Genome Sequences of Two Pseudomonas aeruginosa Isolates with Defects in Type III Secretion System Gene Expression from a Chronic Ankle Wound Infection

ABSTRACT Effector proteins translocated into host cells by the Pseudomonas aeruginosa type III secretion system (T3SS) are critical for phagocytic avoidance and systemic spread of the microorganism. The T3SS genes are present in virtually all P. aeruginosa strains. When examined in environmental isolates and clinical specimens, expression of the T3SS genes is the rule. Isolates from the airways of cystic fibrosis (CF) patients are one exception, and these isolates usually carry mutations that disable T3SS gene expression. In this study, we describe two P. aeruginosa isolates, one pigmented brown and one green, from a keratitis-ichthyosis-deafness (KID) syndrome patient with a chronic cutaneous ankle wound. Similar to most isolates from CF, both of the KID isolates were defective for T3SS gene expression. Providing the primary activator of T3SS transcription (exsA) in trans restored T3SS function. Since the exsA sequences were identical to that of a reference strain with active T3SS gene expression, we examined the cAMP-Vfr system, a critical regulator of T3SS gene expression. Vfr is a cAMP-dependent transcription factor that activates exsA expression. Whereas T3SS activity was corrected in the brown isolate by restoring cAMP synthesis, the same was not observed for the green isolate. These findings suggest that distinct mechanisms resulted in loss of T3SS gene expression in the KID isolates. The mutations responsible for the T3SS defects were not clearly evident by comparison of the whole-genome sequences to a reference strain. Our findings suggest that loss of T3SS gene expression may be a trait common to both CF and non-CF chronic infections. IMPORTANCE A common feature of microorganisms that cause chronic infections is a stealthy lifestyle that promotes immune avoidance and host tolerance. During chronic colonization of cystic fibrosis (CF) patients, Pseudomonas aeruginosa acquires numerous adaptations that include reduced expression of some factors, such as motility, O antigen, and the T3SS, and increased expression of other traits, such as biofilm formation. In this study, we report loss of T3SS gene expression in non-CF chronic isolates. This finding suggests that loss of the T3SS may be a common and important trait that contributes to persistence and may open avenues to explore the significance further using non-CF chronic infection models.

1. You note that MNPAB and MNPAG both belong to the same ST, thus I would like to know how closely related they are to one another. Their genome lengths differ by >350Kb, suggesting there might be differences in mobile genetic element content (maybe a mega-plasmid, or perhaps multiple prophages or ICEs) between them. These should be investigated and described.
2. I agree with the reviewers that the use of PA103 as a reference genome needs to be further motivated/explained, or perhaps a different, more closely related reference sequence should be used to identify mutations in MNPAB and MNPAG. I also would like to know what genetic differences (i.e. mutations) are identified when MNPAB and MNPAG are compared directly to one another, and if any of these differences could explain the observed difference in PexsD-lacZ reporter activity, secreted ExoU, and ExsA expression in the strains upon CyaB expression.
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The ASM Journals program strives for constant improvement in our submission and publication process. Please tell us how we can improve your experience by taking this quick Author Survey. In this study, Karash and colleagues examined the loss of type 3 secretion system (T3SS) activity in two clinical isolates of P. aeruginosa taken from a chronic ankle wound in a person with the genetic disorder keratitis-ichthyosis-deafness (KID) syndrome. The authors used a combination of comparative genomics and targeted in vitro assays to identify the mechanism of T3SS in these isolates. Providing ExsA in trans restored T3SS activity in both isolates and, looking further upstream in the regulatory cascade, providing CyaB in trans to restore cAMP production led to T3SS expression in one of the two isolates. This suggests that the loss of T3SS activity is attributable to two different mutations, although the authors were unable to identify the causative mutations by comparing each genome to a reference (PA103) and complementing a few of the likely candidate allelic variants (fimV and bifA). Overall, the authors conclude that similar to its evolution during chronic infection of the CF airways, the evolution of mutations that inactivate T3SS are important for P. aeruginosa persistence in this chronic skin infection that occurred in a person with KID syndrome.
Critique: This study provides useful and interesting insight into host adaptation of P. aeruginosa outside of the context of chronic CF respiratory infections, which is comparatively understudied. The loss of T3SS activity provides some evidence that P. aeruginosa may employ a common route of evolution toward persistence in a human host environment, whether it is in CF (reviewed in PMID: 26946977, non-CF bronchiectasis (e.g. PMID: 28446558), or in KID syndrome. Aside from a couple of technical suggestions regarding the use of a reference genome versus de novo assemblies (described in the "Major comments" section), my main critique of this paper is that it stops short of identifying the causative mutations leading to T3SS inactivation in these two isolates. However, considering the comparative gap in the literature regarding how P. aeruginosa evolves to persist in human hosts outside of the context of CF, I think it is informative to describe the isolates from KID syndrome and their genomes will be valuable to researchers broadly interested in P. aeruginosa evolution.
Major comments: (1) Issues related to use of a reference genome (PA103) 1a) It is not clear to me how distant the two clinical isolates are from the reference genome that they used to map their reads to and call mutations (PA103). It would be helpful if the authors included a phylogenetic tree based on core genome SNPs (e.g. using roary) with each clinical isolate, PA103, and a couple of other P. aeruginosa reference strains from each of the main clades (e.g. PAO1, PA14, and PA7; clades described in Figure 1A of PMID: 26483767).
1b) By mapping reads to a reference genome (PA103), the authors may miss mutations occurring in genes that are not present in the reference genome. If the two clinical isolates are more closely related to each other than to PA103, then the authors should use one of the de novo assembled clinical isolate genomes as a reference genome and map reads from the other clinical isolate to this, in order to identify variants that may explain the loss of T3SS activity. breseq is an easy-to-use tool for this type of analysis (PMID: 24838886).
Minor comments: (2) The authors should provide a supplemental table with the mutations they identified in these two strains.
(3) The authors describe an experiment providing BifA in trans (lines 153-154), but say "data not shown". The authors should include a figure with this data.

Reviewer #2 (Comments for the Author):
This study by Karash et al. describes two Pseudomonas aeruginosa strains, isolated from a chronic cutaneous wound from a keratitis-ichthyosis-deafness (KID) syndrome patient, in terms of their type III secretion system (T3SS) activity. Through assessment of an exsD transcriptional reporter and secretion of T3SS substrates, the authors show that both isolates are deficient in expression and activation of the T3SS, but that provision of the transcriptional activator ExsA in trans rescues this defect. Upstream in the regulatory cascade for T3SS activity, ectopic production of the cAMPdependent activator Vfr has little effect; however, CyaB expression restores T3SS expression and activity in one isolate, MNPAB. Nevertheless, CyaB has no impact on T3SS activity in the other isolate MNPAG. The authors conclude that the loss of type III secretion in these non-CF clinical isolates is through distinct mechanisms, proposing that T3SS deficiency may be a more frequent occurrence in chronic non-CF P. aeruginosa isolates than previously appreciated. The study is straightforward, with claims supported adequately by the data presented. This work, in tandem with the corresponding deposited sequences, sets the groundwork for furthering our understanding of the regulatory cascades governing the T3SS in P. aeruginosa in the context of human infection. Minor comments: 1) Please describe or reference the plasmids used to ectopically express exsA, vfr and cyaB. Are the genes under the control of a constitutive promoter? 2) Please add marker sizes for the immunoblots and Coomassie-stained gels 3) The description of the genome sequences could be more informative with a figure and perhaps some expansion. PA103 is claimed to be a "related" strain; however, the large number of polymorphisms precludes identification for the factor(s) responsible for the T3SS defects. Would the large number of polymorphisms prevent inclusion of a table documenting those of interest? 4) A comment on the major differences, for example in virulence factor genes, between the isolates and PA103 would be informative, since the differences in predicted CDS number appears to amount to hundreds.

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Spectrum00340-21 Review
Comments for the authors This study by Karash et al. describes two Pseudomonas aeruginosa strains, isolated from a chronic cutaneous wound from a keratitis-ichthyosis-deafness (KID) syndrome patient, in terms of their type III secretion system (T3SS) activity. Through assessment of an exsD transcriptional reporter and secretion of T3SS substrates, the authors show that both isolates are deficient in expression and activation of the T3SS, but that provision of the transcriptional activator ExsA in trans rescues this defect. Upstream in the regulatory cascade for T3SS activity, ectopic production of the cAMP-dependent activator Vfr has little effect; however, CyaB expression restores T3SS expression and activity in one isolate, MNPAB. Nevertheless, CyaB has no impact on T3SS activity in the other isolate MNPAG. The authors conclude that the loss of type III secretion in these non-CF clinical isolates is through distinct mechanisms, proposing that T3SS deficiency may be a more frequent occurrence in chronic non-CF P. aeruginosa isolates than previously appreciated. The study is straightforward, with claims supported adequately by the data presented. This work, in tandem with the corresponding deposited sequences, sets the groundwork for furthering our understanding of the regulatory cascades governing the T3SS in P. aeruginosa in the context of human infection.
Minor comments: 1) Please describe or reference the plasmids used to ectopically express exsA, vfr and cyaB. Are the genes under the control of a constitutive promoter? 2) Please add marker sizes for the immunoblots and Coomassie-stained gels 3) The description of the genome sequences could be more informative with a figure and perhaps some expansion. PA103 is claimed to be a "related" strain; however, the large number of polymorphisms precludes identification for the factor(s) responsible for the T3SS defects. Would the large number of polymorphisms prevent inclusion of a table documenting those of interest? 4) A comment on the major differences, for example in virulence factor genes, between the isolates and PA103 would be informative, since the differences in predicted CDS number appears to amount to hundreds. Re: Spectrum00340-21R1 (Genome sequences of two Pseudomonas aeruginosa isolates with defects in type III secretion system gene expression from a chronic ankle wound infection) Dear Dr. Timothy L Yahr: Thank you for submitting a revised version of your manuscript. Your manuscript has now been accepted, and I am forwarding it to the ASM Journals Department for publication. You will be notified when your proofs are ready to be viewed.
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