A bovine epithelial cell line (MDBK) was purchased from the American Type Culture Collection (Manassas, VA) and maintained on Dulbecco's modified Eagle medium (Invitrogen, Carlsbad, CA) supplemented with 10% heat-inactivated fetal bovine serum (Gemini Bio-products, Woodland, CA) as described before (16
Mycobacterium avium subsp. paratuberculosis ATCC 19698, a bovine clinical isolate from an animal with Johne's disease, was grown at 37°C on either modified Middlebrook 7H9 broth or 7H11 agar (Difco Laboratories, Sparks, MD) supplemented with 2 mg/liter of mycobactin J (Allied Monitor, Fayette, MO), 10% (vol/vol) oleic acid-albumin-dextrose-catalase (Hardy Diagnostics, Santa Maria, CA), and 0.05% Tween 80 (Sigma, St. Louis, MO). For the invasion assay, individual colonies were selected and resuspended in Hanks' balanced salt solution (HBSS) to give turbidity equivalent to a 0.5 McFarland standard. To minimize clumping, the bacterial suspension was passed through a 26-gauge needle 10 times and then allowed to settle for 5 min. Only the top fraction of the suspension containing dispersed bacteria was used for the assays.
Construction of an M. avium subsp. paratuberculosis transposon library.
To identify genes associated with invasion, a transposon library was constructed by transformation of M
strain 19698 competent cells with the temperature-sensitive plasmid pTNGJC-KAN, a plasmid containing the transposon Tn5367 harboring a kanamycin-resistant gene, as previously described (27
). Briefly, M
competent cells were centrifuged at 3,000 × g
for 15 min at 4°C and washed in a solution of 10% glycerol and 0.1% Tween 80. Washing and centrifugation were repeated three times, and the pellet was resuspended in 1 ml of 10% glycerol (Sigma). Bacteria were electroporated with plasmid pTNGJC-KAN by use of a GenePulser X cell (Bio-Rad, Hercules, CA), plated onto 7H11 agar plates with 400 μg/ml of kanamycin (Sigma), and incubated at 30°C for 3 weeks. Twenty colonies were randomly selected and screened by PCR for the presence of the kanamycin gene by use of the primers 5′-TAATGTCGGGCAATCAGGTG-3′ (forward) and 5′-TGTTCAACAGGCCAGCCA-3′ (reverse). PCR cycling was as follows: 35 cycles of 95°C for 30 s, 57°C for 1 min, and 72°C for 1 min. Prior to the first cycle, a temperature of 95°C was held for 5 min, and at the end of the last cycle, a temperature of 72°C was maintained for 10 min. The confirmed kanamycin-resistant colonies were then grown in 7H9 broth containing kanamycin at a nonpermissive temperature (40°C) for an additional week. The suspensions were diluted and placed onto 7H11 agar with kanamycin at 37°C. Colonies were picked randomly and collected in individual wells of a 96-well tissue culture plate, generating a library of 1,980 individual transposon mutants. Because pTNGJC-KAN contains a temperature-sensitive mycobacterial origin of replication, the shift in temperature eliminated the plasmid, and all surviving kanamycin-resistant cells necessarily contained the transposon in random positions of the bacterial chromosome.
Approximately 600 transposon mutants were individually screened for the ability to enter MDBK cells, as previously described (11
). Briefly, 105
MDBK cells/ml growing in 24-well plates were infected with individual mutants (multiplicity of infection [MOI], 100) and incubated at 37°C in a 5% CO2
incubator for 4 h. The cell monolayers were then washed three times with HBSS to remove extracellular bacteria and then treated with 1 ml of tissue culture medium supplemented with 200 μg/ml of amikacin (Sigma) for 2 h at 37°C. Following treatment, the monolayers were washed twice with HBSS, and the viable intracellular bacteria were released by incubation with 0.5 ml of 0.1% Triton X-100 (Sigma) in sterile water for 10 min. After the addition of 0.5 ml of Middlebrook 7H9 broth, samples were disrupted by vigorous pipetting. Lysates were collected and viable bacteria were quantified by plating for CFU onto Middlebrook 7H11 agar containing mycobactin J. The percentages of invasion of mutants and wt bacterium were calculated as the percentages of the inoculated bacteria that were recovered from the cell lysate.
Identification of transposon-inactivated mutants.
Chromosomal DNA of the low-invasion mutants was extracted and purified as previously described (15
). The isolation of DNA sequences flanking transposon insertions was carried out using a nonspecific nested suppression PCR method previously described by Tamme et al. (23
). Briefly, a first round of PCR was conducted at a low annealing temperature by use of 1.6 μM of a transposon-specific primer (5′CCA TCA TCG GAA GAC CTC-3′) and a polymerase lacking exonuclease activity (FideliTag; USB, Cleveland, OH) to generate a mixture of products, including fragments of the desired flanking sequence. PCR was carried out from 100 ng of genomic DNA in a total volume of 30 μl in 3% (vol/vol) dimethyl sulfoxide and 15 μl of FideliTag PCR master mix containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 3 mM MgCl2
, 0.4 mM deoxynucleoside triphosphates, and FideliTag. PCR cycling conditions were as follows: 35 cycles of 95°C for 30 s, 50°C for 1 min, and 72°C for 4 min. Prior to the first cycle, a temperature of 95°C was held for 5 min, and at the end of the last cycle a temperature of 72°C was maintained for 10 min. Only 1 μl of the first-round amplification was then used as a template for the second-round PCR (nested PCR) using a primer that is 6 nucleotides (GACCCC) longer at the 3′ end than the primer used in the first round of PCR and a polymerase exhibiting exonuclease activity (Pfu
DNA polymerase; Stratagene, La Jolla, CA). Reactions were carried out in a total volume of 50 μl in 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2 mM MgCl2
, 0.4 mM deoxynucleoside triphosphates, 5% (vol/vol) dimethyl sulfoxide, 1.6 μM of the primer, and 2.5 U of Pfu
DNA polymerase. The PCR cycling was the same as for the first PCR, except the annealing cycle was 30 s at 56°C. The PCR products of the second amplification were run in a 1% agarose gel, and each PCR band that appeared in the gel was excised and purified using a DNA gel extraction kit (Stratagene) before cloning into the pCR2.1 TOPO vector (Invitrogen). Samples were sequenced using the M13 reverse primer at the Center for Gene Research and Biotechnology, Oregon State University, Corvallis, OR. Database searches and sequence comparisons were performed using the BLAST server from the National Center for Biotechnology Information. To determine a function for the identified genes, we searched for M
homologues with TubercuList (http://genolist.pasteur.fr/TubercuList/
) and BLAST.
Amplification of MAP3464 and 16S rRNA genes from genomic DNA.
MAP3464 and 16S rRNA genes were amplified from genomic DNA by use of specific primers and the GC-rich PCR system following the manufacturer's instructions (Roche, Penzberg, Germany). The primers for the amplification of the MAP3464 gene were 5′-TTTGAATTCATGCCCACCCCTCCGGACGT-3′ (forward) and 5′-TCGAAGCTTTTAAGTTGCGGCGCTGGTGTG-3′ (reverse). For the amplification of the 16S rRNA, primers 5′-CGAACGGGTGAGTAACACG-3′ (forward) and 5′-TGCACACAGGCCACAAGGGA-3′ (reverse) were used. PCR amplification was carried out at 35 cycles of 95°C for 30 s, 62°C for 30 s, and 72°C for 2 min.
RNA extraction, cDNA synthesis, and reverse transcription-PCR (RT-PCR).
To determine whether the MAP3464 gene was upregulated when the bacterium was exposed to the host cell, MDBK cells were incubated with wt M
in HBSS at 37°C for 15, 30, and 60 min. The supernatant was collected and bacteria were pelleted at 3,500 × g
for RNA preparation, as previously described (10
). Total RNA from bacteria incubated at 37°C in HBSS for 60 min was also prepared as a control. Bacterial pellets were mixed with 1 ml of TRIzol reagent (Invitrogen), and RNA was isolated by rapid mechanical agitation in a bead beater. To remove cellular debris, cells were centrifuged at 13,000 × g
for 5 min at 4°C. The supernatant was removed and added to a 2-ml phase lock gel heavy (Eppendorf, Hamburg, Germany) containing 300 μl of chloroform-isoamyl alcohol (24:1). Samples were centrifuged for 10 min at 4°C, and the aqueous layer was collected, extracted with the same volume of phenol-chloroform, and precipitated with isopropanol. The pellet was then washed with 75% ethanol and dried at room temperature for 10 min. RNA samples were treated with DNase I (Clontech, Palo Alto, CA) for 1 h at 37°C, followed by precipitation with ethanol. Total RNA was quantified by measuring absorbance at 260 nm, and quality was determined by measuring the 260/280-nm absorbance ratio. Ratios of ≥1.8 were considered acceptable. RNA was then electrophoresed on a 1% denaturing agarose gel to confirm quality.
Total RNA was reverse transcribed using the SuperScript first-strand synthesis system for RT-PCR following the manufacturer's instructions (Invitrogen). Total RNA (1.5 μg) was incubated with 1 μl of a 10 mM concentration of deoxynucleotide triphosphate mix and 1 μl of random hexamers at 65°C for 5 min. Samples were then mixed with 2 μl of 10× RT buffer, 4 μl of 25 mM MgCl2, 2 μl of 0.1 M dithiothreitol, 1 μl of RNase out recombinant RNase inhibitor (40 U/μl) and incubated at 25°C for 2 min. Finally, 1 μl of SuperScript II reverse transcriptase (50 U/μl) was added to each tube, and the samples were incubated at 42°C for 50 min. The reaction was terminated at 70°C for 15 min. The tube was placed on ice and centrifuged briefly, and 1 μl of RNase H was added to each sample and incubated for 20 min at 37°C. For RT-PCR, the MAP3464 and 16S RNA genes were amplified from 1 μl of cDNA as described above. Ten microliters of the PCR products was separated on a 1% agarose gel and visualized by staining with ethidium bromide. The 16S RNA gene was used as a constitutively expressed control to ensure that the same amount of RNA was added to each reaction mixture.
Quantitative real-time RT-PCR.
Quantitative fluorogenic amplification of cDNA was performed using an iCycler real-time detection system and SYBR green technology (Bio-Rad, Hercules, CA) as previously described (27
). Briefly, PCRs were carried out in 50-μl reaction mixtures consisting of 25 μl of IQ SYBR green supermix, 1 μl of each primer (10 μM), 1 μl of cDNA, and 23 μl of water. Each PCR amplification consisted of denaturation at 95°C for 30 s, annealing at 62°C for 30 s, and extension at 72°C for 2 min. Real-time PCR efficiency was determined using a dilution series of cDNA template with a fixed concentration of primers. Slopes calculated by the LightCycler software were used with the following formula to calculate efficiency (E
. Cycle threshold (CT
) is defined as the fraction cycle number at which the fluorescence reaches 10 times the standard deviation of the baseline and was quantified as described in the user bulletin for the ABI PRISMS 7700 sequence detection system. Since the 16S RNA gene is constitutively expressed, target genes from both control and experimental groups were normalized to the expression level of the 16S RNA gene. To determine the change (n
-fold) in gene expression, the following formula was used: change (n
, where ΔCT
(target) − CT
(16S) and Δ(ΔCT
) is ΔCT
(experimental) − ΔCT
(control). Standard deviations were calculated for the samples.
Complementation of the ΔOx mutant.
To complement the ΔOx mutant, the MAP3464, MAP3465, and MAP3466 genes were PCR amplified using wt genomic DNA as a template. The sequences of the upstream and downstream primers were as follows: MAP3464 forward, 5′-TTT GGA TCC ATG CCA CCC TCC GGA CGT-3′; MAP3464 reverse, 5′-TTT GGA TCC TTA AGT TGC GGC GCT GGT GTG-3′; MAP3465 forward, 5′-TTT GAA TTC ACA CCA GCG CCG CAA CTT AAT-3′; MAP3465 reverse, 5′-TTT GAA TTC GGC CGG CCT CAG CGT TTC AG-3′; MAP3466 forward, 5′-AAA GTT AAC CTG GGT GTG ATG ACT CGC TCC-3′; and MAP3466 reverse, 5′-AAA GTT AAC TCA GCG CCG CAA CCG GAT GCG-3′. The MAP3464, MAP3465, and MAP3466 PCR products were cloned into the pGEM vector (Promega) and verified by sequencing using the T7 promoter. Subsequently, the PCR products were digested with BamHI, EcoRI, and HpaI, respectively, prior to cloning into pMAV261-AprII, a vector containing the hsp60 promoter and an apramycin-resistant gene. Plasmids pMAV261-MAP3464 and pMAV261-MAP3464-65-66 were electroporated into ΔOx competent cells as described before. The transformants were then plated onto 7H11 agar plates containing 200 μg/ml of apramycin and kanamycin. To confirm transformation, the MAP3464 gene was identified in the complemented mutant clones by PCR amplification as described before.
Cell surface biotinylation.
Biotinylation of bacterial surface proteins for Western blotting was performed using a protein biotinylation system (Amersham, Little Chalfont, Buckinghamshire, United Kingdom). Briefly, three 125-cm2 flasks of 90 to 95% confluent MDBK cells were infected at 37°C for 1 h with wt M. avium subsp. paratuberculosis or with the ΔOx mutant. The supernatant was collected and extracellular bacteria were pelleted at 3,500 × g for 20 min and resuspended in 40 mM bicarbonate buffer, pH 8.6, at a concentration of 1 McFarland standard. Three milliliters of the suspension was surface labeled by incubation with 40 μl of biotinamidohexanoic acid N-hydroxysuccinimide ester (20 mg/ml) (Sigma) in dimethylformamide for 1 h at 4°C. Unreacted biotinylation reagent was removed by three successive washes in ice-cold phosphate-buffered saline buffer, and biotinylated bacteria was subsequently lysed with 200 μl of ice-cold lysis buffer (250 mM NaCl, 25 mM Tris [pH 7.5], 5 mM EDTA [pH 8], 1% NP-40, and a protease inhibitory cocktail) by rapid mechanical agitation in a bead beater. wt and ΔOx bacteria grown in 7H9 medium biotinylated and lysed in the same manner were used as controls. Cell lysates were centrifuged at 10,000 × g for 2 min at 4°C, and protein concentration in the clarified supernatant was measured using the Bradford assay (Bio-Rad). Total proteins (0.5 μg) were analyzed by electrophoresis on a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel (Bio-Rad) at 120 V for 2 h and transferred to a nitrocellulose membrane in a trans-blot semidry apparatus (Bio-Rad). Protein-containing membranes were blocked overnight in 5% (wt/vol) dry milk in Tris-buffered saline (TBS). After several washes in TBS with 0.1% Tween (TBST), the membrane was probed with 1:1,500 (vol/vol) of horseradish peroxidase (HRP)-conjugated streptavidin for 1 h. The membranes were washed again with TBST and proteins were visualized with an enhanced chemiluminescence detection kit (Amersham).
Immunoprecipitation and Western blotting.
MDBK cells growing in a six-well plate were infected with wt M. avium subsp. paratuberculosis or with the ΔOx mutant at 37°C for 15, 30, 60, or 120 min (MOI, 1:100). Uninfected MDBK cells were used as the control. Monolayers were then washed with HBSS and lysed with 1 ml of sterile water in the presence of 0.05 M phenylmethylsulfonyl fluoride in isopropanol. Cells were gently scraped off the plates, passed through a 21-gauge needle 10 times, and centrifuged at 3,500 × g for 10 min at 4°C. The protein concentration in the supernatant was measured using the Bradford assay (Bio-Rad). Equal amounts of cell lysates were incubated for 1 h at 4°C with 10 μl of α-phosphotyrosine or α-phosphothreonine monoclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA). Immunocomplexes were captured by adding 30 μl Protein A/G Plus-agarose immunoprecipitation beads (Santa Cruz Biotechnology) and gently rocking at 4°C for 30 min. Beads were washed several times with phosphate-buffered saline, and the captured proteins were resolved on a 12% SDS-PAGE gel at 120 V for 2 h and transferred to a nitrocellulose membrane using a semidry transfer apparatus (Bio-Rad). Protein-containing membranes were blocked overnight with 5% dry milk in TBS. After several washes in TBST, the membranes were probed with α-Cdc42, α-RhoA, and α-Rac antibodies (Santa Cruz Biotechnology) in TBS. Blots were washed with TBST and incubated with HRP-conjugated secondary antibody (Amersham) in TBS for 1 h. Proteins were visualized with an enhanced chemiluminescence detection kit (Amersham) followed by autoradiography.
Affinity precipitation of active Cdc42, in-gel tryptic digestion, and mass spectrometry.
Affinity precipitation of active Cdc42 from MDBK-infected cells was carried out using the EZ-Detect Cdc42 activation kit according to the manufacturer's instructions (Pierce, Rockford, IL). Briefly, MDBK cells growing in 125-ml flasks were infected with wt M. avium subsp. paratuberculosis or with the ΔOx mutant for 30 min at 37°C. After infection, the cells were rinsed once with ice-cold TBS and lysed with a cold lysis/binding/wash buffer containing 1 mM phenylmethylsulfonyl fluoride. Cells were scraped from the plate and clarified by centrifugation at 10,000 × g for 15 min in a microcentrifuge. Equal amounts of cell lysates were incubated in a spin column with 20 μg of the p21-binding domain (PDB) of activated kinase 1 (Pak1) in the presence of a SwellGel-immobilized glutathione disc (GST-Pak1-PDB) at 4°C for 1 h. After incubation, the mixture was centrifuged at 7,200 × g for 30 s to remove unbound proteins. The resin was washed three times with lysis/binding/wash buffer, and the sample was eluted in 50 μl of 2× SDS sample buffer without β-mercaptoethanol after boiling at 95°C for 5 min. Samples were centrifuged at 7,200 × g for 2 min and analyzed on a 12% SDS-PAGE gel. As a positive control, cell lysates from uninfected cells were treated with 0.1 mM GTPγ at 30°C for 15 min to activate endogenous Cdc42.
Coomassie dye-stained 1-D gel electrophoresis-separated proteins were digested using an in-gel tryptic digestion kit following the manufacturer's instructions (Pierce, Rockford, IL). Protein bands were excised from the one-dimensional (1-D) acrylamide gel and destained at 37°C for 1 h with 200 μl of 50 mM ammonium bicarbonate in a 50% (vol/vol) acetonitrile solution (destaining solution). Reduction and alkylation of the cystine residues from proteins were performed by adding 50 mM Tris[2-carboxyethyl]phosphine and 100 mM iodoacetamide, respectively. Samples were washed twice by incubation with the destaining solution at 37°C for 30 min, dehydrated by incubation with 50 μl of 100% acetonitrile for 15 min at room temperature, and dried at room temperature for 10 min. For peptide extraction, protein bands were digested with 20 ng of sequencing-grade modified trypsin and 25 μl of 25 mM ammonium bicarbonate at 37°C for 4 h. The digestion mixture was removed and placed in a clean tube for liquid chromatographic separation and mass spectrometry using a Waters nanoAcquity high-pressure liquid chromatography instrument connected to a Waters Q-Tof Ultima global mass spectrometer (Milford, MA). Briefly, 5 μl of each sample was loaded onto a Waters symmetry C18 trap at 4 μl/min. The peptides were then eluted from the trap onto a Waters Atlantis analytical column (10 cm by 75 μm) at 350 nl/min. Peptide “parent ions” were monitored as they eluted from the analytical column with 0.5-s survey scans from m/z 400 to 2,000. Up to three parent ions per scan that had sufficient intensity and had two, three, or four positive charges were chosen for mass spectrometry. The raw data were processed with MassLynx 4.0 to produce pkl files, a set of smoothed and centroided parent ion masses with the associated fragment ion masses. The pkl files were searched with Mascot 2.0 (Matrix Science Ltd, London, United Kingdom) database-searching software and the Swiss protein database, using mass tolerances of 0.2 for the parent and fragment masses. Peaks Studio (Bioinformatics Solutions Inc., Ontario, Canada) was also used to search the data, using mass tolerances of 0.1.
Each experiment was repeated at least three times, and the results were expressed as means ± standard deviations of the means. The significance of the difference between the experimental and control group was analyzed by Student's t test. P values of <0.05 were considered significant.