All animal studies complied with National and Nankai University guidelines regarding the use of animals in research. All animal experiments protocol was approved by the institutional animal care and use committee of the college of life sciences of Nankai University with the permit number NK-04-2012.

The bacterial strains used in this study are listed in Table S1. Bacteria were cultured in Luria–Bertani (LB) broth (10 g/l tryptone, 5 g/l yeast extract, 5 g/l Nacl, pH 7.0–7.5; Oxoid Ltd, USA) or LB agar (LB broth containing 15 g/l agar) under aerobic condition at 37°C. Antibiotics were used at the following concentrations: for P. aeruginosa, gentamicin at 100 μg/ml, carbenicillin at 150 μg/ml; for E. coli, ampicillin at 100 μg/ml. All antibiotics were purchased from BBI life sciences, Shanghai, China.

Plasmids used in this study are listed in Table S1. For DNA manipulation, standard protocols or manufacturer's instructions of commercial products were followed. A region containing the waaP gene and 50 bp upstream of the start codon was amplified by PCR and cloned into the SalI, XbaI sites of pRKaraRed, resulting in pE2106, where the waaP gene is under the control of an arabinose-inducible promoter and fused with a His tag on the C terminus. An IPTG-inducible expression vector that expresses antisense RNA (asRNA) with paired termini (PT) against waaP was constructed as previously described (Nakashima and Tamura, 2009). The fragment of PT and a transcription terminator T0 (Nakashima and Tamura, 2009) was synthesized and cloned into the EcoRI-HindIII sites of pMMB67EH, resulting in pE1992. A fragment covering 35 bp upstream of the start codon and the first 65 bp of the waaP gene was PCR amplified and cloned into NcoI-XhoI sites inside of PT fragment of pE1992, resulting in pP2804.

HL60 and A549 cells were cultured in RPMI-1640 medium with 10% fetal bovine serum (FBS) at 37°C with 5% CO₂, supplemented with penicillin G (100 U/ml) and streptomycin (100 μg/ml; Hyclone, USA). HL60 cell differentiation was performed as previously described (Sun et al., 2014).

A549 cells (1 × 10⁴) were seeded into each well of a 96-well plate, and incubated at 37°C with 5% CO₂ for 16–20 h. The cells were washed twice with Hank's balanced salt solution (HBSS; Hyclone, USA) before bacterial infection. Overnight bacterial culture was sub-cultured in fresh LB broth and grown to an OD₆₀₀ of 1.0. Bacteria were washed once and resuspended in HBSS to a concentration of 1 × 10⁸ CFU/ml. One hundred microliters bacterial suspension was added to A549 cells and incubated for 10 min, followed by addition of 1 × 10⁶ differentiated HL60 cells. Twenty minutes later, antibiotics at indicated concentrations were added to each well and incubated at 37°C. After 50 min, the number of live bacteria was determined by serial dilution and plating. The bacterial survival rate was calculated by dividing the number of live bacteria in the presence of antibiotic by that in the absence of antibiotic.

ROS level was measured as previously described (Sun et al., 2014) with minor modification. A549 cells in each well of a 96-well plate were washed twice with warm HBSS. Then, 100 μl HBSS containing 100 μM luminol and 5 units of horseradish peroxidase (Sigma) was added to each well. For differentiated HL60 cells, 1 × 10⁶ cells in 100 μl of HBSS containing 100 μM luminol and 5 units of horseradish peroxidase were added to each well and incubated for 10 min at 37°C. PAO1 in HBSS (1 × 10⁷ CFU) were added into each well, and the ROS level was monitored every 2–5 min for 2 h with a luminometer (Varioskan Flash, Thermo Scientific).

Total RNA was isolated using TRIzol (Thermo Fisher Scientific, USA) and a Direct-Zol RNA Miniprep (Zymo Research, USA). Total RNA from cells and bacteria mixtures were immediately treated with a MICROB Enrich (Life Technologies, USA) kit to remove RNA from eukaryotic cells. cDNA was synthesized with PrimeScript Reverse Transcriptase (TaKaRa, Dalian, China) and random primers. cDNA was mixed with 10 μl SYBR Premix Ex TaqTM II (TaKaRa) and 4 pmol forward and reverse primers (Table S1) in a total volume of 20 μl. The 30S ribosomal protein gene rpsL was used as an internal control in each real time PCR assay. Real time PCR was performed with a CFX Connect Real-Time system (Bio-Rad, USA).

Total RNA was isolated using the Trizol Reagent (Thermo Fisher Scientific, USA). The quantity and integrity were determined using a NanoDrop spectrophotometer (Thermo Scientific) and a Bioanalyzer 2100 system (Agilent). For mRNA sequencing, a Ribo-Zero rRNA Removal Kit (Illumina, San Diego, CA, USA) was used to selectively remove rRNA. Random oligonucleotides and SuperScript III were used to synthesize the first strand cDNA. Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase treatment. After adenylation of the 3′ ends of the DNA fragments, Illumina PE adapter oligonucleotides were ligated to prepare for hybridization. To select cDNA fragments around 300 bp in length, the library fragments were purified using the AMPure XP system (Beckman Coulter, Beverly, CA, USA). DNA fragments with ligated adaptor molecules on both ends were selectively enriched using Illumina PCR Primer Cocktail in a 15 cycle PCR reaction. The products were purified with the AMPure XP system and quantified using the Agilent high sensitivity DNA assay on a Bioanalyzer 2100 system (Agilent). The DNA library was then sequenced on a NextSeq 500 platform (Illumina) by Shanghai Personal Biotechnology Cp. Ltd.

Sequence reads were mapped onto PAO1 reference genome (NC_002516.2) using a CLC genomics Workbench 8.0 (CLC Bio-Qiagen, Aarhus, Denmark). The count data of expression values were then analyzed using a DESeq package of R/Bioconductor. The differentially expressed genes were identified by performing a negative binomial test using the DESeq software, with the cut-off fold-change larger than 2. The raw sequence reads were normalized by dividing with size factors, then Log2 (N + 1) transformed. The data have been deposited in the NCBI Short Read Archive (SRA) database with an accession number SRP100146.

Bacteria with or without incubation with eukaryotic cells were diluted to 10⁶ CFU/ml in HBSS. One microliter 20 mM DiOC2(3) was incubated with the bacteria in a 1 ml volume and incubated for 20 min at room temperature in dark, followed by flow cytometry analyses (Accuri C6, BD). DiOC2(3) was excited at 484 nm and emissions at 530 nm (Green fluorescence) and 610 nm (Red fluorescence) were measured. Membrane potential was indicated by ratios of Green and Red fluorescence (Novo et al., 1999).

Antisense RNA (AsRNA) construction was performed as previously described (Nakashima and Tamura, 2009). A fragment containing 35 bp upstream of the start codon and the first 65 bp of the waaP gene was amplified using PAO1 genomic DNA as the template. The fragment was cloned into pE1992, in which an RNA complementary to the waaP mRNA is driven by an inducible tac promoter. The construct was confirmed by DNA sequencing. To examine the inhibitory effect of the AsRNA, the waaP gene including its upstream 50 bp was fused with a 6 × HIS tag at the 3′ terminus (waaP-His) and cloned into pRKaraRed (Liang and Liu, 2010; Verstraeten et al., 2015), where expression of the gene is driven by an arabinose inducible promoter (P_BAD). The two plasmids were co-transformed into PAO1, and the levels of WaaP-His was determined by Western blot.

P. aeruginosa strains were grown in LB overnight at 37°C and then diluted 50-fold in fresh LB with 1 mM IPTG and 0.2% (vol/vol) arabinose, and cultured for 3 h. Samples from equivalent numbers of bacterial cells were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, USA) and probed with a rabbit polyclonal antibody against 6 × HIS (Cell Signaling Technology, USA). The luminescence signals were detected with an ECL-plus kit (Millipore, USA).

Mouse acute pneumonia was performed as previously described (Sun et al., 2014). P. aeruginosa strains were grown in LB overnight and then subcultured into fresh medium and grown at 37°C with aeration to an OD₆₀₀ of 1.0. Bacteria were washed and adjusted to 2 × 10⁹ CFU/ml in phosphate-buffered saline (PBS). Each female BALB/c mouse (6–8 weeks old) was anesthetized with an intraperitoneal injection of 5 μl/g 7.5% chloral hydrate. Then 20 μl bacterial suspension was intranasally inoculated into each anesthetized mouse, 10 μl per nostril. Bronchi alveolar lavage fluid (BALF) was collected as previously described (Wu et al., 2012). Briefly, 6 h post infection, mice were sacrificed by inhalation of CO₂. Two microliters PBS containing 0.05 mM EDTA was injected into lungs through trachea by a vein detained needle (BD, Angiocath). After 1 min detaining, fluid was collected.

To examine whether host environment affects P. aeruginosa susceptibility to antibiotics, we infected mice with a wild type P. aeruginosa strain PAO1 itranasally and isolated bacteria from BALF 6 h post infection. The bacteria were treated with tobramycin, which is used in clinic to treat patients infected by P. aeruginosa. As a comparison, similar numbers of bacteria grown in LB were treated with tobramycin. The number of bacteria in each sample was listed in Table S2. Interestingly, the isolated bacteria showed higher susceptibility to tobramycin than those grown in vitro (Figure 1), suggesting that host environment might affect bacterial susceptibility to antibiotics.

To explore the mechanism of the increased bacterial susceptibility, we treated wild type PAO1 with tobramycin in the presence or absence of mammalian cells in vitro. Previously in a murine acute pneumonia model, it was demonstrated that neutrophils are recruited to the lung after infection with P. aeruginosa (Diaz and Hauser, 2010; Sun et al., 2014). These results indicate that the invading bacteria contact with epithelial cells first, followed by encountering neutrophils which generate reactive oxygen species (ROS) and release proteases and bactericidal peptides locally (Weiss, 1989). To mimic the in vivo infection scenario, we infected the human alveolar adenocarcinoma (A549) cells with PAO1 for 10 min, and then added neutrophil-like differentiated HL60 cells (designated as dHL60 hereafter). The response of dHL60 cells to P. aeruginosa was monitored by measuring ROS production. As shown in Figure 2A, dHL60 cells generated significantly higher amount of ROS than A549 cells upon encountering P. aeruginosa. Meanwhile, live bacteria number was determined 50 min after the addition of dHL60 cells. As shown in Figure S1, presence of A549 and dHL60 cells did not affect bacterial survival (Figure S1).

We next treated PAO1 with 2 μg/ml tobramycin for 10 min followed by the addition of dHL60 cells, when the ROS production reached the peak (Figure 2A). Fifty minutes later, the bacterial survival rates were determined (Figure 2B). As a comparison, similar numbers of bacteria grown in LB were treated with tobramycin for the same period of time. Compared to bacteria alone, incubation with A549 and dHL60 cells reduced the bacterial survival rate by ~80-fold (Figure 2C).

To dissect the role of each type of cell in the increased bacterial susceptibility, PAO1 were incubated with A549 or dHL60 cells individually, followed by treatment with tobramycin. A549 cells did not affect the bacteria survival rate, whereas dHL60 cells reduced the survival rate by ~20-fold (Figure 2C). These results suggest that dHL60 cells play a major role in the increased bacterial susceptibility to tobramycin, and the presence of A549 cells further increased the bacterial susceptibility.

As shown in Figure 2, dHL60 cells generate large amount of ROS upon encountering bacteria. To examine whether ROS plays a role in sensitizing bacteria to tobramycin, we used 6 N-Acetyl-L-cysteine (NAC) to neutralize ROS (Curtin et al., 2002). NAC alone did not alter bacterial survival rate in the absence of dHL60 cells, however, in the presence of dHL60 cells, NAC increased the survival rate in a dose dependent manner (Figure 3). To confirm the role of ROS, we treated bacteria with various concentrations of H₂O₂ in the presence or absence of tobramycin. At the concentration of 0.25 mM, H₂O₂ alone did not affect bacterial survival, but reduced the survival rate in the presence of tobramycin (Figure S2). These results suggest that ROS contributes to the increased bacterial susceptibility to tobramycin.

To further explore the mechanism of the increased bacterial susceptibility to tobramycin, we compared the transcription profiles among the following samples: PAO1, PAO1+tobramycin, PAO1+A549+dHL60, PAO1+A549+dHL60+tobramycin. In a previous transcriptome analysis of PAO1 in response to tobramycin, bacteria were treated with 2 μg/ml tobramycin for 30 min in BBL cation-adjusted Mueller-Hinton broth medium (CAMHB) before RNA isolation (Kindrachuk et al., 2011). We tested the survival rates of PAO1 after treatment with 2 μg/ml tobramycin for various periods of time in HBSS. After 20-min treatment, the bacterial survival rate was ~80% (Figure S3A) and we chose this time point for RNA isolation. Next, we determined the bacterial survival rates in the presence of A549 and dHL60 cells after treatment with various concentrations of tobramycin for 20 min. As shown in Figure S3B, treatment with 0.3 μg/ml tobramycin resulted in 80% live bacteria. Thus, this condition was chosen for RNA isolation. To compare the bacterial responses to the same concentration of tobramycin with or without A549 and dHL60 cells, we included a bacterial sample treated with 0.3 μg/ml tobramycin for 20 min in HBSS. The experimental conditions for RNA sample collection are summarized in Table 1.

The transcriptome analysis results were presented as RNA expression levels relative to the PAO1 incubated in HBSS (Table 1, Table S3). Previously, it had been demonstrated that lethal concentrations of tobramycin highly activated the expression of genes involved in heat shock response, whereas bacteriostatic concentrations of tobramycin had minor effect (Kindrachuk et al., 2011). Consistent with the report, heat shock response genes ibpA, groES, grpE, hslV, and hslU were all up regulated after treatment with 2 μg/ml of tobramycin for 20 min in HBSS, however, 0.3 μg/ml of tobramycin did not significantly affect the expression of these genes (Table 1). Interestingly, treatment with 0.3 μg/ml of tobramycin in the presence of A549 and dHL60 cells induced the expression of those heat shock genes even higher than treatment by 2 μg/ml of tobramycin in HBSS (Table 1). Real time PCR analysis confirmed the mRNA levels of ibpA and groES (Figure 4). These results suggest that contact with the host cells intensified bacterial response to tobramycin, which is consistent with our bacterial survival results.

We hypothesized that the increased bacterial susceptibility to tobramycin might be due to decreased membrane potential (Taber et al., 1987). Indeed, incubation with A549 and dHL60 decreased bacterial membrane potential (Figure 5). To dissect the role of each type of cell in the decreased membrane potential, we incubated the bacteria with individual cell line. Incubation with dHL60 but not A549 cells decreased membrane potential (Figure 5). These results suggest that dHL60 played a major role in the decreased membrane potential, which might contribute to increased bacterial susceptibility to tobramycin.

Besides membrane potential, the cellular entry of positively charged aminoglycoside antibiotic depends on its interaction with negatively charged LPS (Schindler and Teuber, 1975; Moore et al., 1986; Schnaitman and Klena, 1993). In our transcriptome analysis results, we found LPS synthesis genes as well as the LPS core kinase gene waaP were up regulated in response to the dHL60 and A549 cells (Table 1). Real time PCR assay confirmed the results (Figures 6A,B). We thus hypothesized that the up regulation of the LPS core kinase WaaP might increase the phosphorylation of LPS, which facilitates the entrance of tobramycin. Since waaP is an essential gene in P. aeruginosa (Walsh et al., 2000; Zhao et al., 2002; Delucia et al., 2011), we overexpressed an antisense RNA to reduce its expression level (Figure 7A). Indeed, reduction of WaaP level reduced bacterial susceptibility to tobramycin in the presence of dHL60 and A549 cells (Figure 7B).

If up regulation of WaaP contributes to the increased bacterial susceptibility to tobramycin, the bacterial should be susceptible to other aminoglycoside but not a different type of antibiotics. Indeed, incubation with dHL60 and A549 cells increased bacterial susceptibility to neomycin, but not the macrolide antibiotic azithromycin (Figure 7C). Reduction of WaaP level also reduced bacterial susceptibility to neomycin but did not affect the susceptibility to azithromycin in the presence of dHL60 and A549 cells (Figure 7D).

The in vivo environment is much more complicated than our in vitro cell infection condition. In addition to epithelial and phagocytic cells, bacteria encounter a variety of molecules, such as mucin and defensins as well as iron limiting environment. In order to investigate whether the host in vivo environment affects the expression of waaP, we isolated bacteria from BALF 6 h post infection. mRNA levels of the T3SS gene pcrV and iron acquisition gene hasR, which are known to be activated during acute pneumonia (Shaver and Hauser, 2004; Damron et al., 2016) were higher in the bacteria isolated from BALF (Figure 8A), suggesting an adaptation to host environment. Consistent with our in vitro cell infection results, the expression of waaP was induced during infection (Figure 8A). And the isolated bacteria showed higher susceptibility to neomycin, but not to azithromycin (Figures 8B,C). Overall, these results suggest that host environment could affect bacterial susceptibility to aminoglycoside antibiotics.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.