All experiments involving animals have been approved by the ethics committee of the Instituto Nacional de Tecnologia Agropecuaria (INTA) where they were conducted. This ethics committee works according with the National Institutes of Health Guide for the Care and Use of Animals Laboratory .
Bacterial Strains and Growth Conditions
For this study, we used the WT Salmonella enterica serovar Typhimurium strain ATCC 14028. Bacterial strains were grown in Luria-Bertani (LB) or M9 minimal medium containing casamino acids and glucose. Appropriate antibiotics were added to the following final concentrations: 100 μ g ml-1 ampicillin, 25 μ g ml-1 kanamycin, and 10 μ g ml-1 chloramphenicol.
DNA Manipulation and plasmid construction
Plasmid pET24D-YqiC (encoding full length YqiC protein) was generated by polymerase chain reaction (PCR) from Salmonella enterica serovar Typhimurium ATCC 14028 chromosome DNA using primers 5'-AACCATGGTTGACCCGAAAAAAATT-3' and 5'-TTCTCGAGCTCTTGTTGTGGATCGAC-3' and the product was cloned in Nco I and Xho I sites of pET24D vector (Novagen) in frame with the T7 promoter. The product included a six-histidine tag fused to the C-terminal end of the protein. To construct plasmid pBBR-yqiC, a 1210 bp fragment containing yqiC gene and flanking regions from S. Typhimurium was amplified by PCR using the primers 5'-GGCTTCAATGGTCACGGTAA-3' and 5'-GCAATATGGACGAGGAGCATC-3'. The resulting fragment was then cloned into the EcoRI site of the broad-host-range plasmid pBBR1MCS1 .
Expression and Purification of Recombinant Protein
pET24D plasmid encoding the sequence of yqiC was transformed in E. coli BL21 (lambda DE3). The cells were grown in LB at 37°C to an OD 600 of 0.5 and induced with 1 mM isopropyl β-D thiogalactoside (IPTG) for 4 h. Cells were harvested by centrifugation at 3000 × g for 20 min, resuspended in binding buffer (Qiagen), and disrupted by sonication with a probe tip sonicator. Total cell lysate was centrifuged at 14000 × g for 30 min to remove non-soluble protein, cell debris, and unbroken cells. Binding and elution from nickel nitrilotriacetic acid-agarose resin were carried out under native conditions according to the manufacturer's instructions (Qiagen). Eluted proteins were dialyzed against phosphate-buffered saline (pH 7.4). Proteins were assayed with a Coomassie blue-based staining solution.
Phospholipids were purchased from Avanti Polar Lipids (Birmingham, AL) and from Sigma. L-α-dipalmitoylphosphatidylcholine (DPPC) and L- α-dipalmitoylphosphatidic acid (DPPA) were cosolubilized in chloroform in different molar ratios, dried under N2, resuspended in buffer 50 mM Tris-HCl pH 8.0 or 50 mM sodium acetate pH 4.0 and sonicated to yield small unilamellar vesicles (SUV).
Purified YqiC was cross-linked with ethylene glycol bis (succinimidylsuccinate) (EGS) (Sigma) used at concentrations of 0.5, 1.0, and 5.0 mM. The reactions were carried out for 30 min at room temperature in phosphate-buffered saline and stopped by addition of 50 mM Tris-HCl, pH 8.0. Cross-linked products were analyzed by SDS-PAGE.
Determination of the Molecular Weight by Static Light Scattering
The average molecular weight (M w) of YqiC was determined on a Precision Detector PD2010 light scattering instrument tandemly connected to a high-performance liquid chromatography system and an LKB 2142 differential refractometer. The sample was loaded on a Superdex 75 column and eluted with PBS buffer. The 90° light scattering and refractive index signals of the eluting material were analyzed with Discovery32 software, supplied by Precision Detector. The 90° light scattering detector was calibrated using bovine serum albumin (66.5 kDa) as a standard.
Circular Dichroism Spectroscopy
The circular dichroism (CD) spectra of YqiC in the far-UV region (250-200 nm) were measured on a Jasco J-810 spectrophotometer using quartz cuvettes with a path length of 0.1 cm. The CD spectra were analyzed with K2D software http://www.ogic.ca/projects/k2d2/ to evaluate the secondary structure content.
Turbidity measurements were taken on a Multiskan Spectrum double-beam spectrophotometer (Thermo Electro Corp.) by using 1 cm matched silica cuvettes at 400 nm. The SUV concentration was 250 μ M. The lipid:protein ratio for the turbidity assays was kept at 50:1.
Vesicle Internal Content Mixing
Small unilamellar vesicles were prepared containing either 5 mM terbium chloride, 50 mM sodium citrate,10 mM Tris/HCl (pH 7.4), or 50 mM sodium dipicolinate (DPA) and 10 mM Tris-HCl (pH 7.4). The vesicles concentration was 100 μM. In both cases, no encapsulated material was removed by gel filtration of the vesicles using Sephadex G-25 (Pharmacia) equilibrated with iso-osmolar 50 mM NaCl, 1 mM EDTA, and 10 mM Tris-HCl (pH 7.4). Zero percent and 100% fluorescence (aqueous content mixing) were taken as the intrinsic fluorescence intensity of the Tb/DPA-labeled liposome mixture and the fluorescence obtained after vesicle lysis with 0.2% n-dodecyl maltoside in assay buffer without EDTA as described by Duzgunes et al. Fluorescence measurements were carried out at 25°C using a Molecular Devices SpectroMAX GeminiEM spectrofluorometer. The extent of vesicles aqueous content mixing was determinated according to the following equation:
Where F0 is the value of initial fluorescence of the vesicles, Ft is the value of fluorescence after incubation for t minutes with the protein, and Fmax is the value of fuorescence after addition of 0.2% of n-dodecyl maltoside.
Polyclonal anti-YqiC primary antibodies were obtained in mice immunized with purified YqiC. Immobilon-NC Transfer Membranes (Millipore) containing transferred proteins were blocked in 5% nonfat milk PBS for 1 h, and incubated with either a 1:200 dilution of polyclonal anti-YqiC or 1:200 anti-MBP mouse polyclonal antibodies. The secondary antibody used was goat anti-mouse IgG (Fc Specific) Peroxidase Conjugate (Sigma) at 1:1000 dilution. Positive signals were detected with Chemiluminiscent ECL Plus Western Blotting Detection System (Amersham Biosciences) on a Storm Image and Detection system (Molecular Dynamics).
Wild-type S. Typhimirium strain was grown in 80 mL LB medium to an OD600 of 1 and harvested by centrifugation at 4000 × g. The pellet was resuspended in 3 ml 20 mM Tris-HCl (pH 8.0) and 150 mM NaCl and mechanically lysed in a FastPrep instrument. Cell debris was removed by centrifugation for 30 min at 8000 × g. Subsequently, membranes were sedimented by ultracentrifugation for 1 h at 100,000 × g (4°C). The pellet was resuspended in a volume equivalent to that of the supernatant. Samples from the supernatant and pellet fraction were analyzed by immunoblotting.
Construction of yqiC S. Typhimurium mutant strain
Elimination of the yqiC gene was achieved by using Lambda Red-mediated recombination described previously . A lineal DNA fragment that contains the sequence for a chloramphenicol resistance cassette plus flanking regions of yqiC was constructed by PCR using the pair of primers 5'-CGCACTACAATAAGAGCTAACACTTACCAGTTCAGGGAAAGTGTAGGCTGGAGCTGCTTCG-3' and 5'-TGGATCGACTGGCGGAATGGCGGGCGCAGGTTTTACTTCTCATATGAATATCCTCCTTA-3'. 5 μg of this construction were introduced into strain LB5010 by electroporation. Chloramphenicol resistant colonies were then verified by PCR using a set of primers that hybridize within the insertion cassette and with an adjacent chromosomal region. Finally, isogenic strain was constructed by P22-mediated transduction of the mutant DNA into S. Typhimurium ATCC 14028. The substitution of the yqiC gene in this strain was verified by PCR and by the lack of expression of YqiC protein using western blot assay. The S. Typhimurium ΔyqiC::CAT mutant was named 14028 ΔyqiC::CAT.
To determine the 50% lethal dose (LD50) of the S. Typhimurium strains used, groups of seven 6-8 weeks old, female, BALB/c mice were infected intraperitoneally (i.p.) with serial 10-fold dilutions (from 1 × 101 to 1 × 105 CFU) of the wild type S. Typhimurium ATCC 14028 or 14028 ΔyqiC::CAT, and deaths were recorded for 28 days. For oral infections with S. Typhimurium ATCC 14028, 14028 ΔyqiC::CAT and 14028 ΔyqiC::CAT trans-complemented with pBBR-yqiC, mice were starved for food and water for 4 h. Following starvation, 105 CFU of each specific strain in 100 μl of phosphate-buffered saline (pH 7.4) were administered by oral gavage to each mouse. Survival of infected mice was recorded over 30 days. Inoculation doses were verified by serial dilution and plating into LB agar.
Cell invasion and intracellular replication
J774 murine macrophages and HeLa human epithelial cell lines were seeded at a density of 2 × 105 cells per well in 24-well culture plates. Stationary phase cultures of S. Typhimurium ATCC 14028, 14028 ΔyqiC::CAT and complemented strain 14028 ΔyqiC::CAT + pBBR-yqiC grown at 28°C overnight were added to the cells at a multiplicity of infection (MOI) of 10. Culture plates containing infected cells were centrifuged at 1000 rpm for 10 min and incubated at 37°C for 30 min to allow bacterial uptake and invasion. The extracellular bacteria were removed by washing thrice with PBS and incubating with 100 μg/ml gentamycin for 1 h. Thereafter, the cells were incubated with 25 μg/ml gentamycin for the rest of the experiment. After 1, 6 and 24 h, the cells were lysed with 1 mL of 0.1% Triton-X 100 per well and bacterial counts were determined by plating serial dilutions of the lysates on LB agar plates with appropriate antibiotic followed by incubation at 28°C.