Comparative genome analyses of Staphylococcus aureus from platelet concentrates reveal rearrangements involving loss of type VII secretion genes
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Staphylococcus aureus has been involved in transfusion-transmitted fatalities associated with platelet concentrates (PCs) due to its heightened pathogenicity enhanced by genome-encoded virulence and antibiotic resistance genes. This may be facilitated by mobile genetic elements (MGEs) that can cause rearrangements. Several factors contribute to S. aureus virulence, including the type VII secretion system (T7SS), composed of six core genes conserved across S. aureus strains. In this study, we conducted comparative genome analyses of five S. aureus isolates from PCs (CI/BAC/25/13 /W, PS/BAC/169/17 /W and PS/BAC/317/16 /W were detected during PCs screening with the BACT/ALERT automated culture system, and ATR-20003 and CBS2016-05 were missed during screening and caused septic transfusion reactions). Multiple alignments of the genomes revealed evidence of rearrangements involving phage Sa3int in PS/BAC/169/17 /W and PS/BAC/317/16 /W. While the former had undergone translocation of its immune evasion cluster (IEC), the latter had lost part of the phage, leaving behind the IEC. This observation highlights S. aureus genome plasticity. Unexpectedly, strain CBS2016-05 was found to encode a pseudo-type VII secretion system (T7SS) that had lost five of the conserved core genes ( esxA , esaA, essA, esaB and essB ) and contained a 5′ truncated essC . Since these genes are essential for the function of the T7SS protein transport machinery, which plays a key role in S. aureus virulence, CBS2016-05 probably compensates by recruiting other export mechanisms and/or alternative virulence factors, such as neu-tralizing immunity proteins. This study unravels genome rearrangements in S. aureus isolated from PCs and reports the first S. aureus isolate lacking conserved T7SS core genes.
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The work presented is clear and the arguments well formed. This study would be a valuable contribution to the existing literature. The reviewers have highlighted minor concerns with the work presented. Please ensure that you address their comments.
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The reviewers have highlighted minor concerns with the work presented. Please ensure that you address their comments.
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Comments to Author
The authors have addressed my comments.
Please rate the manuscript for methodological rigour
Satisfactory
Please rate the quality of the presentation and structure of the manuscript
Satisfactory
To what extent are the conclusions supported by the data?
Strongly support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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Comments to Author
The authors addressed the concerns raised in the first round of revisions and I have only very minor comments on the revised manuscript, detailed below: Fig 1 - The genomic island such as the vSaa island are labelled as SaPIs in Fig 1 and in text (eg. Line 319). From my understanding, SaPIs are phage mobilizable pathogenicity islands whereas the vSa islands are not phage mobilizable and are therefore not classed as SaPIs (10.4161/bact.20632). Fig S1-2 - The genome alignments in these figures are hard to analyse as they are in different orientations. If possible using this program, it would be beneficial to the reader to flip these alignments so each genome is facing the same direction for the alignemnet? Fig 4c - The annotation on the right of Fig 4c is split across lines.
Please rate …
Comments to Author
The authors addressed the concerns raised in the first round of revisions and I have only very minor comments on the revised manuscript, detailed below: Fig 1 - The genomic island such as the vSaa island are labelled as SaPIs in Fig 1 and in text (eg. Line 319). From my understanding, SaPIs are phage mobilizable pathogenicity islands whereas the vSa islands are not phage mobilizable and are therefore not classed as SaPIs (10.4161/bact.20632). Fig S1-2 - The genome alignments in these figures are hard to analyse as they are in different orientations. If possible using this program, it would be beneficial to the reader to flip these alignments so each genome is facing the same direction for the alignemnet? Fig 4c - The annotation on the right of Fig 4c is split across lines.
Please rate the manuscript for methodological rigour
Good
Please rate the quality of the presentation and structure of the manuscript
Good
To what extent are the conclusions supported by the data?
Strongly support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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This study would be a valuable contribution to the existing literature. This is a study that would be of interest to the field and community. The reviewers have highlighted major concerns with the work presented. Please ensure that you address their comments. Please deposit the data underlying the work in the Society’s data repository Figshare account here: https://microbiology.figshare.com/submit. Please also cite this data in the Data Summary of the main manuscript and list it as a unique reference in the References section. When you resubmit your article, the Editorial staff will post this data publicly on Figshare and add the DOI to the Data Summary section where you have cited it. This data will be viewable on the Figshare website with a link to the preprint and vice versa, allowing for greater discovery of your work, and the …
This study would be a valuable contribution to the existing literature. This is a study that would be of interest to the field and community. The reviewers have highlighted major concerns with the work presented. Please ensure that you address their comments. Please deposit the data underlying the work in the Society’s data repository Figshare account here: https://microbiology.figshare.com/submit. Please also cite this data in the Data Summary of the main manuscript and list it as a unique reference in the References section. When you resubmit your article, the Editorial staff will post this data publicly on Figshare and add the DOI to the Data Summary section where you have cited it. This data will be viewable on the Figshare website with a link to the preprint and vice versa, allowing for greater discovery of your work, and the unique DOI of the data means it can be cited independently.
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Comments to Author
Chi et al. conducted a comparative genomic analysis of several S. aureus isolates reported in literature, with 5 from contaminated platelet concentrates, and by doing so, revealed their characteristics. Two strains in particular had escaped typical detection methods, one of these isolates interestingly had a truncated T7SS and the other one had a genomic rearrangement which involved phage PhiSa3. Main comments: The authors have done extensive genomic comparison analysis in this work, and their results are clearly presented. The methodology is explained thoroughly for reproducibility, codes and analysis clearly stated and provided. WGS data made available on NCBI and links are provided. Figures are great, except for Figure 3b. This figure panel is very difficult to read. This could be split up for …
Comments to Author
Chi et al. conducted a comparative genomic analysis of several S. aureus isolates reported in literature, with 5 from contaminated platelet concentrates, and by doing so, revealed their characteristics. Two strains in particular had escaped typical detection methods, one of these isolates interestingly had a truncated T7SS and the other one had a genomic rearrangement which involved phage PhiSa3. Main comments: The authors have done extensive genomic comparison analysis in this work, and their results are clearly presented. The methodology is explained thoroughly for reproducibility, codes and analysis clearly stated and provided. WGS data made available on NCBI and links are provided. Figures are great, except for Figure 3b. This figure panel is very difficult to read. This could be split up for the ease of readers? I have a few specific questions/comments to improve the discussion of their findings in the context of literature: - Mutants of T7SS has been generated and used in literature to look at functionality. In this context, could the authors compare their findings to that of literature with T7SS mutants generated in previous studies, and discuss the virulence traits found in such mutants, and that of their truncated T7SS? Has mutants similar to this truncation been shown in literature? What are your hypotheses to what this truncation could lead to in a host setting. Any evolutionary benefits to the truncation expected? How could it have happened - was it a homologous recombination event that may have lead to excision, thus loss? - Garrett et al from the Palmer lab has recently shown a reverse domain arrangement in a T7SS tslA. Could the authors maybe discuss their findings in the context of such recent work? - Could you expand on the points (Line 394) from Bowman and Palmer et al. on S. warneri further in the context of your strain. Are there any similarities of this organism in genomic context to your T7SS truncation? Could you do comparative analysis of the genomic location of T7SS and surroundings, using S. warneri as an additional figure panel to compare? Other comments: - The authors use "confirming genomic plasticity" Line 73 in their manuscript - This is an unnecessary statement, when there are only a few isolates studied within the work, with one strain with genomic rearrangements, I do not think the authors "confirm" this. This should be reworded to remove this claim. - Line 78 - 80: Please re-word this section to simplify the claims made in this manuscript. This is based on genomic analysis, without phenotypic confirmation. - Lines 83-85: Include references for the statements. - Lines 92-3: U.S.A, Canada, Europe, 93 and Africa [6-8] - Is there a reference missing here? - Lines 104-5: "There does not appear to be a distinction between commensal and pathogenic strains regarding virulence genes." Could the authors please clarify what they mean by this statement more clearly? Is this for the strains they are looking at in this study, or is it a general statement? - Lines 241-43: "Two adjacent co-linear blocks of combined size from 43,519 to 45,137 bp were present in all the analyzed PC isolates but lacked the homolog sequence in strain PS/317 (Figure 1)." Please re-word this sentence to be more clear. - 243-44: "Examination of this region revealed the presence of genomic features consistent with phage ɸSa3." Can the authors please expand further on this statement and spell out exactly what they observed here? - 245-51: "… found no general evolutionary pattern between S. aureus population from blood and other sources…." - What is the context of this. Can you please clarify what "patterns" you looked at to come to this conclusion. I am not sure one can make this statement without additional analysis. - 339-40 "…diverse structural repertoire…" - what do you mean by structural here? Please expand. (Also on, Line 352) - Line 353-356: "The mobility and lifecycle of phages have been well studied. During the lysogenic life cycle, a bacteriophage incorporates its genetic DNA into the host bacterial chromosome and replicates with it without destroying the bacterial cell [67]." This is a very loose statement, and needs to be re-written in a more sophisticated scientific language. - Figure 3b: This figure panel is very difficult to read. This could be split up for the ease of readers?
Please rate the manuscript for methodological rigour
Very good
Please rate the quality of the presentation and structure of the manuscript
Good
To what extent are the conclusions supported by the data?
Strongly support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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Comments to Author
In this manuscript, the authors describe the variations in virulence factors carried by Staphylococcus aureus species isolated from platelet concentrates, linked to disease in transfusion patients. Comparative genomics was used to identify differences between strains in regions associated with virulence, including the Type VII Secretion System (T7SS), chromosomal islands and the different prophages that are found to be lysogenised at the Sa3 site of integration. The authors describe the first documented occurrence of a S. aureus isolate that lacks a functional T7SS. Whilst this work is experimentally sound, I believe the conclusions drawn from these findings are flawed due to the absence of significant literature relating to the field. I believe a more thorough discussion of the relevant literature …
Comments to Author
In this manuscript, the authors describe the variations in virulence factors carried by Staphylococcus aureus species isolated from platelet concentrates, linked to disease in transfusion patients. Comparative genomics was used to identify differences between strains in regions associated with virulence, including the Type VII Secretion System (T7SS), chromosomal islands and the different prophages that are found to be lysogenised at the Sa3 site of integration. The authors describe the first documented occurrence of a S. aureus isolate that lacks a functional T7SS. Whilst this work is experimentally sound, I believe the conclusions drawn from these findings are flawed due to the absence of significant literature relating to the field. I believe a more thorough discussion of the relevant literature is required to give context to these findings, which I have detailed below. Major: The T7SS in Actinomycetota and in some instances, in Bacillota, has been associated with virulence, however, the T7SS is also harboured by many non-pathogenic bacteria. In S. aureus the T7SS is associated with a virulence phenotype, with substrates of the system, such as EsxA and EsxC, causing a significant immune response (PMID: 15657139). However, in recent years, there have been several strides taken in understanding how this system functions at a molecular level. It is now known that EsxA is a core secreted component that is required for the secretion of all other substrates (PMID: 25040609), The system is responsible for the secretion of large protein toxins which can be used to target other bacteria, such as the nuclease EsaD (PMID: 27723728; 27795323) and EsxC is one of targeting factors required to target EsaD to the T7SS (PMID: 37815362). Whilst the nuances of the system are still unclear, there is a clear role of the system in targeting both the host and other bacteria, whether by immune stimulation or interbacterial competition. However, the authors make no mention of the role that this system plays in interbacterial competition. As there is a role of the T7SS in bacterial competition, perhaps the loss of the T7SS in platelet concentrates is due to the absence of competiting bacteria and so is lost to minimise the metabolic burden on the cell? I believe this work would be greatly improved by expanding on the discussion of the T7SS literature in this respect. Line 27/106 - The T7SS is not composed of 12 genes. The authors references the 6 core, conserved genes which is fine, however, the additional genes cited are a substrate cluster composed of a nuclease toxin (EsaD - PMID: 27723728; 27795323), a chaperone (EsaE - PMID: 27723728), three targeting factors (EsxCBD - PMID: 37815362) and an immunity protein (EsaG - PMID: 27723728; 36307446). Downstream of EsaG are several paralogous copies of EsaG, as well as other known or predicted T7-associated immunity encoding genes (PMID: 35960642). This substrate cluster is only found in EssC1 variants of the system (REF- PMID: 26969225) and so if the authors wish to include this, should highlight that this cluster is only found in certain variants of the system and should mention the downstream cluster of orphan immunity-encoding genes. Line 181/195/204/218 - The reason for the use of a different reference strain in each experiment is unclear. Could the authors clarify the reason for this? Line 245-251/Fig. 2 - This is an interesting finding, but I was wondering if the authors could include the clonal complexes that each strain belongs to, in order to contextualise how closely these strains are related, eg. If all belong to CC8 or if there are a wide range of CC represented here. Fig 4a - Incorrect labels: > esxC is labelled as esaC; >esaE is labelled as esxD; >several genes are labelled 'T7SS' which is not correct; >I suspect that either the genes annotated as esxCBD in CBS2016-05 are either not of the correct representative size or are incorrectly annotated as they are much larger than the other Esx genes. >EsaG is consistently used incorrectly in this figure. EsaG is specifically the immunity protein which protects against the toxin, EsaD, and is often annotated as a DUF600 protein. Therefore, I suspect that the genes annotated as EsaG in all the strains except USA300 and ATR are incorrect. Line 316-22/Fig. 7 - It is clear from Fig. 7 that the phages that are lysogenised in each strain are different phages, however the term phiSa3 is used throughout the paragraph, implying that this is the same phage. The terminology used in this paragraph should be updated to make it clear that the prophages are lysogenised at the same locus but are not the same phages (PMID: 34126612). I believe when referring to the integration sight rather than the specific phage it is Sa3int. Can the gene diagram of the reference also be flipped to align with the other phage gene diagrams, and the same for the PS/169. Line 376 - The role of the T7SS in interbacterial antagonism should be included here. Line 386 - 'significantly reduced function' - in the data produced this strain not only lacks all other core components of the T7SS, but also the N-terminal transmembrane helices of EssC, leaving only ATPase domains in the cytoplasm, if produced at all. Therefore it would not be possible for anything to be secreted at even reduced levels by the T7SS in this strain as any components that spans the membrane are totally absent. Line 399 - The statement that the orphan immunity proteins encoded at this locus are used to 'inhibit host antimicrobial responses', is incorrect and not supported by the cited literature. The review that is cited highlights the orphan immunity proteins encoded at the T7SS gene clusters which are thought to protect against the T7-secreted polymorphic toxins secreted by competing bacteria, not against host antimicrobial peptides which insert in the membrane. As such the concluding statement 'supporting its virulence' is incorrect. Minor: Lines 51-57 - Including accession numbers for each strain would make it easier for the reader to locate these sequences. Line 83 - 'common part' - rephrase Lines 110 - 112/374-378 - Some references don't seem to cite the original soruces of these findings: Line 110 - PMID: 15657139 Line 111 - PMID: 15657139; 17680693; 25040609; 27130157 Line 374 - PMID: 26969225 Line 378 - PMID: 18554323; 15657139 Line 128 - There are also T7SS substrates associated with the vSaa island (PMID: 38114483). Line 218 - For the vSaa, a v is used in place of the 'nu'. Line 239 - the wording here is confusing as the use of pathogenetic island could be mistaken for pathogenicity islands which are also discussed in the manuscript. Removing this and taking specifically about prophage would be much clearer. Table 1/S1 - The lines in some columns do not align making it difficult for the reader to decipher the information presented. Fig S1 - Can the authors rotate the genomes of eg ATR, so all genomes are in the same orientation as the information in the comparisons is lost when all the comparisons lines cross over. All figures - the annotations used throughout for the strains (and in some cases the genes etc.) are not consistent.
Please rate the manuscript for methodological rigour
Good
Please rate the quality of the presentation and structure of the manuscript
Poor
To what extent are the conclusions supported by the data?
Partially support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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