure font consistency.

Line 83: Change to either 'Methods of Fungal EV Extraction' or 'Fungal EV Extraction Method.'

Line 149: Use bold type consistently.

Line 158: Check the font type.

Line 205: Change to 'the capacity of EVs to interact with.'

Line 245: Replace 'common fungi' with 'opportunistic fungus.'

Lines 276 and 285: Change 'fungi' to 'fungus.'

Line 286: Change 'fungi' to 'fungal.'

Line 351: Replace with 'immunity-related pathway.'

Author Response

Thank you for all the suggestions and comments. Follow the responses:

[Major comments]

1. Improving the Structure for Readability: The authors may want to reconsider the overall structure of this review to enhance its readability. Currently, the review is divided into four sections:

1) General introduction of fungal extracellular vesicles

2) Fungal EV extraction methods

3) Cargo delivered by Fungal EVs

4) Impacts of fungal EVs on host immunity.

 

While the first three sections are well-structured, the last section is somewhat difficult to follow due to its distracted and unfocused presentation. I recommend creating subsections for each category of fungal pathogen, such as 4.1 Candida EVs and host immunity, 4.2 Cryptococcus EVs and host immunity, etc. This restructuring will provide readers with specific, comprehensible information about EVs related to each fungal pathogen, and can replace the current Figure 3 (or Table 1). In addition, the ‘1. Introduction’ section seems to be dispensable for this type of review.

We modified the organization of the text to be more clear to read. 

2. Adding a Future Perspective Section: The authors might consider adding a section on future perspectives to discuss unknown questions and directions in studying fungal EVs. This would further contribute to the review's depth and relevance.

We agree and we added a section for this:

Challenges and perspectives of fungal EVs

The study of fungal EVs has advanced a lot in recent years, but several challenges are faced in this process. Regarding the isolation of EVs, despite the studies using similar isolation techniques, there is a large variation in the fungal growth conditions, which can cause variation in the size and molecular/biochemical composition of the EVs and consequently have a different activation of the host's immune response.

Concerning the functional capacity of EVs, there are a multitude of options for in vitro and in vivo models, which present particular characteristics of each one. Several studies are necessary to determine the real effect of EVs from each fungus on the immune response. With the advancement of laboratory techniques, more and more information can be obtained about the composition and function of fungal EVs. Furthermore, new bioengineering technologies will make it possible to explore the modulation of EVs and obtain new therapeutic targets and diagnostic markers.

3. Tense Consistency: Please ensure that the present tense is used for published results throughout the manuscript.

We reviewed the tense consistency.

Comments on the Quality of English Language

I suggest the following corrections:

We reviewed and corrected all the suggestions below.

Line 2: Replace 'Fungi extracellular vesicles' with 'Fungal extracellular vesicles.'

Line 17: Change ‘fungi EVs’ to 'fungal EVs.'

Line 73: Change to 'sporotrichosis (Ikeda et al., 2018). In the upcoming years,'

Lines 77-79: Ensure font consistency.

Line 83: Change to either 'Methods of Fungal EV Extraction' or 'Fungal EV Extraction Method.'

Line 149: Use bold type consistently.

Line 158: Check the font type.

Line 205: Change to 'the capacity of EVs to interact with.'

Line 245: Replace 'common fungi' with 'opportunistic fungus.'

Lines 276 and 285: Change 'fungi' to 'fungus.'

Line 286: Change 'fungi' to 'fungal.'

Line 351: Replace with 'immunity-related pathway.'

Round 2

Reviewer 2 Report

The authors have addressed my major comments.

N/A