Convergent Within-Host Adaptation of Pseudomonas aeruginosa through the Transcriptional Regulatory Network

ABSTRACT Bacteria adapt to their host by mutating specific genes and by reprogramming their gene expression. Different strains of a bacterial species often mutate the same genes during infection, demonstrating convergent genetic adaptation. However, there is limited evidence for convergent adaptation at the transcriptional level. To this end, we utilize genomic data of 114 Pseudomonas aeruginosa strains, derived from patients with chronic pulmonary infection, and the P. aeruginosa transcriptional regulatory network. Relying on loss-of-function mutations in genes encoding transcriptional regulators and predicting their effects through the network, we demonstrate predicted expression changes of the same genes in different strains through different paths in the network, implying convergent transcriptional adaptation. Furthermore, through the transcription lens we associate yet-unknown processes, such as ethanol oxidation and glycine betaine catabolism, with P. aeruginosa host adaptation. We also find that known adaptive phenotypes, including antibiotic resistance, which were identified before as achieved by specific mutations, are achieved also through transcriptional changes. Our study has revealed novel interplay between the genetic and transcriptional levels in host adaptation, demonstrating the versatility of the adaptive arsenal of bacterial pathogens and their ability to adapt to the host conditions in a myriad of ways. IMPORTANCE Pseudomonas aeruginosa causes significant morbidity and mortality. The pathogen's remarkable ability to establish chronic infections greatly depends on its adaptation to the host environment. Here, we use the transcriptional regulatory network to predict expression changes during adaptation. We expand the processes and functions known to be involved in host adaptation. We show that the pathogen modulates the activity of genes during adaptation, including genes implicated in antibiotic resistance, both directly via genomic mutations and indirectly via mutations in transcriptional regulators. Furthermore, we detect a subgroup of genes whose predicted changes in expression are associated with mucoid strains, a major adaptive phenotype in chronic infections. We propose that these genes constitute the transcriptional arm of the mucoid adaptive strategy. Identification of different adaptive strategies utilized by pathogens during chronic infection has major promise in the treatment of persistent infections and opens the door to personalized tailored antibiotic treatment in the future.


Preparing Revision Guidelines
To submit your modified manuscript, log onto the eJP submission site at https://msystems.msubmit.net/cgi-bin/main.plex. Go to Author Tasks and click the appropriate manuscript title to begin the revision process. The information that you entered when you first submitted the paper will be displayed. Please update the information as necessary. Here are a few examples of required updates that authors must address: • Point-by-point responses to the issues raised by the reviewers in a file named "Response to Reviewers," NOT IN YOUR COVER LETTER. • Upload a compare copy of the manuscript (without figures) as a "Marked-Up Manuscript" file. • Each figure must be uploaded as a separate file, and any multipanel figures must be assembled into one file. • Manuscript: A .DOC version of the revised manuscript • Figures: Editable, high-resolution, individual figure files are required at revision, TIFF or EPS files are preferred ASM policy requires that data be available to the public upon online posting of the article, so please verify all links to sequence records, if present, and make sure that each number retrieves the full record of the data. If a new accession number is not linked or a link is broken, provide production staff with the correct URL for the record. If the accession numbers for new data are not publicly accessible before the expected online posting of the article, publication of your article may be delayed; please contact the ASM production staff immediately with the expected release date.
For complete guidelines on revision requirements, please see the journal Submission and Review Process requirements at https://journals.asm.org/journal/mSystems/submission-review-process. Submission of a paper that does not conform to mSystems guidelines will delay acceptance of your manuscript.
I would like to thank the authors for their thorough response to the raised concerns. My points were answered in detail, both in the rebuttal letter and in the newly added and revised text passages in the manuscript. Although the approach presented is quite unique, I think the analysis is now much easier to follow. The significance and relevance of the gene expression changes predicted in this work are clearly supported by the integration of additional phenotypic data, e.g. with regards to antibiotic susceptibility/resistance. Most ambiguities have been resolved. The manuscript presents interesting and important insights into the role of convergent evolution and the interplay of (patho-)adaptive mutations and their transcriptional consequences. With best regards,

Hanah Margalit
List of revisions (Please note that the line numbering we refer to is the one adjacent to the text)

Reviewer 2 I would like to thank the authors for their thorough response to the raised concerns. My points were answered in detail, both in the rebuttal letter and in the newly added and revised text passages in the manuscript. Although the approach presented is quite unique, I think the analysis is now much easier to follow. The significance and relevance of the gene expression changes predicted in this work are clearly supported by the integration of additional phenotypic data, e.g. with regards to antibiotic susceptibility/resistance. Most ambiguities have been resolved. The manuscript presents interesting and important insights into the role of convergent evolution and the interplay of (patho-)adaptive mutations and their transcriptional consequences.
I have only a few minor comments/suggestions: P. 3, line 28: Wording -maybe "patients with CF" instead of "CF" Done (page 3, line 28).

P. 3 line 30: In the context of evolution of tolerance and the subsequent evolution of resistance, the work of Levin-Reisman et al (https://doi.org/10.1126/science.aaj2191) and Santi et al (https://doi.org/10.1128/mBio.03482-20) would fit in quite nicely.
We have added references to these papers (page 3, line 30).
P. 5 line 20: Do I understand correctly that of the 163 nodes representing TFs/SFs in the TRN used, only the 29 TFs/SFs shown in Fig. S1b have LOF mutations in more than one progenitor-progeny pair? I think this number should be mentioned in the results section. The finding that LOF mutations occur in <20% of the included TFs/SFs is not uninteresting.
We added these results to the manuscript (page 5, lines 26-28). The mexR gene did not undergo an LOF mutation in this patient. However, mexR is predicted to have reduced expression via the TRN in the relevant progenitor-progeny pair. This possibly explains the predicted increased expression of mexAB-oprM. We have added this information to the manuscript (page 8, line 13).   We have added the number of data points, there are 28 progenitor-progeny pairs with mucA LOF mutations (Fig. S1b, Table S2) and 158 pairs without such mutations. Note that the figure was slightly changed. The previous version did not exclude progenitor-progeny pairs with preexisting mucA LOF mutations. After excluding these pairs from the analysis, the difference between the groups is even more pronounced than before.
Supplementary Results: Page 3: Predicted reduced synthesis of HCN is quite an interesting finding, since hcn genes are, among others, regulated by the quorum sensing master regulators RhlR and LasR (hotspot for patho-adaptive mutations). I think the possible link to QS/mutations in LasR could be mentioned here.
We addressed this possible link ( Supplementary Information page 3, lines 11-15). Notably, the Heterogeneity scores of all genes in this pathway are very high, meaning their predicted changes in expression are the result of different genetic events in different progenitorprogeny pairs. Therefore, even if the predicted changes in some pairs are linked to LOF mutations in lasR, they could not explain the changes across all pairs. The review we cited by Poole addresses this subject (1). According to the review, the MexAB-OprM efflux pump accommodates the broadest range of β-Lactams, including carabapenems, penicillin-ticarcillin, and aztreonam. We added some of the references cited by Poole on this subject to strengthen the connection (2)(3)(4)(5).