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PA1426 regulates Pseudomonas aeruginosa quorum sensing and virulence: an in vitro study

Tan, Fang; She, Pengfei; Zhou, Linying; Li, Shijia; Zeng, Xianghai; Xu, Lanlan; Liu, Yaqian; Hussain, Zubair; Wu, Yong

Author Information
doi: 10.1097/JBR.0000000000000088

Abstract

Introduction

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.[1]P. aeruginosa typically forms biofilms, which are very common in chronic lung infection.[2]

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,[3] promote its virulence, as well as secreted virulence factors/proteins, such as pyocyanin, pyoverdine, protease, elastase, hydrogen cyanide, and so on.[4]

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).[5] 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.[1]

The QS are arranged in a hierarchical network that plays a key role in regulating P. aeruginosa virulence gene expression and biofilm formation.[6] The las system is the primary system in this hierarchical QS system and dominates the other systems, including the rhl, pqs and iqs systems.[6] The rhl system represents a second positive feedback loop, and is regulated by both the las and pqs systems.[4] 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.[7] The QS not only plays an important role in regulating the expression of various P. aeruginosa virulence factors, but also affects host immune function.[8]

Dötsch et al[9] 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.[10] 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[11] 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,[10] 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.[11] 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.[12] 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).

Supernatant preparation

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.

Pyocyanin assay

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).[13]

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).[13]

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).[14]

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).[15]

Rhamnolipid assay

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).[14]

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).[16] 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 assay

Motility was assayed using a variety of media types and inoculation quantities.[17] 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-seq

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.[18] The featureCounts software[19] 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.[20]

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/)[21] 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).

Statistical analysis

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.

Results

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).

Figure 1
Figure 1:
Growth curves for the wild-type strain (PAO1), knockout strain (ΔPA2146) and complemented strain (ΔPA2146/pRK415-PA2146). Strains was diluted with LB to OD630nm = 0.05, and incubated at 37°C with shaking at 180r/min for 24 hours. The OD630nm was determined at set time intervals. OD = optical density.

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).

Figure 2
Figure 2:
PA2146 knockout reduced biofilm formation. (A) Pseudomonas aeruginosa (P. aeruginosa) was grown in a 6-well plate at 37°C for 24 hours, after which the biofilms were stained with crystal violet. (B) P. aeruginosa was grown in 96-well plates at 37°C for 24 hours. The biofilms were stained with crystal violet and quantified using the OD measured at 570 nm (OD570nm). Data are presented as mean±standard deviation. The results are representative of three independent experiments. ∗∗∗ P < 0.001, vs wildtype or complemented strain (Student's t test). (C) P. aeruginosa was grown on a sterile glass cover slide in a 6-well plate at 37°C for 24 hours, and biofilms on slides were stained with 0.25% crystal violet. Images were taken at a magnification of 100×. OD = optical density.

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.

Figure 3
Figure 3:
PA2146 knockout increased protease and elastase production. (A) The total protease activity was assessed by incubating cell-free supernatants with skim milk. (B) The turbidity of the solutions was measured at OD630nm. (C) LasA elastase activity, 1 mL of 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 OD440nm was measured. (D) LasB elastase activity, 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 OD495nm was measured. P < 0.05, ∗∗ P < 0.01, vs wildtype or complemented strain (Student's t-test). OD = optical density.

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.

Figure 4
Figure 4:
ΔPA2146 exhibited increased virulence compared with the wild-type strain. (A) Representative images of pyocyanin production in supernatant of Pseudomonas aeruginosa (P. aeruginosa) PAO1 and its derivatives. The presence of the blue-green pigment indicates pyocyanin production. (B) Pyocyanin content, 3 mL of chloroform was added to 5 mL of P. aeruginosa cell-free supernatant and mixed, the clear liquid at the bottom was collected and mixed with 1 mL of 0.2 M HCl to acidify the solution, centrifuged, and the OD of the pink upper aqueous phase was measured at 520 nm. (C) Rhamnolipid content, P. aeruginosa cell-free supernatant was combined with ethyl acetate and vortexed 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). The chloroform phase was then mixed with 0.2 HCl. The upper acid phase contained the rhamnolipids complexed with methylene blue, was evaluated by measuring the OD at 638 nm. (D) Hemolytic activity, 200 μL of the 4% red blood cell suspension was mixed with 200 μL of PAO1 cell-free supernatant. 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, and the OD at 450 nm (OD450nm) was measured. ∗∗ P < 0.01, ∗∗∗∗ P < 0.0001, vs wildtype or complemented strain (Student's t-test). OD = optical density.

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.[22] Swimming and swarming are mainly dependent on flagella, and twitching is mediated by flagella and type IV fimbriae.[23] 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.

Figure 5
Figure 5:
PA2146 affects Pseudomonas aeruginosa (P. aeruginosa) motility. (A) Swimming: The swimming medium was composed of 0.3% agar, 1% Tryptone and 0.5% NaCl. Swimming plates were inoculated with P. aeruginosa grown overnight on an LB agar plate at 37°C using a sterile toothpick and incubated at 30°C for 14 to 16 hours. (B) Swarming: The swarming medium consisted of 0.5% agar, 0.8% nutrient broth and 0.5% glucose. 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. (C) Twitching: The twitching medium was composed of LB broth 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. Data are the averages of three independent experiments. P < 0.05, vs wildtype or complemented strain (Student's t-test). OD = optical density.

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.[24]HcnB and HcnC are also QS-related genes.[25]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.[26] 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.[26] 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
Gene_ID Symbol Description log2FoldChange P value
Increased expression
PA0997 pqsB Hypothetical protein PA0997 6.2418 0
PA0996 pqsA Anthranilate--CoA ligase 6.0623 5.14E-308
PA0998 pqsC Hypothetical protein PA0998 4.8493 4.67E-198
PA1000 pqsE Thioesterase PqsE 4.4865 1.90E-195
PA0999 pqsD 2-Heptyl-4 (1H)-quinolone synthase PqsD 4.4104 2.85E-234
PA3361 lecB Fucose-binding lectin PA-IIL 4.3756 7.23E-17
PA3479 rhlA Rhamnosyltransferase subunit A 3.8016 9.93E-17
PA3332 Hypothetical protein PA3332 3.6273 4.70E-47
PA4133 Cbb3-type cytochrome C oxidase subunit I 3.622 2.21E-70
PA1001 phnA Anthranilate synthase component I 3.5961 2.50E-133
PA1664 Hypothetical protein PA1664 3.3663 1.48E-41
PA3330 Short-chain dehydrogenase 3.3556 2.93E-67
PA4129 Hypothetical protein PA4129 3.3456 8.27E-44
PA4128 Hypothetical protein PA4128 3.3243 4.28E-56
PA3328 FAD-dependent monooxygenase 3.3159 1.71E-58
PA3333 fabH2 3-Oxoacyl-ACP synthase 3.3114 6.92E-61
PA4130 Sulfite/nitrite reductase 3.2996 3.31E-42
PA3329 Hypothetical protein PA3329 3.2421 1.86E-55
PA4141 Hypothetical protein PA4141 3.1664 4.46E-68
PA3331 Cytochrome P450 3.1095 8.14E-61
PA1665 Hypothetical protein PA1665 2.9407 2.18E-68
PA1666 Hypothetical protein PA1666 2.901 2.48E-76
PA3334 Acyl carrier protein 2.7617 6.84E-39
PA3478 rhlB Rhamnosyltransferase subunit B 2.7364 3.59E-35
PA1659 Hypothetical protein PA1659 2.6256 1.73E-47
PA1660 Hypothetical protein PA1660 2.5578 6.90E-66
PA1663 Transcriptional regulator 2.5032 2.83E-68
PA4131 Iron-sulfur protein 2.5031 8.07E-22
PA4134 Hypothetical protein PA4134 2.4551 3.79E-38
PA0523 norC Nitric oxide reductase subunit C 2.2761 2.01E-21
PA1662 ClpA/B-type protease 2.2458 1.20E-37
PA2461 Hypothetical protein PA2461 2.1919 1.16E-43
PA4211 phzB1 Phenazine biosynthesis protein 2.1662 6.50E-18
PA3327 Non-ribosomal peptide synthetase 2.1364 2.97E-50
PA2195 hcnC Hydrogen cyanide synthase subunit HcnC 2.131 4.08E-36
PA0526 Hypothetical protein PA0526 2.1042 1.51E-17
PA3291 Hypothetical protein PA3291 2.0665 4.37E-08
PA1657 Hypothetical protein PA1657 2.0436 1.11E-40
PA2194 hcnB Hydrogen cyanide synthase subunit HcnB 2.0101 2.35E-20
Decreased expression
PA4881 Hypothetical protein PA4881 −6.2323 4.00E-135
PA3229 Hypothetical protein PA3229 −5.9723 0
PA1942 Hypothetical protein PA1942 −5.1781 1.17E-249
PA2493 mexE Resistance-nodulation-cell division (RND) multidrug efflux membrane fusion protein MexE −5.1525 7.73E-140
PA2495 oprN Multidrug efflux outer membrane protein OprN −4.935 1.75E-270
PA2494 mexF Resistance-nodulation-cell division (RND) multidrug efflux transporter MexF −4.7991 8.71E-233
PA1970 Hypothetical protein PA1970 −4.7375 7.86E-102
PA4623 Hypothetical protein PA4623 −4.2391 5.24E-115
PA2491 Oxidoreductase −3.7758 1.10E-264
PA3230 Hypothetical protein PA3230 −3.7118 9.29E-148
PA2758 Transcriptional regulator −2.9759 7.08E-117
PA1744 Hypothetical protein PA1744 −2.7826 6.49E-26
PA4356 xenB Xenobiotic reductase −2.6635 1.05E-98
PA2490 Hypothetical protein PA2490 −2.4798 6.07E-41
PA2432 bexR Bistable expression regulator BexR −2.4047 0.0036463
PA2698 Hydrolase −2.3602 0.0016415
PA2697 Hypothetical protein PA2697 −2.1830 0.00026316
PA5530 MFS dicarboxylate transporter −2.0073 2.33E-34
PA1743 Hypothetical protein PA1743 −2.0043 5.71E-17
PA2365 Hypothetical protein PA2365 −2.0024 3.10E-22

Figure 6
Figure 6:
KEGG analysis of genes that were differentially expressed between PAO1 and ΔPA2146. The degree of KEGG enrichment is measured by Rich factor, FDR and the number of genes enriched in this pathway. The Rich factor refers to the ratio of the number of differentially expressed genes at the beginning of the pathway to the total number of genes located at the beginning of the pathway among all of the annotated genes. FDR is the P value after multiple hypothesis testing and correction, and its value range is [0,1]: the closer to zero, the more significant the enrichment. The vertical axis shows the name of the pathway, and the horizontal axis shows the Rich factor corresponding to the pathway. FDR magnitude is represented by the color of the dots: the smaller the FDR, the more red the dot. The number of differential genes contained in each pathway is represented by the size of the dots. FDR = False Discovery Rate, KEGG = Kyoto Encyclopedia of Genes and Genomes, OD = optical density.

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
Protein ID Description ΔPA2146/PAO1 P value
Increased expression
AAG03555 Hypothetical protein PA0165 10.911359 0.001308681
AAG05051 Probable ClpA/B-type protease 6.52423 0.021392242
AAG05046 Hypothetical protein PA1567 5.449608 0.020324073
AAG05047 Conserved hypothetical protein 5.17326 0.016558395
AAG04347 Basic amino acid, basic peptide and imipenem outer membrane porin OprD precursor 5.134186 0.000213079
AAG03552 Histidine porin OpdC 3.036086 0.014357004
AAG03666 Conserved hypothetical protein 2.796365 0.003932005
AAG03455 Hypothetical protein PA0065 2.433829 0.044330121
AAG03516 Hypothetical protein PA0126 2.221117 0.001531744
AAG05062 Hypothetical protein PA1673 2.221089 0.003199647
Decreased expression
AAG05883 Multidrug efflux outer membrane protein OprN precursor 0.005072 0.006577391
AAG05881 Resistance-Nodulation-Cell Division (RND) multidrug efflux membrane fusion protein MexE precursor 0.008848 0.012132249
AAG05882 Resistance-Nodulation-Cell Division (RND) multidrug efflux transporter MexF 0.011571 0.013083203
AAG05880 Transcriptional regulator MexT 0.071387 0.001651352
AAG05879 Probable oxidoreductase 0.080687 0.004098222
AAG07744 Xenobiotic reductase 0.096788 0.011674137
AAG06201 Probable glutathione S-transferase 0.146393 3.69655E-05
AAG07742 Conserved hypothetical protein 0.229912 0.000496482
AAG07549 Probable short-chain dehydrogenase 0.253845 0.001640279
AAG06200 Probable ATP-binding component of ABC transporter 0.308949 0.000306991
AAG08048 Lipid A 3-O-deacylase 0.31148 0.010179438
AAG05258 Probable acyl carrier protein 0.351472 0.044247468
AAG06990 Conserved hypothetical protein 0.389761 0.022215822
AAG07563 Peptidyl-prolyl cis-trans isomerase C2 0.399258 0.027142187
AAG08746 Two-component sensor PhoR 0.419506 0.011096041
AAG04479 Hypothetical protein PA1090 0.484327 0.013227326
AAG03884 Hypothetical protein PA0495 0.486182 0.002171862

Figure 7
Figure 7:
Top 20 KEGG pathways with the most differentially expressed proteins in ΔPA2146 vs PAO1. The x-axis shows the pathways that the differentially expressed proteins are involved in, and the y-axis shows the number of differentially expressed proteins involved in that pathway. The more differentially expressed proteins corresponding to the expected pathway, the more important the pathway. KEGG = Kyoto Encyclopedia of Genes and Genomes.

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,[8] 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.

Figure 8
Figure 8:
Increased expression of genes related to QS in ΔPA2146 compared with PAO1. PAO1 and ΔPA2146 were cultured for 24 hours, and the transcription of QS-related genes and PQS synthesis genes was measured by qPCR. PQS = Pseudomonas Quinolone Signal, qPCR = Quantitative Real-time PCR, QS = quorum sensing.

Discussion

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.[27]

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.[28] 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.[29]PA2146 knockout also lead to high production of rhamnolipids, which may cause host macrophage and lymphocyte necrosis and is related to host immunity.[6]

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.[30] 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.[31] 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.[31]

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.[32] 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.[31] 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[35] and pyocyanin,[36] so when PA2146 is knocked out, the expression of pyocyanin and LasB will increase. PQS also activates rhl system,[31] while elastase and rhamnolipid production are mainly regulated by the las and rhl systems.[37] 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,[38] 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,[39] and the pump can remove chloramphenicol and quinolone from the intracellular environment.[40] 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).[41] 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.[42] MexEF-OprN activation reduces virulence factor expression and swarming behavior.[43] 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.

Acknowledgments

None.

Author contributions

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.

Financial support

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.

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Keywords:

bacterial virulence; PA2146; Pseudomonas aeruginosa; QS; quorum sensing

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