2.2. PA14_27940 Deletion Alters the Motility of P. aeruginosa

P. aeruginosa’s motility plays a key role in the bacterial colonization of surfaces and biofilm formation, thus its reduction is usually associated with a reduced virulence phenotype [30]. The former work showed that a transposon insertion mutant in PA14_27940 presented a lower motility than the parental strain [26]. Consequently, we analyzed the motility of the ΔPA14_27940 mutant. As shown in Figure 2, the mutant showed an impaired swarming phenotype: the mutant’s motility diameter was 43.3 mm, while PA14′s was 66.7 mm. Swimming motility was affected in the mutant as well, both in the motility pattern—regular circle for the PA14 and splash for the ΔPA14_27940 mutant—and the motility diameters—33 and 15 mm for the wild-type strain and the mutant, respectively (Figure 2). In light of these results, we can assert that the deletion of PA14_27940 entails a reduced motility in P. aeruginosa PA14, which indicates that the regulator encoded by this gene is implicated, directly or indirectly, in this virulence trait, reinforcing its candidacy as a potential target of antivirulence co-adjuvants in antipseudomonal therapies.

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Figure 2

Motility phenotype of P. aeruginosa ΔPA14_27940 mutant. The image illustrates the swarming (A) and swimming (B) phenotype of P. aeruginosa PA14 strain and ΔPA14_27940 mutant. The plates are shown in the order: PA14 and ΔPA14_27940. The diameters of the strains are reported in the figure at the bottom of the images. The swimming shapes are identified following the classification included in [31]. Both swimming and swarming were statistically significantly lower in ΔPA14_27940 mutant than in PA14 (p < 0.05).

2.3. Effect of PA14_27940 in the Production of P. aeruginosa Virulence Determinants

Pyocyanin production, elastase activity and biofilm formation are important virulence factors for the infective process of P. aeruginosa [32,33,34,35,36], whose expression was impaired in the transposon insertion mutant of the gene PA14_27940 [26]. Hence, we evaluated these phenotypes in the ΔPA14_27940 mutant, with regard to the PA14 strain. As it is shown in Figure 3, although some slight decrease was observed in the ΔPA14_27940 mutant for all virulence features, these differences were not statistically significant. Ergo, our results support that the clean deletion of PA14_27940 does not affect either pyocyanin, elastase or biofilm production. Hence, this regulator is not likely to be involved in these phenotypes, in spite of our data obtained by analyzing the insertion mutant of this gene [26].

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Figure 3

Quantification of virulence factors in P. aeruginosa ΔPA14_27940 mutant. The figure exposes elastase activity data (A), biofilm formation assay data (B) and pyocyanin production data (C) of P. aeruginosa PA14 strain and ΔPA14_27940 mutant. Error bars indicate standard deviations of the results from eight independent experiments in the biofilm formation assay and from three experiments in the other analysis. Differences between bars are not statistically significant (p > 0.05; by t-test, in all cases).

The analysis of the phenotype of insertion mutants is a valuable tool for global genetic analyses, and it has historically provided relevant results in the study of the bacterial intrinsic resistome [37]. However, our results support that, on occasion, the observed phenotype is not a direct consequence of the gene in question; in contrast, other features—such as polar effects—might compromise the phenotype. Therefore, our data strongly suggest that an ulterior analysis of phenotypes of interest in insertion mutants should require the construction of clean in-frame deletion mutants of the gene in question, before jumping to conclusions regarding said phenotypes.

2.4. The Loss of PA14_27940 Impedes the Induction of armZ, Hence Reducing the Induction of mexXY by Tobramycin

As stated above, the ΔPA14_27940 mutant presents increased susceptibility to aminoglycosides, erythromycin, tigecycline and tetracycline, among other drugs, while susceptibility to beta-lactams and quinolones was not altered (Table 1). This phenotype is similar to that observed in a MexXY deficient mutant [10]. Since mexXY expression is induced by its antibiotic substrates, and this induction is required for the intrinsic resistance of P. aeruginosa to aminoglycosides, we measured the level of expression of mexX in the presence of subinhibitory concentrations of tobramycin in the ΔPA14_27940 mutant and in the PA14 parental strain. As shown in Figure 4, although mexX expression was induced in both strains, the level of induction was significantly lower (p < 0.05) in the ΔPA14_27940 mutant than in the parental PA14 strain. Albeit this lower induction might contribute to the increased susceptibility to aminoglycosides of ΔPA14_27940, the fact that mexX is still inducible suggests that other elements, besides mexX induction, might contribute to the hypersusceptibility phenotype.

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Figure 4

Induction of mexX by subinhibitory concentrations of tobramycin in P. aeruginosa ΔPA14_27940 mutant. The level of mexX expression in PA14 and ΔPA14_27940 was measured in presence of subinhibitory concentrations of tobramycin (1/8 of its respective MICs). Error bars indicate standard deviations of the results from three independent experiments. The induction level of the ΔPA14_27940 mutant was statistically significantly lower than the one of PA14 (p < 0.05).

As stated above, the anti-repressor ArmZ is a critical element in mexXY induction of the expression by the substrates of the efflux pump and hence in P. aeruginosa intrinsic resistance to aminoglycosides [10,13]. Consequently, we measured its expression in the presence of subinhibitory concentrations of tobramycin in the wild-type PA14 strain and in the ΔPA14_27940 mutant.

As shown in Figure 5, subinhibitory concentrations of tobramycin induced armZ in the wild-type strain, while the expression of this gene was not induced in the ΔPA14_27940 mutant, with the differences being statistically significant. Under these conditions, while MexZ—the repressor of the expression of mexXY—is sequestered by ArmZ in the wild-type PA14 strain [38], thus allowing the induction of mexXY expression in the presence of tobramycin, and the lack of armZ induction in the ΔPA14_27940 mutant keeps the level of MexZ, and as a consequence, the induction of the expression of mexXY is hampered. Ergo, these results could explain the hypersusceptibility phenotype of the mutant.

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Figure 5

Induction of armZ by subinhibitory concentrations of tobramycin in P. aeruginosa ΔPA14_27940 mutant. The level of armZ expression in PA14 and ΔPA14_27940 was measured in presence of subinhibitory concentrations of tobramycin (1/8 of their respective MICs). Error bars indicate standard deviations of the results from three independent experiments. The induction level of the ΔPA14_27940 mutant was statistically significantly lower than the one of PA14 (p < 0.05).

3.2. Bacterial Growth Conditions

Unless otherwise stated, all strains were grown in rich Luria Bertani Broth (LBB) Lennox (Conda, Torrejón de Ardoz, Spain) at 37 °C and 250 rpm or in Luria Bertani Agar (LBA) (LBB with 1.5% agar (Conda, Torrejón de Ardoz, Spain)) at 37 °C. Different antibiotics were added to the growth media when required: 100 μg/mL ampicillin for the selection of E. coli DH5α and E. coli S17-1 clones containing pGEM^®-T Easy or RGD001, respectively, and 350 μg/mL carbenicillin for the selection of P. aeruginosa clones containing the deletion of PA14_27940 [42].

3.3. Construction of ΔPA14_27940 P. aeruginosa Mutant

The deletion of the gene PA14_27940 in P. aeruginosa PA14 strain was performed by homologous recombination as herein described. An insert that comprised 500 bp upstream and 500 bp downstream of the gene, flanked by HindIII restriction sites, was obtained by PCR using the primers PA14_27940.ups_fw, PA14_27940.ups_rv, PA14_27940.dns_fw and PA14_27940.dns_rv (Table 3). Then, the PCR product was introduced in a HindIII-digested and dephosphorylated pEX18Ap vector [42] by using T4 DNA ligase (New England Biolabs, NEB, Ipswich, MA, USA) and incubating O/N at 16 °C. The resultant plasmid, RGD001, was introduced by transformation in E. coli S17-1, a donor strain that was submitted to conjugation and mutant selection with P. aeruginosa PA14 strain, as described elsewhere [42], using 350 μg/mL of carbenicillin and 10% of sucrose to select the deletion-containing bacteria. The deletion of the gene was verified by PCR with primers PA14_27940.comp_fw-PA14_27940.comp_rv and PA14_27940.ups_fw-PA14_27940.dns_rv (Table 3).

3.4. Whole-Genome Sequencing and Bioinformatics Analysis

Genomic DNA extractions and DNA quality check were performed by the Translational Genomics Unit, Instituto Ramón y Cajal de Investigación Sanitaria (IRYCIS), Hospital Ramón y Cajal (HRYC), Madrid, Spain. Genomic DNA of each population was extracted by Chemagic™ DNA Bacterial Kit H96 (CMG-799 Chemagic™), using the equipment Chemagic™ 360/MSMI (PerkinElmer, Waltham, MA, USA), and DNA quality check was performed by Agilent 2200 TapeStation System bioanalyzer (Santa Clara, CA, USA). The whole-genome sequencing (WGS) was performed by the Oxford Genomics Centre. Constructed libraries were paired-end (2 × 350) and sequenced with NovaSeq6000 Illumina system (San Diego, CA, USA). The average number of reads per sample represented a coverage of 300×. WGS data were analyzed using the CLC Genomics Workbench 21.0.5 software (QIAGEN, Hilden, Germany). Genomic information was trimmed, and the reads were aligned against the GenBank P. aeruginosa UCBPP-PA14 reference chromosome (NC_008463.1). ΔPA14_27940 mutant’s genome was also filtered against the sequence of the PA14 strain used in our laboratory. The genome is accessible with the BioProject database ID code PRJNA1012553.

3.5. Analysis of Susceptibility to Antibiotics

The susceptibility of the different strains to tobramycin, streptomycin, amikacin, kanamycin, tigecycline, tetracycline, ciprofloxacin, ceftazidime, imipenem, aztreonam, fosfomycin, erythromycin and chloramphenicol was determined using antibiotic gradient strips (MIC Test Strip, Liofilchem^®, Roseto degli Abruzzi, Italy) in Mueller Hinton Agar (MHA) (Sigma, St. Louis, MO, USA), whereas susceptibility to colistin and polymyxin B was determined in MHA II. Plates were incubated 24 h at 37 °C to read the MIC values.

3.6. Motility Assays

Motility assays were carried out in fresh agar plates containing 25 mL of medium. Swarming assays were performed in a medium that contained 0.5% Casamino Acids, 0.5% Bacto agar, 0.5% filtered glucose, 3.3 mM K₂HPO₄ and 3 mM MgSO₄, as formerly depicted [43]. Swimming assays were made in 20 g/l LBB with 0.3% agar [44]; in a medium containing 1% tryptone, 0.5% yeast extract and 0.5% NaCl. A 4 µL inoculum (OD600 = 1) of the bacterial strains was placed on the center of the agar surface. Three replicates of each strain were incubated for 24 h at 37 °C. After the incubation, the motility zone of the swarming and swimming assays was measured.

3.7. Elastase Activity and Pyocyanin Production

To measure elastase activity and pyocyanin production, the bacterial strains were first cultured in 10 mL of LBB at 37 °C for 24 h. After incubation, 1 mL samples were collected, centrifuged for 10 min at 7000 rpm, and the supernatants were filtered through 0.2 µm pore-size filters (Whatman, Maidstone, UK). The elastase assay was performed as follows: 100 µL of each supernatant was incubated for 2 h with 1 mL of 5 mg/mL of Congo red elastin (Sigma-Aldrich, St. Louis, MO, USA) in TrisHCl 100 mM (pH = 7.5) and CaCl 1 mM, at 37 °C and 250 rpm. Later, samples were centrifuged for 10 min at 13,000 rpm. A total of 100 µL of each supernatant was placed in a 96-well microtiter plate (Nunc) and the elastase activity was quantified by reading the optical density at 495 nm (OD₄₉₅) in a Tecan Spark 10 M plate reader (Tecan, Mennedorf, Switzerland). Two technical and three biological replicates were included in the assay. On the other side, the pyocyanin production was determined by placing 100 µL of the above-said filtered supernatants in a 96-well microtiter plate (Nunc) and measuring the OD₆₉₀, using the same plate reader. Three biological replicates of each strain were included.

3.8. Biofilm Formation

The study of biofilm formation was performed using 96-well microtiter plates (Falcon 3911 Microtest III flexible assay plate (Fisher, Hampton, VA, USA)) as previously described [26,45]. Briefly, each strain was grown at 37 °C for 24 h and 250 rpm in LBB. The O/N bacterial cultures were diluted 1:100 and inoculated into the microtiter plate (100 µL/well). The plate was kept at 37 °C for 48 h. Next, each well was incubated with 25 µL of a solution of 0.1% crystal violet in ethanol for 5 min, and the excess dye was washed with distilled water for 4 times. After, in order to detach the biofilm from the wells, 150 µL of Triton X-100 (0.25%) was added to each well and 100 µL of each sample was transferred to a 96-well microtiter plate (Nunc, Roskilde, Denmark). The quantification of biofilm formation was performed by measuring the OD₅₇₀ in a Tecan Spark 10 M plate reader (Tecan).

3.9. Quantification of Intracellular Fosfomycin

Assays to test the intracellular concentration of fosfomycin in bacterial cells were conducted using a bioassay as previously stated [46]. P. aeruginosa PA14 wild-type strain, as well as P. aeruginosa ΔglpT and ΔfosA, were used as controls. Briefly, 10⁹ cells/mL bacterial suspensions containing 2 mg/mL of fosfomycin were incubated at 37 °C (60 min) and washed sequentially three times in a buffer containing 10 mM Tris [pH 7.3], 0.5 mM MgCl₂ and 150 mM NaCl to remove the remaining extracellular Fosfomycin. After washing, cells were suspended in 0.6 mL of 0.85% NaCl. The number of CFU/mL in each sample was determined by plating dilutions of the suspensions onto LB agar that were incubated 20 h at 37 °C. The samples were boiled for 5 min at 100 °C to release the intracellular fosfomycin, and cellular debris were removed by centrifugation (11,900 g, 10 min). A total of 40 μL of each supernatant was poured on sterilized disks (9 mm; Macherey-Nagel, Düren, Germany), and the disks were deposited onto LB agar plates, previously overlaid with OmniMAX E. coli. In parallel, disks containing different amounts of fosfomycin were also used in the same conditions to make a titration curve. The plates were incubated for 20 h at 37 °C. The diameter of inhibition zones was measured and compared with the ones of the controls containing different amounts of fosfomycin to quantify the concentration of this antibiotic in each sample. The fosfomycin concentration was represented as micrograms per 10⁷ cells.

3.10. RNA Extraction and qRT-PCR

RNA was extracted from three cultures of each strain. First, 20 mL of LBB medium were inoculated from O/N cultures to a final OD₆₀₀ of 0.01 and grown until exponential phase (OD₆₀₀ = 0.6) was reached, in presence and absence of a subinhibitory concentration of tobramycin (1/8 of MIC of each strain). Then, the cultures were spun down, and the resulting pellets were collected and used to perform RNA extraction, following the protocol of RNeasy mini Kit (QIAGEN). The residual DNA was removed by two different DNA treatments: the first one with DNase (QIAGEN) and the second one with Turbo DNA-free (Ambion, Seoul, Republic of Korea). The absence of DNA was checked by PCR, using primers rplU_fw and rplU_rv (Table 3). After, cDNA was obtained from 2.5 to 5 μg of RNA in a final volume of 20 μL, using the High-Capacity cDNA reverse transcription kit (Applied Biosystems, Waltham, MA, USA). qRT-PCR was performed in an ABI Prism 7300 Real-time PCR system (Applied Biosystems), using Power SYBR green PCR master mix (Applied Biosystems). A total of 50 ng of cDNA was used for the reaction. Primer3 Input software (https://bioinfo.ut.ee/primer3-0.4.0/primer3/, access date 6 June 2023) was utilized to design the primers (Table 3), and their efficiency was analyzed by qRT-PCR using serial dilutions of cDNA. Said primers were used at a concentration of 10 μM. All mRNA expression values were determined as the average of three independent biological replicates, each one with three technical replicates. Primers rplU_fw and rplU_rv were used to quantify the expression of the housekeeping gene rplU that was used to normalize the results, and the relative expression of each gene was calculated with the 2^−ΔΔCt method [47].