The quorum sensing regulator RhlR positively controls the expression of the type III secretion system in Pseudomonas aeruginosa PAO1

Luis Fernando Montelongo-Martínez; Miguel Díaz-Guerrero; Verónica Roxana Flores-Vega; Martín Paolo Soto-Aceves; Roberto Rosales-Reyes; Sara Elizabeth Quiroz-Morales; Bertha González-Pedrajo; Gloria Soberón-Chávez; Miguel Cocotl-Yañez

doi:10.1371/journal.pone.0307174

. 2024 Aug 15;19(8):e0307174. doi: 10.1371/journal.pone.0307174

The quorum sensing regulator RhlR positively controls the expression of the type III secretion system in Pseudomonas aeruginosa PAO1

Luis Fernando Montelongo-Martínez ¹, Miguel Díaz-Guerrero ^1,², Verónica Roxana Flores-Vega ³, Martín Paolo Soto-Aceves ⁴, Roberto Rosales-Reyes ⁵, Sara Elizabeth Quiroz-Morales ⁴, Bertha González-Pedrajo ², Gloria Soberón-Chávez ⁴, Miguel Cocotl-Yañez ^1,^*

Editor: Rajesh P Shastry⁶

PMCID: PMC11326643 PMID: 39146292

Abstract

Pseudomonas aeruginosa is an opportunist bacterium that causes acute and chronic infections. During acute infections, the type III secretion system (T3SS) plays a pivotal role in allowing the bacteria to translocate effectors such as ExoS, ExoT, and ExoY into host cells for colonization. Previous research on the involvement of quorum sensing systems Las and Rhl in controlling the T3SS gene expression produced ambiguous results. In this study, we determined the role of the Las and Rhl systems and the PqsE protein on T3SS expression. Our results show that in the wild-type PAO1 strain, the deletion of lasR or pqsE do not affect the secretion of ExoS. However, rhlI inactivation increases the expression of T3SS genes. In contrast to the rhlI deletion, rhlR inactivation decreases both T3SS genes expression and ExoS secreted protein levels, and this phenotype is restored when this mutant is complemented with the exsA gene, which codes for the master regulator of the T3SS. Additionally, cytotoxicity is affected in the rhlR mutant strain compared with its PAO1 parental strain. Overall, our results indicate that neither the Las system nor PqsE are involved in regulating the T3SS. Moreover, the Rhl system components have opposite effects, RhlI participates in negatively controlling the T3SS expression, while RhlR does it in a positive way, and this regulation is independent of C4 or PqsE. Finally, we show that rhlR, rhlI, or pqsE inactivation abolished pyocyanin production in T3SS-induction conditions. The ability of RhlR to act as a positive T3SS regulator in the absence of its cognate autoinducer and PqsE shows that it is a versatile regulator that controls different virulence traits allowing P. aeruginosa to compete for a niche.

Introduction

Pseudomonas aeruginosa is a human opportunist pathogen that causes acute and chronic infections. This bacterium possesses a vast arsenal of virulence factors including pyocyanin, rhamnolipids, and elastase, that allows it to compete for a favorable niche [1,2]. One of the main virulence determinants in P. aeruginosa acute infection is the type III secretion system (T3SS), involved in avoiding phagocytosis and inducing macrophage apoptosis, among others roles [3–5]. Mutants defective in the T3SS machinery are less virulent in a mouse model infection [6,7].

The T3SS resembles a molecular syringe, known as injectisome, which forms a channel that crosses the bacterial envelope and the host cell membrane, enabling bacteria to inject effectors such as ExoS, ExoT and ExoY into the host cell cytoplasm [5,8,9]. ExoS and ExoT share 76% of identity in amino acid sequence and inhibit phagocytosis by disrupting actin cytoskeletal arrangement and signal transduction cascades essential for phagocytic function [10]. ExoY possesses adenylate cyclase activity that increases cAMP intracellular levels damaging the cellular function [11]. The genes encoding the structural and regulatory elements of the injectisome are organized together in five operons, whereas the genes that encode for the effectors and associated chaperones are distributed in the chromosome [12]. Expression of the T3SS genes is tightly regulated and is induced when P. aeruginosa is in contact with host cells or when extracellular calcium concentrations are reduced (S1 Fig) [13,14]. In addition, all T3SS genes are regulated at the transcriptional level by the main activator ExsA, a member of the AraC family of transcriptional regulators [15,16]. Each of the five operon promoters and the promoters of the effectors contain an ExsA binding motif located ~45 bp upstream of the transcriptional start site [17]. exsA is expressed within exsCEBA operon, whose transcription is positively controlled by PsrA and negatively by MvaT [18,19]. Also, exsA contains an internal promoter that is transcriptionally activated by Vfr and negatively regulated by MvaT and MvaU [20,21]. Furthermore, it was recently reported that spermidine modulates the T3SS by affecting exsCEBA operon expression [22,23]. An additional layer of regulation includes a partner-switching mechanism that involves the antiactivator ExsD and the antiantiactivator ExsC, which together control ExsA activity [24].

In P. aeruginosa, the synthesis of several virulence factors is regulated at the transcriptional level by the quorum sensing (QS) response, which is a cellular communication process based on the production and secretion of signal molecules named autoinducers (AI) that when are extracellularly accumulated, enter into the bacterium and bind to a transcriptional regulator that turns on virulence genes expression [25]. This bacterium harbors three QS systems named Las, Rhl, and Pqs that, in certain conditions such as rich medium, are hierarchically organized with the Las system placed at the top of this regulatory network [26–28]. The first two QS systems are based on the synthesis and detection of N-acyl-homoserine lactones while the Pqs system is based on producing and detecting alkyl-quinolones molecules. In the Las system, LasR is the transcriptional regulator that binds to N-3-oxo-dodecanoyl-homoserine lactone (C12), which is synthesized by LasI, to activate the expression of some virulence factors, and also of rhlR and rhlI, which encode to the transcriptional factor RhlR and the N-butyryl-homoserine lactone (C4) synthase, RhlI [29,30]. RhlR binds to C4 and activates the expression of genes involved in pyocyanin and rhamnolipids synthesis, among others [31,32]. In the Pqs system, PqsR is the transcriptional regulator that binds to 2-heptyl-3-hydroxy-1H-quinolin-4-one (PQS) or 2-heptyl-1H-quinolin-4-one (HHQ) and controls pyocyanin production by turning on the expression of pqsE [28,33]. In this regard, it has been reported that in addition to C4, RhlR can work by forming a complex with PqsE to control the expression of a different subset of genes; therefore, there are RhlR regulons dependent and independent of C4 [32,34–36]. Moreover, it has been reported that RhlR can control the expression of genes in the absence of its autoinducer [37]. Thus, these characteristics make RhlR a more versatile regulator than LasR.

Since the Las and Rhl QS systems control about 6% of gene expression [30], it has been investigated whether these two systems also control the T3SS gene expression. The first report established that exoS expression increased when rhlI or rhlR were inactivated, suggesting that the Rhl system negatively controls the T3SS; while exoS transcription was not affected when lasI was inactivated [38]. In a second study, it was reported that rhlI inactivation increased the expression of some T3SS genes and ExoS was secreted in the early stages of growth. In this study, the authors reported that lasR inactivation did not affect the expression of the T3SS genes indicating that the Rhl system, but not the Las system, is a negative regulator of the T3SS. Moreover, it was also reported that the exsCEBA operon, which encodes for ExsA activator, was not up-regulated in the rhlI mutant strain, suggesting that the Rhl system individually controls each promoter of the T3SS genes [39]. On the other hand, in recent reports, it was documented that even though a lasR/rhlR double mutant strain abolishes the production of elastase, pyocyanin, and rhamnolipids, the T3SS was not affected [40]. Furthermore, when the PAO1, PA14 and two clinical isolates were treated with the AiiM lactonase, which can degrade C12 and C4, the virulence factors production such as elastase, pyocyanin, and HCN was reduced but the secretion of exotoxins ExoS and ExoU was similar to that of the wild-type strain [41]. These data suggested that the T3SS expression is not regulated by the QS response. Additionally, it has been reported that rhlI mutants display higher virulence compared with rhlR mutants, and in the latter, the virulence is attenuated [32,42]. Therefore, there are some discrepancies in the virulence and regulation of the T3SS by the QS systems.

Herein, we determine the effect of the QS systems on the expression of the T3SS. We found that neither lasR nor pqsE inactivation affects the ExoS protein levels. However, regarding the Rhl system, our results showed that the inactivation of rhlI up-regulates the expression of T3SS genes, while rhlR inactivation down-regulates it. Moreover, the rhlR mutant strain showed reduced cytotoxicity, which is in line with previous reports where virulence is affected when rhlR is inactivated. Also, here we show that constitutive expression of the PA2592 gene, which codes for a putative spermidine-binding protein, partially restores ExoS secretion in the rhlR mutant strain in the early stationary phase. Finally, we show that even though RhlR controls T3SS expression in the absence of C4 and PqsE, both are required for the positive control of RhlR on pyocyanin synthesis in T3SS-induction conditions.

Materials and methods

Bacterial strains and growth conditions

The bacterial strains and plasmids used in this study are listed in supplemental material S1 Table. Pseudomonas aeruginosa MPAO1 strain was used in all the experiments (referred to as PAO1) whereas Escherichia coli DH5a strain was used for standard techniques of cloning and propagation. Unless otherwise noted, P. aeruginosa and E. coli strains were grown in LB (Lysogenic- Broth) medium at 37°C and 225 rpm. For expression assays, LB medium was used as a non-induction medium, and LB medium supplemented with 5 mM EGTA and 20 mM MgCl₂ was used for T3SS induction [43]. When necessary, antibiotics at the following final concentrations were used for P. aeruginosa: tetracycline (Tc) 120 μg/mL, streptomycin (Sm) 200 μg/mL, carbenicillin (Cb) 200 μg/mL, apramycin (Apc) 150 μg/mL, gentamicin (Gm) 100 μg/mL. For E. coli: tetracycline (Tc) 15 μg/mL, streptomycin (Sm) 30 μg/mL, ampicillin (Amp) 200 μg/mL and gentamicin (Gm) 15 μg/mL.

DNA manipulation techniques

The genomic DNA template was obtained from strain PAO1 using the GeneJET DNA purification system (Thermo Scientific). The high-fidelity DNA polymerase enzyme Phusion (Thermo Scientific) was used to amplify the DNA regions. DNA fragments were obtained from agarose gel bands and DNA was purified using the Wizard SV Gel and PCR Clean-Up System protocol (Promega). Restriction enzymes (New England Biolabs) and T4 DNA ligase enzyme (Promega) were used according to manufacturer instructions. Plasmids were purified using Wizard Plus SV Minipreps DNA Purification Systems (Promega) and manipulated according to standardized techniques [44]. Synthesis of oligonucleotide and DNA sequencing were performed at Unidad de Síntesis y Secuenciación de DNA (USSDNA) by Instituto de Biotecnología at Universidad Nacional Autónoma de México (UNAM). Oligonucleotide pairs used for the PCR reactions are listed in S2 Table.

Construction of transcriptional fusions

Mini-CTX-lux plasmid [45] was used to construct all transcriptional fusions. The promoter sequences were amplified at 60°C using PAO1 genomic DNA and specific oligonucleotides pair (S2 Table) that include the -35/-10 binding sites for RNA polymerase and the ExsA-binding motif (S2 Fig). PCR products were purified and cloned into the XhoI and HindIII sites in mini-CTX-lux. Also, the pCTX plasmid was used as a negative control for lux expression, which was constructed by cloning ~400 bp of the rhlR structural region, previously amplified with primers pair rt-rhlR-F3 and rt-rhlR-R2, into the SmaI site from mini-CTX-lux. Plasmids were mobilized into P. aeruginosa strains and chromosomal integration were confirmed by PCR reactions, using a forward primer, corresponding to the region of each cloned promoter, and a LuxRv reverse primer recognizing the luxC gene of mini-CTX-lux plasmid.

Construction of pExsA and pUC2592 plasmids

A 975 bp and 1,232 bp corresponding to the structural region of the transcriptional regulator exsA and the gene PA2592 were amplified using the primers pair ExsA_Fw/ExsA_Rv and 5UpEcIPA2592Fw/3DwBmIPA2592Rv, respectively (S2 Table). The corresponding products were purified and cloned into the BamHI-HindIII and EcoRI-BamHI sites in pUCP20 plasmid [46], obtaining pExsA and pUC2592 plasmids. Then, one μg of the purified pExsA or pUC2592 plasmid was introduced by electroporation into the PAOΔrhlR strain.

Generation of mutant strains

Gene deletions were performed by homologous recombination to the bacterial chromosome of plasmid-borne insertion-deletion as previously described [47] with minor modifications. Briefly, allele replacement of the lasR gene from PAO1 was achieved by constructing the pEX-lasR::Apc deletion plasmid as follows: a 487 bp fragment and a 644 bp fragment corresponding to the upstream and downstream region of the lasR gene were amplified from PAO1 genomic DNA with primers pair 6709–2015_H3lasRUp/8653-2015_lasR5Apra and 6514–2018_lasR3Apra/6710-2015_H3lasRDown, respectively. Also, an apramycin resistant cassette was amplified from the pIJ773 plasmid [48] with primers 9522–2014_F-Apra and 9523–2014_R-Apra. The three PCR products were purified and used as a template in a nested PCR. The PCR product was digested with HindIII and cloned into the HindIII site of the pEX18 plasmid [49], which is unable to replicate in P. aeruginosa, resulting in the pEX-lasR::Apc plasmid. This plasmid was mobilized into the wild-type PAO1 strain to replace the lasR gene with the apramycin resistance marker by double allelic exchange, thus obtaining the lasR single mutant PAOΔlasR strain. Also, the pEX-lasR::Apc plasmid was mobilized into the PAOΔrhlR strain [46] to obtain the double mutant strain PAOΔlasRΔrhlR. For rhlI deletion, the pEX-rhlI::Aa plasmid [46] was used to construct the PAOΔrhlI^Apc, and the resistance marker was subsequently removed using the pFLP2 plasmid, as previously reported [49], obtaining the PAOΔrhlI strain. To obtain the double mutant strain PAOΔrhlIΔpqsE, the pEX-pqsE::Gm plasmid [36] was mobilized into the PAOΔrhlI strain. Finally, the genes exsA, exsD, pscB and part of pscC were deleted in PAO1 strain using the plasmid pJET1.2_T3SS::Apc. This plasmid was constructed as follows: a 608 bp fragment and a 773 bp fragment corresponding to the upstream region of the exsA gene and downstream region of the pscB gene were amplified from PAO1 genomic DNA with primers pair 6767–2023_T3SSUp/6768-2023_T3SS3aaApra and 6769–2023_T3SS5aaApra/6770-2023_T3SSDown, respectively. Also, an apramycin resistant cassette was amplified as previously described. The three PCR products were purified and used as a template in a nested PCR and cloned into the pJET1.2 plasmid (Thermo-Fischer), resulting in the pJET1.2_T3SS::Apc plasmid, which was then mobilized into the PAO1 strain to obtain the double recombinant PAOΔT3SS ^Apc strain. The resistance marker was subsequently removed using the pFLP2 plasmid obtaining PAO1ΔT3SS strain. In each case, the candidate clones were positive selected on LB medium with the respective antibiotics and confirmed by PCR and sequencing of the modified region.

Luminescence assays

Bacterial overnight pre-cultures were diluted at an O.D.₆₀₀ = 0.05 in 125 mL flasks with 15 mL of non-induction and/or induction medium, which were incubated until reaching the final cell density corresponding to the log phase (O.D.₆₀₀ = 0.8) or early stationary phase (O.D.₆₀₀ = 2.0). For each biological assay, 200 μL samples of each culture were collected at the desired cell density and loaded in triplicate into 96-well flat clear bottom black polystyrene plates (Costar). The ratio of relative units of luminescence (R.L.U.) produced was quantified using the Synergy HT Plate Reader (Biotek) and normalized over the O.D.₆₀₀ value at the time of sample collection (R.L.U./O.D.₆₀₀). The results represent the mean ±S.D. of three biological experiments.

Western blot assays

Bacterial overnight pre-cultures were diluted at an O.D.₆₀₀ = 0.05 in 125 mL flasks with 15 mL of induction or non-induction medium, which were incubated at 37°C and 225 rpm, until the desired cell density was reached. Subsequently, 1 mL samples were collected and centrifuged at 4°C (14,000 rpm, 2 min), and proteins of the supernatant were precipitated with 100 μL of trichloroacetic acid (TCA) 100% at 4°C, overnight. Then, samples were centrifugated at 4°C (14,000 rpm, 30 min), and pellets were resuspended with the volume corresponding to 30 μL of SDS-PAGE loading buffer previously normalized to the cell density value at the time of sample collection. Resuspended samples were denatured at 90°C for 5 minutes. For electrophoresis separation, 5 μL of each sample was loaded in 12% SDS-PAGE gels following Bio-Rad protocols. Proteins were transferred to 0.2 μm nitrocellulose membranes and blocked with 5% BSA in Tris-HCl buffer pH 7.4 supplemented with 0.1% Tween-20 detergent (TBS-T) at 4°C, for 2 h. The membranes were washed for 15 min with 15 mL of TBS-T, three times. Subsequently, the first antibody anti-ExoS [40] diluted 1:10,000 was added and incubated at 4°C for 1 h. The membranes were washed, and then a second antibody was added: 1:10,000 anti-IgG-GAR (Jackson Immunoresearch) or 1:5000 anti-rabbit IgG conjugated to alkaline phosphatase (Abcam) and incubated at 4°C for 1 h. Finally, the membranes with anti-IgG-GAR were washed and a reaction 1:1 solution of HRP chemiluminescent Immobilon Western kit (Millipore) was added and the bands were developed on X-ray film (Carestream MXB-Blue film). Membranes with anti-rabbit IgG conjugated to alkaline phosphatase (anti-IgG-AP) were washed and developed using a 1-step NBT/BCIP solution (Thermo Scientific). GroEL was used as a loading control since it is constitutively expressed in the conditions used [50]. For GroEL detection the same membranes were washed with 15 mL of stripping buffer (25 mM glycine, 1% SDS, pH 2.0) at 4°C for 1 h with shaking and washing. The membranes were blocked and incubated for 12 h at 4°C, washed, and incubated with 1:10,000 anti-GroEL polyclonal antibody (Sigma). Finally, the membranes were washed with TBS-T and incubated with 1:10,000 anti-IgG-GAR or 1:5,000 anti-IgG-AP. Bands were revealed on an X-ray film or using a 1-step NBT/BCIP solution.

Cytotoxicity assays

Strains of P. aeruginosa were grown on in shaking, 180 rpm at 37°C in LB broth. The bacteria were adjusted at an MOI of 600 nm to infect 6x10⁵ HeLa cells (ATCC^® CCL-2TM). To synchronize the infection, the plates were centrifuged at 1,400 rpm for 2 min at room temperature, and incubated at 37°C, 5% CO_2, and 5% humidity for 30 min. Next, the infected cells were washed and incubated for 24 hours at 37°C, 5% CO₂, and 5% humidity. The supernatants of infected cells were collected and centrifuged at 14,000 rpm for 2 minutes. The clarified supernatants were used to quantify the cytosolic enzymatic activity of lactate dehydrogenase (Promega). For this, 50 μL of the supernatants were taken, and mixed with 50 μL of the substrate in 96-well flat-bottomed plates (Nunc). The mixture was incubated for 10–20 minutes at room temperature, and the enzymatic activity was quantified spectrophotometrically at 490 nm. Cytotoxicity was quantified using the following formula: % CTX = [(sample cytotoxicity-spontaneous cytotoxicity) / (total cytotoxicity-spontaneous cytotoxicity)] x 100% [51].

Pyocyanin quantification

Bacterial overnight pre-cultures were diluted at an O.D.₆₀₀ = 0.05 and incubated in 125 mL flasks with 15 ml of induction medium for 24 h with continuous shaking (225 rpm) at 37°C. Pyocyanin production was quantified from cell- free supernatants at 695 nm and normalized by the O.D.₆₀₀ at 24 h, as reported previously [32].

Densitometry analysis

Densitometry analysis of at least three Western blot assays was carried out using the ImageJ software [52]. Relative ExoS protein levels were normalized by the levels of GroEL, used as a loading control. ExoS protein levels of PAO1 strain were considered as 100% and ATCC 9027 as 0%.

Statistical analysis

Means and standard deviation from at least three biological replicates were analyzed using the Graph Prism 9.0 statistical software with a confidential level of 95% (α = 0.05%). Data were considered statistically significant if the difference value was p<0.05.

Results

RhlR, but not LasR, decreases ExoS secretion

In order to determine whether the QS regulators, LasR and RhlR, are able to control the expression of the T3SS, we measured ExoS secretion by Western blot using PAOΔlasR and PAOΔrhlR, and a double mutant PAOΔlasRΔrhlR strain, and compared it with the wild-type PAO1 strain. In addition, ATCC 9027 strain was used as a negative control since this strain has a natural deletion of the T3SS [40]. Western blot assays were carried out in induction conditions at an O.D.₆₀₀ of 0.8 and 2.0 which correspond to the log phase and early stationary phase, respectively. As shown in Fig 1A, in the log phase, ExoS protein secretion levels were similar in the PAOΔlasR strain compared to the wild-type PAO1 strain, while in the PAOΔrhlR strain ExoS is barely detected. In addition, and similar to rhlR inactivation, ExoS secreted protein levels were almost abolished in the double mutant PAOΔlasRΔrhlR strain. These results were similar to those of the early stationary phase where ExoS protein secretion levels were not affected by lasR deletion but decrease in both the rhlR mutant strain and the double lasR/rhlR mutant strain (Fig 1B). Thus, since RhlR seems to have a positive effect on exoS expression, we verified that this phenotype was complemented with rhlR using pGMYC plasmid carrying this gene. As shown in Fig 1, the rhlR mutant strain with pGMYC plasmid fully restored the ExoS secretion, but not with the empty plasmid pUCP20 used as a negative control. With regard to the double mutant strain, rhlR expression restored the phenotype to that of the lasR mutant strain where ExoS is secreted (S3 Fig). Since these results contrast with the previous report where RhlR seemed to act as a negative regulator of exoS expression [38], we used another rhlR mutant strain with a different marker, PAO1ΔrhlR^Gm [53], and Western blot was carried out to detect ExoS protein, producing similar results as the assays using the initial strain PAO1ΔrhlR (Fig 2). Together, these results suggest that RhlR positively regulates the expression of the T3SS.

Fig 1 — ExoS identification was performed by Western blot assay using anti-ExoS polyclonal antibody on supernatants of strains grown in induction conditions at log phase (a) and early stationary phase (b). PAO1 was used as a positive control, whereas ATCC 9027, lacking the T3SS, was used as a negative control. GroEL was detected using polyclonal antibody anti-GroEL and used as a loading control. The densitometry graphs show the mean ± S.D. of ExoS levels of at least three biological replicates. Significant differences were obtained by ordinary one-way ANOVA and Tukey’s multiple comparisons (α = 0.05%). Different letters indicate significant differences, while equal letters indicate no significant differences.

Fig 2 — ExoS identification was performed by Western blot assay using anti-ExoS polyclonal antibody on supernatants of strains grown in induction conditions at log phase (a) and early stationary phase (b). PAO1 was used as a positive control, whereas ATCC 9027, lacking the T3SS, was used as a negative control; also, PAOΔ*rhlR*^Gm strain was included. GroEL was detected using polyclonal antibody anti-GroEL and used as a loading control. The densitometry graphs show the mean ± S.D. of ExoS levels of at least three biological replicates. Significant differences were obtained by ordinary one-way ANOVA and Tukey’s multiple comparisons analysis (α = 0.05%). Different letters indicate significant differences, while equal letters indicate no significant differences.

RhlR positively regulates the expression of exoS, spcS and the exsCEBA operon

Since rhlR inactivation almost abolishes ExoS secretion, we constructed an exoS transcriptional fusion (PexoS::lux) fusing the exoS promoter to the luxCDABE operon. Also, three additional transcriptional fusions were constructed using the promoter of the spcS gene which encodes for the ExoS chaperone (PspcS::lux), the promoter of the first gene of the exsCEBA operon (PexsC::lux), and the internal promoter of exsA (PexsA::lux). First, we tested whether these transcriptional fusions were activated in the T3SS-induction conditions as described in the methods section. As shown in S4 Fig, all fusions, except PexsA::lux, were activated when Ca⁺ concentrations were reduced with EGTA during induction conditions. Then, PexoS::lux, PspcS::lux, and PexsC::lux plasmids were mobilized into the rhlR mutant strain and its derivatives, and luminescence was measured at an O.D.₆₀₀ of 0.8. As shown in Fig 3, the luminescence of the three transcriptional fusions was reduced in the rhlR mutant background compared to the wild-type strain, and the activity was restored when PAOΔrhlR strain was complemented with pGMYC but not with the empty plasmid. The above results suggest that RhlR activates the expression of the T3SS genes.

Fig 3 — Expression of the *exsCEBA* operon (PexsC::lux), *exoS* (PexoS::lux) and *spcS* (PspcS::lux) genes were evaluated by lux-transcriptional fusions. Strains were incubated in 15 ml of induction medium at 37°C and 225 rpm. Relative luminescence units (R. L.U.) were quantified and normalized to the O.D.₆₀₀ at the time of cell collection. Results represent the mean ± S.D. of three biological experiments performed in three replicates each time. Significant differences were obtained by ordinary one-way ANOVA and Tukey’s multiple comparisons analysis (α = 0.05%). Different letters indicate significant differences, while equal letters indicate no significant differences.

exsA expression restores ExoS secretion and the T3SS genes expression in the rhlR mutant strain

Since ExsA is the master regulator of the T3SS, we explored whether RhlR controls the T3SS expression via ExsA activity or whether it is an ExsA-independent regulation. In the first scenario, exsA expressed under a constitutive promoter must restore exoS transcription and also ExoS secretion in the rhlR mutant strain, but if this was not the case, then RhlR must control each operon or gene of the T3SS. Thus, we constructed a plasmid expressing exsA under a constitutive promoter, named pExsA, and it was mobilized into the PAOΔrhlR strain. Then, we carried out a Western blot assay to detect ExoS protein in this strain. As shown in Fig 2, exsA constitutive expression in the rhlR mutant strain resulted in the detection of ExoS protein at an O.D.₆₀₀ of 0.8 and 2.0. Next, we measured the transcriptional activity of exoS, exsCEBA, and spcS in this background and compared it to the wild-type strain. As shown in Fig 3 the expression of the three transcriptional fusions was restored in the rhlR mutant strain when exsA is expressed and these levels are higher than in the wild-type strain. Since these results did not discard the possibility that RhlR and ExsA could act independently, we determined whether rhlR overexpression in a strain defective in the T3SS, named PAOΔT3SS, was able to restore exsCEBA and exoS expression in the log and early stationary phase. As shown in S5 Fig, the transcriptional activity of both fusions, PexsC::lux and PexoS::lux, in the PAOΔT3SS strain overexpressing rhlR was similar to those of the mutant strain and the mutant strain with the empty plasmid pUCP20, whereas in the wild-type strain, both transcriptional fusions were activated. These results show that RhlR is unable to activate the T3SS expression in the absence of the master regulator ExsA and indicate that RhlR controls the T3SS by controlling exsA expression from the exsCEBA operon.

Cytotoxicity is affected when rhlR is inactivated

PAO1 displays lower cytotoxicity compared to the PA14 strain due to its genome containing the exoS gene instead of exoU; however, ExoS is able to cause cell rounding and apoptosis in eukaryotic cells [5,54]. Thus, we carried out a cytotoxicity assay to determine whether rhlR inactivation, which reduces T3SS gene expression, also reduces cytotoxicity. Cell cultures from PAO1, PAOΔrhlR, and its derivates were used to infect the HeLa eukaryotic cell line, and cytotoxicity was measured as described in the methods. As shown in Fig 4, in the rhlR mutant strain and complemented with the empty plasmid cytotoxicity was reduced compared to PAO1 strain or PAOΔrhlR mutant strain complemented with rhlR. Moreover, exsA expression increased cytotoxicity in the rhlR mutant strain. These results are in agreement with our initial observations indicating that RhlR is a positive regulator of the T3SS and that this regulation is achieved by controlling exsA expression.

Fig 4 — Selected strains at MOI of 100 were used to infect 6x10⁵ HeLa cells (ATCC® CCL-2TM) in 24-well boxes. Infection conditions were synchronized and incubated at 37°C, 5% CO2 and 5% humidity for 24 hours. Clarified supernatants were used to quantify the enzymatic activity of lactate dehydrogenase in 96-well flat-bottomed plates at 490 nm. Results represent the mean ± S.D. of three biological experiments performed in duplicates each time. Significant differences were obtained by ordinary one-way ANOVA and Tukey’s multiple comparisons analysis (α = 0.05%). Different letters indicate significant differences, while equal letters indicate no significant differences.

RhlR regulates T3SS expression in the absence of C4 or PqsE

Since RhlR activity is usually dependent on binding to C4 and on the activity of PqsE [32], we carried out Western blot analysis at an O.D.₆₀₀ of 0.8 and 2.0 to detect ExoS when rhlI, pqsE, or both genes are inactivated. As shown in Fig 5, ExoS secretion in the three mutant strains was similar to that in the wild-type strain at an O.D.₆₀₀ of 0.8 and 2.0. We next measured the activity of the exsCEBA promoter in these three mutant strains at an O.D.₆₀₀ of 0.8 using the PexsC::lux plasmid. As expected, the activity of exsCEBA promoter was similar in the three mutant strains compared to the wild-type PAO1 strain (S6 Fig). Since it has been reported that rhlI inactivation increases the expression of exoS and exoT at an O.D.₆₀₀ >1 [38,39], we constructed an exoT transcriptional fusion (PexoT::lux). Then, PexoS::lux and PexoT::lux were mobilized into the rhlI mutant strain, and promoter activity of these two transcriptional fusions and the exsCEBA transcriptional fusion (PexsC::lux) were measured at an O.D.₆₀₀ of 2.0. As shown in Fig 6, the expression of the three transcriptional fusions was higher in PAOΔrhlI than in the wild-type strain indicating that, as previously reported, RhlI seems to negatively regulate the expression of the T3SS. These results suggest that RhlR without C4 is a positive exsCEBA regulator, while RhlR coupled with C4 acts as a negative regulator of its transcription.

Fig 5 — ExoS identification was performed by Western blot assays using an anti-ExoS polyclonal antibody on supernatants of strains grown in induction conditions at log phase (a) and early stationary phase (b). PAO1 was used as a positive control whereas ATCC 9027, lacking the T3SS, was used as a negative control. GroEL was detected using polyclonal antibody anti-GroEL and used as a loading control. The densitometry graphs show the mean ± S.D. of ExoS levels of at least three biological replicates. Significant differences were obtained by ordinary one-way ANOVA and Tukey’s multiple comparisons analysis (α = 0.05%). Different letters indicate significant differences, while equal letters indicate no significant differences.

Fig 6 — Expression of the *exsCEBA* operon (PexsC::lux), *exoS* (PexoS::lux) and *exoT* (PexoT::lux) genes was evaluated by lux-transcriptional fusions. Strains were incubated in 15 ml of induction medium at 37°C and 225 rpm until reaching an O.D.₆₀₀ of 2.0. Relative luminescence units (R. L.U.) were quantified and normalized to the O.D.₆₀₀ at the time of cell collection. Results represent the mean ± S.D. of three biological experiments performed in three replicates each time. Significant differences were obtained by multiple unpaired t-test and are indicated with asterisks (* p<0.05; ** p<0.01; *** p<0.001).

PA2592 expression partially restores ExoS secretion in the rhlR mutant strain

Our data indicates that RhlR positively regulates T3SS, probably by regulating exsA expression; however, we could not detect a las-rhl box in the exsCEBA promoter indicating that the regulation must be indirect. Previously it was reported that spermidine works as a signal that modulates T3SS expression [22,23]. Interestingly, PA2592 which codes for a putative spermidine-binding protein was downregulated when rhlR was inactivated, and the promoter region of this gene contains a las-rhl box that could be recognized by RhlR [55]. Therefore, we determined whether the expression of PA2592 from a constitutive promoter was able to restore ExoS secretion in the rhlR mutant strain. We constructed the plasmid pUC2592, as described in the methods section, and it was mobilized into PAOΔrhlR strain. Then, Western blot assay against ExoS was carried out at an O.D.₆₀₀ of 0.8 and 2.0. Fig 7 shows that ExoS secretion was partially restored only in the early stationary phase. This result suggests that RhlR regulation of PA2592 is involved in T3SS expression but that an additional pathway is also involved.

Fig 7 — ExoS identification was performed by Western blot assay using anti-ExoS polyclonal antibody on supernatants of strains grown in induction conditions at log phase (a) and early stationary phase (b). PAO1 was used as a positive control whereas ATCC 9027, lacking the T3SS, was used as a negative control. GroEL was detected using polyclonal antibody anti-GroEL and used as a loading control. The densitometry graphs show the mean ± S.D. of ExoS levels of at least three biological replicates. Significant differences were obtained by ordinary one-way ANOVA and Tukey’s multiple comparisons analysis (α = 0.05%). Different letters indicate significant differences, while equal letters indicate no significant differences.

Pyocyanin production is dependent on C4 and PqsE in T3SS-induction conditions

As shown above, T3SS expression is regulated by RhlR in the absence of C4 or PqsE. Thus, we explored whether pyocyanin, whose synthesis is positively controlled by QS systems and particularly by RhlR, requires C4 and PqsE in T3SS-inducing conditions as required in LB medium [40,56]. The strains, including the rhlR mutant strain overexpressing exsA, were grown in T3SS-inducing conditions, and pyocyanin production was measured. As shown in Fig 8, lasR inactivation slightly reduced pyocyanin synthesis compared with the PAO1 strain. As expected, rhlR inactivation abolished pyocyanin production and it was restored in the PAOΔrhlR strain when rhlR was expressed from pGMYC plasmid but not with the plasmid expressing exsA nor pUCP20 empty plasmid. Furthermore, rhlI and pqsE inactivation also abolishes pyocyanin synthesis since C4 and the PqsE protein have been reported to be necessary for the RhlR activity on pyocyanin production [56]. These results are similar to those previously reported in LB medium indicating that T3SS-inducing conditions do not affect pyocyanin regulation by the QS systems.

Fig 8 — Results represent the mean ± S.D. of three biological experiments performed in duplicates each time. Significant differences were obtained by ordinary one-way ANOVA and Tukey’s multiple comparison analysis (α = 0.05%). Different letters indicate significant differences, while equal letters indicate no significant differences.

Discussion

One of the main virulence determinants in P. aeruginosa is the T3SS which allows translocating effector proteins into the host cell to avoid phagocytosis [3,24]. PAO1 strains code for three exotoxins ExoY, ExoT, and ExoS while PA14 strain contains ExoU instead of ExoS, which makes it more cytotoxic [57,58]. The master regulator of this system is ExsA whose activation is coupled to a cascade of three interacting proteins (ExsC, ExsD, and ExsE) (S1 Fig) [24]. In addition, exsA expression is regulated at the transcriptional and post-transcriptional levels. The latter includes the Gac-Rsm system where RsmA is a post-transcriptional regulator that positively controls the translation of exsA [59]. Furthermore, transcription of exsA involves the activation of the exsCEBA operon by PsrA and it is negatively controlled by MvaT [18,19], while the internal exsA promoter is activated by Vfr and VqsM, and negatively regulated by MvaT and MvaU [20,21,60]. In addition to the gene regulation of the T3SS, other factors such as spermidine concentrations affect exsCEBA operon expression and, therefore, the T3SS activation [22,23].

Since QS systems are involved in regulating several virulence traits such as pyocyanin, elastase, and rhamnolipids, among others, different research groups have tried to elucidate whether QS systems are involved in controlling T3SS expression as well. Early reports placed the Rhl system, and particularly RhlI, as a negative regulator for the T3SS [38,39]. However, in recent years it has been reported that the QS systems seem not to be involved in controlling this secretion system [40,41]. Thus, our objective was to define the role of the QS system elements in T3SS. First, we verified that the T3SS is not induced in LB medium and is activated when EGTA is added during induction conditions (S4 and S7 Figs). Therefore, all experiments were carried out under T3SS-inducing conditions. Our results showed that LasR is not involved in regulating the T3SS since ExoS protein levels in the PAOΔlasR mutant are similar to the ones in the wild-type strain. These results are in agreement with previous reports where expression of T3SS genes is not affected by lasI or lasR deletion [38,39]. Regarding the Rhl system, we found that the inactivation of rhlR almost abolishes ExoS secretion. Moreover, exoS, spcS, and exsCEBA expression are reduced when rhlR is inactivated, confirming that RhlR is able to regulate not only exoS expression but also other elements of the T3SS, including the master regulator ExsA. Since these results are in contrast to the negative role of RhlR previously proposed [38], we confirmed the positive role of RhlR using another rhlR mutant strain, obtaining similar results. This discrepancy on the role of RhlR could be related to the different culture conditions used to activate the T3SS. Moreover, cytotoxicity was affected when rhlR was inactivated which supports the positive regulation of RhlR on the T3SS.

Additionally, in this study, we used a PAOΔlasRΔrhlR double mutant strain and found that ExoS protein secretion levels are diminished compared to the wild-type strain. This phenotype is explained by the rhlR deletion, and this was confirmed when the mutant was complemented with rhlR and ExoS secretion was fully restored, resembling a lasR mutant strain. In our previous work, we reported that the ExoS secretion was not affected when lasR and rhlR were inactivated [40]. The mutant strain used in that previous work contained the markers tetracycline and streptomycin (PAOΔlasR^TcΔrhlR^Sm), while the double mutant strain constructed in this work contains the markers apramycin and streptomycin; however, these changes do not explain the differences in the Western blot results. Therefore, we sequenced both genomes in order to find any mutation that could explain these discrepancies. We found that the PAOΔlasR^TcΔrhlR^Sm strain possesses a single point mutation in the mvaT gene that lies in the DNA-binding motif [61] and changes the TGG codon for Trp¹¹⁹ to a TAG stop codon. This result was confirmed by sequencing the mvaT gene (S8 Fig). MvaT is a negative regulator for the T3SS that binds to the exsA internal promoter and also to the exsCEBA operon promoter [19,21], and it has been reported that mvaT inactivation derepresses exsA expression [21], explaining why in the previously reported PAOΔlasR^TcΔrhlR^Sm strain ExoS is detected [40].

RhlR binds to its canonical AI C4 to control the expression of its target genes, however, in recent years it has been reported that RhlR is also able to regulate a set of genes independently of C4 and that this regulation is modulated by PqsE [32,34–36]. With regard to the T3SS, it was reported that rhlI inactivation up-regulates the expression of some T3SS genes, including exoS and exoT expression and ExoS is secreted earlier compared to the wild-type strain [38,39]. Here we showed that, as previously reported, rhlI inactivation up-regulates the expression of exoS and exoT and also the exsCEBA operon, indicating that RhlI is a negative regulator of the T3SS. However, our results show that even though the expression of T3SS is increased in the rhlI mutant strain, secretion of ExoS is similar to that of the wild-type strain suggesting an additional level of regulation for its secretion. On the other hand, ExoS protein levels in the pqsE mutant strain were similar to those of the wild-type strain, and exsCEBA transcription was not affected by pqsE inactivation, suggesting that PqsE has no role in regulating the T3SS. In addition, ExoS secreted protein levels in the PAOΔrhlIΔpqsE double mutant strain were similar to those of the wild-type strain at 0.8 and 2.0 O.D.₆₀₀, indicating that RhlR is able to activate the T3SS in the absence of RhlI and PqsE. In this regard, it has been reported that RhlR is able to bind to the rhlAB promoter and repress its transcription in the absence of C4 [37], demonstrating that RhlR could act as a positive or negative regulator in the presence or the absence of C4 or PqsE. These results also explain why the virulence is reduced when rhlR is inactivated but maintained in a rhlI mutant strain [32,42].

Since the exsCEBA promoter region lacks a site for RhlR, which suggests that this regulation must be in an indirect way, we search for a las-rhl box in the previously reported T3SS regulators such as PsrA/RpoS, ArtR, or RetS (also known as RtsM) [5]. However, we could not identify a probable las-rhl box in the promoter regions of these regulators. In addition, it has been reported that rpoS expression is not regulated by RhlR [62]. Moreover, previous work showed that spermidine is a signal that modulates exsCEBA operon expression and, in this context, PA2592 was identified as a probable gene regulated by RhlR that codes for a putative spermidine-binding protein [22,23,55]. Here, we demonstrated that constitutive expression of PA2592 in the PAOΔrhlR mutant strain partially restored ExoS secretion only in the early stationary phase. This result indicates that PA2592 has a role in modulating intracellular spermidine concentrations and therefore in regulating the exsCEBA operon expression. However, since its expression was unable to fully restore ExoS secretion in the log phase or early stationary phase, it suggests that RhlR controls other elements involved in regulating exsA expression.

Finally, since RhlR can control the T3SS expression in the absence of C4 and PqsE, we determined whether C4 and PqsE were still required for pyocyanin production in T3SS-induction conditions as been reported in LB [40,56]. Our results showed that lasR inactivation slightly reduced pyocyanin production, but it was abolished in the rhlR, rhlI, and pqsE mutant strains indicating that regulation by the QS systems on pyocyanin synthesis is maintained in T3SS-induction conditions.

Overall, this work permits placing RhlR as a dual regulator, which negatively or positively controls T3SS expression depending upon binding to its C4 autoinducer. This can be related to turning on the T3SS that is necessary at the onset of an acute infection when C4 levels are low but turning it off when C4 levels increase, which also allows to activate the production of other virulence factors dependent on C4 and PqsE such as pyocyanin (Fig 9). These characteristics makes RhlR a very versatile protein that regulates different virulence traits promoting bacterial niche colonization.

Fig 9 — RhlR with C4 and PqsE positively controls pyocyanin production. In addition, RhlR is a positive regulator of the T3SS; however, this positive regulation is exerted in the absence of C4 and PqsE.

Supporting information

S1 Fig. T3SS regulation.

ExsA is the main activator of the T3SS genes. Its activity is controlled by a partner-switching mechanism. During non-induction conditions, ExsD binds to ExsA preventing T3SS activation. Inducing conditions lead to ExsE secretion allowing ExsC to bind ExsD and releasing ExsA, which in turn activates the T3SS expression. Furthermore, exsA expression is controlled by additional transcriptional regulators including PsrA, Vfr, MvaT, VqsM, and the post-transcriptional RsmA regulator.

(TIFF)

pone.0307174.s001.tiff^{(2.4MB, tiff)}

S2 Fig. DNA regions used to construct the transcriptional fusions with the lux reporter.

DNA regions include -35 and -10 sequences, ExsA binding sites (BS) and/or sites for additional transcriptional regulators previously reported. Nucleotides in base pair (bp) are indicated according to the transcriptional start site (+1).

(TIFF)

pone.0307174.s002.tiff^{(1.6MB, tiff)}

S3 Fig. Effect of rhlR expression in the PAOΔlasRΔrhlR double mutant strain on ExoS secretion by Western blot assay at log phase.

ExoS identification was performed by Western blog assay using anti-ExoS polyclonal antibody on supernatants of strains grown in induction conditions. GroEL, detected using polyclonal antibody anti-GroEL, was used as a loading control.

(TIFF)

pone.0307174.s003.tiff^{(1MB, tiff)}

S4 Fig. Activation of transcriptional fusions in induction conditions.

Transcriptional fusions activity in PAO1 strain were evaluated in non-induction (LB) and induction conditions (LB + 5 mM EGTA, 20 mM MgCl2). 200 μL of strains were incubated, in triplicate, at an initial O.D.₆₀₀ of 0.05 directly into the wells of a clear-bottomed polystyrene plate, which was incubated at 37°C without shaking until log phase. Negative control (pCTX), exsCEBA operon (PexsC::lux), exsA (PexsA::lux), exoS (PexoS::lux), and spsC (PspcS::lux). Relative luminescence units (R. L.U.) were quantified and normalized to the O.D.₆₀₀ at the time of data collection. Results represent the mean ± S.D. of three biological experiments performed in triplicate each time. Significant differences were obtained by two-way ANOVA and Šídák’s multiple comparisons analysis. Asterisks indicate statistical significance (n.s. = not significant, * p<0.05; ** p<0.01; *** p<0.001).

(TIFF)

pone.0307174.s004.tiff^{(559.5KB, tiff)}

S5 Fig. RhlR is unable to activate exsCEBA and exoS transcription in the absence of ExsA.

The transcriptional activity of exsCEBA operon (PexsC::lux) and exoS (PexoS::lux) was evaluated in the PAOΔT3SS strain and its derivates with pGMYC or pUCP20 plasmid and in the wild-type PAO1 strain. Strains were incubated in 15 ml of induction medium at 37°C and 225 rpm until reaching an O.D.₆₀₀ of 0.8 (a) and 2.0 (b). Relative luminescence units (R. L.U.) were quantified and normalized to the O.D.₆₀₀ at the time of cell collection. Results represent the mean ± S.D. of three biological experiments performed in three replicates each time. Significant differences were obtained by two-way ANOVA and Tukey’s multiple comparison analysis (α = 0.05%). Different letters indicate significant differences, while equal letters indicate no significant differences.

(TIFF)

pone.0307174.s005.tiff^{(221.8KB, tiff)}

S6 Fig. Effect of rhlI and pqsE inactivation on exsCEBA transcription (PexsC::lux).

Strains were incubated in 15 ml of induction medium at 37°C and 225 rpm. Relative luminescence units (R. L.U.) were quantified and normalized to the O.D.₆₀₀ at the time of cell collection. Results represent the mean ± S.D. of three biological experiments performed in three replicates each time. Significant differences were obtained by ordinary one-way ANOVA and Tukey’s multiple comparison analysis (α = 0.05%). Different letters indicate significant differences, while equal letters indicate no significant differences.

(TIFF)

pone.0307174.s006.tiff^{(54.5KB, tiff)}

S7 Fig. T3SS is activated only during induction conditions.

ExoS identification was performed by Western blot assay using anti-ExoS polyclonal antibody on supernatants of strains grown in non-induction (a) and induction conditions (b). PAO1 was used as a positive control whereas ATCC 9027, lacking the T3SS, was used as a negative control. GroEL was detected using polyclonal antibody anti-GroEL and used as a loading control.

(TIFF)

pone.0307174.s007.tiff^{(261KB, tiff)}

S8 Fig. Identification of mvaT point mutation G355A in PAOΔlasR^TcΔrhlR^Sm strain.

Comparison of the alignment of nucleotide (a) and amino acid (b) sequences of strains PAO1 vs PAOΔlasR^TcΔrhlR^Sm showing a point mutation G355A that generates a stop codon at position Trp119.

(TIFF)

pone.0307174.s008.tiff^{(300.5KB, tiff)}

S1 Table. Strains and plasmids used in this study.

(DOCX)

pone.0307174.s009.docx^{(30KB, docx)}

S2 Table. Oligonucleotides used in this study.

(DOCX)

pone.0307174.s010.docx^{(14.6KB, docx)}

S1 Dataset

(PDF)

pone.0307174.s011.pdf^{(180.6KB, pdf)}

S1 Raw images

(PDF)

pone.0307174.s012.pdf^{(2.7MB, pdf)}

Acknowledgments

LFM‐M is a doctoral student of Programa de Maestría y Doctorado en Ciencias Bioquímicas, Universidad Nacional Autónoma de Míxico (UNAM), and thanks CONAHCYT. VRF-V is a master student of Biomedicina Molecular, Centro de Investigación y de Estudios Avanzados del IPN (CINVESTAV-IPN), and thanks CONAHCYT. We thank Dr. Annia Rodríguez Hernández from the Cellular and Molecular Pharmacology Department of the University of California San Francisco (UCSF), for critical reading of the manuscript, and Abigail González Valdez and Norma Espinosa for technical support.

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

MC-Y research was supported by Consejo Nacional de Humanidades, Ciencias y Tecnologías (CONAHCYT) FORDECYT‐ PRONACES grant 53366 and Programa de Apoyo a Proyectos de Investigación e Innovación Tecnológica (PAPIIT) DGAPA, Universidad Nacional Autónoma de México (UNAM), grant number IA204221 and IA200823. GS-Ch and BG-P research were supported by DGAPA, PAPIIT UNAM grant IN201222 and IN229023, respectively. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS One. doi: 10.1371/journal.pone.0307174.r001

Decision Letter 0

Rajesh P Shastry

20 Feb 2024

PONE-D-24-00964The quorum sensing regulator RhlR positively controls the expression of the type III secretion system in Pseudomonas aeruginosa PAO1.PLOS ONE

Dear Dr. Cocotl-Yanez,

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

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Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The current study investigates the QS essential proteins namely Las, Rhl, and Pqs QS receptors, and inducers' role in the T3SS activation in P. aureginosa. I find these researches that correlate the QS activation to other bacterial activity is an essential area and unfortunately, there are no sufficient studies. However, I have a lot of concerns regarding this study as it requires further deep investigations, I still recommend its publication as it could shed the light in this direction.

1- What is the 1ry antibody used in the western blot

2- I think it would be nice if you can quantify the expresed proteins (use flourescent 2ry antibody and quantify the flourecence, for instance) to obtain statistical significance

3- Line 52, please re-write "Mutant strains defective in this system are severely attenuated in their virulence" with clarification

4- Please clarify the methodology section to be more clear to non-experts, as clarifying the importance of Groel protein as reference one

5- The discussion is well understood, however I recommend writing more detailed conclusion

6- I may recommend the authors to draw a represenative graph illustrating their findings

7- Furthermore, I may advise the authors to extend their findings showing the impact on the bacterial virulence in vitro by quantification of pyocyanin as an example to QS or immunstining to the exoS proteins in macrophages or HeLa cells, as an example

Reviewer #2: This paper examines the role of the global regulator RhlR in the regulation of Pseudomonas aeruginosa type 3 secretion system (T3SS). The transcriptional regulator RhlR was initially characterised at the end of the last century as the receptor for the quorum-sensing signal molecule: N-butanoyl-L-homoserine lactone (C4). However, it is now known that this protein has multiple mechanisms of action, both C4-dependent and C4-independent. The authors re-examine what the role of QS might be in the regulation of T3SS in light of today's knowledge.

Briefly, by means of transcriptional fusions and western-blot experiments, evidence is brought to support the hypothesis that RhlR positively regulates the expression of ExoS (the main effector of T3SS) in a C4-independent manner. Preliminary evidence is also brought to support the hypothesis that RhlR can regulate exoS transcription by positively controlling the promoter of the exsCEBA operon, which in turn encodes for the complex regulatory system that controls T3SS gene expression.

Overall, the authors show that: i) RhlR positively regulates the expression of exoS regardless of the presence of the C4 inducer; ii) this mechanism involves activation of the exsCEBA operon promoter; iii) constitutive transcription in trans of exsA, the main activator of T3SS, restores the expression of exoS in a rhlR mutant.

This is interesting work because it disproves the common belief that QS negatively regulates T3SS expression and draws attention to the multiple mechanisms by which RhlR can control P. aeruginosa virulence. Unfortunately, the work remains somewhat superficial and does not go deeper into the mechanism underlying the RhlR-dependent regulation of exoS and exCEBA genes.

Overall, the work is a bit preliminary and should be enriched with further experiments. Below there are some major issues that the authors need to address in order to get the work published:

1) The regulation of T3SS is very complex, the authors should explain it a little better in the introduction, possibly by including a figure in supplementary showing the exsCEBA operon and the exoS and spcS genes, also indicating the promoters cloned into transcriptional fusions with the lux genes. Concerning T3SS, it would be useful to cite Horna & Ruiz's review (doi:10.1016/j.micres.2021.126719).

2) In Figures 1, 2 and 3, it is not clear whether the experiments were conducted under inducing or not-inducing conditions. In any case, the authors should conduct the experiments shown in Figures 1, 2 and 3 under both conditions.

3) The names given to transcriptional fusions (pS, pP, pC, pA) are too contracted and do not help understanding. Authors should name the promoters as per convention (es. promoter of exoS gene = PexoS. Fusion between PexoS and luxABCDE = PexoS::luxABCDE, the latter could be abbreviated as PexoS::lux).

4) The PexsCEBA promoter is activated by RhlR in a C4- and PqsE-independent manner. Thus, this regulation appears to be indirect. The authors speculate that the regulation might depend on the spermidine binding protein PA2592 (lines 457-463). The authors should construct this mutant and test this hypothesis.

5) The PexsCEBA promoter is regulated by other regulators such as PrsA/RpoS, ArtR, RtsM (Horna & Ruiz of 2021 and references therein). Is there a relationship between these regulators and RhlR? The authors should answer this question referring to literature data or by conducting experiments.

**********

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Reviewer #1: Yes: Wael A. H. Hegazy

Reviewer #2: No

**********

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PLoS One. 2024 Aug 15;19(8):e0307174. doi: 10.1371/journal.pone.0307174.r002

Author response to Decision Letter 0

4 Apr 2024

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Yes

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No.

R: We carried out an additional statistical analysis to be more rigorous with our results, this analysis has been included in the revised version.

Reviewer #2: Yes

3. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

4. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: Yes

5. Review Comments to the Author

1- What is the 1ry antibody used in the western blot

R: The first antibody used was anti-ExoS, the information was included in Materials and methods and in the figure legends.

2- I think it would be nice if you can quantify the expresed proteins (use flourescent 2ry antibody and quantify the flourecence, for instance) to obtain statistical significance

R: Thank you for your suggestion. We carried out a densitometry analysis to obtain statistical significance using at least three Western blot assays and it was included in the Western blot figures. Also, the methodology was included (lines 264-268).

3- Line 52, please re-write "Mutant strains defective in this system are severely attenuated in their virulence" with clarification

R: Thanks for your suggestion. The sentence was modified for clarification (lines 51-52)

4- Please clarify the methodology section to be more clear to non-experts, as clarifying the importance of Groel protein as reference one

R: The methodology section was reviewed and modified to be clear.

5- The discussion is well understood, however I recommend writing more detailed conclusion.

R: We have written a more detailed conclusion according to your suggestion (lines 562-565)

6- I may recommend the authors to draw a represenative graph illustrating their findings

R: Thank you for your recommendation. A figure summarizing our findings has been included (Fig 9).

R: As you suggested, we quantified pyocyanin production in T3SS-induction conditions. This was included in the revised manuscript (lines 258-262 and 458-476).

Overall, the work is a bit preliminary and should be enriched with further experiments. Below there are some major issues that the authors need to address in order to get the work published:

R: Thank you for your suggestion. In this revised version we have included two additional supplementary figures related to the regulation of the T3SS and the transcriptional fusions constructed in this work (Fig S1 and S2). Also, the cite Horna and Ruiz was included.

R: All experiments were conducted under inducing conditions since in LB the T3SS is not active. We have included this information in the revised version and also, we have included a supplementary figure where we showed that ExoS is not detected in non-induction conditions (lines 281-282, 498-500) (Fig S6).

R: Names of plasmids were changed in the text according to your suggestion.

R: In this work, our main objective was to define the role of the quorum sensing systems on the T3SS expression. Therefore, we did not determine the target of RhlR to control the T3SS expression. However, according to your suggestion, in this revised version, we explored whether PA2595 has a role in the T3SS control. Our approach was to evaluate whether the expression of PA2592 under a constitutive promoter was able to restore ExoS secretion in the rhlR mutant strain. These results were included as a new section (lines 432-456). Also, the discussion was modified according to these results (lines 551-560).

R: Thank you for your comment. There is not a relationship previously reported between RhlR and PsrA, ArtR, or RetS (RtsM). Also, we searched for a las-box in these and other regulators of the T3SS reported by Horna & Ruiz but we could not find any consensus sequence for RhlR. We discussed these findings in the discussion section (lines 547-551).

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Wael A. H. Hegazy

Reviewer #2: No

Attachment

Submitted filename: Response to Reviewers.docx

pone.0307174.s013.docx^{(22.1KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0307174.r003

Decision Letter 1

Rajesh P Shastry

14 May 2024

PONE-D-24-00964R1The quorum sensing regulator RhlR positively controls the expression of the type III secretion system in Pseudomonas aeruginosa PAO1.PLOS ONE

Dear Dr. Cocotl-Yanez,

Please submit your revised manuscript by Jun 28 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Rajesh P. Shastry, Ph.D

Academic Editor

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #3: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #3: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #3: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #3: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #3: Yes

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6. Review Comments to the Author

Reviewer #1: (No Response)

Reviewer #3: In this MS Cocotl-Yanez et al. delve into the role of QS in regulating T3SS in Pseudomonas aeruginosa. Since QS has branching global effects on virulence in this pathogen, this is a very relevant topic and the results provided by this group shed some light on T3SS regulation and provide highly needed clarifications to some experimental discrepancies found regarding this topic. The authors make a sound analysis on the regulation of the T3SS and provide solid data that support most of their conclusions. Although I consider it a high quality work and I would support this MS for its acceptance, there are a few concerns that I think need to be addressed regarding some of their conclusions:

Major concerns:

The authors conclude that, as overexpressing exsA restores ExoS production in a rhlR mutant, RhlR controls the T3SS expression via ExsA. This is not necessarily the case, they can both act independently, and overexpressing one of the two independent activators may still correct the lack of the other. Their data proves that RhlR does not act downstream of ExsA, but there is still the question if RhlR acts upstream of ExsA as they claim or if they act in different signaling pathways. This would be clarified if they introduced their rhlR overexpression plasmid in a exsA mutant background. If overproducing RhlR can also restore ExoS production they might be independent activators. On the other hand, if ExoS production is not restored, it may indicate that both regulators act hierarchically as they describe.

I have trouble relating ExoS quantifications in figure 5b with their associated western blot data. What I see in the graphs is not an increase in ExoS protein levels in the rhlI mutant background as claimed in the main text (L399) and glimpsed in the western blot (in any case average levels in the graphs seem to be higher in the pqsE mutant although not significant). Also the statistical analysis does not seem to indicate that any of the mutants besides rhlR is significantly different from the wild type to make any of these statements. Please clarify these discrepancies.

Minor concerns:

Also regarding ExsA and RhlR relationship, it would be interesting to see if the internal PexsA promoter is also affected by the rhlR mutation.

L118: I fail to understand why are there discrepancies on T3SS regulation by the Las system. It is clear why Rhl role in T3SS needs further clarification, but all evidence discussed up to this point about Las system seems to agree that it is not involved. I would suggest changing this to “there are some discrepancies in the virulence and regulation of the T3SS by QS systems” so it is more accurate.

L302: The first section of the results jumps from figure 1 to figure 3. If the authors consider that discussing this mutant at this point of the MS is important I would suggest to rename it as figure 2. Otherwise they can try to introduce this result at a later point.

L395: As they also mentioned in the introduction, RhlR is known to be able to act independently of C4. I would rephrase this to “can be dependent” or other formulation that better reflects that this is not an absolute dependence.

L401-404: It is a bit hard to understand why this result is discussed here, and it also introduces a bit of difficulties in following the reasoning of the experimental procedure. I would move this phrase to a different point, maybe after discussing the results on Fig 6, so it is easier to follow and shows in a clear way why there are differences between exsC promoter activities in Fig 6 and S5.

L438, 442, 551, and 553: The authors probably mean a rhl box or a las-rhl box?

L461: The authors should clarify how their results show dependence on C4 and PqsE. By looking at figure 8 one can see PqsE dependence, but this is not discussed in the text, and C4 dependence is equally not properly described in this section despite being shown in the picture. Please reformulate this whole section so it is clear to the reader why your data supports your claims (which does).

Minor errata and things the authors may want to check before submitting their final version:

L73: control.

L127: show.

L170: remove the primer sequence since it is already in the supplementary table and no other primer sequences are shown.

L265: A citation may be missing here for “as reported previously”.

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #1: Yes: Wael A. H. Hegazy

Reviewer #3: Yes: Francisco Javier Marcos-Torres

**********

PLoS One. 2024 Aug 15;19(8):e0307174. doi: 10.1371/journal.pone.0307174.r004

Author response to Decision Letter 1

17 Jun 2024

June 17, 2024

Rajesh P. Shastry, Ph.D

Academic Editor

PLOS ONE

In this document, we respond to the comments and suggestions made by the reviewer for the manuscript PONE-D-24-00964R1 entitled ‘The quorum sensing regulator RhlR positively controls the expression of the type III secretion system in Pseudomonas aeruginosa PAO1’ by Luis Fernando Montelongo-Martínez, Miguel Díaz-Guerrero, Verónica Roxana Flores-Vega, Martín Paolo Soto-Aceves, Roberto Rosales-Reyes, Sara Elizabeth Quiroz-Morales, Bertha González-Pedrajo, Gloria Soberón-Chávez, Miguel Cocotl-Yañez.

Best regards

Miguel Cocotl-Yañez

Corresponding author.

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

Reviewer #3: (No Response)

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #3: Yes

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #3: Yes

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #3: Yes

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #3: Yes

6. Review Comments to the Author

Reviewer #1: (No Response)

Major concerns:

R: Thank you for your suggestion. We overexpressed rhlR in a strain defective in the T3SS and transcription of exsCEBA and exoS were measured, the results are described in lines 378-387.

R: Thank you for your observation. We have corrected it (lines 416-417) and also it was discussed (lines 556-561).

Minor concerns:

Also regarding ExsA and RhlR relationship, it would be interesting to see if the internal PexsA promoter is also affected by the rhlR mutation.

R: Thanks for your comment. We found that exsA internal promoter is barely expressed and is not activated by induction conditions, thus we focused on determining the role of the QS systems on the T3SS genes that were activated in induction conditions.

R: It was changed (Line 118)

R: Figure 3 was renamed as Figure 2.

R: It was changed (Line 414)

R: The section was modified according to your suggestion.

L438, 442, 551, and 553: The authors probably mean a rhl box or a las-rhl box?

R: Thank you. It was changed to las-rhl box.

R: Thank you for your comment. The section was modified and also the results were discussed (Lines 586-591).

Minor errata and things the authors may want to check before submitting their final version:

L73: control. R: Corrected

L127: show. R: Corrected

L170: remove the primer sequence since it is already in the supplementary table and no other primer sequences are shown. R: Corrected

L265: A citation may be missing here for “as reported previously”. R: Corrected

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #1: Yes: Wael A. H. Hegazy

Reviewer #3: Yes: Francisco Javier Marcos-Torres

Attachment

Submitted filename: Response to Reviewers.docx

pone.0307174.s014.docx^{(21KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0307174.r005

Decision Letter 2

Rajesh P Shastry

2 Jul 2024

The quorum sensing regulator RhlR positively controls the expression of the type III secretion system in Pseudomonas aeruginosa PAO1.

PONE-D-24-00964R2

Dear Dr. Cocotl-Yanez,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Rajesh P. Shastry, Ph.D

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Reviewer #1: From the first revision; all the raised points have been adressed and it can be published in the current form

Reviewer #3: With this new version of the MS, Cocotl-Yanez et al have made a remarkable work addressing all previous concerns and strengthening their conclusions. Considering the quality of the resulting work I fully recommend for its publication without further modifications.

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Wael A. H. Hegazy

Reviewer #3: Yes: Francisco Javier Marcos-Torres

**********

PLoS One. doi: 10.1371/journal.pone.0307174.r006

Acceptance letter

Rajesh P Shastry

6 Aug 2024

PONE-D-24-00964R2

PLOS ONE

Dear Dr. Cocotl-Yanez,

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team.

At this stage, our production department will prepare your paper for publication. This includes ensuring the following:

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If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps.

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on behalf of

Dr. Rajesh P. Shastry

Academic Editor

PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. T3SS regulation.

(TIFF)

pone.0307174.s001.tiff^{(2.4MB, tiff)}

S2 Fig. DNA regions used to construct the transcriptional fusions with the lux reporter.

(TIFF)

pone.0307174.s002.tiff^{(1.6MB, tiff)}

S3 Fig. Effect of rhlR expression in the PAOΔlasRΔrhlR double mutant strain on ExoS secretion by Western blot assay at log phase.

(TIFF)

pone.0307174.s003.tiff^{(1MB, tiff)}

S4 Fig. Activation of transcriptional fusions in induction conditions.

(TIFF)

pone.0307174.s004.tiff^{(559.5KB, tiff)}

S5 Fig. RhlR is unable to activate exsCEBA and exoS transcription in the absence of ExsA.

(TIFF)

pone.0307174.s005.tiff^{(221.8KB, tiff)}

S6 Fig. Effect of rhlI and pqsE inactivation on exsCEBA transcription (PexsC::lux).

(TIFF)

pone.0307174.s006.tiff^{(54.5KB, tiff)}

S7 Fig. T3SS is activated only during induction conditions.

(TIFF)

pone.0307174.s007.tiff^{(261KB, tiff)}

S8 Fig. Identification of mvaT point mutation G355A in PAOΔlasR^TcΔrhlR^Sm strain.

Comparison of the alignment of nucleotide (a) and amino acid (b) sequences of strains PAO1 vs PAOΔlasR^TcΔrhlR^Sm showing a point mutation G355A that generates a stop codon at position Trp119.

(TIFF)

pone.0307174.s008.tiff^{(300.5KB, tiff)}

S1 Table. Strains and plasmids used in this study.

(DOCX)

pone.0307174.s009.docx^{(30KB, docx)}

S2 Table. Oligonucleotides used in this study.

(DOCX)

pone.0307174.s010.docx^{(14.6KB, docx)}

S1 Dataset

(PDF)

pone.0307174.s011.pdf^{(180.6KB, pdf)}

S1 Raw images

(PDF)

pone.0307174.s012.pdf^{(2.7MB, pdf)}

Attachment

Submitted filename: Response to Reviewers.docx

pone.0307174.s013.docx^{(22.1KB, docx)}

Attachment

Submitted filename: Response to Reviewers.docx

pone.0307174.s014.docx^{(21KB, docx)}

Data Availability Statement

All relevant data are within the manuscript and its Supporting Information files.

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PERMALINK

The quorum sensing regulator RhlR positively controls the expression of the type III secretion system in Pseudomonas aeruginosa PAO1

Luis Fernando Montelongo-Martínez

Miguel Díaz-Guerrero

Verónica Roxana Flores-Vega

Martín Paolo Soto-Aceves

Roberto Rosales-Reyes

Sara Elizabeth Quiroz-Morales

Bertha González-Pedrajo

Gloria Soberón-Chávez

Miguel Cocotl-Yañez

Roles

Abstract

Introduction

Materials and methods

Bacterial strains and growth conditions

DNA manipulation techniques

Construction of transcriptional fusions

Construction of pExsA and pUC2592 plasmids

Generation of mutant strains

Luminescence assays

Western blot assays

Cytotoxicity assays

Pyocyanin quantification

Densitometry analysis

Statistical analysis

Results

RhlR, but not LasR, decreases ExoS secretion

Fig 1. Effect of lasR and rhlR inactivation on ExoS secretion.

Fig 2. Effect of exsA expression in the rhlR mutant strain on ExoS secretion.

RhlR positively regulates the expression of exoS, spcS and the exsCEBA operon

Fig 3. Effect of rhlR inactivation on transcription of T3SS genes and exsA expression in the rhlR mutant strain.

exsA expression restores ExoS secretion and the T3SS genes expression in the rhlR mutant strain

Cytotoxicity is affected when rhlR is inactivated

Fig 4. Effect of rhlR inactivation on cytotoxicity.

RhlR regulates T3SS expression in the absence of C4 or PqsE

Fig 5. Effect of rhlI and pqsE inactivation on ExoS secretion.

Fig 6. Effect of rhlI inactivation on transcription of T3SS genes during early stationary phase.

PA2592 expression partially restores ExoS secretion in the rhlR mutant strain

Fig 7. Effect of PA2592 expression in the rhlR mutant strain.

Pyocyanin production is dependent on C4 and PqsE in T3SS-induction conditions

Fig 8. Pyocyanin production by PAO1 and its derivatives mutant strains in T3SS-induction conditions.

Discussion

Fig 9. Regulatory model by RhlR on T3SS-induction conditions.

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Rajesh P Shastry

Roles

Author response to Decision Letter 0

Decision Letter 1

Rajesh P Shastry

Roles

Author response to Decision Letter 1

Decision Letter 2

Rajesh P Shastry

Roles

Acceptance letter

Rajesh P Shastry

Roles

Associated Data

Supplementary Materials

Data Availability Statement

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