Pseudomonas aeruginosa (P. aeruginosa) is a common opportunistic pathogen that is widely distributed in nature, including on human skin and in the human intestinal tract. It can cause skin infections (of intact or burned skin), respiratory tract infections, urinary tract infections, and more, as well as bacteremia, endocarditis and cystic fibrosis in immunocompromised persons.P. aeruginosa typically forms biofilms, which are very common in chronic lung infection.
The strong environmental adaptability and complex mechanisms of virulence regulation in P. aeruginosa are important reasons for its high levels of drug resistance and the high rate of mortality associated with infection with this pathogen. Many P. aeruginosa components, including fimbriae, flagella and lipopolysaccharides, promote its virulence, as well as secreted virulence factors/proteins, such as pyocyanin, pyoverdine, protease, elastase, hydrogen cyanide, and so on.
P. aeruginosa can sense the change of cell density according to the concentration of specific signal molecules, when the concentration of these signal molecules reaches a certain threshold, the expression of a specific set of genes is triggered by activating a cognate receptor, this regulatory system is called the bacterial quorum sensing (QS). In P. aeruginosa, there are currently four known interconnected QS systems: las, rhl, pqs and iqs systems. QS systems are activated by the N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12-HSL), N-butyryl-L-homoserine lactone (C4-HSL), 2-heptyl-3-hydroxy-4-quinoqone (Pseudomonas quinolone signal, PQS) and 2-(2-hydroxyphenyl)-thiazole-4-carbaldehyde (IQS) signaling molecules, respectively.
The QS are arranged in a hierarchical network that plays a key role in regulating P. aeruginosa virulence gene expression and biofilm formation. The las system is the primary system in this hierarchical QS system and dominates the other systems, including the rhl, pqs and iqs systems. The rhl system represents a second positive feedback loop, and is regulated by both the las and pqs systems. The pqs system is the third QS system and is regulated by PQS, while the fourth QS system, iqs, can partly assume the functions of the las system under conditions of phosphate-depletion stress. The QS not only plays an important role in regulating the expression of various P. aeruginosa virulence factors, but also affects host immune function.
Dötsch et al used RNA-seq to identify a series of genes that are highly expressed in P. aeruginosa biofilms. We analyzed the expression of the eight genes that exhibited the most significant differences in expression between biofilm and planktonic cells using Quantitative Real-time PCR (qPCR), and specifically focused on PA2146, which exhibited the greatest difference in expression (Additional Fig. 1, http://links.lww.com/JR9/A25). In our previous research, we found that compared to the wildtype, PAO1ΔPA2146 significantly increased pyocyanin production, but inhibited interleukin-6 (IL-6) secretion in neutrophils and cytokine production in macrophages. In a mouse acute pneumonia infection model, PAO1ΔPA2146 inhibited cytokine secretion in the lungs but increased the infiltration of inflammatory cells. These results indicated PA2146 is associated with host immune response. In this study, we further examined the role of PA2146 in P. aeruginosa biofilm formation and virulence factor expression.
Materials and methods
Bacterial strains, plasmids, and culture conditions
P. aeruginosa PAO1 (ATCC 15692) provided by Mingqiang Qiao (Nankai University, Tianjin, China) was cultured in Luria-Bertani (LB) medium at 37°C with shaking at 200r/min. The pRK415-PA2146 plasmid was used for gene complementation, and complemented strains ΔPA2146/pRK415-PA2146 were selected for growth on medium containing 10 μg/mL tetracycline.
PA2146 knockout and complementation
We previously constructed a PAO1 PA2146 knockout strain, ΔPA2146, briefly, the upstream and downstream fragments of PA2146 were amplified and the products were then fused by overlapping PCR, and the fused fragment was cloned into pLP12 (KnoGen Biotech, Guangzhou, China) using recombinant enzyme Exnase II (ClonExpress II, Vazyme) to generate recombinant plasmid pLPPA2146. Next, pLPPA2146 was transferred into Escherichia coli β2163, co-cultured with PAO1 on LB agar containing 0.3 mM daptomycin with 0.3% D-glucose, and the insertion mutation was selected on LB agar containing 36 μg/mL tetracycline with 0.3% D-glucose. The ΔPA2146 strain was selected on LB agar with 0.4% L-arabinose. In order to construct a complementary strain, the PA2146 promoter and coding regions were integrated into the attb site of the ΔPA2146 mutant genome using pRK415. The DNA regions of the target genes were PCR-amplified using the primer pair PA2146-up and PA2146-down (Additional Table 1, http://links.lww.com/JR9/A26). The resulting complemented mutant strain was verified by PCR analysis and named ΔPA2146/pRK415-PA2146.
Bacterial growth curves
Strains were inoculated into LB medium and grown overnight at 37°C with shaking at 180r/min. The overnight culture was then inoculated into fresh medium at optical density (OD)630nm = 0.05, and the fresh culture was incubated at 37°C with shaking at 180r/min for 24 hours. the OD630nm was determined at set time intervals.
Assessment of biofilm biomass
An overnight PAO1 culture was diluted 100-fold, and 200 μL of the diluted suspension was added into the wells of a 96-well plate. The plate was incubated at 37°C for 24 hours, after which the medium was discarded and the plate was washed twice with saline to remove planktonic cells. Next, 200 μL of a 0.25% crystal violet (Solarbio, Beijing, China) solution was added to the wells, and the plate was incubated for 15 min at room temperature to stain the biofilm. Then, the 96-well plate was washed twice with normal saline to remove the excess dye. The plate was then dried at 50°C for 30 minutes, and the bound dye in each well was dissolved with 95% ethanol for 20 minutes. The OD of the samples at 570 nm (OD570nm) was measured. All of these experiments were conducted in triplicate. For biofilm morphology, 40 μL of overnight culture was added to a 6-well cell culture plate (Corning Costar, Cambridge, MA, USA) containing 1960 μL of LB broth and an 18mm×18 mm sterile glass cover slide. After 24 hours of incubation at 37°C without shaking, the glass slide was washed once gently with saline to remove planktonic cells. Finally, biofilms on slides were stained with 0.25% crystal violet, and visualized and photographed using a light microscope (Olympus CX31, Olympus, Tokyo, Japan).
An overnight PAO1 culture was diluted 100-fold with LB medium, incubated at 37°C for 24 hours with shaking, and centrifuged. The supernatant was collected and filtered through 0.22-μm sterile filter (Millipore, Bedford, MA, USA) to obtain cell-free P. aeruginosa culture supernatant, which was then stored at -20°C or used immediately.
Three milliliters of chloroform was added to 5 mL of PAO1 cell-free supernatant and mixed well. Next, the clear liquid at the bottom was collected and mixed with 1 mL of 0.2 M HCl to acidify the solution. Then the solution was centrifuged, and the OD of the pink upper aqueous phase was measured at 520 nm using a Microplate Reader (PerkinElmer, Walsham, MA, USA).
Total protease assay
A total of 0.5 mL of PAO1 cell-free supernatant was mixed with 0.5 mL of a 1.25% skimmed milk solution. The mixture was incubated at 37°C for 30 minutes, and the OD at 600 nm (OD600nm) was measured using a Microplate Reader (PerkinElmer).
LasA elastase assay
The PAO1 cell-free supernatant was combined with 1 mL of a 0.05 M Tris-HCl solution (pH 7.5) and 0.3% azocasein and incubated at 4°C for 4 hours, after which 10% trichloroacetic acid (TCA) was added to the solution to stop the reaction. The mixture was centrifuged at 4000×g for 5 minutes, the clear supernatant was collected and mixed with 1 M NaOH, and the OD at 440 nm (OD440nm) was measured using a Microplate Reader (PerkinElmer).
LasB elastase assay
A total of 500 μL of PAO1 cell-free supernatant was combined with 500 μL of 100 mM Tris-HCl buffer containing 10 mg of Elastin–Congo red (ECR) and incubated at 37°C with shaking for 6 hours. The mixture was then centrifuged to remove insoluble ECR, and the OD at 495 nm (OD495nm) was measured using a Microplate Reader (PerkinElmer).
PAO1 cell-free supernatant was combined with an equal volume of ethyl acetate and vortexed vigorously to obtain an organic phase containing rhamnolipids. The rhamnolipids were extracted and dissolved by adding a mixture of chloroform and freshly prepared methylene blue solution (pH 8.6 ± 0.2) and vortexing. The chloroform phase was then mixed with 0.2 HCl and vortexed. The upper acid phase, which contained the rhamnolipids complexed with methylene blue, was evaluated by measuring the OD at 638 nm (OD638nm) using a Microplate Reader (PerkinElmer).
Hemolytic activity assay
Blood samples of 2 to 3 healthy individuals undergoing physical examination were collected from the Clinical Laboratory of the Third Xiangya Hospital and washed three times with PBS (pH 7.4). The red blood cells (RBCs) were then mixed and diluted to 4% with PBS. Next, 200 μL of the RBC suspension was mixed with 200 μL of PAO1 cell-free supernatant and incubated at 37°C for 1 hour. TritonX-100 (1%) was used as a positive control, and PBS was used as a negative control. After incubation for 1 hour, the solution was centrifuged at 500 × g for 5 minutes, 200 μL of the supernatant was added to a 96-well plate, and the OD at 450 nm (OD450nm) was measured using a Microplate Reader (PerkinElmer). This study was approved by the Institutional Review Board of the Third Xiangya Hospital, Central South University (approval No. 2019-S021) and conducted in accordance with the Declaration of Helsinki. Blood was isolated from clinical samples routinely collected from patients, and the identification of patients was not needed. Therefore, the need for written informed consent was waived and oral informed consent was obtained.
RNA extraction and qPCR
An overnight PAO1 culture was diluted with LB to OD630nm = 0.1 and then incubated at 37°C for another 24 hours. The suspension was then centrifuged at 4000 × g for 5 minutes to collect the bacteria. The E.Z.N.A. Total RNA Kit II (Omega Bio-tek, Norcross, GA, USA) was used to extract the RNA, and cDNA was prepared using TransScript All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (Transgene, Beijing, China). qPCR was performed using TransStartTM Green qPCR SuperMix UDG (Transgene). The reaction conditions were as follows: 94°C for 30 seconds, followed by 40 cycles of 94°C for 5 seconds, 58°C for 15 seconds, and 72°C for 10 seconds. The 2−ΔΔCt method was used to calculate relative gene expression. The primer information is shown in (Additional Table 1, http://links.lww.com/JR9/A26).
Motility was assayed using a variety of media types and inoculation quantities. The swimming medium was composed of 0.3% agar (Hope Bio-Technology, Qingdao, China), 1% Tryptone (Solarbio) and 0.5% NaCl (Xilong Scientific, Guangzhou, China). Swimming plates were inoculated with bacteria grown overnight on an LB agar plate at 37°C using a sterile toothpick and incubated at 30°C for 14 to 16 hours. The swarming medium consisted of 0.5% agar, 0.8% nutrient broth (Hope Bio-Technology) and 0.5% glucose (Sigma-Aldrich, St Louis, MO, USA). P. aeruginosa cultures were diluted to an OD600nm of 0.1, and 2 μL of the diluted bacterial solution was spotted to the center of the swarming agar plates, which were then incubated at 37°C for 14 to 16 hours. The twitching medium was composed of LB broth (Solarbio) with 1% agar. Twitching plates were stab-inoculated from an overnight culture grown on an LB agar plate using a sharp toothpick that was inserted until it touched the bottom of the petri dish, and were then incubated at 37°C for 24 hours.
RNA was isolated from planktonic cultures of the PAO1 and ΔPA2146 strains as described above (three biological replicates were performed, for a total of six samples). The purified RNA was then sent to Applied Protein Technology (APTBIO) for library preparation, sequencing, and data analysis. Libraries were prepared using the first-strand protocol, and paired-end sequencing of rRNA-depleted libraries was performed on an Illumina HiSeq instrument. The reads were trimmed using fastp software (HaploX, Shenzhen, China) and then aligned with the P. aeruginosa PAO1 genome using HISAT2 software. The featureCounts software was used to analyze gene expression levels for each sample by counting the number of genes with different expression levels and quantifying the expression level of individual genes. DESeq2 was used for differential gene expression analysis.
Label-free relative quantitative proteomics and KEGG pathway analysis
As mentioned above, proteins were extracted from planktonic cultures of strains PAO1 and ΔPA2146 (three biological replicates, for six samples in total). The proteins were reduced and alkylated, and digested by trypsin. After high pH reversed-phase peptide fractionation, the data were collected by liquid chromatography-tandem mass spectrometry (LC-MS/MS) (AB Sciex, Framingham, MA, USA). After protein identification and quantification, bioinformatics analyses were carried out. Proteins were defined as differentially expressed if they exhibited a fold change greater than 1.5-fold, and the P value was less than 0.05. Blast2GO (https://www.blast2go.com/) software was used to annotate the GO function of the differentially expressed proteins. Differentially expressed proteins were subsequently annotated in the KEGG pathway database (http://www.genome.jp/kegg/pathway.html). The analysis was performed using KAAS software (KEGG Automatic Annotation Server).
The quantitative data are presented as means±standard deviation. Student's t-test was used for inter-group comparisons. Statistical analyses were performed using SPSS 21.0 (IBM, Armonk, NY, USA). P values <0.05 were considered significant.
The effect of gene knockout on bacterial growth
The cell concentration was determined by detecting the OD630nm of the PAO1 and ΔPA2146 strains in LB broth. The growth curve showed that there was no significant difference in growth rate among the PAO1, ΔPA2146 and complemented strains (Fig. 1).
The ΔPA2146 strain exhibited reduced biofilm formation compared with PAO1
Crystal violet staining showed that the ΔPA2146 strain formed thinner biofilms than PAO1 and ΔPA2146/pRK415-PA2146 (Fig. 2A). Semi-quantitative analysis based on dissolving the crystal violet in absolute ethanol showed that ΔPA2146 formed less biofilm mass than PAO1 and ΔPA2146/pRK415-PA2146 (Fig. 2B). Microscopic observation of biofilms grown on glass slides and stained with crystal violet showed that ΔPA2146 formed obviously thinner biofilms than the wild-type strain (Fig. 2C).
ΔPA2146 exhibited increased virulence compared with PAO1
Compared with PAO1, ΔPA2146 supernatant showed greater degradation of the proteins in skim milk, resulting in clearer milk (Fig. 3A). The OD630nm data also showed that the skim milk mixed with ΔPA2146 supernatant was significantly less turbid than that mixed with supernatant from the other two strains (P < 0.01; Fig. 3B), indicating that PA2146 knockout significantly increased the production of total protease. Further testing showed that the LasA and LasB elastases were expressed at significantly higher levels in ΔPA2146 than in PAO1 or ΔPA2146/pRK415-PA2146 (P < 0.05; Fig. 3C, D), indicating that PA2146 inhibits P. aeruginosa LasA and LasB elastase production.
After 24 hours of incubation, the color of the culture medium changed obviously to green (Fig. 4A). Further testing showed that the pyocyanin concentration was significantly higher in ΔPA2146 than in PAO1 or PA2146/pRK415-PA2146 (P < 0.01; Fig. 4B). ΔPA2146 also exhibited a significantly higher concentration of rhamnolipids compared with the other two strains (P < 0.01; Fig. 4C). Furthermore, ΔPA2146 produced significantly higher levels of hemolysin than PAO1 or PA2146/pRK415-PA2146, the supernatants of which showed only slight hemolytic activity (P < 0.01; Fig. 4D) at 24 hours. These results indicate that PA2146 inhibits the production of P. aeruginosa virulence factors.
ΔPA2146 exhibited increased motility compared with PAO1
Motility is very important for P. aeruginosa invasion and diffusion, which are closely related to its virulence and pathogenicity. The mode of motility varies in different media, such as swimming in a fluid film; swarming on a semi-solid viscous surface; and twitching on a solid surface. Swimming and swarming are mainly dependent on flagella, and twitching is mediated by flagella and type IV fimbriae. Compared with the wild-type strain PAO1, the ΔPA2146 strain demonstrated significantly enhanced swimming, swarming and twitching motility, and the complemented strain showed phenotypes similar to those observed in the wild-type strain (P < 0.05; Fig. 5). These results suggest that PA2146 negatively regulates P. aeruginosa motility.
PA2146 deletion increased the expression of QS-related genes and reduced MexEF-OprN efflux pump proteins
The RNA-seq results showed that, compared with the wild-type strain, the expression of 125 genes in the ΔPA2146 strain was increased (log2FoldChange >1), while the expression of 73 genes was decreased (log2FoldChange >1). The genes whose expression increased the most significantly were pqsB, pqsA, pqsC, pqsE and pqsD, all of which are components of the PQS operon (Table 1). This strongly indicates that PA2146 inhibits the expression of the PQS operon. The expression of rhlA and rhlB, which are also important components of the QS, was increased in ΔPA2146 compared with the wild-type strain. PhnA and phzB1 are important genes in the phenazine synthesis pathway, and are part of the QS. PhnA is an essential gene for PQS synthesis.HcnB and HcnC are also QS-related genes.MexE, MexF and OprN comprise the MexEF-OprN efflux pump operon, and their expression was reduced in the ΔPA2146 strain compared with the wild-type strain. LecB lectin is a carbohydrate-binding protein produced by P. aeruginosa that is also called PA-IIL, and binds to many cell receptors in different hosts. There is evidence that LecB lectin is an important virulence factor, as a P. aeruginosa lacking LecB was less pathogenic than the wild-type strain. KEGG analysis showed that the genes that exhibited differential expression between ΔPA2146 and PAO1 are primarily involved in the biofilm formation, quorum sensing and phenazine biosynthesis pathways. These results suggest that PA2146 may mainly affect biofilm formation, QS, and phenazine biosynthesis in P. aeruginosa (Fig. 6). This is consistent with the experimental results showing that biofilm formation, virulence factor expression and pyocyanin expression were altered by PA2146 knockout.
Table 1 -
Differentially expressed genes between PAO1 and ΔPA2146
||Hypothetical protein PA0997
||Hypothetical protein PA0998
||2-Heptyl-4 (1H)-quinolone synthase PqsD
||Fucose-binding lectin PA-IIL
||Rhamnosyltransferase subunit A
||Hypothetical protein PA3332
||Cbb3-type cytochrome C oxidase subunit I
||Anthranilate synthase component I
||Hypothetical protein PA1664
||Hypothetical protein PA4129
||Hypothetical protein PA4128
||Hypothetical protein PA3329
||Hypothetical protein PA4141
||Hypothetical protein PA1665
||Hypothetical protein PA1666
||Acyl carrier protein
||Rhamnosyltransferase subunit B
||Hypothetical protein PA1659
||Hypothetical protein PA1660
||Hypothetical protein PA4134
||Nitric oxide reductase subunit C
||Hypothetical protein PA2461
||Phenazine biosynthesis protein
||Non-ribosomal peptide synthetase
||Hydrogen cyanide synthase subunit HcnC
||Hypothetical protein PA0526
||Hypothetical protein PA3291
||Hypothetical protein PA1657
||Hydrogen cyanide synthase subunit HcnB
||Hypothetical protein PA4881
||Hypothetical protein PA3229
||Hypothetical protein PA1942
||Resistance-nodulation-cell division (RND) multidrug efflux membrane fusion protein MexE
||Multidrug efflux outer membrane protein OprN
||Resistance-nodulation-cell division (RND) multidrug efflux transporter MexF
||Hypothetical protein PA1970
||Hypothetical protein PA4623
||Hypothetical protein PA3230
||Hypothetical protein PA1744
||Hypothetical protein PA2490
||Bistable expression regulator BexR
||Hypothetical protein PA2697
||MFS dicarboxylate transporter
||Hypothetical protein PA1743
||Hypothetical protein PA2365
The effect of PA2146 on global protein expression was evaluated by label-free relative quantitative proteomics. The main changes in ΔPA2146 protein expression levels compared with the wild-type strain were the decreased expression of AAG05883 (multidrug efflux outer membrane protein OprN), AAG05881 (RND multidrug efflux membrane fusion protein MexE precursor) and AAG05882 (RND multidrug efflux transporter MexF) (Table 2). KEGG pathway analysis showed that the proteins that were differentially expressed in ΔPA2146 vs PAO1 were mainly located in important pathways related to bacterial secretion system, biofilm formation, the two-component system (Fig. 7).
Table 2 -
Differentially expressed proteins between PAO1 and ΔPA2146
||Hypothetical protein PA0165
||Probable ClpA/B-type protease
||Hypothetical protein PA1567
||Conserved hypothetical protein
||Basic amino acid, basic peptide and imipenem outer membrane porin OprD precursor
||Histidine porin OpdC
||Conserved hypothetical protein
||Hypothetical protein PA0065
||Hypothetical protein PA0126
||Hypothetical protein PA1673
||Multidrug efflux outer membrane protein OprN precursor
||Resistance-Nodulation-Cell Division (RND) multidrug efflux membrane fusion protein MexE precursor
||Resistance-Nodulation-Cell Division (RND) multidrug efflux transporter MexF
||Transcriptional regulator MexT
||Probable glutathione S-transferase
||Conserved hypothetical protein
||Probable short-chain dehydrogenase
||Probable ATP-binding component of ABC transporter
||Lipid A 3-O-deacylase
||Probable acyl carrier protein
||Conserved hypothetical protein
||Peptidyl-prolyl cis-trans isomerase C2
||Two-component sensor PhoR
||Hypothetical protein PA1090
||Hypothetical protein PA0495
Verification of differentially expressed genes
ΔPA2146 exhibited increased expression of bacterial pyocyanin, rhamnolipid and LasA and LasB elastase, as well as enhanced virulence, compared with the wild-type strain. These virulence factors are controlled by the QS, and RNA-seq analysis suggested that the expression of related genes in the QS system also increased (Table 1). qPCR analysis showed that the expression of important genes in the QS system and genes related to PQS synthesis was increased in ΔPA2146 compared with the wild-type strain (Fig. 8), which is consistent with the RNA-seq results.
PA2146 is a gene of unknown function located on the sense strand of the P. aeruginosa genome (2361706–2361873). The gene encodes a 55-amino acid protein. PA2146 expression increased when PAO1 formed a biofilm. The ΔPA2146 strain could still form biofilms, but the biomass of biofilms was significantly reduced, suggesting that PA2146 promotes biofilm formation in this study. This observation also shows that biofilm formation is very complicated and not controlled by a single gene. Indeed, biofilm formation is known to be regulated by diguanosine-5’-monophosphate, small RNAs and QS.
Bacterial pathogenicity is closely related to virulence. To further evaluate the effect of PA2146 knockout on PAO1 virulence, virulence factor/protein secretion and bacterial motility were tested. The ΔPA2146 strain exhibited significantly increased total protease expression compared with PAO1, as well as a significant increase in the expression of LasA and LasB in this study. Elastase is a metalloenzyme that exhibits hydrolytic activity toward connective tissue. Both LasA and LasB are elastases, which can degrade elastin and cause lung parenchymal damage and bleeding. In addition, LasA and LasB are also related to P. aeruginosa transmission and infection, and elastase secretion is mainly positively regulated by the las system.PA2146 knockout also lead to high production of rhamnolipids, which may cause host macrophage and lymphocyte necrosis and is related to host immunity.
In this study, ΔPA2146 produced significantly more pyocyanin than the wild-type strain. In vitro experiments have shown that pyocyanin has many potential effects on various organ systems, including respiratory system, cardiovascular system, urinary system and central nervous system. Phenazine production in P. aeruginosa is a complex process, operon phz1 and phz2 drive the production of phenazine-1-carboxylic acid (PCA), which is further converted to pyocyanin by two modifying enzymes PhzM and PhzS. It has been reported that pyocyanin production is related to QS, and that LasR, RhlR, RsaL, PqsE and PqsR and the signaling molecules, 2-heptyl-4(1H)-quinolone (HHQ) and PQS all help regulate the production of pyocyanin.
The QS is a density-dependent bacterial gene regulation system. QS allows bacteria to sense, communicate and coordinate with each other through secreted chemical signaling molecules. QS is also closely related to bacterial virulence and environmental adaptability. When 3-oxo-C12-HSLs, C4-HSLs and PQS signaling molecules bind to their corresponding transcriptional regulators, LasR, RhlR and PqsR, they can form a complex that can regulate the expression of P. aeruginosa virulence factor/protein synthesis-related genes, thereby regulating P. aeruginosa virulence. QS can influence the transcription of hundreds of downstream genes, these genes are controlled by different QS systems and are induced or inhibited by multiple QS regulators. The changes in the QS may be the main reason for the increase in virulence factor expression observed in the PA2146 knockout strain.
RNA-seq analysis showed that knocking out PA2146 resulted in an increase in the synthesis of genes related to PQS, and these results were confirmed by qPCR in the present study. The expression of the MexEF-OprN efflux pump operon was also decreased in the PA2146 knockout strain at the gene and protein levels in the present study. One of the reasons for the increased expression of virulence factors in ΔPA2146 may be that PA2146 is a negative regulator of the pqs system, meaning that when PA2146 is knocked out, the pqs system is derepressed, leading to increased expression of pqsABCDE and synthesis of PQS signaling molecules. Pyocyanin synthesis is positively regulated by the rhl and pqs systems.[33,34] The pqs system can increase the expression of lasB elastase and pyocyanin, so when PA2146 is knocked out, the expression of pyocyanin and LasB will increase. PQS also activates rhl system, while elastase and rhamnolipid production are mainly regulated by the las and rhl systems. Therefore, rhamnolipid and elastase expression would be expected to increase when PA2146 is knocked out. Mutant strains that produce high levels of rhamnolipids exhibit enhanced detachment of individuals cells from the biofilm, so overproduction of rhamnolipids can promote the collapse of the biofilm. This may explain why ΔPA2146 exhibited increased virulence but decreased biofilm formation.
RNA-seq analysis and proteomics sequencing showed that the expression of mexEF-oprN efflux pump components was decreased in the ΔPA2146 strain. Transcription of the MexEF-OprN efflux pump operon is positively regulated by the transcriptional activator MexT, and the pump can remove chloramphenicol and quinolone from the intracellular environment. Studies have shown that overexpression of the MexEF-OprN multi-drug efflux system affects P. aeruginosa intercellular signal transduction and reduces the levels of extracellular virulence factors (pyocyanin, elastase and rhamnolipids). Overexpression of MexEF-OprN leads to a decrease in the expression of several QS regulatory genes. A study performed in Caenorhabditis elegans showed that overproduction of MexEF-OprN can decrease P. aeruginosa virulence and inhibit the type VI secretion system. These phenotypes are caused by a delay in PQS production due to extrusion of kynurenine (HHQ), a PQS precursor, through the efflux pump. MexEF-OprN activation reduces virulence factor expression and swarming behavior. Therefore, another reason for the increased virulence of ΔPA2146 may be the decrease in expression of the mexEF-oprN efflux pump, leading to reduced efflux of HHQ, and therefore a relative increase in PQS synthesis, which in turn increased the virulence through upregulation of the pqs system.
However, the limitation of this study is that it is not clear which pathway plays a dominant role, the evidences of this study are not straightforward enough, and the specific mechanism by which PA2146 regulates QS and its association with other regulatory factors and pathways are still unclear and require further study.
In summary, in this study we found that knocking out PA2146 in P. aeruginosa PAO1 increased virulence, increased the expression of pqs QS system-related genes and decreased the expression of mexEF-oprN efflux pump–related genes. These results suggest that PA2146 inhibits the QS by directly promoting synthesis of PQS signaling molecules or reducing efflux of the PQS precursor HHQ by reducing the expression of mexEF-oprN efflux pump components.
FT, YW and PS designed the experiments. FT performed most of the experiments, analyzed the results, and wrote the manuscript. SL, LZ, XZ, LX, YL, ZH performed the supporting experiments. YW conceived and supervised the study. All the authors read and approved the final manuscript.
This study was supported by the National Natural Science Foundation of China (grant No. 82072350 to YW).
Institutional review board statement
This study was approved by the Institutional Review Board of the Third Xiangya Hospital, Central South University, China (approval No. 2019-S021) and conducted in accordance with the Declaration of Helsinki. Blood samples were isolated from clinical samples routinely collected from patients, and the identification of patients was not needed. Therefore, the need for written informed consent was waived and oral informed consent was obtained.
Conflicts of interest
The authors declare no conflict of interest.
. Azam MW, Khan AU. Updates on the pathogenicity status of Pseudomonas aeruginosa. Drug Discov Today
. Winstanley C, O’Brien S, Brockhurst MA. Pseudomonas aeruginosa evolutionary adaptation and diversification in cystic fibrosis chronic lung infections. Trends Microbiol
. Tognon M, Köhler T, Gdaniec BG, et al. Co-evolution with Staphylococcus aureus leads to lipopolysaccharide alterations in Pseudomonas aeruginosa. ISME J
. Moradali MF, Ghods S, Rehm BH. Pseudomonas aeruginosa lifestyle: a paradigm for adaptation, survival, and persistence. Front Cell Infect Microbiol
. Schuster M, Sexton DJ, Hense BA. Why quorum sensing
controls private goods. Front Microbiol
. Lee J, Zhang L. The hierarchy quorum sensing
network in Pseudomonas aeruginosa. Protein Cell
. Lee J, Wu J, Deng Y, et al. A cell-cell communication signal integrates quorum sensing
and stress response. Nat Chem Biol
. Turkina MV, Vikström E. Bacteria-host crosstalk: sensing of the quorum in the context of Pseudomonas aeruginosa infections. J Innate Immun
. Dötsch A, Eckweiler D, Schniederjans M, et al. The Pseudomonas aeruginosa transcriptome in planktonic cultures and static biofilms using RNA sequencing. PLoS One
. She P, Liu Y, Luo Z, et al. PA2146 gene knockout is associated with Pseudomonas aeruginosa pathogenicity in macrophage and host immune response. Front Cell Infect Microbiol
. Keen NT, Tamaki S, Kobayashi D, et al. Improved broad-host-range plasmids for DNA cloning in gram-negative bacteria. Gene
. Stepanovic S, Vukovic D, Dakic I, et al. A modified microtiter-plate test for quantification of staphylococcal biofilm formation. J Microbiol Methods
. Banerjee M, Moulick S, Bhattacharya KK, et al. Attenuation of Pseudomonas aeruginosa quorum sensing
, virulence and biofilm formation by extracts of Andrographis paniculata. Microb Pathog
. Pattnaik S, Ahmed T, Ranganathan SK, et al. Aspergillus ochraceopetaliformis SSP13 modulates quorum sensing
regulated virulence and biofilm formation in Pseudomonas aeruginosa PAO1. Biofouling
. Jack AA, Khan S, Powell LC, et al. Alginate Oligosaccharide-Induced Modification of the lasI-lasR and rhlI-rhlR Quorum-Sensing Systems in Pseudomonas aeruginosa. Antimicrob Agents Chemother
. Fan F, She P, Zhou L, et al. Bactericidal and anti-biofilm activity of the retinoid compound CD437 against Enterococcus faecalis. Front Microbiol
. Zhang Y, Qin J, Tan B, et al. The P-type ATPase PA1429 regulates quorum-sensing systems and bacterial virulence. Front Microbiol
. Kim D, Langmead B, Salzberg SL. HISAT: a fast spliced aligner with low memory requirements. Nat Methods
. Liao Y, Smyth GK, Shi W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics
. Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol
. Götz S, García-Gómez JM, Terol J, et al. High-throughput functional annotation and data mining with the Blast2GO suite. Nucleic Acids Res
. Yang A, Tang WS, Si T, et al. Influence of physical effects on the swarming motility of Pseudomonas aeruginosa. Biophys J
. Henrichsen J. Bacterial surface translocation: a survey and a classification. Bacteriol Rev
. Yang N, Cao Q, Hu S, et al. Alteration of protein homeostasis mediates the interaction of Pseudomonas aeruginosa with Staphylococcus aureus. Mol Microbiol
. Wang W, Huang X, Yang H, et al. Antibacterial activity and anti-quorum sensing
mediated phenotype in response to essential oil from Melaleuca bracteata leaves. Int J Mol Sci
. Thuenauer R, Landi A, Trefzer A, et al. The Pseudomonas aeruginosa lectin LecB causes integrin internalization and inhibits epithelial wound healing. mBio
. Taylor AE, Gibson WH, Granger HJ, et al. The interaction between intracapillary and tissue forces in the overall regulation of interstitial fluid volume. Lymphology
. Konstantinović J, Yahiaoui S, Alhayek A, et al. N-Aryl-3-mercaptosuccinimides as antivirulence agents targeting Pseudomonas aeruginosa Elastase and Clostridium Collagenases. J Med Chem
. Castillo-Juárez I, Maeda T, Mandujano-Tinoco EA, et al. Role of quorum sensing
in bacterial infections. World J Clin Cases
. Hall S, McDermott C, Anoopkumar-Dukie S, et al. Cellular effects of pyocyanin, a secreted virulence factor of Pseudomonas aeruginosa. Toxins (Basel)
. Higgins S, Heeb S, Rampioni G, et al. Differential regulation of the phenazine biosynthetic operons by quorum sensing
in Pseudomonas aeruginosa PAO1-N. Front Cell Infect Microbiol
. Francis VI, Stevenson EC, Porter SL. Two-component systems required for virulence in Pseudomonas aeruginosa. FEMS Microbiol Lett
. Lundgren BR, Thornton W, Dornan MH, et al. Gene PA2449 is essential for glycine metabolism and pyocyanin biosynthesis in Pseudomonas aeruginosa PAO1. J Bacteriol
. Welsh MA, Eibergen NR, Moore JD, et al. Small molecule disruption of quorum sensing
cross-regulation in pseudomonas aeruginosa causes major and unexpected alterations to virulence phenotypes. J Am Chem Soc
. Li WR, Ma YK, Shi QS, et al. Diallyl disulfide from garlic oil inhibits Pseudomonas aeruginosa virulence factors by inactivating key quorum sensing
genes. Appl Microbiol Biotechnol
. D’Angelo F, Baldelli V, Halliday N, et al. Identification of FDA-approved drugs as antivirulence agents targeting the pqs quorum-sensing system of Pseudomonas aeruginosa. Antimicrob Agents Chemother
. Wang M, Zhao L, Wu H, et al. Cladodionen is a potential quorum sensing
inhibitor against Pseudomonas aeruginosa. Mar Drugs
. Díaz De Rienzo MA, Stevenson PS, Marchant R, et al. Pseudomonas aeruginosa biofilm disruption using microbial surfactants. J Appl Microbiol
. Köhler T, Epp SF, Curty LK, et al. Characterization of MexT, the regulator of the MexE-MexF-OprN multidrug efflux system of Pseudomonas aeruginosa. J Bacteriol
. Köhler T, Michéa-Hamzehpour M, Henze U, et al. Characterization of MexE-MexF-OprN, a positively regulated multidrug efflux system of Pseudomonas aeruginosa. Mol Microbiol
. Köhler T, van Delden C, Curty LK, et al. Overexpression of the MexEF-OprN multidrug efflux system affects cell-to-cell signaling in Pseudomonas aeruginosa. J Bacteriol
. Olivares J, Alvarez-Ortega C, Linares JF, et al. Overproduction of the multidrug efflux pump MexEF-OprN does not impair Pseudomonas aeruginosa fitness in competition tests, but produces specific changes in bacterial regulatory networks. Environ Microbiol
. Lamarche MG, Déziel E. MexEF-OprN efflux pump exports the Pseudomonas quinolone signal (PQS) precursor HHQ (4-hydroxy-2-heptylquinoline). PLoS One