Dual-functional quorum sensing signal synthases DspII and DspI coordinate virulence switch in Pseudomonas aeruginosa

Domain: neuroscience
Published: 2026-01-22
Editorial outcome: major_revision

Abstract
The transition between motility-associated acute infection and biofilm-associated chronic infection is crucial for bacterial proliferation and survival within hosts. This process of transition can be influenced by various host and environmental cues, however, whether bacteria could actively modulate the switch remains poorly understood. Here we identify a novel enoyl-CoA hydratase/isomerase, PA0744 (designated as DspII), which is co-upregulated with the previously characterized enoyl-CoA hydratase/isomerase DspI in a cell density-dependent manner to induce chronic-to-acute virulence switch in Pseudomonas aeruginosa. The two proteins form a heterocomplex that synthesizes the DSF-family quorum sensing signal cis-2-decenoic acid (CDA), which positively regulates the expression of the phosphodiesterase RbdA, and subsequently, activates biofilm dispersion and swarming motility through the c-di-GMP/FleQ pathway. Intriguingly, the DspII-DspI complex also synergistically antagonizes the GacA-upregulated sRNAs, RsmY and RsmZ, leading to overproduction of type III secretion system (T3SS) and augment of acute pathogenesis. Notably, the affinity of DspII-DspI interaction and consequently T3SS gene expression are fine-tuned by CDA levels. This study unveils the novel enzymatic role of the DspII-DspI complex in CDA biosynthesis as well as its non-enzymatic role in T3SS gene regulation, which presents a simple yet sophisticated bacterial signal-driven auto-regulatory mechanism that governs the chronic-to-acute virulence switch in P. aeruginosa.

Introduction
Bacterial pathogenesis progresses through multiple stages, which initiate with colonization at host infection sites by planktonic, hyper-virulent pathogenic cells, resulting in the early stage of acute infection1. This early stage is characterized by the expression of acute virulence factors such as the type III secretion system (T3SS) that facilitate host invasion and immune evasion2,3. However, upon encountering unfavorable conditions or certain environmental cues, such as immune response or other bio-stresses, the pathogens may reprogram their virulence either mounting attack by boosting virulence factor production4, or retracting by switching from acute to chronic infection5. The transition from acute to chronic infection is marked by the attenuation of motility (e.g., flagellar biosynthesis) and the activation of biofilm-associated factors (e.g., exopolysaccharides (EPSs)), leading to the formation of a sessile, matrix-encased community known as biofilm6. Within this protective biofilm structure, bacteria establish persistent infections that are extraordinary resistant to both host immune defenses and antimicrobial treatments7,8. Notably, this is a reversible process and bacterial cells within biofilms are common source of new infections, as they could also be activated under certain environmental conditions to switch back from chronic persistence to acute infection mode9.

Pseudomonas aeruginosa, a versatile opportunistic pathogen capable of causing serious acute and chronic infections in humans10, serves as an exemplary model for studying the environmental cues and mechanisms that influence the virulence switch. Diverse host and environmental cues such as oxygen levels, nutrient availability, metal ion concentrations, antibiotics, and other factors11–15, could influence the transition between acute and chronic infections. Central to this switch is the second messenger cyclic di-GMP (c-di-GMP). Elevated intracellular c-di-GMP content promotes biofilm formation, shifting the planktonic-associated acute infection to biofilm-associated chronic persistence, whereas depleted intracellular c-di-GMP level promotes biofilm dispersal, switching the pathogen from chronic persistence to acute infection mode16.

cis-2-decenoic acid (CDA), a self-produced fatty acid-based quorum sensing (QS) signaling molecule belonging to the diffusible signal factor (DSF) family, has been demonstrated to induce biofilm dispersion and reactivate motility in P. aeruginosa, thereby promoting the chronic-to-acute virulence switch17. The enoyl-CoA hydratase/isomerase DspI is essential for CDA biosynthesis, and recently, it was reported that CDA is recognized by the sensor/response regulator hybrid DspS and then induces the chronic-to-acute virulence switch by decreasing the intracellular c-di-GMP content9,18,19. Despite these advances, it remains largely unclear how CDA is synthesized and whether DspI is the sole synthase for CDA. In addition, detailed molecular mechanisms underlying the regulation of virulence switch by CDA are largely unknown. In this study, we identified a novel enoyl-CoA hydratase/isomerase, PA0744 (designated as DspII), which complexes with DspI for CDA biosynthesis. Intriguingly, we further uncovered a non-enzymatic role of the DspII-DspI complex in regulating T3SS gene expression by directly binding to a response regulator GacA and the promoter of a small RNA RsmZ. More interestingly, formation of the DspII-DspI complex and interaction between the complex and DNA promoter are auto-modulated by CDA. These findings unveil the novel dual-functional roles of the DspII-DspI complex in modulating chronic-to-acute virulence switch and the sophisticated regulatory network.

Results
An novel enoyl-CoA hydratase/isomerase PA0744 is potentially involved in chronic-to-acute virulence switch
Systematic genomic analysis of P. aeruginosa PAO1 revealed 12 genes encoding probable enoyl-CoA hydratase/isomerases (Supplementary Fig. 1A). Except PA1748 which did not show significant similarity with the previously characterized enoyl-CoA hydratase/isomerase DspI (PA0745), the other 10 uncharacterized ones share about 26%~40% similarity with DspI (Supplementary Fig. 1A and 1B). Given the established role of DspI in CDA biosynthesis20,21, we hypothesized potential functional redundancy among these proteins in synthesizing CDA and regulating biofilm dispersion. We generated 12 mutants with single gene deletion and determined their function in biofilm dispersion by comparing the production of biofilm with the PAO1 wild-type (WT) strain. In addition to DspI, we found that the loss of another enoyl-CoA hydratase/isomerase, PA0744, led to a substantial accumulation of biofilm production (Supplementary Fig. 1C), suggesting that PA0744 is also essential for biofilm dispersion and potentially required for the chronic-to-acute virulence switch.

PA0744 is encoded by the gene immediately downstream of dspI (PA0745), sharing about 27.85% similarity with DspI at the peptide sequence level (Supplementary Fig. 1A and 1B), and notably, they constitute a contiguous cluster with three metabolic genes PA0743, PA0746, and PA0747, which encodes probable 3-hydroxyisobutyrate dehydrogenase, probable acyl-CoA dehydrogenase, and probable aldehyde dehydrogenase, respectively (Figs. 1A, B). In addition, an adjacent gene PA0748 is predicted to encode a probable local transcriptional regulator for the gene cluster of PA0743-PA0747 (Fig. 1A, B). To verify if PA0743-PA0747 genes are transcribed together, we performed reverse transcriptase PCR (RT-PCR) and confirmed polycistronic transcription of PA0743-PA0747 (Fig. 1C). Given the close genetic relationship of these genes, we were curious whether PA0743, PA0746, PA0747, and PA0748 contributed to the chronic-to-acute virulence switch as well. To answer this, we knocked out each of these genes and compared their production of biofilm and ability of swarming compared to the WT strain. Unexpectedly, only the mutants ΔPA0744 and ΔdspI displayed increased biofilm accumulation and impaired swarming motility, while deletions of adjacent genes PA0743, PA0746, PA0747, and PA0748 did not alter biofilm or swarming profiles (Fig. 1D, E), indicating functional specificity of PA0744 and dspI in modulating the chronic-to-acute virulence switch within the operon.

Furthermore, the double-deletion mutant ΔPA0744ΔdspI showed similar levels of biofilm production and swarming motility with the single-deletion mutants ΔPA0744 and ΔdspI, respectively, and complementation of the deleted gene(s) fully restored biofilm dispersion and swarming motility in all the mutants ΔPA0744, ΔdspI, and ΔPA0744ΔdspI (Fig. 1F, G), suggesting that PA0744 and DspI probably function in the same pathway.

PA0744 (DspII), co-expressed with DspI in a cell density-dependent manner, is essential for CDA biosynthesis
Given that CDA is a QS signal, we investigated whether PA0744 and dspI are expressed in a cell density-dependent manner. First, reverse transcription-quantitative PCR (RT-qPCR) assay showed that the expression of PA0744 and dspI was reciprocally regulated, i.e., loss of one almost fully inactivated the expression of the other gene (Fig. 2A). Moreover, promoter activity assay using a lacZ fusion showed that both PA0744 and DspI were essential for the expression of the whole PA0743-PA0747 operon (Fig. 2B). Time-course RT-qPCR assay during the growth of PAO1 revealed the synchronous upregulation of PA0744 and dspI along with the increase of cell density (Fig. 2C), confirming the cell density-dependent co-expression of PA0744 and dspI.

Owing to the same enzymatic annotation and the physiological function of PA0744 and DspI, we speculated that PA0744 participates in CDA biosynthesis. As expected, accumulation of biofilm in the ΔPA0744 strain was prevented with the co-culturing of the WT strain (Fig. 2D), implying the importance of PA0744 in producing a diffusible signal for cell-to-cell communication. The signal was subsequently confirmed as CDA because CDA was no longer detected by LC-MS in the mutant ΔPA0744 and exogenous CDA restored biofilm dispersion in ΔPA0744 (Fig. 2E, F). Considering the same function of PA0744 as DspI in CDA biosynthesis and biofilm dispersion, we named PA0744 as DspII (Dispersion inducer II) hereafter.

Because deletion of dspII and dspI respectively resulted in almost complete repression of the other gene (Fig. 2A), we next expressed DspI in the ΔdspII mutant and expressed DspII in the ΔdspI mutant to ensure that DspI and DspII were normally produced in ΔdspII and ΔdspI, respectively, to further clarify the contribution of DspII and DspI to CDA biosynthesis. However, CDA biosynthesis was not restored in both reconstructed strains (Fig. 2G). To further dissect the functional interdependence, we expressed DspII, DspI, and DspII-DspI in the mutant ΔdspIIΔdspI which serves as a pure background without any production of either DspII or DspI. As shown in Fig. 2G, expression of DspII or DspI alone in the double mutant ΔdspIIΔdspI was incapable of CDA biosynthesis, whereas the presence of both DspII and DspI restored CDA biosynthesis in ΔdspIIΔdspI. Consistently, biofilm dispersion and swarming motility were re-activated by the co-presence of DspII and DspI in ΔdspIIΔdspI (Fig. 2H, I). These data indicate that DspII and DspI function in the same pathway or as a complex for CDA biosynthesis and the chronic-to-acute virulence switch.

Domain architecture analysis using InterPro22 showed that DspII contains an N-terminal ClpP/crotonase domain and an additional C-terminal ClpP/crotonase domain while DspI contains only one (Fig. 2J). Conserved glutamate residues for the activity of enoyl-CoA hydratase/isomerases were predicted in both DspII and DspI. DspI was predicted to contain two active glutamate residues Glu126 and Glu146, while DspII has one active glutamate residue Glu143 (Fig. 2J). To verify the enzymatic activity of these residues, we generated DspII and DspI variants, DspIIE143A, DspIE126A, and DspIE146A, and found that these variants were incapable of recovering CDA biosynthesis in ΔdspII and ΔdspI mutants (Fig. 2K). Moreover, biofilm dispersion and swarming motility in the ΔdspII and ΔdspI mutants cannot be re-activated with the complementation of their enzymatically dead variants (Fig. 2L, M), indicating that these glutamate residues are essential for the enzymatic activity of DspII and DspI to synthesize CDA and regulate the chronic-to-acute virulence switch.

CDA induces biofilm dispersion and swarming motility by inversely regulating Psl and flagellar biosynthesis
We moved to explore detailed mechanisms about how CDA could induce the chronic-to-acute virulence switch by shifting the sessile lifestyle to the motile lifestyle. Given that Psl and Pel are two important EPSs stabilizing the biofilm structure in non-mucoid P. aeruginosa strains23, we evaluated the biosynthesis of Psl and Pel in mutants ΔdspII and ΔdspI by measuring the promoter activity of their synthetic operons with fluorescent reporters. We found that activities of the psl promoter in both ΔdspII and ΔdspI mutants were substantially higher than the WT strain, particularly at the late growth stages with high cell density (Fig. 3A). In contrast, the pel promoter in both ΔdspII and ΔdspI mutants showed a decreased activity compared to the WT strain (Fig. 3B). Consistently, RT-qPCR assays showed significantly increased expression levels of the pslA gene in both ΔdspII and ΔdspI mutants compared to the WT strain (Fig. 3C). These results suggested that the loss of DspII or DspI lead to the continuous biosynthesis of Psl, the primary EPS used to develop biofilm and maintain its structure in PAO124, which prevents biofilm dispersion. To confirm this, we deleted pslA to disrupt Psl biosynthesis in the ΔdspII and ΔdspI mutants, which consequently abolished the accumulation of biofilm (Fig. 3D). In contrast, biofilm still accumulated in ΔdspII and ΔdspI mutants which were unable to synthesize Pel (Fig. 3D). Regarding to swarming motility, RT-qPCR assays showed that the flagellar synthetic genes flgB and flgF were significantly repressed in the ΔdspII and ΔdspI mutants (Fig. 3E), meaning that DspII and DspI activate swarming motility by upregulating flagellar biosynthesis. We thus compared the flagellar morphology of ΔdspII, ΔdspI, and WT strains under a transmission electron microscope (TEM), which showed that the average flagellar lengths of the ΔdspI and ΔdspII cells were significantly shorter than the WT cells (Figs. 3F, G). Collectively, these data demonstrated that CDA induces biofilm dispersion and swarming motility by negatively regulating Psl biosynthesis and positively regulating flagellar biosynthesis.

CDA induces biofilm dispersion and swarming motility through the c-di-GMP/FleQ pathway
The second messenger c-di-GMP has been identified as a critical intracellular molecule that orchestrates acute-to-chronic virulence switch when its content elevates in response to specific external cues25. We first evaluated c-di-GMP contents in ΔdspII, ΔdspI, and WT strains by measuring the expression of a c-di-GMP-responsive indicator CdrA26, which suggested that c-di-GMP contents in two mutants were dramatically higher than that in the WT strain (Fig. 4A). LC-MS quantification confirmed that c-di-GMP contents were increased in the ΔdspII and ΔdspI mutants (Fig. 4B). c-di-GMP is synthesized by diguanylate cyclase (DGC) and degraded by phosphodiesterase (PDE)27. To validate if reduced abilities of biofilm dispersion and swarming in ΔdspII and ΔdspI mutants were ascribed to increased c-di-GMP levels, we expressed an exogenous PDE W909_14950 (14950), which is from Dickeya oryzae EC1 and has been well-characterized and demonstrated to function efficiently in P. aeruginosa to lower c-di-GMP levels without nonspecific effects28, in both mutants and found that biofilm dispersion and swarming motility were recovered (Figs. 4C, D).

There are at least 42 DGCs and PDEs in P. aeruginosa29. We next moved to further explore how c-di-GMP content was increased in ΔdspII and ΔdspI mutants. We constructed promoter-gfp fusions for all the DGC and PDE genes and compared their expression in the ΔdspI and WT strains. The results identified 2 putative DGCs (PA0290 and PA2072) and 2 PDEs (PipA and RbdA) with more than 20% higher or lower expression, which might potentially increase the c-di-GMP levels in the ΔdspI mutant (Supplementary Data 1). Upregulation of PA0290 and PA2072 and downregulation of PipA and RbdA were further confirmed in the ΔdspII mutant (Fig. 4E, F; and Supplementary Fig. 2A to 2C). However, deletion of the DGC enzymes PA0290 or PA2072 and overexpression of the PDE enzyme PipA in ΔdspII and ΔdspI mutants did not prevent biofilm accumulation (Supplementary Fig. 2D–F). In contrast, overexpression of RbdA restored biofilm dispersion and swarming motility in the ΔdspII and ΔdspI mutants (Figs. 4G, H). RbdA is a transmembrane PDE which contains multiple domains (Fig. 4I). We further overexpressed RbdAΔEAL, a RbdA variant lacking the EAL domain and the phosphodiesterase activity, in the ΔdspII and ΔdspI mutants. As shown in Fig. 4J, K, the variant did not restore biofilm dispersion and swarming motility of two mutants any longer. Together, these results indicate that CDA decreases c-di-GMP content and induces the chronic-to-acute virulence switch by upregulating the PDE RbdA in P. aeruginosa.

The second messenger c-di-GMP fulfills its physiological functions through interactions with specific proteins named effectors30. At least 12 effectors have been already identified in P. aeruginosa29. To determine which effector connects the chronic-to-acute virulence switch with the CDA-repressed c-di-GMP levels, we deleted 12 candidate effector genes in the WT, ΔdspII, and ΔdspI strains and measured biofilm production. Among 12 candidates, only the deletion of fleQ abolished the accumulation of biofilm in both the ΔdspII and ΔdspI mutants, whereas deletion of other effector genes had no effect on the biofilm production in these two mutants (Supplementary Fig. 3). We treated WT and ΔfleQ strains with CDA and found that CDA inhibited the activity of the psl promoter only in the WT strain but not the ΔfleQ mutant (Fig. 4L). Furthermore, deletion of fleQ abolished the upregulation of Psl synthetic genes and downregulation of flagellar synthetic genes in the ΔdspII and ΔdspI mutants (Fig. 4M and Supplementary Fig. 4). These results suggest the essential role of the c-di-GMP effector FleQ in mediating CDA’s induction on the chronic-to-acute virulence switch. The involvement of the c-di-GMP/FleQ pathway was further confirmed by the overexpression of RbdA in the backgrounds of ΔdspIIΔfleQ and ΔdspIΔfleQ, which no longer restored biofilm dispersion and swarming motility (Fig. 4N, O).

DspII and DspI synergistically upregulate T3SS gene expression and acute pathogenesis
To further validate the chronic-to-acute virulence switch, we evaluated the pathogenicity of ΔdspII and ΔdspI mutants by performing cytotoxicity assay using the A549 cell line and acute infection assay using the model of Galleria mellonella31,32. It was shown that deletion of dspII or dspI led to attenuated cytotoxicity (Fig. 5A) and increased survival of G. mellonella (Fig. 5B and Supplementary Fig. 5), confirming that DspII and DspI not only induce biofilm dispersion and swarming motility but also contribute to acute pathogenesis in P. aeruginosa. Since T3SS is the key virulence factor causing acute infection, we examined T3SS gene expression in the ΔdspII and ΔdspI mutants. RT-qPCR assays showed that the mRNA levels of selected T3SS genes, including exsA, exsC, exoS, and pcrV, were significantly reduced in ΔdspII and ΔdspI mutants compared to the WT strain (Fig. 5C). T3SS gene expression is activated by the master transcription factor ExsA, which is transcribed from two promoters, i.e., PexsCEBA and PexsA, under the control of various transcription factors33 (Fig. 5D). Thus, we focused on ExsA and found that reduced expression of ExsA in both the ΔdspII and ΔdspI mutants was confirmed at the protein level using western blot assay (Fig. 5E). We also measured the activity of promoters, PexcCEBA and PexsA, and found that upregulation of exsA was due to activated transcription of PexsCEBA instead of PexsA by DspII and DspI (Fig. 5F, G). Moreover, we found that DspII and DspI did not influence T3SS gene expression, i.e., activity of PexcCEBA and ExsA production, when ExsA is not or saturated expressed (Fig. 5H, I), which demonstrated that DspII and DspI positively regulate T3SS gene expression through the master activator ExsA.

Like biofilm dispersion, we wondered if DspII and DspI regulate T3SS interdependently and whether the regulation is controlled by CDA. We next measured the activity of PexcCEBA in strains including ΔdspII(dspI), ΔdspI(dspII), ΔdspIIΔdspI(dspI), and ΔdspIIΔdspI(dspII), which only produce DspII or DspI. It was shown that the activity of PexcCEBA in these strains remained repressed at the level as that in ΔdspII, ΔdspI, and ΔdspIIΔdspI strains (Fig. 5J). Only the strains producing both DspII and DspI displayed as high activity of PexcCEBA as that in the WT strain (Fig. 5J), meaning that upregulation of T3SS gene expression also requires the cooperation of DspII and DspI. Intriguingly, exogenous supplementation of CDA was incapable of recovering the T3SS gene expression and complementation of enzymatically dead variants DspIIE143A, DspIE126A, and DspIE146A significantly induced T3SS gene expression in both ΔdspII and ΔdspI mutants (Fig. 5K, L), indicating that DspII and DspI might activate T3SS gene expression through a novel CDA-independent mechanism that is different from the regulation of biofilm dispersion and swarming motility. Moreover, it was fascinating to notice that the activity of PexcCEBA in the WT strain was reduced with the increased concentration of exogenous CDA (Fig. 5K), showing an inhibitory effect of CDA on the DspII and DspI-induced T3SS gene expression.

DspII and DspI activate T3SS gene expression by antagonizing the GacA-upregulated small RNAs RsmY and RsmZ
Considering the contribution of c-di-GMP to inhibit T3SS gene expression as we recently found34, we initially hypothesized that the increased c-di-GMP content in ΔdspII and ΔdspI mutants led to the downregulation of T3SS genes. However, decreasing c-di-GMP content by overexpressing PDEs 14950 and RbdA or additional deletion of fleQ did not restore the level of T3SS gene expression in ΔdspII and ΔdspI mutants (Supplementary Fig. 6), meaning that DspII and DspI activate T3SS gene expression bypassing the c-di-GMP/FleQ pathway. Another important pathway modulating T3SS gene expression in P. aeruginosa is the Gac/Rsm pathway. In this pathway, the two-component system GacS/GacA positively regulates the expression of two small RNAs (sRNAs) named RsmY and RsmZ, and subsequently, the sRNAs sequester RsmA which is a post transcriptional regulator involved in the activation of T3SS gene translation, leading to the reduced translation of T3SS proteins including ExsA35,36 (Fig. 6A). The expression of the response regulator GacA was not changed with the deletion of dspII or dspI (Fig. 6B), while the expression of two sRNAs RsmY and RsmZ was obviously increased (Fig. 6C, D). This results suggests that DspII and DspI might repress RsmY and RsmZ to liberate free RsmA for the upregulation of T3SS genes. We then generated ΔrsmZ, ΔrsmY, and ΔrsmZΔrsmY mutants. It was shown that T3SS gene expression was still reduced by deleting dspII and dspI in the genetic backgrounds of ΔrsmZ or ΔrsmY, while the expression was no longer reduced in the genetic background of ΔrsmZΔrsmY (Fig. 6E, F). Since RsmY and RsmZ repress T3SS gene expression by sequestering RsmA, we next overexpressed RsmA in the ΔdspII and ΔdspI mutants and the results showed that T3SS gene expression was reactivated (Fig. 6G, H). These results together demonstrate that DspII and DspI repress the expression of RsmY and RsmZ, which releases RsmA to elevate T3SS gene expression in P. aeruginosa.

Because GacA is a critical transcriptional activator of RsmY and RsmZ and its expression was consistent among WT, ΔdspII, and ΔdspI strains, we were curious about the connection of GacA, DspII, and DspI in controlling RsmY and RsmZ expression. Deletion of gacA dramatically reduced the transcription of both RsmY and RsmZ (Fig. 6I, J). In addition, deletion of dspII or dspI did not increase the transcription of RsmY and RsmZ any longer in the ΔgacA background (Fig. 6I, J), meaning that DspII and DspI repress RsmY and RsmZ in a GacA-dependent manner. Consistently, T3SS gene expression was no longer reduced when dspII and dspI were deleted in the ΔgacA background (Fig. 6K, L), which confirmed the GacA-dependent regulation of T3SS gene expression by DspII and DspI.

DspII and DspI antagonize the GacA-activated transcription of RsmZ and RsmY by directly interacting with the RsmZ promoter and the GacA protein
Our findings suggested that DspII and DspI only diminished the transcription of RsmY and RsmZ when GacA is present to first drive their transcription. GacA activates transcription of RsmY and RsmZ by binding at their promoters. Given that DspII and DspI activate T3SS gene expression independently of CDA, we hypothesized that two proteins might be potential roadblocks to impede GacA-induced transcription of RsmY and RsmZ by directly binding at their promoters. We purified DspII and DspI (Supplementary Fig. 7) and performed electrophoretic mobility shift assay (EMSA) to examine the binding potentials of two proteins at the promoters of rsmY and rsmZ. DspII and DspI did not interact with the promoter of rsmY (PrsmY) (Supplementary Fig. 8A and 8B), while it was surprisingly found that both DspII and DspI interacted with the promoter of rsmZ (PrsmZ) (Supplementary Fig. 8A and 8B; Figs. 7A, B), albeit DNA-binding motif was not predicted in both proteins. This result indicates that DspII and DspI repress the transcription of RsmZ by directly binding at its promoter.

To further characterize the binding details of the DspII-PrsmZ and DspI-PrsmZ complexes, we performed multiple rounds of EMSA which tested the binding of DspII or DspI with truncated promoter probes. A 320-bp DNA sequence containing the promoter PrsmZ was used as a full-length probe (Fig. 7C). The R fragments were truncated from the 3’ end, resulting in R1 (103 bp), R2 (184 bp), R3 (225 bp), R4 (272 bp), and R5 (320 bp) (Fig. 7C). As shown in Fig. 7D, both DspII and DspI bound to fragments R2 to R5 but not the fragments R1, suggesting that R2 contained the binding site of DspII and DspI. Therefore, R2 was further truncated into 123-bp, 143-bp, 163-bp, and 184-bp fragments. EMSA showed that DspII and DspI bound to the 143-bp, 163-bp, and 184-bp fragments (Fig. 7E), indicating that the sequence between 123-143 contained the binding site. To determine whether additional binding sites existed upstream of the 123-143 region, we truncated the full-length probe from the 5’ end, resulting in F1 (95 bp), F2 (176 bp), F3 (225 bp), F4 (285 bp), and F5 (320 bp). EMSA results showed that DspII and DspI bound to F3, F4, and F5 but not F1 and F2 (Fig. 7F), meaning that there was no binding site upstream of the 123-143 region. Taken together, the binding site of DspII and DspI reside in the region of 5’-TTAAGTAACTTATTGAAATTA-3’, which partially overlaps with the binding site of MvaT and locates at downstream of the binding site of GacA35,37 (Fig. 7C). This result not only determines the binding details of DspII-PrsmZ and DspI-PrsmZ complexes but also explains that DspII and DspI act as roadblocks to antagonize the GacA-dependent upregulation of RsmZ.

Since CDA exhibited an inhibitory effect on DspII/DspI-induced T3SS gene expression (Fig. 5K), we moved to explore whether CDA influenced the binding affinity of DspII and DspI at the promoter PrsmZ. It was shown that the binding between DspII or DspI and PrsmZ became weaker when the CDA concentration was increased (Supplementary Fig. 8C), implying that high concentration of CDA inhibits T3SS gene expression partially due to the reduced binding affinity of DspII and DspI with the promoter of rsmZ. Given that the enzymatically dead DspII and DspI variants cannot fully restore T3SS gene expression in the ΔdspII and ΔdspI mutants (Fig. 5L), we wondered whether these enzymatically active sites participate in DNA binding. We purified these variants and performed EMSA to examine their binding affinity with PrsmZ. As expected, decreased binding affinity of these variants with PrsmZ was observed compared to the wild-type DspII and DspI (Fig. 7G). These results indicate that all three enzymatically active residues in DspII and DspI are important for DNA binding. Possibly owing that DspI contains one more enzymatically active residue than DspII, it displayed higher DNA-binding affinity than the DspII protein as we observed in all the EMSA results.

To further understand how DspII and DspI antagonize the GacA-activated transcription of RsmZ and RsmY by directly interacting with the RsmZ promoter and the GacA protein, we first tried to examine whether co-presence of DspII and DspI can bind at the promoter of RsmY since upregulation of T3SS gene expression requires the cooperation of DspII and DspI. However, EMSA did not show any shifted PrsmY in the co-presence of DspII and DspI (Supplementary Fig. 9A). Because the DNA sequence that is recognized by DspII and DspI in PrsmZ is not present in PrsmY, it is reasonable why DspII and DspI cannot bind to PrsmY. We next investigated whether DspII or DspI affect the binding affinity of GacA at PrsmY but the EMSA result showed that neither DspII nor DspI affects the DNA-binding affinity of GacA (Supplementary Fig. 9B). We moved to explore if DspII or DspI can potentially interact with GacA and thus reduce the transcription activity of PrsmY. Interestingly, both DspII and DspI were found to directly interact with GacA using a pull-down assay (Fig. 7H). This result indicated that DspII and DspI antagonize the GacA-activated transcription of RsmY by directly interacting with the GacA protein.

DspII interacts with DspI and the binding affinity is associated with CDA concentrations
Given the interdependence of DspII and DspI in synthesizing CDA, inducing biofilm dispersion and swarming motility, and activating T3SS gene expression, we hypothesized that DspII and DspI might interact directly. First, we conducted bacterial two-hybrid assay to detect potential interactions between DspII and DspI. It showed that co-presence of DspII and DspI enabled the normal growth of colonies on the selective medium (Fig. 8A), suggesting that DspII interacts with DspI. A pull-down assay was also performed, confirming the physical interaction between DspII and DspI (Fig. 8B). We further investigated the effect of CDA on DspII and DspI interaction. As shown in Fig. 8C, the binding affinity between DspII and DspI was enhanced with an increasing concentration of CDA below 10 μM, while the binding affinity was decreased when the concentration of CDA was higher than 10 μM. These findings demonstrated that DspII interacts with DspI to form a complex and CDA modulates the affinity of interaction in a concentration-dependent manner.

To understand the interaction details of the DspII-DspI complex, we employed HDOCK and PLIP servers38,39 to predict the interaction model and the result showed three types of interactions including hydrophobic interactions, hydrogen bonds, and salt bridges (Fig. 8D and Supplementary Data 2). To validate the interaction between DspII and DspI, we selectively generated substitutions of four amino acids residues involved in the three types of interactions in the DspI protein (Fig. 8E). The resultant DspI# (DspIR57A/R96A/E231A/R232A) showed a dramatically decreased binding affinity with DspII compared to the wild-type DspI (Fig. 8F). In addition, complementation of the DspI# variant in the ΔdspI mutant was unable to re-activate the biosynthesis of CDA, biofilm dispersion, swarming motility, and T3SS gene expression (Fig. 8G–J). Together, these results demonstrated that the interaction between DspII and DspI is essential for both the enzymatic and non-enzymatic functions to mediate the chronic-to-acute virulence switch.

Methods
Bacterial strains, plasmids, primers, and growth conditions
Strains and plasmids used in this study are listed in Supplementary Data 3. Primers used in this study are summarized in Supplementary Data 4. Strains were normally cultured in Luria-Bertani (LB) medium (10g/L Tryptone, 5g/L Yeast extract and 5g/L NaCl) or on LB-agar plates at 37°C. Antibiotics were added as following concentrations when necessary: gentamicin, 50μg/ml; kanamycin, 50μg/ml; ampicillin, 100μg/ml.

Construction of gene-deletion mutants and gene complementation or overexpression
For gene deletion, 500–1000-bp upstream and downstream regions of target genes were amplified and ligated to pK18mobsacB which was pre-digested with BamHI and HindIII. Using pRK2013 as the helper plasmid, constructed plasmids for gene deletion were delivered into PAO1 or its derivatives by tri-parental mating. Desired mutants were selected on LB plate containing 10% sucrose and confirmed by PCR and sequencing. For gene complementation, the open reading frames of target genes and their promoter sequences were amplified from the PAO1 genome and cloned into the plasmid pUC18T-mini-Tn7T-Gm which was pre-digested with BamHI and HindIII. The constructed plasmids were introduced into PAO1 mutants with the help of the plasmid pTNS2 by electroporation. Desired colonies were selected on Pseudomonas Isolation Agar (PIA) plates with gentamicin and confirmed by PCR and sequencing. For gene overexpression, target genes were ligated into pUCP18 which was pre-digested with HindIII and BamHI. Constructed plasmids were introduced into corresponding strains by tri-parental mating.

Biofilm assay
Biofilm biomass was measured as previously described46. Briefly, 100μl bacterial culture with OD of 0.02 was inoculated in LB medium in 96-well polypropylene microtiter dishes. After incubation under static condition for 16h, cell density was determined, and the liquid culture was removed carefully. 0.1% crystal violet was added to each well and incubated for 15min at room temperature (~25°C). After staining, wells were washed carefully to remove the unbound crystal violet. Biofilms were dissolved in 95% ethanol and A570 of each well was measured using a microplate reader (BioTek, United States). Biofilm biomass was normalized by cell density (OD600). All experiments were conducted with three independent biological repeats.

Swarming motility assay
Swarming motility was performed as described previously47. Plates used for swarming assay were prepared with 8g/L nutrient broth, 5g/L glucose, and 5g/L bacto-agar. The overnight cultures were diluted to OD600 of 1.0 and 1μl culture was spotted at the center of swarming plates. After incubation at 37°C for 16-20h, diameters of swarming zones were measured. All experiments were conducted with three independent biological repeats.

Transmission electron microscopy (TEM)
TEM observation of bacterial flagellum was performed as preciously described48. Bacterial cells were collected from swarming plates and diluted in the MilliQ water. Cells were stained with 2% aqueous solution of phosphotungstic acid (pH 7.4) for 20min before fixing in parlodion (Mallinckrodt, Inc., United States) carbon-coated grid (300 mesh). After that, samples were examined by the transmission electron microscope Talos F200S (Thermo Fisher Scientific, United States).

Promoter activity assay
Promoter activity was assessed by measuring GFP fluorescence or β-galactosidase activity. Target gene promoters were amplified and ligated to pPROBE-NT-gfp or mini-CTX-lacZ vectors. The resulting constructs were transformed into P. aeruginosa strains and verified by colony PCR. For GFP fluorescence assay, overnight bacterial cultures were diluted to an OD of 0.02 and then sub-cultured in 96-well plates under static conditions. Cell densities (OD600) and fluorescence were measured every 4h using a microplate reader (BioTek, United States). Results were shown as the ratio of fluorescence and OD600 at different time points. For β-galactosidase activity assays, bacterial cells were harvested at an OD of 1.5 via centrifugation and then resuspended in Z buffer (60mM Na2HPO4, 40mM NaH2PO4, 10mM KCl, 1mM MgSO4, 50mM β-mercaptoethanol). A 100μl aliquot of bacterial suspension was diluted in 500μl Z buffer, followed by the addition of 20μl chloroform and 20μl 0.1% SDS. Cells were lysed by thorough vortexing and incubated in a 30 °C water bath for 20min. Subsequently, 100μl of 4% (w/v) o-nitrophenyl-β-D-galactoside (ONPG) was then added to the suspension, which was then incubated for 10min. The reaction was terminated with 250μl of 1M Na2CO3. The promoter activity in Miller Units was calculated and relative promoter activity was presented.

RT-PCR and RT-qPCR
Overnight bacterial cultures were 1:50 diluted with fresh LB medium and grown when OD reached 1.5. Total RNA was extracted using the SV Total RNA separation and purification system (Promega, United States) following manufacturer’s instructions. After determining the RNA quality and concentration, first-strand cDNAs were reverse transcribed from 1μg RNA using HiScript III 1st Strand cDNA Synthesis Kit (with gDNase) (Vazyme, China). Subsequently, RT-PCR was performed using 2× Phanta Max Master Mix (Vazyme, China) with RNA or cDNA as templates. For RT-qPCR analysis, the ChamQ Universal SYBR qPCR master mix (Vazyme, China) was used, and the amplification was monitored in the ABI QuantStudio 6 Flex real-time system. Relative gene expression level was calculated by using the 2-ΔΔCT method and the 50S ribosomal protein gene rplU was selected as the reference to standardize all the samples49. All experiments were conducted with three independent biological repeats.

CDA quantification using LC-MS
CDA quantification was carried out as reported previously with slight modifications17. The overnight culture was 1:50 diluted with fresh LB medium and incubated for 20h. Supernatant was collected, and 1/1000 volume of acetic acid was added before CDA extraction with an equal volume ethyl acetate. The organic phase was dried by vacuum freeze and residues were dissolved with 1ml methanol. Samples were analyzed using Q Exactive Focus Hybrid Quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific, United States) in negative ionization mode. CDA separation was achieved by a 2.1×100-mm HSS T3 1.8 μm column (Waters, United States) using a gradient system consisting of methanol and MilliQ water with 1/1000 volume of formic acid. CDA was quantified using selected-ion monitoring (SIM) mode. All measurements were performed with three independent biological repeats.

c-di-GMP quantification using LC-MS
The quantification of c-di-GMP was carried out following a previously reported protocol with minor adjustments50. The overnight culture was diluted 1:100 into fresh LB medium and incubated for 20h. 10ml cell culture were pelleted and resuspend in 1ml sterilized MilliQ water. After perchloric acid was added, cell suspensions were vortexed thoroughly and then incubated on ice for 30min. After centrifugation, supernatants were transferred to a new tube and 1/5 volume of 2.5M KHCO3 was added. The samples were centrifuged to remove perchlorate salt precipitates. Supernatant was collected for LC-MS analysis. LC-MS was undertaken using an Q Exactive Focus Hybrid Quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific, United States) with a 2.1×100-mm HSS T3 1.8 μm column (Waters, United States). All measurements were performed with three independent biological repeats.

Bacterial two-hybrid assay
Bacterial two-hybrid assay was carried out to detect protein-protein interaction according to protocol of BacterioMatch II Two-Hybrid System Vector Kit (Agilent Technologies, United States). The ORFs of dspI and dspII were cloned into pBT and pTRG, respectively. The recombinant pBT-dspI and pTRG-dspII plasmids were co-transformed into the reporter strain XL1-Blue MRF Kan. The overnight bacterial cultures were serially diluted using M9+His dropout broth and spotted on nonselective medium and selective medium (5mM 3-AT plus 12.5μg/ml streptomycin). Plates were incubated at 37°C for 24h.

Pull-down assay
Pull-down assay was carried out using Pierce GST Protein Interaction Pull-Down Kit (Thermo Fisher Scientific, United States) according to the manufacturer’s instructions. Briefly, bacterial cells were collected by centrifugation and resuspended with lysis buffer. The purified GST-tagged protein was incubated with glutathione agarose for 4h at 4°C and then washed gently to remove unbound proteins. The glutathione agarose was then incubated with cell lysate containing His-tagged protein at 4°C for 16h. The column was washed five times, and the proteins were eluted using 10mM glutathione elution buffer. The eluted samples were incubated with loading buffer and heated at 95°C for 10min prior to western blot analysis.

Protein purification
Plasmids for protein expression were delivered into BL21(DE3). For protein expression, 1ml bacterial overnight culture was inoculated into 1L LB medium. When bacterial cells grow to OD of 0.5-0.8, IPTG was added to induce protein expression, and the cell culture was incubated at 16°C overnight. Cell pellets were harvested and lysed by sonication. After centrifugation, the supernatant was filtered with a 0.22-μm filter prior to incubation with glutathione beads (Smart-Lifescience, China) or NiNTA beads (Smart-Lifescience, China) for 30min. After washing with PBS, proteins were eluted by 10mM glutathione elution buffer or 200mM imidazole elution buffer. The GST-tag and His-tag of the purified proteins were further digested using PreScission Protease (Smart-Lifescience, China), respectively, following the manufacture’s construction.

Electrophoretic gel mobility shift assay (EMSA)
DNA sequences of target promoter regions were amplified by PCR using the primers listed in Supplementary Data 4. DNA fragments were labeled with biotin using a Biotin 3′ End DNA Labeling Kit (Thermo Fisher Scientific, United States). Protein-DNA interaction was analyzed using LightShift Chemiluminescent EMSA Kit (Thermo Fisher Scientific, United States) following the manufacture’s construction. Briefly, purified protein and the labeled probes were added into the binding reactions and incubated at 25°C for 30min. The reaction mixtures were mixed with 5× Loading Buffer and loaded onto the polyacrylamide gel. Following electrophoresis, DNA probes were transferred to the nylon membrane (Millipore, United States) and crosslinked using UV irradiation. Membranes were sequentially washed with Blocking Buffer, Wash Buffer, and Substrate Equilibration Buffer as specified in the manufacturer’s protocol. Protein-DNA complexes were visualized using Working Substrate Solution and imaged with a cooled CCD camera (Tanon, China). For competitive EMSA assays, 50ng or 150ng of unlabeled PrsmZ DNA fragments were added to the reaction mixtures.

Western blot analysis
Overnight bacterial cultures were 1:50 diluted in fresh LB medium and grown at 37°C when OD reached 1.5. 10-ml aliquots of bacterial cultures were centrifuged, and pellets were suspended in 200µl RIPA lysis buffer (Biosharp, China) and followed by incubation at 25°C for 30min. After centrifugation, the supernatants were mixed with 5× SDS-PAGE loading buffer and heated at 98°C for 10min. Proteins were separated using SmartPAGE™ 4-20% Precast Protein Gel (Smart-Lifescience, China) and transferred to a PVDF membrane (Millipore, United States) via electroblotting. Subsequently, Membranes were blocked for 1h at room temperature with PBST (PBS containing 1% Tween-20) containing 5% skim milk, then probed with anti-ExsA, anti-β-RNAP, anti-GST, and anti-His primary antibodies, respectively. After washing, membranes were incubated with HRP-conjugated goat anti-mouse secondary antibody (Abbkine, United States). Protein bands were visualized using the ECL kit according to the manufacture’s protocol and imaged with a cooled CCD camera (Tanon, China).

Cytotoxicity assay
Bacterial cytotoxicity was assessed by measuring the activity of lactate dehydrogenase (LDH) which is released from the human lung epithelial cell A54951. Briefly, 1.5×104 A549 cells were seeded into a 96-well plate containing DMEM medium (Gibco, United States) supplemented with 10% FBS (Gibco, United States) and cultured overnight at 37°C under 5% CO2 and 95% humidity. P. aeruginosa strains were cultured in LB medium to an OD of 1.5. Bacterial cells were centrifuged and resuspended in DMEM with 1% FBS. A549 cells were washed twice with PBS buffer and infected with bacteria at a multiplicity of infection (MOI) of 50 for 4h. Cytotoxicity was quantified by CytoTox 96® Non-Radioactive Cytotoxicity Assay kit (Promega, United States) according to the manufacturer’s instructions.

Galleria mellonella killing assay
The acute virulence of P. aeruginosa was evaluated using a G. mellonella infection model adapted from a previous study52. Overnight bacterial cultures were 1:50 diluted in fresh LB medium and grown to an OD of 1.5. Bacterial cells were pelleted, resuspended in PBS, and adjusted to 1×105 CFU. Ten G. mellonella larvae were injected with 20μl bacterial suspension into the secondary right proleg using a syringe and then incubated at 30°C. The survival rates were monitored every 2h.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This manuscript presents a comprehensive and well-executed study identifying a novel enoyl-CoA hydratase/isomerase, DspII, that functions in concert with the previously characterized DspI in Pseudomonas aeruginosa. The authors demonstrate that these two proteins form a functional complex essential for the biosynthesis of cis-2-decenoic acid (CDA), a quorum-sensing signal molecule that promotes the transition from chronic to acute infection. Notably, the study reveals a dual functionality for the DspII/DspI : in addition to their enzymatic role in CDA synthesis, they also exerts a regulatory effect on the expression of type III secretion system (T3SS) genes. The authors propose this effect is exerted by DNA binding and by antagonizing GacA-mediated transcription of the small RNA RsmZ.\n\nThe findings are highly significant and novel, particularly in uncovering an unexpected transcriptional regulatory role for quorum-sensing synthases. The manuscript provides novel mechanistic insights into the virulence switch in P. aeruginosa and broadens the functional landscape of DSF-family quorum sensing enzymes. The experiments are logically structured, rigorous, and well controlled. The manuscript is clearly written, and the data are solid.\n\nGeneral/major comments:\n- The PA0744 gene (encoding DspII) is longer (1104 bp) than dspI (819 bp). Does DspII contain additional domains that might explain its non-enzymatic DNA-binding function? Domain prediction analysis (e.g., InterPro, Pfam) would strengthen this point.\n- The separation between CDA-mediated biofilm dispersion and CDA-independent T3SS activation is clearly shown. However, the mechanism by which DspII–DspI antagonizes GacA activity is not fully explored. A catalytic mutant could help test whether DNA binding/regulatory activity is independent of enzymatic activity.\n- EMSAs with the reconstituted DspII–DspI complex may show increased DNA affinity or revel binding to rsmY promoter.\n- While cdrA is a valid reporter of intracellular c-di-GMP, LC-MS/MS quantification could validate these results and exclude secondary effects.\n\nMinor comments:\n• Line 172: biofilm was still accumulated → biofilm still accumulated\n• Line 142: the protein production of DspII → DspII protein expression\n• Line 230: reached to a peak → reached a peak\n• Line 248: was also not observed → was not observed either\n• Line 305: the rsmZ but not rsmY promoter → the rsmZ promoter, but not the rsmY promoter\n• Line 370: The fluorescence decreased after 16 h → Fluorescence decreased after 16 h\n• Line 470: and their derivatives were cultured → and its derivatives were cultured\n• Line 540: statistically significance → statistical significance\n• Line 610: EMSA results showed that DspI and DspII repressed the rsmZ promoter → EMSA results showed that the DspI–DspII complex repressed the rsmZ promoter", "reviewer_2": "This manuscript by Huang and colleagues describes the function of a gene product, DspII, that is involved in production of cis-2-decenoic acid. It's thorough and the experiments are logical and logically presented. There are a few issues:\n1. The figures are quite challenging to read with very small text and in cases where there are multiple lines (eg, 3B,5B, etc.) these are difficult to distinguish. Figure 3A - it is impossible to discern what the authors are showing.\n2. The discussion of Pel in PAO1 is misplaced. (Lines 154-181). The decrease in transcription is marginal. Pel is not known to contribute to biofilm formation in PAO1, so it's not clear at all what is being shown by the authors here.\n3. Lines 191-193. Why this PDE from D. oryzae?\n4. Lines 212-225. FleQ deletion is known to abrogate Psl production in PAO1. The fact that it does so in the DspI/II strains does not make it follow that FleQ mediates CDA's induction of Psl.\n5. Lines 341-354. There is no evidence presented that the components have to be in complex to achieve the production of CDA. It's an inference the authors make, but it does not support the assertion (line 348-349) that the \"direct interaction is essential for the chronic-to-acute switch\".\n6. Issues of word choice or language -\n- line 19-20. There is no such thing as a \"process of virulence\"\n- line 39 - pathogenesis does not undergo development\n- line 51 - what is compounded?\n- lines 88-89 - 25.81 - 40.16% is overly precise.", "reviewer_3": "The manuscript titled ”Dual-Functional DSF-family Quorum Sensing Signal Synthases DspII and DspI Coordinate Chronic-to-Acute Virulence Switch in Pseudomonas aeruginosa” is interesting. This manuscript is about the molecular mechanisms of the chronic-to-acute virulence switch in Pseudomonas aeruginosa, dual function of the DspII-DspI complex, Enzymatic: biosynthesis of the quorum sensing signal cis-2-decenoic acid (CDA) as well as non-enzymatic: direct regulation of gene expression (T3SS) via promoter binding, Regulation of c-di-GMP and its effect on biofilm and motility, Interaction between the Gac/Rsm regulatory system and DspII/DspI. And finally, the Importance of quorum sensing in bacterial pathogenicity and adaptation.\n\nThe experiments design is robust based in the use of gene knockouts, gene expression assays, protein-DNA binding assays, cytotoxicity tests, and Galleria infection models. Moreover, it will be able the therapeutic potential by identifies novel targets to prevent the transition to acute infection.\n\nThe figures are well organized, with clear labels and explanatory legends. But, there are some defects or inconsistencies:\n- Figure 3d (c-di-GMP modulation by CDA): Although a reduction in c-di-GMP is shown, it is not quantified with additional controls (e.g., independent PDEs) to distinguish whether CDA acts directly or via global regulation.\n- Extended Figure 4a (protein purification): SDS-PAGE gels of DspII and DspI purification are shown, but no molecular weight marker is labeled. This makes it difficult to confirm band purity or expected size.\n- Figure 6b (protein-DNA interaction, EMSA): The observed shift for DspII appears subtle and may raise doubts about binding strength, but the article does not discuss this limitation in detail.\n- Final schematic model (Fig. 7):\n• It summarizes the proposed mechanism well but does not include:\n• The biphasic effect of CDA (as described in the text).\n• The unresolved indirect repression of RsmY, which the article\n- Lack of structural validation: No visual information is presented on the DspII-DspI structure or interaction predictions, which would strengthen the functional complex hypothesis.\n- No images of the infection model (Galleria mellonella) are included in the figures, which could have provided direct visual evidence of the virulence phenotype.\n\nMoreover, I have some general comments to take in consideration of the authors:\n1. Unresolved indirect regulation of RsmY: repression is observed, but the underlying mechanism remains unclear. Please, the authors must elucidate the indirect repression mechanism of RsmY by DspII/DspI.\n2. High specificity of the CDA control system to P. aeruginosa, which limits its generalization to other species. To explore additional downstream targets of CDA: such as other PDEs, DGCs, or effectors involved in c-di-GMP modulation.\n3. Low sequence similarity between DspII and DspI, raising questions about the evolutionary basis of their functional interaction\n4. Determine the prediction structure structure by bioinformatics tools of the DspII-DspI complex to understand their interaction and CDA-dependent regulation.\n5. The authors could Investigate clinical applications: such as inhibitors of DspII/DspI or their interaction to prevent acute virulence switching."}, "author_response": "Re: NCOMMS-25-48364\nDear Editor and Reviewers:\nThank you for your thoughtful review of our manuscript entitled “Dual-Functional DSF-family Quorum Sensing Signal Synthases DspII and DspI Coordinate Chronic-to-Acute Virulence Switch in Pseudomonas aeruginosa”. We are grateful for the insightful comments, which have helped us significantly strengthen the study. In this revised version, we have performed extensive additional experiments to provide deeper mechanistic insights. Below, we provide a point-by-point response to the comments.\n\nEditorial comment:\nAs you will see from the reports copied below, the reviewers find your work novel and of interest. However, they raise important concerns that we agree limit the strength of the study, and therefore we ask you to address them with additional experimental work. In line with the reviewers’ comments, your revision must provide further mechanistic insight, in particular by expanding the analyses of the DspI/II complex and its interactions.\n\nOur Response: We thank the Editor and Reviewers for handling our manuscript and for the encouraging and valuable suggestions. In response, we have conducted new experiments during the revision to further elucidate the mechanism of the interaction between DspII and DspI, the repression of rsmY by DspII-DspI, and so on. We believe these additions have substantially enhanced the mechanistic depth and overall impact of our work.\n\nReviewer #1 (Remarks to the Author):\nThis manuscript presents a comprehensive and well-executed study identifying a novel enoyl-CoA hydratase/isomerase, DspII, that functions in concert with the previously characterized DspI in Pseudomonas aeruginosa. The authors demonstrate that these two proteins form a functional complex essential for the biosynthesis of cis-2-decenoic acid (CDA), a quorum-sensing signal molecule that promotes the transition from chronic to acute infection. Notably, the study reveals a dual functionality for the DspII/DspI : in addition to their enzymatic role in CDA synthesis, they also exerts a regulatory effect on the expression of type III secretion system (T3SS) genes. The authors propose this effect is exerted by DNA binding and by antagonizing GacA-mediated transcription of the small RNA RsmZ.\n\nThe findings are highly significant and novel, particularly in uncovering an unexpected transcriptional regulatory role for quorum-sensing synthases. The manuscript provides novel mechanistic insights into the virulence switch in P. aeruginosa and broadens the functional landscape of DSF-family quorum sensing enzymes. The experiments are logically structured, rigorous, and well controlled. The manuscript is clearly written, and the data are solid.\n\nOur Response: We thank the reviewer for the positive feedback and insightful suggestions.\n\nGeneral/major comments:\n- The PA0744 gene (encoding DspII) is longer (1104 bp) than dspI (819 bp). Does DspII contain additional domains that might explain its non-enzymatic DNA-binding function? Domain prediction analysis (e.g., InterPro, Pfam) would strengthen this point.\n\nOur Response: We thank the reviewer for this suggestion. We performed a domain architecture analysis using InterPro, which revealed that DspII contains two ClpP/crotonase domains, whereas DspI contains only one (New Figure 2J). This analysis also identified key enzymatic residues (Glu126 and Glu146 in DspI, Glu143 in DspII) essential for CDA biosynthesis. We confirmed the functional importance of these residues by generating alanine substitution mutants, which lost the ability to produce CDA and regulate the chronic-to-acute virulence switch (New Figures 2K, 2L, 2M). Interestingly, these Glu residues also contribute to the non-enzymatic DNA-binding activity of both proteins (New Figure 7G). DspI, which contains two such residues, exhibited stronger DNA binding than DspII, which contains only one. This new result also answered the question from Reviewer #3 why the observed shift for DspII appears subtle than DspI in the EMSA data.\n\n- The separation between CDA-mediated biofilm dispersion and CDA-independent T3SS activation is clearly shown. However, the mechanism by which DspII-DspI antagonizes GacA activity is not fully explored. A catalytic mutant could help test whether DNA binding/regulatory activity is independent of enzymatic activity.\n\nOur Response: Thanks for the comment. (1) We agree that decoupling the enzymatic and regulatory roles is crucial. To this end, we generated enzymatically dead variants (DspIE126A, DspIE146A, and DspIIE143A) of DspI and DspII. As expected, these mutants did not produce CDA or restore biofilm dispersion and swarming motility (New Figures 2K, 2L, 2M). However, they significantly rescued T3SS gene expression in the ΔdspII and ΔdspI mutants (New Figure 5L), confirming that the regulatory function is independent of enzymatic activity. We note that the rescue was partial, as these residues also contribute to DNA binding (New Figure 7G). (2) Furthermore, we performed additional EMSAs to explore how DspII and DspI antagonize GacA-dependent transcription of rsmY. We showed that the DspII-DspI complex did not bind to the rsmY promoter (New Figure S9A), DspII and DspI did not affect GacA’s binding to this promoter (New Figure S9B). Surprisingly, we found that both DspII and DspI physically interact with GacA (New Figure 7H). This suggests a dual mechanism: DspII-DspI directly binds GacA to dampen its transcriptional activity toward both rsmY and rsmZ, while also binds to the rsmZ promoter to further repress rsmZ transcription. This updated model is summarized in the New Figure 9.\n\n- EMSAs with the reconstituted DspII-DspI complex may show increased DNA affinity or revel binding to rsmY promoter.\n\nOur Response: Thanks for the comment. As noted above, we showed that DspII-DspI complex cannot bind to the rsmY promoter and DspII/DspI proteins do not influence the DNA-binding affinity of GacA (New Figure S9). Instead, we found that DspII and DspI can directly interact with GacA (New Figure 7H), likely reducing its transcriptional activity of GacA and thereby repressing rsmY expression.\n\n- While cdrA is a valid reporter of intracellular c-di-GMP, LC-MS/MS quantification could validate these results and exclude secondary effects.\n\nOur Response: We agree and have now quantified intracellular c-di-GMP levels directly using LC-MS. The results confirm a significant increase in c-di-GMP in ΔdspII and ΔdspI mutants, which is restored in the complemented strains (New Figure 4B).\n\nMinor comments:\n• Line 172: biofilm was still accumulated → biofilm still accumulated\n• Line 142: the protein production of DspII → DspII protein expression\n• Line 230: reached to a peak → reached a peak\n• Line 248: was also not observed → was not observed either\n• Line 305: the rsmZ but not rsmY promoter → the rsmZ promoter, but not the rsmY promoter\n• Line 370: The fluorescence decreased after 16 h → Fluorescence decreased after 16 h\n• Line 470: and their derivatives were cultured → and its derivatives were cultured\n• Line 540: statistically significance → statistical significance\n• Line 610: EMSA results showed that DspI and DspII repressed the rsmZ promoter → EMSA results showed that the DspI-DspII complex repressed the rsmZ promoter\n\nOur Response: We thank the reviewer for carefully identifying these grammatical and phrasing issues. The manuscript was thoroughly proofread.\n\nReviewer #2 (Remarks to the Author):\nThis manuscript by Huang and colleagues describes the function of a gene product, DspII, that is involved in production of cis-2-decenoic acid. It's thorough and the experiments are logical and logically presented. There are a few issues:\n\nOur Response: We thank the reviewer for the positive feedback and insightful suggestions.\n\n1. The figures are quite challenging to read with very small text and in cases where there are multiple lines (eg, 3B,5B, etc.) these are difficult to distinguish. Figure 3A - it is impossible to discern what the authors are showing.\n\nOur Response: Thanks for the comment. These figures were revised to improve readability using curves with distinguishable colors.\n\n2. The discussion of Pel in PAO1 is misplaced. (Lines 154-181). The decrease in transcription is marginal. Pel is not known to contribute to biofilm formation in PAO1, so it's not clear at all what is being shown by the authors here.\n\nOur Response: Thanks for the comment. We agree with the reviewer that Pel is not the major EPS for biofilm development in PAO1. Since both Psl and Pel are known to contribute to the biofilm development in non-mucoid P. aeruginosa, we also included Pel for examination. Our result confirmed that CDA induces biofilm dispersion by repressing the expression of Psl genes. We now explicitly state that CDA-induced biofilm dispersion occurs primarily through repression of Psl, consistent with the knowledge that Psl is the primary EPS used to develop biofilm and maintain its structure in PAO1 (Colvin et al., 2012).(Lines 182-183)\n\n3. Lines 191-193. Why this PDE from D. oryzae?\n\nOur Response: Given that native PDEs might coordinate with other cellular activities and thus exhibit regulatory function independently of changing the intracellular c-di-GMP content, we chose to confirm the role of c-di-GMP by expressing an exogenous PDE W909_14950 from D. oryzae, which has been well-characterized and demonstrated to function efficiently in P. aeruginosa to lower c-di-GMP levels in our previous study (Zhou et al, 2022), to eliminate unexpected effects might be caused by native PDEs. The reason for using this exogenous PDE was added: “we expressed an exogenous PDE W909_14950 (14950), which is from Dickeya oryzae EC1 and has been well-characterized and demonstrated to function efficiently in P. aeruginosa to lower c-di-GMP levels without nonspecific effects28” (Lines 207-209).\n\n4. Lines 212-225. FleQ deletion is known to abrogate Psl production in PAO1. The fact that it does so in the DspI/II strains does not make it follow that FleQ mediates CDA's induction of Psl.\n\nOur Response: We thank the reviewer for highlighting this overstatement. To test the role of FleQ, we exogenously added CDA to WT and ΔfleQ strains and measured Psl expression. CDA reduced Psl expression in the WT but not in the ΔfleQ mutant (New Figure 4L), confirming that FleQ is required for CDA-mediated repression of Psl. (Lines 235-236)\n\n5. Lines 341-354. There is no evidence presented that the components have to be in complex to achieve the production of CDA. It's an inference the authors make, but it does not support the assertion (line 348-349) that the \"direct interaction is essential for the chronic-to-acute switch\".\n\nOur Response: Thanks for the comments. We predicted the structural model of DspII-DspI complex and confirmed the interaction between DspII and DspI by mutating critical residues (R57/R96/E231/R232) in DspI for their interaction (New Figures 8D, 8E, 8F). We further demonstrated that the ΔdspI mutant was unable to synthesize CDA as well as regulate biofilm dispersion, swarming motility, and T3SS gene expression with the complementation of the DspI variant DspI# (DspIR57A/R96A/E231A/R232A) (New Figure 8G, 8H, 8I, 8J). These newly obtained results supported that direct interaction is essential for the chronic-to-acute virulence switch. (Lines 393-404)\n\n6. Issues of word choice or language -\n- line 19-20. There is no such thing as a \"process of virulence\"\n- line 39 - pathogenesis does not undergo development\n- line 51 - what is compounded?\n- lines 88-89 - 25.81 - 40.16% is overly precise.\n\nOur Response: We have carefully addressed all the language issues pointed out by the reviewer throughout the manuscript.\n\nReviewer #3 (Remarks to the Author):\nThe manuscript titled “Dual-Functional DSF-family Quorum Sensing Signal Synthases DspII and DspI Coordinate Chronic-to-Acute Virulence Switch in Pseudomonas aeruginosa” is interesting. This manuscript is about the molecular mechanisms of the chronic-to-acute virulence switch in Pseudomonas aeruginosa, dual function of the DspII-DspI complex, Enzymatic: biosynthesis of the quorum sensing signal cis-2-decenoic acid (CDA) as well as non-enzymatic: direct regulation of gene expression (T3SS) via promoter binding, Regulation of c-di-GMP and its effect on biofilm and motility, Interaction between the Gac/Rsm regulatory system and DspII/DspI. And finally, the Importance of quorum sensing in bacterial pathogenicity and adaptation.\n\nThe experiments design is robust based in the use of gene knockouts, gene expression assays, protein-DNA binding assays, cytotoxicity tests, and Galleria infection models. Moreover, it will be able the therapeutic potential by identifies novel targets to prevent the transition to acute infection.\n\nOur Response: We thank the reviewer for the positive feedback and insightful suggestions.\n\nThe figures are well organized, with clear labels and explanatory legends. But, there are some defects or inconsistencies:\n- Figure 3d (c-di-GMP modulation by CDA): Although a reduction in c-di-GMP is shown, it is not quantified with additional controls (e.g., independent PDEs) to distinguish whether CDA acts directly or via global regulation.\n\nOur Response: Thanks for the comment. There are at least 42 DGCs and PDEs which probably participate in the metabolism of c-di-GMP in P. aeruginosa. We examined the expression of all known DGCs and PDEs in the ΔdspI mutant and only those with expression changes > 20% were focused and further verified, which led us to determine the rbdA as a critical one upregulated by CDA to decrease c-di-GMP content. However, we were unable to precisely quantify the involvement of other potential DGC or PDE in modulating the c-di-GMP pool in the presence of CDA and it is of high possibility that CDA modulates c-di-GMP metabolism via global regulation. We have discussed this: “Notably, our screening result revealed that, apart from RbdA, several other DGCs and PDEs including those known to modulate c-di-GMP content exhibited expression changes (Supplementary Table 1). This finding highlights the intricate regulatory network through which CDA influences intracellular c-di-GMP dynamics via global regulation with RbdA as the main player. Future investigations could explore the involvement of additional DGCs and PDEs targeted by CDA, as their collective actions may contribute to the chronic-to-acute virulence switch.” (Lines 461-465)\n\n- Extended Figure 4a (protein purification): SDS-PAGE gels of DspII and DspI purification are shown, but no molecular weight marker is labeled. This makes it difficult to confirm band purity or expected size.\n\nOur Response: SDS-PAGE gels of DspII, DspI, and their variants including molecular weight markers were shown in New Figure S7.\n\n- Figure 6b (protein-DNA interaction, EMSA): The observed shift for DspII appears subtle and may raise doubts about binding strength, but the article does not discuss this limitation in detail.\n\nOur Response: We agree with the reviewer that all EMSA results showed a weaker binding affinity between PrsmZ and DspII than PrsmZ and DspI. During revision, we found that the catalytic sites of DspII and DspI are important for DNA binding (as shown in the new EMSA result in response to the first comment from Reviewer #1, New Figure 7G). Since DspII only has one catalytic site Glu143, while DspI has two catalytic sites Glu126 and Glu146, fewer catalytic sites in DspII explains its weaker DNA-binding affinity than DspI. (Lines 357-365)\n\n- Final schematic model (Fig. 7):\n• It summarizes the proposed mechanism well but does not include:\n• The biphasic effect of CDA (as described in the text).\n• The unresolved indirect repression of RsmY, which the article\n\nOur Response: The model has been revised to include the biphasic effect of CDA (enhances DspII-DspI interaction and inhibits the binding of DspII and DspI at PrsmZ) and the newly discovered repression mechanism of RsmY (direct binding of DspII and DspI with GacA) (New Figure 9).\n\n- Lack of structural validation: No visual information is presented on the DspII-DspI structure or interaction predictions, which would strengthen the functional complex hypothesis.\n\nOur Response: We employed HDOCK and PLIP servers to predict the interaction of DspII and DspI and introduced mutations of the critical residues predicted for DspII-DspI interaction. The newly obtained results (New Figures 8D to 8J) not only identified the critical residues to support the interaction between DspII and DspI but also confirmed the importance of the interaction in synthesizing CDA and modulating the virulence switch. (Lines 393-404)\n\n- No images of the infection model (Galleria mellonella) are included in the figures, which could have provided direct visual evidence of the virulence phenotype.\n\nOur Response: Representative images of the G. mellonella larvae following 14-h inoculation of P. aeruginosa strains were shown in New Figure S5.\n\nMoreover, I have some general comments to take in consideration of the authors:\n1. Unresolved indirect regulation of RsmY: repression is observed, but the underlying mechanism remains unclear. Please, the authors must elucidate the indirect repression mechanism of RsmY by DspII/DspI.\n\nOur Response: As also suggested by Reviewer #1, we tried to further explore the repression mechanism of rsmY by DspII and DspI. We excluded the possibility of binding at PrsmY by the DspII-DspI complex (New Figure S9A) and preventing GacA’s binding to PrsmY by DspII or DspI (New Figure S9B). While surprisingly, we found that DspII/DspI can directly interact with the GacA protein (New Figure 7H), which reduces the transcriptional activity of GacA and thus inhibits the transcription of rsmY. Together, we are now able to show the mechanism of how DspII and DspI antagonize the GacA-dependent induction of rsmZ and rsmY. (Lines 366-378)\n\n2. High specificity of the CDA control system to P. aeruginosa, which limits its generalization to other species. To explore additional downstream targets of CDA: such as other PDEs, DGCs, or effectors involved in c-di-GMP modulation.\n\nOur Response: We screened all the known DGCs and PDEs and then verified the contribution of those with expression changes > 20% to regulate biofilm dispersion (Table S1). Only RbdA was confirmed as the critical one that mediates CDA’s regulation on c-di-GMP metabolism. However, the contribution of all the DGCs and PDEs was hardly to be precisely determined. Considering the expression of multiple DGCs and PDEs are influenced by CDA, it is of high possibility that CDA modulates c-di-GMP metabolism via global regulation with RbdA as the main player. We have discussed this in Lines 461-465. With regards to the effector, we have examined all 12 known effectors in P. aeruginosa and found that FleQ is the target effector that mediates CDA’s regulation on biofilm dispersion and swarming motility (Figure S3).\n\n3. Low sequence similarity between DspII and DspI, raising questions about the evolutionary basis of their functional interaction\n\nOur Response: Thanks for the comment. Low sequence similarity between DspII and DspI suggests that they originated from an ancient gene duplication event. To provide evolutionary basis for their functional interaction, we performed distribution and evolutionary analysis (New Figure S10), which showed that the dspII-dspI gene clusters are confined to three specific clades, meaning that the gene cluster was formed on at least three independent occasions in evolutionary history. We added this result and discussed that the occurrence of the dspII-dspI gene cluster implies a strong selective advantage potentially reflecting a pre-existing functional synergy between two proteins that was stabilized and enhanced through coordinated, localized regulation (Lines 443-452).\n\n4. Determine the prediction structure by bioinformatics tools of the DspII-DspI complex to understand their interaction and CDA-dependent regulation.\n\nOur Response: We employed HDOCK and PLIP servers to predict the structure of the DspII-DspI heterocomplex and generated mutations at the critical residues that are predicted for DspII-DspI interaction. The new results (New Figures 8D to 8J) not only identified the critical residues to support the interaction between DspII and DspI but also confirmed the importance of the interaction in synthesizing CDA and modulating the virulence switch. (Lines 393-404)\n\n5. The authors could Investigate clinical applications: such as inhibitors of DspII/DspI or their interaction to prevent acute virulence switching.\n\nOur Response: We thank the reviewer for this forward-looking suggestion. We fully agree that our findings may open up new therapeutic avenues. As now detailed in a new paragraph in the Discussion section (Lines 507-520), we discuss the potential of targeting the DspII-DspI complex. We propose two main strategies: 1) inhibiting CDA synthesis or degrading CDA to prevent CDA-mediated dispersion, and 2) more promisingly, disrupting the protein-protein interaction itself to simultaneously block both CDA production and T3SS activation, thereby \"locking\" the bacterium in a less virulent state. We also outline our future direction to screen for such inhibitory compounds. We believe this addition significantly strengthens the translational impact of our work."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have performed additional experiments to address the reviewer’s comments and have provided satisfactory responses.", "reviewer_2": "The authors have addressed my prior concerns with the manuscript."}, "author_response": ""}]

Decision letter
Re: NCOMMS-25-48364
Dear Editor and Reviewers:
Thank you for your thoughtful review of our manuscript entitled “Dual-Functional DSF-family Quorum Sensing Signal Synthases DspII and DspI Coordinate Chronic-to-Acute Virulence Switch in Pseudomonas aeruginosa”. We are grateful for the insightful comments, which have helped us significantly strengthen the study. In this revised version, we have performed extensive additional experiments to provide deeper mechanistic insights. Below, we provide a point-by-point response to the comments.

Editorial comment:
As you will see from the reports copied below, the reviewers find your work novel and of interest. However, they raise important concerns that we agree limit the strength of the study, and therefore we ask you to address them with additional experimental work. In line with the reviewers’ comments, your revision must provide further mechanistic insight, in particular by expanding the analyses of the DspI/II complex and its interactions.