Analysis of Bacterial Phosphorylcholine-Related Genes Reveals an Association between Type-Specific Biosynthesis Pathways and Biomolecules Targeted for Phosphorylcholine Modification

ABSTRACT Many bacterial surface proteins and carbohydrates are modified with phosphorylcholine (ChoP), which contributes to host mimicry and can also promote colonization and survival in the host. However, the ChoP biosynthetic pathways that are used in bacterial species that express ChoP have not been systematically studied. For example, the well-studied Lic-1 pathway is absent in some ChoP-expressing bacteria, such as Neisseria meningitidis and Neisseria gonorrhoeae. This raises a question as to the origin of the ChoP used for macromolecule biosynthesis in these species. In the current study, we used in silico analyses to identify the potential pathways involved in ChoP biosynthesis in genomes of the 26 bacterial species reported to express a ChoP-modified biomolecule. We used the four known ChoP biosynthetic pathways and a ChoP transferase as search terms to probe for their presence in these genomes. We found that the Lic-1 pathway is primarily associated with organisms producing ChoP-modified carbohydrates, such as lipooligosaccharide. Pilin phosphorylcholine transferase A (PptA) homologs were detected in all bacteria that express ChoP-modified proteins. Additionally, ChoP biosynthesis pathways, such as phospholipid N-methyltransferase (PmtA), phosphatidylcholine synthase (Pcs), or the acylation-dependent phosphatidylcholine biosynthesis pathway, which generate phosphatidylcholine, were also identified in species that produce ChoP-modified proteins. Thus, a major finding of this study is the association of a particular ChoP biosynthetic pathway with a cognate, target ChoP-modified surface factor; i.e., protein versus carbohydrate. This survey failed to identify a known biosynthetic pathway for some species that express ChoP, indicating that a novel ChoP biosynthetic pathway(s) may remain to be identified. IMPORTANCE The modification of bacterial surface virulence factors with phosphorylcholine (ChoP) plays an important role in bacterial virulence and pathogenesis. However, the ChoP biosynthetic pathways in bacteria have not been fully understood. In this study, we used in silico analysis to identify potential ChoP biosynthetic pathways in bacteria that express ChoP-modified biomolecules and found the association between a specific ChoP biosynthesis pathway and the cognate target ChoP-modified surface factor.

Reviewer #2 (Comments for the Author): General: This submission provides an updated analysis of pathways involved in the expression of phosphorylcholine (ChoP) on the surface of bacteria. As this host-like structure appears on the cell surface in multiple ways and on many diverse species, this in silico analysis provides a useful roadmap for this genetic complexity. I have relatively few comments. 1) Abstract: Line 24-5. It would be clearer if you delete although.....is well studied, this. 2) Abstract: Line 29 bacterial species instead of bacteria 3) Abstract: Line 34. Not clear what Similarly refers to? 4) It would be helpful to point out those structures/species for which there is structural evidence for ChoP expression as opposed to mAb recognition, which as pointed out in the case of Pseudomonas aeruginosa is putative. 5) It might be useful to cite the review on bacterial ChoP by SE Clark (IAI 2013) which discusses some of the biological advantages of cell surface ChoP expression. 6) It might help the readers to explain the structural relationship between PE and ChoP. 7) Are there examples of species with both Lic1 and PptA homologs/pathways? 8) The potential sources of ChoP (i.e. choline uptake v. biosynthesis) could be explained much more clearly. This should include environmental free choline and the transfer of choline from host intermediates involved in phosphatidylcholine biosynthesis (see Fan X, Mol Micro 2001, 2003 which is not spelled out. 9) In Table 1, consider adding the primary site within the host where each species is typically found. 10) A brief discussion of the comparison to phosphocholine biosynthesis to generate PC in eukaryotes would be a useful addition.

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Dear Prof Tang,
Thank you for your letter and for the reviewer's comments concerning our manuscript entitled "Analysis of bacterial phosphorylcholine related genes reveals an association between the type-specific biosynthesis pathways and the biomolecules targeted for phosphorylcholine modification". These comments have been very valuable in improving our revised manuscript. We have responded to each comment, detailed in the point by point response below and in the manuscript, as indicated.

Reviewer #1 (Comments for the Author):
The review of your work has been completed and I do not have any additional suggestions.
I wish good work.

Response
We thank the reviewer.

Reviewer #2 (Public repository details (Required)):
Lots of large datasets appropriate for a depository.

Response
We disagree. The study comprises a series of blast searches using search terms defined by accession number of the sequence of the protein being used as a search term (noted in the main text and table), and key observations from the search results defined (with accession number and % identity) by lists of relevant proteins in the tables. There are no large datasets that are appropriate to deposit. Any person can use the approach we describe to replicate our data.

Reviewer #2 (Comments for the Author):
General: This submission provides an updated analysis of pathways involved in the expression of phosphorylcholine (ChoP) on the surface of bacteria. As this host-like structure appears on the cell surface in multiple ways and on many diverse species, this in silico analysis provides a useful roadmap for this genetic complexity. I have relatively few comments. 1) Abstract: Line 24-5. It would be clearer if you delete although.....is well studied, this.

Response
Lines 26-28: As suggested, this sentence has been modified and now reads, "For example, the well-studied Lic-1 pathway is absent in some ChoP-expressing bacteria, such as Neisseria meningitidis and Neisseria gonorrhoeae." (See pg. 1, line 26-28.) 2) Abstract: Line 29 bacterial species instead of bacteria

Response
Line 31: As suggested, the word "bacteria" has been modified to "bacteria species" and now reads "In the current study, we used in silico analyses to identify the potential pathways

Response
Line 36: As per the suggestion, the word "Similarly" was inadequate and has been replaced with "Additionally". The context now reads as follows: "Pilin phosphorylcholine transferase A (PptA) homologs were detected in all bacteria that express ChoP-modified proteins.
Additionally, ChoP biosynthesis pathways, such as phospholipid N-methyltransferase (PmtA), phosphatidylcholine synthase (Pcs), or the acylation-dependent phosphatidylcholine biosynthesis pathway, which generate phosphatidylcholine, were also identified in species that produce ChoP modified proteins." (See pg. 2, 4) It would be helpful to point out those structures/species for which there is structural evidence for ChoP expression as opposed to mAb recognition, which as pointed out in the case of Pseudomonas aeruginosa is putative.

Response
Line 637: The Table 1    6) It might help the readers to explain the structural relationship between PE and ChoP.

Response
Lines 262-265: The structural relationship between PE and ChoP has been elucidated in line 262-265. The revised sentence now reads as follows: "ChoP is PE with three methyl groups.
In parasites such as Plasmodium falciparum  Table 1, consider adding the primary site within the host where each species is typically found.

Response
Line 637: As per the suggestion, the primary colonization sites of each specie in the host were added in the 10) A brief discussion of the comparison to phosphocholine biosynthesis to generate PC in eukaryotes would be a useful addition.

Response
The manuscript primarily focuses on prokaryotic bacterial phosphorylcholine.
Phosphocholine biosynthesis to generate PC in eukaryotes is beyond the scope of this study.