PONE-D-21-38824The Chlamydia trachomatis inclusion membrane protein L (IncL) associates with host cell lipid dropletsPLOS ONE

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While both reviewers thought the experiments were well done, they also expressed concerns that the data does not support the overall conclusion that CT006 associates with lipid droplets during Chlamydia infection.

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Reviewers' comments:

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Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

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Reviewer #1: Yes

Reviewer #2: No

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

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Reviewer #1: In this manuscript by Bugalhão et al., the authors used several large-scale screens to preliminarily characterize Chlamydia trachomatis inclusion membrane proteins (Incs). They demonstrate that 2 Incs perturb host vesicular trafficking, which is largely in support of previous studies. Using ectopic expression, they demonstrate that several Incs traffic to eukaryotic organelles. Uniquely, they demonstrate that CT006 (IncL) might target lipid droplets. Overall, this is a nice descriptive study that provides some insight into the putative function of CT006. My comments are minor, namely a few missing citations and that it is premature to rename CT006 to IncL. I have included specifics below.

Missing citations:

Mital J (2013) PLOS One- Ectopically expressed several Incs

Weber MM (2015) Infect Immun- demonstrated that CT006 is a bona fide Inc

Based on the data presented herein, I believe it is premature to rename CT006 to IncL. The observations that it might target lipid droplets largely relies on ectopic data and minimal infection experiments. Without a mutant to confirm it is important for lipid droplet recruitment or a binding partner, it is best to keep its designation as CT006.

Reviewer #2: Intravacuolar Chlamydia trachomatis is notorious for interacting with many host mammalian organelles. Identifying bacterial effectors (eg., Inc proteins) exported at the inclusion membrane or secreted into the host cell, that mediate these interactions are important for better understanding the chlamydial pathogenicity. To identify novel C. trachomatis Incs interfering with host vesicular trafficking, thus potentially interacting with host vesicles/organelles, the authors have undertaken a comprehensive approach: they used S. cerevisiae to ectopically express cytosolic domains of Incs to monitor vacuolar protein sorting mistrafficking in yeast. They identified CT223 and CT229 capable of causing sorting defects in yeast. The paper focuses on CT229 localization.

Overall, the assays performed in the manuscript are well-done (no technical flaws) but the interpretation of the results is erroneous and misleading as we cannot conclude from the data that CT229 (renamed IncL) associates with host cell lipid droplets (LD) for several reasons:

1. the authors have expressed CT229 FL or fragments in yeast and mammalian cells: CT229 FL is cytosolic, CT22991-215 is endosomal and CT2291-88 is targeted to LD. Thus positively charged sequences close to the amino-terminal hydrophobic domain CT229 (between aa 1 and 88) are likely responsible for the tropism of this peptide to LD – same situation with positively charged sequences of caveolin that mediate the sorting of caveolin to LD while central hydrophobic domain anchors of caveolin direct the protein to the ER. However, we cannot extrapolate from these data that CT229 physiologically interact with host LD in infected cells, more especially if CT229 FL does not localize to LD in yeast and mammalian cells.

2. The localization of CT229 assessed by IFA in Chlamydia strains expressing IncL-2HA (with anti-HA) or GSK fused to IncL (with anti-GSK) is at the inclusion membrane from 16 to 24hpi (Fig. 6). In the attempt to examine whether CT229 is at least secreted into the host cell for LD interaction, the authors looked at CT229 localization at early time points (2 to 8hpi in Fig. S10), and observed a signal beyond the cytosolic signal of Hsp60. They conclude that CT229 is on extensions of the inclusion membrane. However, without showing a colocalization of CT229 with IncA or IncG that are expressed on extensions of the inclusion membrane, we cannot ascertain that CT229 are on extensions of the inclusion membrane. Irrespective to these data, CT229 is not observed close/around host LD: in Fig. 6. the CT229 red signal from IncL-2HA does not intersect with the green LD signal. The only conclusion of Fig. 6 is that few host LD are in proximity to the inclusion, at the limit of the fluorescence microscopy resolution (~200nm).

3. The Discussion contains too many repetitions of the Results section (to be combined). What must be better discussed is the connection between CT229 with IncA (according to ref. 5, only IncA associate with purified host LD), IncX (in the proposed model in ref. 5) as well with IncG, CT618 and Cap1 (from ref. 10) in case of physiological role of CT229 on host LD.

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Reviewer #1: No

Reviewer #2: No

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The Chlamydia trachomatis inclusion membrane protein CT006 associates with lipid droplets in eukaryotic cells

PONE-D-21-38824R1

Dear Dr. Mota,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Stacey D Gilk, Ph.D.

Academic Editor

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Additional Editor Comments (optional):

Reviewers' comments:

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Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

Reviewer #2: It is indeed safer to keep CT6006 and avoid using IncL at this stage.

For future work on host organelle-inclusion interaction, it will be better to have an antibody against the inclusion membrane. Many labs have some, just ask.

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Reviewer #1: No

Reviewer #2: No