Review Reports - Bacterial Extracellular Vesicles in the Strategic Interplay Between Pathogens and Hosts

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The studies on the functionality of bacterial EVs is currently widespread research topic. Therefore, the submitted review paper is aligned with this trend. The introduction to this manuscript is interesting. Next, the authors move on to describing the history of research on G- and G+ vesicles, mechanisms of vesicle biogenesis, and BEV composition; however, this section lacks detail. I recommend introducing a table summarizing the most important information on BEV characterization methods and techniques for the mentioned bacterial species, along with a characterization of these structures in terms of size and main components. It would also be good to include more examples of their specific composition, both in relation to proteins and other biomolecules. I have also some comments on Figure 1: it is unclear. How does the cell diagram relate to the blue structures below as well as the vesicle? It should be revised or made more clear and informative.

Point 5.2 Interactions between BEVs and Immune Cell is the same as 5.3. This immunological section needs a clearer structure to improve the overall organization of the manuscript.

In addition, this review is somewhat too superficial, and only a few examples are provided in each subsection, while the current literature on bacterial EVs is quite extensive. There is also a lack of descriptions of EVs produced by anaerobic bacteria. More information also on EVs from health-promoting bacteria and their properties could be added, as well as on BEV-mediated interactions within bacterial communities in host niches, including interplay between host-commensal-pathogen.

Author Response

Dear Reviewer,

We sincerely thank you for your positive evaluation of our manuscript and for your constructive suggestions. Your comments have been very helpful for improving the depth, clarity, and organization of the review. We have carefully revised the manuscript according to your suggestions, as detailed below.

Comment 1

Response

Thank you very much for this helpful suggestion. We agree that the original version did not provide sufficient detail regarding BEV characterization methods, size ranges, and cargo composition. To address this issue, we have added a new table in Section 2 to summarize representative BEVs from Gram-negative, Gram-positive, anaerobic, commensal, and health-promoting bacteria.

In the revised manuscript, Table 1 summarizes the bacterial species, Gram type and oxygen relationship, EV type, reported size range, common characterization methods, major cargoes, representative biological functions, and corresponding references. The table now includes representative species such as Escherichia coli, Salmonella enterica serovar Typhimurium, Vibrio cholerae, Pseudomonas aeruginosa, Bacteroides fragilis, Porphyromonas gingivalis, Staphylococcus aureus, Listeria monocytogenes, Lactobacillus acidophilus, and Akkermansia muciniphila. We also expanded the description of BEV cargoes to include proteins and other biomolecules, such as LPS, outer membrane proteins, peptidoglycan fragments, DNA/RNA, quorum-sensing molecules, polysaccharide A, gingipains, fimbriae, bacteriocins, α-hemolysin, β-lactamase, and listeriolysin O. These additions provide a more detailed overview of BEV composition and characterization.

Revised text

We added the following table to the revised manuscript:

Table 1. Representative bacterial extracellular vesicles: characterization methods, size ranges, major cargoes, and biological functions.

Comment 2

I have also some comments on Figure 1: it is unclear. How does the cell diagram relate to the blue structures below as well as the vesicle? It should be revised or made more clear and informative.

Response

Thank you for pointing this out. We agree that the original Figure 1 was not sufficiently clear, especially regarding the relationship between the bacterial cell diagram, the blue cell-like structures, and the vesicle. To improve clarity, we have revised Figure 1 by removing ambiguous elements and reorganizing it into a clearer schematic showing the Gram-negative bacterial envelope, OMV biogenesis, representative cargoes, and biological functions.

The revised Figure 1 now illustrates the bacterial envelope structure, localized outward budding of the outer membrane, vesicle release, representative OMV cargoes, and major biological functions. We also revised the figure legend to clarify that the figure was originally created using BioGDP.com and subsequently modified by the authors according to the reviewer’s comments.

Revised text

Figure 1. Biogenesis, cargo composition, and biological functions of outer membrane vesicles (OMVs) derived from Gram-negative bacteria.
Figure 1 was originally created using BioGDP.com and subsequently modified by the authors to improve clarity and informativeness according to the reviewer’s comments [74].

Comment 3

Point 5.2 Interactions between BEVs and Immune Cell is the same as 5.3. This immunological section needs a clearer structure to improve the overall organization of the manuscript.

Response

Thank you for identifying this organizational problem. We apologize for the duplicated subsection heading in the original manuscript. To improve the structure and readability of the immunological section, we have reorganized Section 5.

In the revised manuscript, Section 5 is now divided into four subsections:

5.1 Interactions between BEVs and Epithelial Cells
5.2 Interactions between BEVs and Immune Cells
5.3 BEVs as Bridges from Innate Immunity to Adaptive Immunity
5.4 BEVs as Mediators of Host–Commensal–Pathogen Interactions

This revised structure distinguishes epithelial-cell responses, immune-cell interactions, the transition from innate immunity to adaptive immunity, and BEV-mediated interactions within host-associated microbial communities. We believe this organization makes the immunological section clearer and more coherent.

Revised text

5.3 BEVs as Bridges from Innate Immunity to Adaptive Immunity

Although BEVs can activate host immune responses, this process does not necessarily represent a disadvantage for bacteria. Instead, BEVs allow bacteria to shape host immunity in a spatially and temporally controlled manner, thereby creating conditions that may favor bacterial survival, colonization, or immune evasion...

Comment 4

Response

Thank you for this important suggestion. We agree that the previous version did not sufficiently cover anaerobic bacteria, health-promoting bacteria, and BEV-mediated ecological interactions in host niches. To address this concern, we expanded the manuscript in two ways.

First, we added representative anaerobic and health-promoting bacteria to Table 1, including Bacteroides fragilis, Porphyromonas gingivalis, Lactobacillus acidophilus, and Akkermansia muciniphila. Their EV types, size ranges, characterization methods, major cargoes, and biological functions are now summarized.

Second, we added a new subsection, Section 5.4 “BEVs as Mediators of Host–Commensal–Pathogen Interactions.” In this section, we discuss BEVs derived from anaerobic commensals and health-promoting bacteria, including Bacteroides fragilis OMVs enriched in polysaccharide A, Porphyromonas gingivalis OMVs carrying gingipains, fimbriae, LPS, outer membrane proteins, and small RNAs, Lactobacillus acidophilus MVs delivering bacteriocins, and Akkermansia muciniphila EVs involved in gut barrier regulation. We also discuss the context-dependent roles of BEVs in mucosal niches, where host cells, commensals, probiotics, and invading pathogens coexist. These revisions expand the manuscript beyond pathogen-derived virulence vesicles and emphasize BEVs as ecological mediators in host-associated microbial communities.

Revised text

5.4 BEVs as Mediators of Host–Commensal–Pathogen Interactions

In addition to vesicles released by classical pathogens, BEVs from anaerobic commensals and health-promoting bacteria have attracted increasing interest. This is particularly relevant in mucosal niches, where host cells, commensals, probiotics, and invading pathogens coexist in the same ecological space. In such environments, BEVs are not merely carriers of virulence factors. They can also act as mobile packages of bacterial information, allowing different members of the microbial community to communicate with host cells and with each other.

Anaerobic bacteria are an important source of BEVs, although they have been less extensively discussed than aerobic or facultative anaerobic pathogens. For example, Bacteroides fragilis, a major anaerobic commensal in the gut, releases OMVs enriched in polysaccharide A...

Closing Response

We sincerely appreciate the reviewer’s constructive comments. In response to these suggestions, we have added a summary table, revised Figure 1, reorganized the immunological section, expanded the discussion of anaerobic and health-promoting bacteria-derived BEVs, and added a new subsection on BEV-mediated host–commensal–pathogen interactions. We believe these revisions have substantially improved the depth, clarity, and organization of the manuscript.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript "Bacterial Extracellular Vesicles in the Strategic Interplay 2
between Pathogens and Hosts" summarizes research of the bacterial vesicles, which represent one of the bacterial communication and defence mechanisms.

The manuscript is accurately written and structured in a good way.

At the same time, I have a number of comments to improve this manuscript:

Lines 169-171. "In Staphylococcus aureus, culture at 40 °C markedly increases the enrichment of virulence factors in vesicles, whereas culture at 34 °C is associated with greater proteomic diversity"

Is there any hypothesis, why do S. aureus produce virulence factors at high temperature and how can this help it to survive?

Lines 182-185. "β-Lactam and peptide antibiotics can strongly promote BEVs release by disrupting the peptidoglycan layer. Subinhibitory concentrations of tetracycline, ampicillin, and ceftriaxone increase OMVs production in E. coli by only approximately 2- to 4-fold, while polymyxin B and colistin can induce an approximately 10-fold increase"

Tetracycline is not β-Lactam or peptide antibiotic. Please, correct this paragraph.

Lines 230-234. "Under antibiotic pressure, MV production by methicillin-resistant Staphylococcus aureus (MRSA) increases by 22.4-fold, and these MVs are enriched in proteins associated with β-lactam antibiotic degradation, suggesting that they may act as a first line of defense against β-lactam antibiotics [30]."

I think the authors are exaggerating the importance of particles in this case, since all bacteria have active efflux systems.

Sections 5.2 and 5.3 have identical headings and can be merged.

Technical comment about the references. Please check their format in the text.

Author Response

Dear Reviewer,

We sincerely thank you for your positive evaluation of our manuscript and for your constructive comments. Your suggestions have helped us improve the accuracy, clarity, and organization of the manuscript. We have carefully revised the manuscript according to your comments, and our point-by-point responses are provided below.

Comment 1

Lines 169–171. “In Staphylococcus aureus, culture at 40 °C markedly increases the enrichment of virulence factors in vesicles, whereas culture at 34 °C is associated with greater proteomic diversity.” Is there any hypothesis, why do S. aureus produce virulence factors at high temperature and how can this help it to survive?

Response

Thank you very much for this insightful comment. We agree that the original manuscript did not sufficiently explain the possible biological meaning of temperature-dependent cargo selection in Staphylococcus aureus MVs. We have revised this paragraph to provide a more cautious interpretation.

In the revised manuscript, we explain that high temperature may be sensed by S. aureus as a host-associated stress cue, particularly under inflammatory or febrile conditions. Under such conditions, selective enrichment of virulence-associated proteins in MVs may help S. aureus damage host cells, modulate local immune responses, or remodel the infection microenvironment. We also emphasize that this explanation is a possible hypothesis rather than a definitive mechanism.

Revised text

Temperature affects both the production and functional properties of BEVs, mainly through two mechanisms. First, heat stress can activate bacterial stress-response pathways. For example, Escherichia coli produces significantly more BEVs under high-temperature conditions, which may be related to stress signaling triggered by protein denaturation [24]. Second, temperature can influence BEVs formation and cargo composition by changing membrane fluidity. In Staphylococcus aureus, culture at 40 °C markedly increases the enrichment of virulence factors in vesicles, whereas culture at 34 °C is associated with greater proteomic diversity [25]. One possible explanation is that high temperature may be sensed by S. aureus as a host-associated stress cue, particularly under inflammatory or febrile conditions. Under such conditions, selective enrichment of virulence-associated proteins in MVs could help S. aureus damage host cells, modulate local immune responses, or remodel the infection microenvironment. In contrast, lower temperatures may favor broader proteomic packaging, which could be more relevant to environmental persistence or colonization outside highly inflammatory niches. Therefore, temperature may influence not only MV yield but also functional cargo selection.

Comment 2

Lines 182–185. “β-Lactam and peptide antibiotics can strongly promote BEVs release by disrupting the peptidoglycan layer. Subinhibitory concentrations of tetracycline, ampicillin, and ceftriaxone increase OMVs production in E. coli by only approximately 2- to 4-fold, while polymyxin B and colistin can induce an approximately 10-fold increase.” Tetracycline is not β-Lactam or peptide antibiotic. Please, correct this paragraph.

Response

Thank you for pointing out this important classification error. We agree that tetracycline is neither a β-lactam antibiotic nor a peptide antibiotic. To avoid this inaccurate classification, we have revised the paragraph and now describe these treatments more broadly as antibiotic stress. We also clarified the different antibiotic classes and mechanisms involved.

In the revised version, ampicillin and ceftriaxone are described as β-lactam antibiotics that interfere with peptidoglycan synthesis, polymyxin B and colistin are described as membrane-targeting peptide antibiotics, and tetracycline is described as an inhibitor of bacterial protein synthesis.

Revised text

Antibiotic stress can also promote BEV release through different mechanisms, including interference with peptidoglycan synthesis, disruption of cell envelope integrity, perturbation of membrane homeostasis, and activation of general stress responses. For example, subinhibitory concentrations of tetracycline, ampicillin, and ceftriaxone increase OMV production in E. coli by approximately 2- to 4-fold, whereas polymyxin B and colistin can induce an approximately 10-fold increase [29]. Among these antibiotics, ampicillin and ceftriaxone are β-lactam antibiotics that interfere with peptidoglycan synthesis, polymyxin B and colistin are membrane-targeting peptide antibiotics, and tetracycline inhibits bacterial protein synthesis. Therefore, antibiotic-induced BEV production may reflect multiple stress pathways rather than a single cell-wall-targeting mechanism. Similarly, treatment with 64 μg/mL ampicillin increases vesicle production in S. aureus by 22.4-fold [30]. This increase is likely linked to antibiotic-induced interference with cell wall synthesis. When peptidoglycan cross-linking is impaired, increased intracellular pressure may drive localized membrane protrusion and accelerate BEV shedding.

Comment 3

Lines 230–234. “Under antibiotic pressure, MV production by methicillin-resistant Staphylococcus aureus (MRSA) increases by 22.4-fold, and these MVs are enriched in proteins associated with β-lactam antibiotic degradation, suggesting that they may act as a first line of defense against β-lactam antibiotics [30].” I think the authors are exaggerating the importance of particles in this case, since all bacteria have active efflux systems.

Response

Thank you for this helpful comment. We agree that the original wording may have overemphasized the role of MVs in antibiotic defense. Bacterial antibiotic resistance is mediated by multiple mechanisms, including efflux systems, altered antibiotic targets, enzymatic antibiotic degradation, and changes in envelope permeability. Therefore, we have revised this statement to present MV-mediated protection as an additional extracellular protective mechanism that may complement, rather than replace or dominate, classical resistance mechanisms.

Revised text

Under antibiotic pressure, MV production by methicillin-resistant Staphylococcus aureus (MRSA) increases by 22.4-fold, and these MVs are enriched in proteins associated with β-lactam antibiotic degradation [30]. These observations suggest that MVs may provide an additional extracellular protective mechanism under antibiotic stress. However, the protective role of MVs should not be interpreted as replacing classical bacterial resistance mechanisms, such as efflux systems, altered antibiotic targets, or enzymatic antibiotic inactivation within bacterial cells. Rather, MVs may provide an additional extracellular layer of protection by carrying β-lactamase-associated proteins and reducing the effective antibiotic pressure in the surrounding microenvironment. In this sense, MV-mediated protection may complement, rather than dominate, conventional antibiotic resistance strategies.

Comment 4

Sections 5.2 and 5.3 have identical headings and can be merged.

Response

Thank you for pointing out this issue. We apologize for the duplicated heading in the original manuscript. To improve the organization of the immunological section, we have revised and reorganized Section 5.

In the revised manuscript, Section 5 is now divided into four subsections:
5.1 Interactions between BEVs and Epithelial Cells
5.2 Interactions between BEVs and Immune Cells
5.3 BEVs as Bridges from Innate Immunity to Adaptive Immunity
5.4 BEVs as Mediators of Host–Commensal–Pathogen Interactions

Accordingly, the duplicated heading has been removed. We believe that the revised structure better distinguishes epithelial-cell interactions, immune-cell interactions, the transition from innate to adaptive immunity, and BEV-mediated interactions within host-associated microbial communities.

Revised text

5.3 BEVs as Bridges from Innate Immunity to Adaptive Immunity

Comment 5

Technical comment about the references. Please check their format in the text.

Response

Thank you for this technical comment. We have carefully checked the in-text citations and the reference list throughout the manuscript. The citation format has been unified, and the reference list has been revised according to the journal style. In addition, unnecessary database notes such as “From NLM” and “From Cnki” have been removed.

Closing Response

We sincerely appreciate the reviewer’s constructive suggestions. In response to these comments, we have clarified the possible biological significance of temperature-dependent cargo enrichment in S. aureus MVs, corrected the classification of antibiotics, revised the statement regarding MV-mediated antibiotic protection, reorganized the immunological section, and checked the reference format throughout the manuscript. We believe these revisions have improved the scientific accuracy and clarity of the manuscript.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

I would like to thank the authors for their responses. They have addressed the majority of comments. I recommend conducting an additional review of the syntax and grammar following the revisions, as well as removing the statement regarding the reviewers under Figure 1. Please also ensure that species names are written in italics thorough manuscript.

Author Response

Comment: I would like to thank the authors for their responses. They have addressed the majority of comments. I recommend conducting an additional review of the syntax and grammar following the revisions, as well as removing the statement regarding the reviewers under Figure 1. Please also ensure that species names are written in italics throughout manuscript.

Response: Thank you very much for your positive feedback and helpful suggestions. We have gone through the revised manuscript again and made additional edits to improve the syntax, grammar, and readability. The statement referring to the reviewer’s comments has been removed from the legend of Figure 1. We have also checked the manuscript carefully and corrected the formatting of bacterial species names throughout the main text, tables, figure legends, and references to ensure that they are written in italics where appropriate.