CarRS Two-Component System Essential for Polymyxin B Resistance of Vibrio vulnificus Responds to Multiple Host Environmental Signals

ABSTRACT Enteropathogenic bacteria express two-component systems (TCSs) to sense and respond to host environments, developing resistance to host innate immune systems like cationic antimicrobial peptides (CAMPs). Although an opportunistic human pathogen Vibrio vulnificus shows intrinsic resistance to the CAMP-like polymyxin B (PMB), its TCSs responsible for resistance have barely been investigated. Here, a mutant exhibiting a reduced growth rate in the presence of PMB was screened from a random transposon mutant library of V. vulnificus, and response regulator CarR of the CarRS TCS was identified as essential for its PMB resistance. Transcriptome analysis revealed that CarR strongly activates the expression of the eptA, tolCV2, and carRS operons. In particular, the eptA operon plays a major role in developing the CarR-mediated PMB resistance. Phosphorylation of CarR by the sensor kinase CarS is required for the regulation of its downstream genes, leading to the PMB resistance. Nevertheless, CarR directly binds to specific sequences in the upstream regions of the eptA and carRS operons, regardless of its phosphorylation. Notably, the CarRS TCS alters its own activation state by responding to several environmental stresses, including PMB, divalent cations, bile salts, and pH change. Furthermore, CarR modulates the resistance of V. vulnificus to bile salts and acidic pH among the stresses, as well as PMB. Altogether, this study suggests that the CarRS TCS, in responding to multiple host environmental signals, could provide V. vulnificus with the benefit of surviving within the host by enhancing its optimal fitness during infection. IMPORTANCE Enteropathogenic bacteria have evolved multiple TCSs to recognize and appropriately respond to host environments. CAMP is one of the inherent host barriers that the pathogens encounter during the course of infection. In this study, the CarRS TCS of V. vulnificus was found to develop resistance to PMB, a CAMP-like antimicrobial peptide, by directly activating the expression of the eptA operon. Although CarR binds to the upstream regions of the eptA and carRS operons regardless of phosphorylation, phosphorylation of CarR is required for the regulation of the operons, resulting in the PMB resistance. Furthermore, the CarRS TCS determines the resistance of V. vulnificus to bile salts and acidic pH by differentially regulating its own activation state in response to these environmental stresses. Altogether, the CarRS TCS responds to multiple host-related signals, and thus could enhance the survival of V. vulnificus within the host, leading to successful infection.

Reviewer #1 (Comments for the Author): Authors describes biological roles of the two-component system CarRS in the pathogenicity of the human pathogen Vibrio vulnificus. They showed the regulation of target genes by CarRS, which are associated with a survival in host, in molecular genetic levels. Overall the story and experimental design and results are quite straightforward, and has a scientific significance.
Reviewer #2 (Comments for the Author): In this study, Ko et al. investigated the two component systems, TCS, of Vibrio vulnificus which provide resistance to polymyxin B, PMB. Using mutant strains of PMB response regulator CarR and downstream CarR regulated genes, the authors find that CarRS TCS provides V. vulnificus with resistance to PMB. The authors show that CarR is a regulator of three identified operons, eptA, tolCV2 and carRS. They find that CarR activates eptA expression and this activation is required for resistance of PMB. They find that tolCV2 did not appear to affect the resistance to PMB. Additionally, they show that carR is requires phosphorylation mediated by carS which leads to downstream activation of eptA operon. Finally, the authors investigate how the carRS operon responds to host related signals, specifically, cations, bile salts and pH changes. They find that CarRS TCS alters its activation in response to all three signals and that CarR affects the ability for V. vulnificus to respond to host stresses. Overall, the experiments are well performed, and conclusions are supported.
Major Comments: In Figure 4, the authors investigate whether CarR phosphorylation is required for gene regulation and PMB resistance. In Figure  4A, the authors show that carS and a carRD55A point mutant have reduced phosphorylation compared to the wild type. The authors also find that carS and carSH220A have lower CarR-P than wild type. The authors conclude that H220 residue of CarS is involved in CarR phosphorylation. However, Figure 4A does not fully support this conclusion. The authors base their conclusions on one representative western blot, which is not very clear. The authors should repeat the experiments in Figure 4A and perform densitometry analysis to strengthen their conclusions.

Minor Comments:
Line 230-231: The authors show that tolCV2 expression is regulated by CarR but does not affect PMB resistance of V. vulnificus. Although the authors state some reasons for this, the authors could expand on how future studies can be performed to understand why tolCV2 is regulated by CarR.
Line 250-251: Authors state that PcarRS activity increases in low levels of cations. However, the data shows that activity is reduced for V. vulnificus in the presence of cations. The authors should explain this discrpency. Figure 7: Legend states luminescence and growth (A600) were measured at time intervals. However, there is no y-axis which shows A600 readings. Are RLUs and A600 proportional to one another?
Staff Comments:

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Authors describes biological roles of the two-component system CarRS in the pathogenicity of the human pathogen Vibrio vulnificus. They showed the regulation of target genes by CarRS, which are associated with a survival in host, in molecular genetic levels.
Overall the story and experimental design and results are quite straightforward, and has a scientific significance.

Major points.
CarRS has been well studied in the related Vibrio species. What is the novelty of manuscript?
Authors need to highlight of scientific significance of their finding.

Minor points
What would be a working mechanisms of eptA? In this study, Ko et al. investigated the two component systems, TCS, of Vibrio vulnificus which provide resistance to polymyxin B, PMB. Using mutant strains of PMB response regulator CarR and downstream CarR regulated genes, the authors find that CarRS TCS provides V. vulnificus with resistance to PMB. The authors show that CarR is a regulator of three identified operons, eptA, tolCV2 and carRS. They find that CarR activates eptA expression and this activation is required for resistance of PMB. They find that tolCV2 did not appear to affect the resistance to PMB. Additionally, they show that carR is requires phosphorylation mediated by carS which leads to downstream activation of eptA operon. Finally, the authors investigate how the carRS operon responds to host related signals, specifically, cations, bile salts and pH changes. They find that CarRS TCS alters its activation in response to all three signals and that CarR affects the ability for V. vulnificus to respond to host stresses. Overall, the experiments are well performed, and conclusions are supported.

Major Comments:
In Figure 4, the authors investigate whether CarR phosphorylation is required for gene regulation and PMB resistance. In Figure 4A, the authors show that ∆carS and a carRD55A point mutant have reduced phosphorylation compared to the wild type. The authors also find that ∆carS and carSH220A have lower CarR-P than wild type. The authors conclude that H220 residue of CarS is involved in CarR phosphorylation. However, Figure 4A does not fully support this conclusion. The authors base their conclusions on one representative western blot, which is not very clear. The authors should repeat the experiments in Figure 4A and perform densitometry analysis to strengthen their conclusions.

Minor Comments:
Line 230-231: The authors show that tolCV2 expression is regulated by CarR but does not affect PMB resistance of V. vulnificus. Although the authors state some reasons for this, the authors could expand on how future studies can be performed to understand why tolCV2 is regulated by CarR.
Line 250-251: Authors state that PcarRS activity increases in low levels of cations. However, the data shows that activity is reduced for V. vulnificus in the presence of cations. The authors should explain this discrpency. However, there is no y-axis which shows A600 readings. Are RLUs and A600 proportional to one another?

On the comments from Reviewer #2
In this study, Ko et al. investigated the two component systems, TCS, of Vibrio vulnificus which provide resistance to polymyxin B, PMB. Using mutant strains of PMB response regulator CarR and downstream CarR regulated genes, the authors find that CarRS TCS provides V. vulnificus with resistance to PMB. The authors show that CarR is a regulator of three identified operons, eptA, tolCV2 and carRS. They find that CarR activates eptA expression and this activation is required for resistance of PMB. They find that tolCV2 did not appear to affect the resistance to PMB. Additionally, they show that carR is requires phosphorylation mediated by carS which leads to downstream activation of eptA operon. Finally, the authors investigate how the carRS operon responds to host related signals, specifically, cations, bile salts and pH changes. They find that CarRS TCS alters its activation in response to all three signals and that CarR affects the ability for V. vulnificus to respond to host stresses. Overall, the experiments are well performed, and conclusions are supported.
Major Comments: In Figure 4, the authors investigate whether CarR phosphorylation is required for gene regulation and PMB resistance. In Figure 4A, the authors show that ∆carS and a carRD55A point mutant have reduced phosphorylation compared to the wild type. The authors also find that ∆carS and carSH220A have lower CarR-P than wild type. The authors conclude that H220 residue of CarS is involved in CarR phosphorylation. However, Figure 4A does not fully support this conclusion. The authors base their conclusions on one representative western blot, which is not very clear. The authors should repeat the experiments in Figure 4A and perform densitometry analysis to strengthen their conclusions.

Response:
We appreciate the Reviewer's valuable comments. As the Reviewer suggested, we repeated the same experiments to clearly detect the phosphorylated form of CarR (CarR-P) using a Phos-tag SDS-PAGE gel and thus to demonstrate that D55 of CarR and H220 of CarS are essential for the CarR phosphorylation. Due to the difficulties in differentiating CarR and CarR-P using the Phos-tag SDS-PAGE gel, we conducted multiple Western blot analyses by changing several experimental conditions. For example, we tried to optimize the temperature for protein preparation, current and voltage for SDS-PAGE, and even machinery for protein transfer to membrane.
As shown in Figure 4A in the revised manuscript, CarR-P was clearly detected in the wildtype strain but not in the carRD55A strain, indicating that D55 of CarR is the phosphorylation site. In addition, the cellular level of CarR-P was decreased in the ΔcarS and carSH220A strains to the level not detectable by immunoblotting ( Figure 4A in the revised manuscript), indicating that H220 of CarS is obviously involved in the CarR phosphorylation. These results were consistent with our previous results of Figure 4A in the original manuscript. To additionally comply with the Reviewer's comment, we performed densitometry analysis using three independent Western blots and calculated the ratio of the cellular level of CarR-P to that of total CarR. The result showed that the cellular level of CarR-P accounts for about 8% of that of total CarR in the wild-type strain under the condition we tested ( Figure 4B in the revised manuscript).
The new results have been incorporated in the Results and Materials and Methods sections of the revised manuscript with appropriate descriptions (lines 166-170, 344-349).

Minor Comments:
Line 230-231: The authors show that tolCV2 expression is regulated by CarR but does not affect PMB resistance of V. vulnificus. Although the authors state some reasons for this, the authors could expand on how future studies can be performed to understand why tolCV2 is regulated by CarR.

Response:
We appreciate the Reviewer for raising this point. As we described in the Discussion section, TolCV2 is expected to act as a virulence factor of V. vulnificus. Because CarRS senses multiple host-related signals such as bile salts and acidic pH (Figure 7), the TCS could contribute to precise expression of tolCV2 under the host environment. Thus, we are planning to perform several in vitro and in vivo experiments to understand the role of TolCV2 during host infection. Additionally, the effect of the tolCV2 regulation by CarRS on the pathogenicity of V. vulnificus will be determined in the future study. We have updated the Discussion section including this as a future direction (lines 237-239).
Line 250-251: Authors state that PcarRS activity increases in low levels of cations. However, the data shows that activity is reduced for V. vulnificus in the presence of cations. The authors should explain this discrpency.

Response:
We are sorry for the confusing description in this part. We compared the PcarRS activities of the V. vulnificus strains by adding either sterile distilled water (solid line; low levels of divalent cations) or 10 mM CaCl2 or 10 mM MgCl2 (dashed or dotted line; high levels of divalent cations) to the culture medium. As shown in Figure 7B, the PcarRS activity of wild type is higher in the low levels of cations, compared with that in the high levels of cations. For clear description, we have updated Figure 7B in the revised manuscript.