Bacterial strains and culture conditions
The bacterial strains used in this study are listed in Table 1. Bacteria were grown in Luria Bertani (LB) broth (DIBICO; CDMX, México) overnight at 37ºC, under aerobic conditions, and constant shaking for the different assays. Routinely, LB agar was used for bacterial isolation and when needed, ampicillin (Sigma-Aldrich; MI, USA), and/or kanamycin (Amresco; OH, USA) were used at 100 mg/mL and 50 mg/mL, respectively.
Construction of the transposon mutant library
Transposon libraries were constructed using an EZ-Tn5<KAN-2>Tnp Transposome kit (Epicentre; WI, USA). Briefly, C. sakazakii ATCC BAA-894 (CS WT) was grown in LB broth at 37ºC until reaching middle-log phase and then was diluted 1:100 in this medium to obtain an optical density of 0.6 at 600 nm (OD600nm). The bacteria were harvested by centrifugation, washed three times with MilliQâ ultrapure water, and resuspended in 100 mL of ice-cold 10% glycerol in MilliQâ ultrapure water. Competent cells (100 mL) were electroporated with 1 μL of EZ-Tn5TM<KAN-2> Tnp TransposomeTM at 1800 V/5 msec using an ECM 399 electroporation system (BTX Harvard apparatus; MA, USA). The cells were recovered in 1 mL of SOC medium (Super Optimal Broth) with 20 mM glucose and then incubated at 37ºC with shaking (200 rev min-1) for 4 h. The bacterial culture was diluted 1:100 in SOC medium, and a 100 μL aliquot was spread onto LB agar plates with 50 mg/mL kanamycin and incubated overnight at 37°C. Selected kanamycin-resistant strains that were considered non-motile and stored in LB broth with 7.5% glycerol including 50 mg/mL kanamycin.
Screening of non-motile strains by motility assays
The non-motility of kanamycin-resistant strains generated by transposon mutagenesis was confirmed when one colony was spotted into the center of each well in 48-well microplates with 0.3% LB agar supplemented with 50 μg/mL kanamycin (Corning Life Sciences; NY, USA). The microplates were incubated overnight at 37°C, and the absence of turbidity absence was a parameter to confirm the motility loss. Non-motile kanamycin-resistant strains were selected and used for the further assays included in this study.
Visualization of C. sakazakii strains by electron microscopy
The presence or absence of flagella around bacterial cells was visualized by transmission electron microscopy (TEM). CS WT and the non-motile kanamycin-resistant mutant strains were grown overnight in LB broth at 37ºC and one drop of the bacterial culture was placed on a 200 mesh formvar-carbon-coated grid (Electron Microscopy Sciences, London) for 5 min . Excess liquid was wiped off, and the grid with bacteria was negatively stained with 1% sodium phosphotungstic acid (pH 7.2) (MP Biomedicals; OH, USA) for 5 min. The samples were washed three times with distilled water and visualized under a JEM-1010 microscope (JEOL; Tokyo, Japan).
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed as described by Laemmli . Bacterial suspensions were adjusted to an absorbance of 1.0 at OD600; the pellet was separated by spinning at 4,000 x g for 5 min and then mixed with 100 µL of SDS-PAGE sample buffer (4% w/v SDS, 10% v/v β-mercaptoethanol, 0.125 mM Tris-HCl pH 6.8, 20% v/v glycerol, and 0.0025% w/v bromophenol blue) 1X and heated at 90°C for 5 min prior to electrophoresis. Twenty-five-microliter aliquots from whole-cell extracts (equal cell numbers) were loaded for 12% SDS-PAGE and run at 100 V for 2 h at room temperature. The proteins were immobilized onto a nitrocellulose membrane and incubated using blocking buffer [TBS containing 0.1% (v/v) Tween 20 and 5% skim milk], reacted for 1 h with rabbit anti-FliC antibodies at a dilution of 1:1,500, washed 3 times with TBS-Tween 0.1%, and then incubated for 1 h with alkaline phosphatase-conjugated goat anti-rabbit antibody at a 1:20,000 dilution (Sigma Aldrich; MO, USA). The membrane was washed and exposed to 5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium (BCIP/NBT) (Merck Millipore; MA, USA).
Identification of transposon insertion sites
Random amplification of transposon ends (RATE)-PCR was performed using primer Tn5PCRF (5′-GCTGAGTTGAAGGATCAGATC-3′) or Tn5PCRR (5′-CGAGCAAGACGTTCCCGTTG-3′). The amplification was carried out with the following thermal cycles: 1 min at 94ºC; 20 cycles of 94ºC for 30 s, 50ºC for 30 s, 72ºC for 3 min; 30 cycles of 94ºC for 30 s, 30ºC for 30 s, 72ºC for 2 min; 30 cycles of 94ºC for 30 s, 50ºC for 30 s, 72ºC for 2 min; and 72ºC for 7 min, as previously described (Álvarez-Ordóñez et al., 2014b). The reaction was performed using Platinum® Taq DNA Polymerase High Fidelity (Invitrogen; CA, USA). The PCR products were purified with Zymoclean Gel DNA Recovery (Zymo Research; CA, USA), cloned into the pJET1.2/blunt vector (Thermo Scientific; CA, USA) and transformed into Escherichia coli BL21. DNA sequencing was carried out with BigDye® terminator v1.1 cycle sequencing (Applied Biosystems; California, USA) using 100 ng of reverse primer and 500 ng of purified DNA template (Table 2). The products were precipitated (EDTA 125 mM and absolute ethanol) and sequenced using an ABI Prism 377 sequencer (Applied Biosystems; California, USA). The data generated from DNA sequencing were analyzed and compared with a reference nucleotide sequence database in NCBI for bacteria using BLAST and aligned via multiple sequence alignment with hierarchical clustering .
Extraction and processing of proteins
Non-motile (CS fliF::Tn5) and C. sakazakii WT (CS WT) strains were grown in N1E-115 preconditioned media overnight at 37°C with shaking and total membrane proteins were obtained using sucrose gradient centrifugation, as described by Thein et al, . The samples were treated with 50 µg of total proteins; dissolved in 1% SDS (Sigma-Aldrich, MI, USA), 50 mM NH4HCO3 (Sigma-Aldrich, MI, USA), and 10 mM dithiothreitol (DTT; Sigma-Aldrich, MI, USA); and incubated at 40°C for 30 min. Subsequently, the proteins were alkylated with 20 mM iodoacetamide (Sigma-Aldrich, MI, USA) and 30 mM Tris-HCl pH 8.6 (Promega, WI, USA) for 30 min at room temperature in the dark. Proteins were precipitated with 9 volumes of ethanol (Sigma-Aldrich, MI, USA) at -20°C overnight and centrifuged at 20,000 x g for 10 min at 4°C; the supernatant was discarded, and the pellet was washed three times with 90% cold ethanol and then dried. The pellets were solubilized in 50 mM NH4HCO3 and 50 mM guanidine hydrochloride (Sigma-Aldrich, MI USA) and then digested overnight with 1 µg of mass spectrometry grade trypsin (Promega, WI, USA) at 37°C. Peptides were desalted with Sep-Pak tC18 cartridges (Waters, MA, USA), dried in a SpeedVac concentrator (Thermo Scientific, MA, USA), and stored at -80°C. The samples were reconstituted in 50 µL of 0.1% formic acid, centrifuged at 20, 000 x g at 4°C for 5 min and injected on a C18 Nano HPLC column for peptide separation.
Nanoflow liquid ehromatography (Nano LC)-quadrupole time-of fight tandem mass
Spectrometry (QTOF) analysis
The peptide solutions (5 µL) were loaded into a Thermo UltiMate 3000 HPLC system (Thermo Scientific, MA, USA) using a 5-µ-pre-column/peptide trap (300 µm i.d. x 5 mm, 100 Å, Thermo Scientific) and an Acclaim PepMap RSLC C18 (3 µm, 100 Å, 75 µm i.d. x 15 cm, nanoViper) separation column. Chromatographic runs were performed at a constant flow of 250 nL/min of a mixture of 0.1% (v/v) formic acid/water (Buffer A, from a Milli-Q system; MA, USA) and 0.1% (v/v) formic acid/acetonitrile (Buffer B, HPLC grade from Sigma-Aldrich; MA, USA) in a linear gradient of 200 min from 1-30% B. The gradient was increased to 90% B at 210 min and held there for 8 min, after which the percentage of B was returned to 1% for column re-equilibration. Electrospray ionization of the eluted peptides was performed with a CaptiveSpray source (Bruker; MA, USA) assisted by a flow of nitrogen boiled on acetonitrile (0.2 bar), and the mass spectra were acquired with a quadrupole time-of-flight mass spectrometer (Impact II, Bruker). Positive ions were analyzed over an m/z range of 300-2200. Prior to every six injections, calibration was performed with ESI-TOF Tuning Mix (Sigma-Aldrich, MI, USA). MS/MS fragmentation was performed for ions with a signal higher than 5000 counts, applying a cycle time of 3 s and excluding +1 charged ions. Exclusion was active after one spectrum for 2 min, unless the precursor intensity was more than three times higher than that in the previous scan. The collision energy depended on the precursor ion charge and mass (e.g., at 700 m/z, 33eV and 27 eV were used for 2+ and 3+ ions, respectively, whereas at 1100 m/z, 65 eV and 55 eV were used for 2+ and 3+ ions).
Database search and analysis of proteomic data
Protein identification was performed by processing the raw files with the Datanalysis-o-TOF-Default script from the Bruker Compass Datanalysis software (version 4.2 SR2, Bruker; MA, USA). The resultant .xml files were then analyzed in the ProteinScape software (version 3.1, Bruker; MA, USA) using Mascot 2.4.1 (Matrix Science; London, UK) with the following parameters: trypsin as the digestion enzyme with two miscleavages allowed, carbamidomethyl Cys as a fixed modification and oxidation on Met as variable modification. Monoisotopic peptide masses were searched with 7-ppm peptide mass tolerance and 0.05 Da fragment mass tolerance. The false discovery rate (FDR) was set to 1%, and peptide decoy and percolator options were active. The C. sakazakii strain ATCC BAA-894 UniProt reviewed and unreviewed databases were used. Proteins with Mascot scores > 13 and at least two peptides per protein were considered positive hits. Only proteins common to at least two analytical replicates were considered successful identifications.
A comparison of the relative protein abundance (heat map) was made using the Label Free Quantification (LFQ) tool of MaxQuant software (version 22.214.171.124) and the Andromeda search engine (version 126.96.36.199) loaded with the C. sakazakii ATCC BAA-894 UniProt databases mentioned above. The search parameters were as follows: carbamidomethyl cysteine as a fixed modification, N-terminal acetylation and methionine oxidation as variable modifications, two missed cleavages of the protease trypsin allowed, Bruker Q-TOF as the analyzer, peptide mass tolerance of 0.07 Da, MS/MS mass tolerance of 40 ppm, and an FDR of 1% at the protein level. Perseus software (version 188.8.131.52) was used to combine the MaxQuant results from three analytical replicates of each sample. Only those proteins identified in the three analytical replicates of both strains were considered for calculations in the fold change. A protein was considered differentially expressed if the fold change was higher than 1.5, if the difference was statistically significant (FDR=0.05, permutation based-FDR, Q<0.015, Student´s t-test) and if the protein was identified based on a minimum of one peptide. The percentage of overlap at the protein level in the triplicate HPLC runs was higher than 93%, and that of the two biological replicates was 80%. To assess the reproducibility of the LFQ analysis, the scatter plot and the correlation coefficient calculation were performed based on the LFQ intensities. The average Pearson correlation coefficient of the technical replicates was of 0.91±0.03.
Mouse neuroblastoma (N1E-115 ATCCÒCRL-2263; Manassas, USA) cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) high glucose 4.5 g/L from GIBCO (Thermo Scientific; MA, USA) and supplemented with 10% fetal bovine serum (FBS) from GIBCO (Thermo Scientific; MA, USA). N1E-115 cell differentiation was performed after induction with DMEM high glucose medium supplemented with 2% FBS and 1.25% dimethyl sulfoxide (DMSO) (Sigma-Aldrich; MI, USA) for 5 days. Briefly, cell monolayers at 70-80% confluence in 24-well plates containing 1 mL of DMEM were infected at an MOI of 100:1 and incubated at 37°C and 5% CO2 for 4 h according to the method of Cruz et al. . Initially, the non-motile (generated in this study) and WT CS strains were cultured overnight in LB at 37°C. The non-attached bacteria were removed, and the bacteria attached to the cell monolayers were removed by adding 1 mL of 0.1% Triton X-100 (Amresco, OH, USA). The bacterial cells that adhered to N1E-115 monolayers were collected, and serial dilutions were plated onto LB agar plates to determine the number of colony forming units (CFU)/mL. The adherence assays were performed three times in triplicate on different days, and the data are expressed as the mean of the averages.
To perform invasion assays, N1E-115 cell monolayers were prepared and infected according to the procedure described in the adherence assay section. The infected cells monolayers were washed with 1X PBS and incubated with DMEM supplemented with 300 µg/mL lysozyme (Sigma-Aldrich, MI, USA) and 100 µg/mL gentamicin (Sigma-Aldrich; MI, USA) for 2 h at 37°C and 5% CO2. The infected cell monolayers were washed three times with 1X PBS, detached with 1 mL of 0.1% Triton X-100, and plated onto LB agar plates. The invasion frequency was calculated as the number of surviving bacteria after treatment with gentamycin and lysozyme divided by the total number of bacteria without treatment with these antibiotics . The invasion assays were performed three times in triplicate on different days, and the data are expressed as the means of the averages.
Statistical significance (p<0.05) was determined using Student’s t-test for adherence and invasion assays.