Bacterial strains, plasmids and growth conditions
Bacterial strains and plasmids used in this study are listed in Additional file 1. For genetic manipulations, E. coli Top10 and DH5α and S. Typhimurium UMR1 (ATCC14028 Nalr; rdar28) were grown on Luria–Bertani (LB) agar plates or broth overnight at 37 °C. To induce the invasion phenotype, S. Typhimurium was grown overnight in LB broth + 0.3 M NaCl in standing culture, diluted 1:100 in fresh medium and grown until OD600 = 0.6. Antibiotics were ampicillin (100 μg ml−1), kanamycin (30 μg ml−1) and chloramphenicol (20 μg ml−1). For induction of bcsZ cloned in pBAD30, 0.1 % L-arabinose was used, if not otherwise stated.
Construction of mutants
BcsZ mutants were generated by homologous recombination [31] replacing the open reading frame (ORF), except for 40 nucleotides from the start and stop codon. For the non-polar bcsZ mutant, the tetRA cassette along with bcsZ homologous overhangs was PCR-amplified from S. Typhimurium TT946 and electroporated into S. Typhimurium UMR1 carrying pKD46 (primers in Additional file 2). Recovered colonies were purified at least twice on LB medium containing the corresponding antibiotics.
A 3xFLAG-tagged bcsC was constructed using the lambda-red recombination system [31]. The 3xFLAG-Km was amplified from pSUB11 [32] and the final construct was verified by sequencing.
Phage transduction of mutant alleles into a novel strain background was carried out with phage P22 HT105/1 int-201. Transductants were colony purified twice on LB agar plates containing 10 mM EGTA and appropriate antibiotics. All constructed mutants were verified by PCR with control primers located in the genes flanking the deleted ORF.
Plasmid construction
BcsZ was amplified using primer pair BcsZclon_N and BcsZclon_C (Additional file 2) from template S. Typhimurium UMR1. The resulting PCR product was digested with restriction endonucleases SacI and SphI, and ligated with SacI/SphI-restricted vector pBAD30. The ligation product was transformed into E. coli DH5α. The BcsZE56A mutant was constructed by overlapping PCR using primers containing the mutation (Additional file 2). Gene integrity was verified by DNA sequencing.
Phenotypic evaluation—Rdar morphotype assay
Five microliters of an overnight culture suspended in PBS (OD600 of 5) were spotted onto LB without salt agar plates supplemented with Congo red (40 μg ml−1) and Coomassie Brilliant Blue (20 μg ml−1) and incubated at 28 °C for up to 48 h. The development of the colony morphology and dye binding was analysed over time. The rdar morphotype indicates expression of the extracellular matrix components cellulose and amyloid curli fimbriae, while a pink, dry and rough (pdar) morphotype indicates cellulose production only. Control strains with expression of curli only, ΔbcsA, and without a distinct morphotype, ΔcsgD, are brown, dry and rough (bdar) and smooth and white (saw), respectively.
Calcofluor binding assay
For a qualitative agar plate assay, five microliters of an overnight culture suspended in PBS (OD600 of 5) were spotted onto LB without salt agar plates supplemented with Calcofluor (fluorescence brightener 28). Plates were incubated at 28 °C for up to 48 h and dye binding was documented at distinct time intervals.
The amount and distribution of cellulose was also assessed by fluorescent microscopy. LB without salt agar plates were incubated at 28 °C for 48 h and bacterial cells from the middle of the colony gently resuspended in 10 μg/ml Calcofluor dissolved in water. Cellulose production indicated by fluorescence intensity and cell aggregation was observed with an Olympus FV1000 confocal microscope.
A quantitative agar plate assay was performed as described [26]. Briefly, plate-grown cells were suspended to an optical density of OD600 = 0.1. Eight microliter was added into each well of a black 96-well microtiter plate with clear bottom (BD falcon) filled with 200 μl LB without salt agar containing 50 μg ml−1 Calcofluor, 0.1 % L-arabinose and 100 μg ml−1 ampicillin. After 24 h and 48 h incubation at 28 °C, the emission intensity at 460 nm was recorded (excitation at 355 nm) with a multilabel reader (VICTOR™ X3, Perkin Elmer).
Biofilm formation in M9 medium
To assess cellulose production in M9 minimal medium, bacteria grown on LB plates overnight were suspended in PBS. The suspension was inoculated in M9 medium adjusted to OD600 = 0.1. The culture was incubated at 28°C for 24 h with 200 rpm shaking after which bacterial clumping is indicative for cellulose production.
Pellicle formation
In S. Typhimurium, pellicle formation, air–liquid interface growth, in standing culture requires cellulose production. After overnight growth at 37 °C, 20 µl of the pre-culture was used to inoculate 180 µl of saltless LB broth in 96 well plates. The plate was incubated at 28 °C for 48 h. Pellicle strength was determined by subsequent addition of glass beads (Ø 0.75–1.00 mm, Retsch) using a tweezer until disruption.
Swimming and swarming motility
Swimming motility was observed in 0.3 % LB agar plates incubated for 5 h at 28 °C and 4 h at 37 °C after inoculation with a single colony from an overnight LB plate culture at 37 °C.
Swarming motility was analysed on 0.5 % LB agar plates supplemented with 0.5 % glucose at 28 and 37 °C after inoculation with a single colony from an overnight LB plate culture at 37 °C. The radius from the inoculation point to the edge of the motility zone was measured after 4 h. All experiments were done at least twice in duplicates.
Cellulase assay
To demonstrate cellulase activity of BcsZ, 5 µl of a suspension of S. Typhimurium UMR1 and derivatives in PBS (OD600 of 5), grown on LB plates overnight, were spotted onto LB without salt agar plates supplemented with 5 % carboxy methyl cellulose. Colonies were removed from the plate after 48 h incubation at 28 °C. 0.1 % Congo red was spread on the plates, incubated for 30 min at room temperature and plates were washed 3 times with 0.9 % NaCl for 15 min. A yellow spot on the red plate background indicated cellulase activity.
Flow cell biofilm experiment
Single colonies were inoculated in 3 mL of LB for overnight growth at 37 °C, 200 rpm. Cultures were adjusted to an OD600 of 0.04 in M9 minimal medium with 0.4 % glucose, 100 μg ml−1 ampicillin and 0.01 % L-Arabinose. The channels of BioFlux 48 well plate were primed with M9 medium with 0.4 % glucose. A bacterial suspension was seeded into the channels starting from the output side at 2 dyn/cm2 for 3 s and incubated at 28 °C for 1 h to allow attachment. The liquid in outlet well was removed, 900 µl fresh medium added to inlet well with flow 0.58 dyn/cm2 for 19 h. To terminate biofilm formation, 500 µl 70 % isopropyl alcohol was added to inlet well with flow of 0.58 dyn/cm2 for 1 h. After fixation, cells were stained with 25 µl propidium iodine added to inlet well at 0.55 dyn/cm2 for 5 min. After 15 min incubation in the dark, the fluorescence images were acquired with Zeiss LSM510META Confocal Microscope with a 10× objective.
Fitness experiment in LB medium
Overnight bacterial plate cultures of wild type and mutant were suspended and mixed in a 1:1 ratio in PBS. Approximately 103 cells were added to 50 ml LB broth to incubate with shaking (220 rpm) at 37 °C. After 6 and 16 h of growth, cell numbers were estimated by differentially plating 10-fold dilutions onto LB agar plates ± appropriate antibiotics for estimation of viable counts (cfu). The competitive index (CI) of wild type towards mutant was calculated as described [33].
Protein localisation assays
To assess whether BcsZ is secreted, cells were grown in LB medium (35 ml in a 50 ml flask) up to OD600 1.5. Aliquots of 1.5 ml supernatant and cell-associated protein from 0.3 ml suspension were analysed for BcsZ expression by Western blot analysis after trichloroacetic acid precipitation.
To assess surface association of BcsZ, proteinase K digestion of whole bacterial cells was performed. In brief, 10 mg of bacteria were harvested after 48 h of growth on LB without salt plates at 28 °C. Cells were resuspended in 1 ml of Tris–HCl buffer pH 7.5, 5 mM CaCl2 and 40 µg/ml chloramphenicol. Aliquots of the bacterial suspension were digested with different concentrations of proteinase K for 2 h at 37 °C. The suspension was adjusted to 10 % trichloroacetic acid and incubated on ice for 30 min. The reaction mixture was centrifuged and the pellet washed three times with 70 % ethanol. The dried pellet was reconstituted in 80 µl SDS sample buffer, boiled for 5 min and loaded on the gel. After protein separation, western blot analysis was performed to detect BcsZ, OmpR (cytoplasmic control protein) and DsbA (periplasmic control protein).
Creation of an antibody against BcsZ
The conserved amino acid sequences specific for BcsZ, KKDYISQQGRVIDPGDARK and DWVRYESKQGWQLKAEK, were synthesized inhouse (Helmholtz Center for Infection Research, Braunschweig, Germany). The two peptides were used for production of a polyclonal antiserum in mice (Neosystem Group SNPE, France). The serum was loaded onto CnBr-activated Sepharose with peptides containing a N-terminal cysteine residue coupled. The column was washed with 0.1 M acetate buffer and fractions were eluted with 0.2 M acetate buffer and immediately neutralized with 1.5 M Tris–HCl, pH 8.8. In a second step, fractions were eluted with 100 mM Triethylamine buffer, pH 11.5 and neutralized immediately with 1.5 M Tris–HCl, pH 4.5. The fractions containing proteins were combined, buffer exchanged to PBS using Centricon-columns YM-30 and the antibody concentrated to 2 mg/ml.
SDS-PAGE and Western blot analysis
Cell extracts were separated on an 4/8 % SDS-PAGE gel and electro-transferred onto a PVDF membrane (Millipore Corp.) at 120 mA for 4 h. Membranes were blocked using 5 % BSA and 5 % non-fat dry milk in TBST [20 mM Tris–HCl (pH 7.5), 150 mM NaCl and 0.05 % Tween-20] overnight. Anti-BcsZ peptide antibody was used at 1:3000 dilution. Detection of CsgD was carried out using polyclonal anti-CsgD peptide antibody (1:5000) as the primary antibody [30]. Anti-OmpR and anti-DsbA antibodies were used as previously described. Goat anti-rabbit immunoglobulin G (Jackson ImmunoResearch Laboratories) conjugated with horseradish peroxidase at a 1:5000 or 1:2000 dilution, respectively, was the secondary antibody. FLAG primary antibody (Sigma) was used at 1:2000 dilution with peroxidase-conjugated AffiniPure Goat Anti-Mouse IgG (Jackson ImmunoResearch) secondary antibody at 1:3000 dilution. After washing, binding of antibody was detected using the ECL light detection reagent (Roche). Visualization of bands was performed using FUJI LAS1000-plus chemiluminescence imaging system (Fuji, Stamford, CT, USA).
Analysis of curli fimbriae expression
The major subunit of curli fimbriae, the CsgA protein, was enriched, subsequently treated with formic acid and detected on a protein gel [8]. Briefly, 3 mg of an overnight culture grown on LB without salt plate at 28 °C was resuspended in PBS and centrifuged. The pellet was re-suspended in TE buffer (10 mM Tris, 1 mM EDTA and 0.2 % SDS; pH = 7.5), boiled for 45 min at 95 °C and centrifuged at 14,000 rpm. The pellet was washed with H2O two times and dried in a Speed Vac for 1 h. The semi-purified curli were taken up in 100 % formic acid, incubated on ice for 15–20 min and formic acid was evaporated. The denatured pellet was dissolved in 200 μl SDS sample buffer, boiled for 15 min at 95 °C and loaded on a 15 % SDS-PAGE gel. CsgA was visualized by Coomassie staining of the gel.
Human epithelial cell invasion assay
The human epithelial cell line HT-29 (ATCC HTB 38, colon, colorectal adenocarcinoma) was grown to confluence in 24-well plates in RPMI-1640 medium (Life Technologies) supplemented with 25 mM HEPES, 2 mM l-glutamine and 10 % fetal calf serum (Sigma/Aldrich) at 37 °C in 5 % CO2. Bacteria were diluted and seeded on confluent HT-29 cells grown in 24-well plates at a multiplicity of infection of 1.7, which corresponds to 107 cfu ml−1. One-hour post infection, medium containing gentamicin at 100 μg ml−1 was added for 1 h to kill extracellular bacteria. Cells were gently washed twice with PBS and disrupted with 1 % Triton X-100 (Sigma Chemical) in PBS. The number of intracellular bacteria was determined by estimation of colony-forming units (cfu) on agar plates. An ΔompR mutant was used as a negative control [14]. The invasion rate is defined as (cfu recovered inside cells after 1 h/cfu at time of inoculation). The relative invasion rate in % is defined as (invasion rate of mutant/invasion rate of wild type) * 100. Presented results are based on at least three biological replicates consisting of four technical replicates each.
Macrophage infection assay
The murine RAW264.7 macrophage cell-line was cultured in RPMI medium (Gibco, UK) supplemented with 10 % fetal bovine serum (Gibco), 10 mM l-glutamine (Sigma), 10 mM HEPES (Sigma). Overnight bacterial plate cultures were opsonized in 10 % pre-immune BALB/c mouse serum for 30 min at 37 °C prior to infection at a MOI of 10. The macrophages were activated overnight with 10 ng/ml IFN-γ prior to infection. The uptake and intracellular proliferation rate of bacteria was assessed after 2 and 16 h, respectively, counting gentamycin protected bacteria by viable counts (cfu) after hypertonic lysis of macrophages [34] For the competition experiment, strains were mixed at a ratio of 1:1 prior to infection. The competitive index (CI) was calculated as described [33].
Mouse experiments
Competition experiments between wild type and mutants were performed in 6–8 week old female BALB/c J mice (Taconic, Denmark). Overnight bacterial plate cultures were mixed at a 1:1 ratio in PBS and approximately 108 cells/100 μl were administered orally. Livers and spleens of 5 mice/group were collected on day 1 and 3 post infection, homogenized and plated onto LB agar plates ± appropriate antibiotics for estimation of viable counts (cfu). Competitive index (CI) of wild type towards mutant was calculated as described [33]. Experiments were performed at the Department of Microbiology, Tumor and Cell Biology (MTC) animal facility, Karolinska Institutet, Stockholm, Sweden in accordance with national and institutional guidelines (ethical permit N133/13).
Phylogenetic analysis
BcsZ was compared to all experimentally verified cellulases of family 8 glycosidases from the CAZy database (http://www.cazy.org/Citing-CAZy.html; [35]) and cellulases representative for the different classes of cellulose biosynthesis operons [25]. Protein sequences were aligned with Clustal X 2.1 using standard parameters, alignments manually curated and the tree was drawn with TreeView version 1.6.6.
Statistical analysis
Prism 5 (GraphPad Software) was used to calculate statistics. Statistical analysis was performed using a paired Student’s t test or using the Kruskal–Wallis assessment with subsequent Dunn’s test.