The wild-type SE strain C50336 was obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Escherichia coli X7213 λpir is stored in our laboratory. The bacterial strains and plasmids used in this study are listed in Table 1. The gene deletion mutant strains were constructed by double exchange of homologous recombination as described previously . The upstream and downstream fragments of target genes were amplified using PCR by employing the primers listed in Table 2. The PCR products were purified using the TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver 4.0 (TaKaRa Biotechnology Co. Ltd., Dalian, China). The pDM4 plasmid was digested by restriction endonucleases Sal Ⅰ and Sac I (TaKaRa). The purified plasmid and upstream and downstream fragments were fused using the ClonExpress MultiS One Step Cloning Kit (Vazyme Biotechnology Co. Ltd., Nanjing, China). The recombinant plasmids were transferred into X7213 λpir cells and sequenced. Single-crossover mutants were obtained by conjugal transfer of the recombinant suicide plasmids into C50336. Deletion mutants were screened on 15% sucrose Luria-Bertani (LB) plates. Construction of gene complementation strains and overexpression strains were performed as described previously . Target genes were cloned into the low copy number plasmid pMMB207 by employing the ClonExpress II One Step Cloning Kit (Vazyme). The recombinant plasmids were mated from X7213 λpir into mutant or WT C50336 by conjugation. Chloramphenicol resistant transconjugants were selected, and the presence of the plasmids was confirmed by PCR analysis with flanking primers and sequencing.
Construction of transposon mutant library and transposon insertion sequencing
Random transposition of TnpSC189 into the C50336 chromosome was achieved by using the delivery plasmid pSC189 . For conjugation, the X7213 λpir donor strain bearing the plasmid pSC189 was cultivated in LB broth containing 50 μg/mL DAP (diaminopimelic acid) and 100 μg/mL kanamycin, while recipient strain C50336 was cultured in ordinary LB broth and grown until mid-logarithmic phase (OD600= 0.6). Equal volumes (100 μL) of bacterial suspensions were mixed after two washes with fresh LB medium. The mixed suspension was plated on nitrocellulose filters placed on the surface of LB agar plates supplemented with DAP (50 μg/mL) and incubated for 12 hours at 37 ℃. The conjugation mixtures were then diluted with sterile phosphate-buffered saline (PBS) and plated on LB agar supplemented with kanamycin (100 μg/mL).
Two-round semi-arbitrary PCR was used to determine the location of the transposon insertion . PrimeSTAR® Max DNA Polymerase (TaKaRa) was employed in a 10-μL reaction volume consisting of 2 × PrimeSTAR Max Premix (5 μL), 0.25 µM of SP1 primer, 0.1 µM of AB1, AB2, and AB3 primers (Table 2), and 1 μL of an overnight LB-grown culture as the source of DNA. The ﬁrst-round reaction conditions were as follows: 1 cycle of 95 ℃ for 5 min, 6 cycles of 95 ℃ for 30 s, 42-36 ℃ for 30 s (1 ℃ reduction per cycle), and 72 ℃ for 3 min, then 26 cycles of 95 ℃ for 30 s, 58 ℃ for 30 s, and 72 ℃ for 3 min, followed by 1 cycle of 72 ℃ for 10 min. The second-round reaction was performed in a total volume of 30 μL containing 15 μL of 2 × PrimeSTAR Max Premix, 0.25 µM of SP2 and ABS primers (Table 2), with 2 μL of the first-round reaction product as the source of DNA. The second-round PCR amplifications were performed as follows: 1 cycle of 95 ℃ for 5 min, 30 cycles of 95 ℃ for 30 s, 64 ℃ for 30 s, and 72 ℃ for 3 min, followed by 1 cycle of 72 ℃ for 10 min. The second-round PCR products were sequenced using the pSC189-seq primer (Table 2) and the sequences were compared with the GenBank DNA sequence database using the BLASTX program.
Mice and cell culture
Specific pathogen-free (SPF) female C57BL/6 mice (age, 6-8 weeks; body weight, 20 ± 2 g) were obtained from the Comparative Medical Center of Yangzhou University (Yangzhou, China). All mice were bred under specific pathogen-free conditions, in the mouse isolators (Suzhou monkey animal experiment equipment Technology Co. Ltd., Suzhou, China). Five mice were raised in a cage, fed with pathogen-free diet and water. All animal experiments were approved by the Animal Welfare and Ethics Committees of Yangzhou University and complied with the guidelines of the Institutional Administrative Committee and Ethics Committee of Laboratory Animals (IACUC license number: YZUDWLL-201811-001). All animals were subjected to a clinical examination to assess their physical appearance and the normality of their behavior, and those presenting signs of disease were removed. All animals were humanely handled. For bone marrow preparations animals were sacrificed by cervical dislocation after anesthesia by intraperitoneal injection of 20% urethane (ethyl carbamate) solution. Animals used for the evaluation of bacterial virulence in this study were euthanized by CO2 delivery from a compressed CO2 gas cylinder directly in the mouse cages to reduce stress. Euthanasia was accomplished by slow exposure to increasing levels of CO2, replacing approximately 15-30% of the cage volume per minute, outside the mouse cages. Flow was kept for 2 min and the animals were maintained in the same cage for an additional 5 min.
BMDMs are primary macrophages obtained from C57BL/6 mouse bone marrow of the tibia and femur as described previously . Three mice were uesd for BMDM preparation in each individual experiment, three independent experiments were performed. Bone marrow cells were cultured at 37 ℃ with 5% CO2 in Dulbecco's modified Eagle's medium (DMEM, Gibco, Grand Island, NY, USA) supplemented with 10% (v/v) fetal bovine serum (FBS, Gibco), 100 U/mL penicillin, 100 μg/mL streptomycin (Gibco), and 25 ng/mL macrophage colony-stimulating factor (M-CSF, PeproTech, Rocky Hill, NJ, USA). Fresh medium was added on day 3. Differentiated BMDMs were collected for experiments on day 6. J774A.1 and HeLa cells were were purchased from American Tissue Culture Collection (ATCC, Manassas, VA, USA) and cultured in complete DMEM containing 10% FBS, penicillin (100 U/mL), and streptomycin (100 μg/mL). Cell viability and number were determined by trypan blue exclusion assays.
Each single SE colony was cultured in LB medium, followed by overnight incubation at 37 ℃ with shaking at 180 rpm. Overnight cultures were diluted 1:100 and cultured for another 3 h to an OD600 of 0.7 to induce SPI-1 expression . J774A.1 cells or BMDMs were seeded into cell culture plates the day before infection. For inflammasome activation, the cells were washed with Dulbecco's PBS (DPBS, Gibco) and pre-treated with 1 μg/mL LPS (Sigma-Aldrich, St. Louis, MO, USA) diluted in Opti-MEM (Gibco) for 5 h. Bacteria strains were washed twice with sterile PBS, added to the cells at an MOI of 20:1, and spun onto the cells at 1000 rpm for 10 min. Cells were incubated at 37 ℃ for 1 h, subsequently washed twice, and incubated for an additional 3 h with Opti-MEM containing 50 μg/mL gentamicin.
In total, seventy female C57BL/6 mice (6-8 weeks old) were randomly assigned to seven groups (n = 10). Each group was intraperitoneally infected with 100 μL of WT C50336 or gene deletion mutant strains diluted in PBS at a dose of 1 × 105 CFU per mouse. The control mice received 100 μL of PBS via the same route. All mice deaths were recorded over 14 dpc.
Cytotoxicity assays and cytokine measurements
J774A.1 cells or BMDMs were seeded at a concentration of 1 × 105 cells per well in 48-well plates. The cells were infected with SE strains as described above, and supernatants were harvested at 4 h after infection and centrifuged at 2000 rpm for 5 min to remove cell debris. LDH release was quantified using the LDH Cytotoxicity Assay Kit (Beyotime Biotechnology Co. Ltd., Haimen, China) according to the manufacturer’s instructions. Quantitative determination of pro-inflammatory cytokines IL-1β and IL-6 in supernatants were performed through an enzyme-linked immunosorbent assay (ELISA) by employing Mouse IL-1 beta/IL-1F2 DuoSet ELISA and Mouse IL-6 DuoSet ELISA (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s manual.
Immunoblotting and antibodies
J774A.1 cells or BMDMs were seeded into 12-well plates at a density of 5 × 105 cells per well and infected with bacteria as described above. After harvesting the supernatants, the remaining cells were directly lysed with 300 μL cell lysis buffer per well for western and IP (Beyotime). For each single well, the supernatant and lysate were pooled separately. The mixtures were centrifuged at 2000 rpm for 5 min to remove cell debris. An equal volume of methanol and a 0.25 volume of chloroform were added, and the solution was vortexed vigorously for 30 s. The samples were centrifuged at 12000 rpm for 5 min. The upper aqueous phase was removed, and an equal volume of methanol was added to each sample. The mixtures were centrifuged at 12000 rpm for 5 min after vortexing for 10 s. The protein pellets were dried at 55 ℃ for 5-10 min, resuspended with 40 μL of 1 × SDS-PAGE Sample Loading Buffer (Beyotime), and boiled for 10 min at 95 ℃. The samples were loaded onto 15% Tris-glycine gels. The proteins were then transferred from the gels onto nitrocellulose membranes, which were blocked with blocking buffer (3% nonfat dry milk in PBS) for 2 h at room temperature. The membranes were subsequently incubated on a rotator overnight at 4 ℃ with a primary antibody (diluted 1:1000 in blocking buffer). After 5 washes with PBST (0.05% Tween 20 in PBS), the membranes were incubated at room temperature for 1.5 h with a secondary antibody diluted 1:5000 in blocking buffer. Images of antibody reactions with an ECL chemiluminescence substrate (Thermo Scientific, Waltham, MA, USA) were acquired using an Amersham Imager 600 Imaging System (GE Healthcare Life Sciences, Pittsburgh, PA, USA).
The primary antibodies used in this study were as follows: anti-caspase-1 p20 antibody (AG-20B-0042, AdipoGen, San Diego, CA, USA), anti-NLRP3/NALP3 antibody (AG-20B-0014, AdipoGen), anti-NLRC4 antibody (ab201792, Abcam, Cambridge, UK), anti-ASC/TMS1 antibody (67824S, Cell Signaling Technology, Danvers, MA, USA), anti-IL-1β antibody (12507S, Cell Signaling Technology), anti-β-actin antibody (A5441, Sigma-Aldrich), anti-Phospho-SAPK/Jnk antibody (4671S, Cell Signaling Technology), anti-Phospho-p44/42 MAPK (ERK1/2) antibody (4377S, Cell Signaling Technology), and anti-Phospho-p38 MAPK antibody (9215S, Cell Signaling Technology). The secondary antibodies were goat anti-mouse IgG-HRP (401215, Sigma-Aldrich) and goat anti-rabbit IgG-HRP (BS13278, Bioworld Technology, Bloomington, MN, USA).
Protein translocation assays
Plasmid pCX340 was used for a TEM-1 β-lactamase fusion protein FRET assay as described previously . The target genes were amplified from the genome of wild-type C50336 and then ligated into the Nde I and Kpn I restriction sites in the multiple cloning region of pCX340 using the ClonExpress II One Step Cloning Kit (Vazyme). All recombinant plasmids were transferred into C50336 by electroporation. Overnight bacterial cultures were diluted to 1:100 in LB broth supplemented with 12.5 μg/mL tetracycline for 3 h and induced with 1 mM IPTG for another 3 h.
HeLa cells were trypsinized and seeded in black, clear-bottomed 24-well plates (Cellvis, Mountain View, CA, USA) at 2 × 105 cells per well. Bacterial strains bearing plasmid pCX340 were washed twice with DMEM, added to the cells at an MOI of 100:1, and spun onto the cells at 1000 rpm for 10 min. The cells were incubated for 3 h, subsequently washed 4 times, and incubated for an additional 4 h with DMEM. The cells were washed with DMEM and covered with 300 μL DMEM plus 60 μL of 6 × CCF2/AM solution freshly prepared with the CCF2/AM loading kit (CCF2/AM final concentration,1 μM; Invitrogen, Carlsbad, CA, USA). The plates were incubated for 2 h at room temperature, and images were acquired using a Leica confocal microscope (Leica Microsystems, Wetzlar, Germany). The appropriate filters were selected to allow for excitation of coumarin (~410 nm) and detection of blue coumarin (~450 nm) and green fluorescein (~520 nm) emissions, enabling the simultaneous observation of green fluorescence emitted by the CCF2 substrate and blue fluorescence emitted by the cleaved CCF2 product. Translocation of β-lactamase fusions into CCF2-loaded cells was determined by counting the number of blue fluorescent cells in the images. Cells that did not exhibit secretion of TEM fusions appeared green. For a particular cell well from three separate experiments, six pictures were taken, and an average of 1200-2000 cells were counted. Each picture was considered an independent observation and used to calculate the percentage of blue fluorescent cells.
Lentivirus infection assays
Lentiviral vectors pGLV5-dam (EF-1aF/GFP&Puro) and pGLV5-dam-His (EF-1aF/GFP&Puro) for the Dam gene encoding a GFP sequence were designed, constructed, amplified, and purified by GenePharma (Shanghai, China). The packaging, collection, and titer determination of the lentivirus were also conducted by GenePharma. In general, 293T producer cells were transfected with optimized packaging plasmids (pGag/Pol, pRev, and pVSV-G) along with lentiviral vectors expressing Dam or Dam-His constructs by lipofectamine. Six hours post-transfection, the transfection mix was replaced with fresh DMEM containing 10% FBS. The virus-containing supernatant was harvested 72 h post-transfection, centrifuged at 4 ℃ and 4000 rpm for 4 min, filtered through a 0.45 μm filter (Millipore, Burlington, MA, USA), and centrifuged at 4 ℃ and 20000 rpm for 2 h. The lentivirus particles were titrated by adding serial dilutions to fresh 293T cells and assessed using GFP expression after 72 h. The viral titers of approximately 1 × 109 infectious units/mL were obtained and stored at −80 ℃ before infection. The negative control lentivirus Lv-NC (109 TU/mL) was also provided by GenePharma.
J774A.1 cells were seeded into 12-well plates at a density of 1 × 105 cells per well. A 100 μL volume of lentivirus was mixed with 400 μL of complete DMEM, and polybrene (GenePharma) was added to a final concentration of 5 μg/mL. The cells were incubated at 37 ℃ for 24 h, the medium was removed, and the cells were cultured in 500 μL of complete DMEM for another 48 h. The efficiency of lentivirus infection was evaluated by detection of GFP fluorescence. The cells were pre-treated with LPS as described above and stimulated with 1.25 mM ATP (Sigma) diluted in Opti-MEM (Gibco) for 1 h. Then supernatants and cell lysates were collected for immunoblotting or ELISA as described above.
Overnight cultures of the wild-type strain C50336, gene deletion mutant strain C50336Δdam, and overexpression strain C50336::dam were diluted to 1:100 in LB broth and cultured to logarithmic phase (OD600= 0.7). J774A.1 cells were seeded at a density of 5 × 106 cells per 10-cm dish. The cells were pre-treated with LPS and infected with SE strains as described above. Each group comprised three independent replicates. The cells were then collected with 1 mL Trizol (Ambion, Carlsbad, CA, USA) and sent to GENEWIZ (Suzhou, China) for analysis. The total RNA was isolated, purified, and quantified by GENEWIZ. The eukaryotic transcriptome libraries were pooled and sequenced on an Illumina platform (Illumina, San Diego, CA, USA). The subsequent procedures and statistical analyses were conducted by GENEWIZ as previously described . All sequences were deposited at NCBI Sequence Read Archive (SRA) with accession number SRP253144.
J774A.1 cells were seeded into 12-well plates at a density of 5 × 105 cells per well and infected with WT C50336, dam deletion mutant, complementation, and overexpression strains as described above. After infection for 4 h, cells were harvested and total RNA was extracted using E.Z.N.A.™ Total RNA Kit I (Omega Bio-tek, Norcross, GA, USA) by following the manufacturer’s instructions. RNase-free DNase I (TaKaRa) was used to remove contaminating DNA in purified total RNA, in accordance with the manufacturer’s instructions. RNA of the infected cells were quantified using One drop™ spectrophotometer (Wins Technology Co. Ltd., Nanjing, China). PrimeScript RT Reagent Kit (TaKaRa) was used to reverse transcribe total RNA into cDNA. Reaction was performed in a total volume of 20 μL, containing 1 μg of total RNA, 1 μL of PrimeScript RT Enzyme Mix I, 4 μL of RT Primer Mix, 4 μL of 5 × PrimeScript Buffer 2. The mixture was incubated at 37 ℃ for 15 min, followed by incubation at 85 ℃ for 5 s, subsequently stored at −20°C until further use.
The Applied Biosystems QuantStudio 6 Flex Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) was used to measure the mRNA expression levels of c-Fos, Braf, Rasa1, Sos2, Nr4a1, Elk4, Mef2c, Cul1, Tgfbr1, Rps6kb1, Rbl1, Id1, Id3, Xiap, Bcl3, Chuk, Dnm1l, Akt3, Itch, Pik3ca, Pik3r1, Pten, Irs2, Sirt1, Klf2, Sgk3, Pdpk1, Mdm2, and Rbl2 as well as of the internal control gene, mouse GAPDH. Primers were designed using Primer Express software v3.0 (Applied Biosystems, Carlsbad, CA, USA) and were listed in Table 3. The qRT-PCR reaction was performed in a total volume of 20 μL, containing 200 ng of cDNA, 10 μL of 2 × SYBR Green Master Mix (TaKaRa), 0.6 μL each of 10 μM forward and reverse primers, and 6.8 μL RNase-free water. The comparative threshold cycle (2–ΔΔC(T) method) was used to calculate relative concentrations. All qRT-PCR reactions were performed in triplicates.
The Z score was calculated for each well in a 48-well plate as described previously . For a given well, the Z score was calculated by subtracting the mean value of the wells on that plate from the value of the given well and dividing by the standard deviation value for all of the plate wells. A Z score ≤ -2 or ≥ 2 was considered significant. All data are presented as mean ± standard error (SEM) of triplicate samples per experimental condition from three independent experiments using GraphPad Prism 5 software (La Jolla, USA). To detect significant differences between experimental groups, an one-way analysis of variance (ANOVA) followed by Bonferroni’s multiple comparison test were conducted. Statistical significance was determined at p values of <0.05 (*), <0.01(**), or <0.001 (***).