Bacterial strain
A. pleuropneumoniae MBHPP147 is a non-hemolytic derivative of the serotype 1 reference strain S4074. Construction of strain MBHPP147 is described in patent EP 0810283A2. Briefly, a streptomycin and nalixidic acid resistant mutant of A. pleuropneumoniae strain 4074 (MBHPP104) was mated with an Escherichia coli SM10 λpir strain carrying a plasmid containing a deleted apxIC gene. Positive exconjugants were selected on solid medium containing nalixidic acid and gentamycin and a mutant with reduced haemolytic activity on solid CBM containing 2% sheep erythrocyte was selected and named MBHPP111. Once the deletion was confirmed, this mutant was then mated with an E. coli S17-1 λpir strain carrying a plasmid containing an in-frame deletion in the apxIIC gene. Exconjugants were selected on solid media supplemented with nalixidic acid and gentamycin. A mutant lacking hemolytic activity on blood agar was selected and was named MBHPP147. In summary, strain MBHPP147 contains deletions in both the apxIC and apxIIC genes. The protoxins ApxIA and ApxIIA are formed and exported but are not acylated, and thus not activated, due to the absence of the ApxC proteins. The bacterial strain was cultured on brain heart infusion (BHI) broth (Oxoid Ltd, Basingstoke, Hampshire, England) or on BHI agar supplemented with 5 or 15 μg/mL nicotinamide adenine dinucleotide (NAD) (BHI-NAD), respectively, at 37°C in 5% CO2. A. pleuropneumoniae MBHPP147 was transformed by electroporation with pMC-Express, a plasmid for GFP expression [15], as described before [20]. Transformants were selected on BHI agar with chloramphenicol (1 μg/ml).
Cell culture
The St. Jude porcine lung epithelial cell line (SJPL) (St. Jude Children’s Hospital, Memphis, TN, USA) [21, 22] was grown in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Burlington, ON, Canada) supplemented with 10% of fetal bovine serum (Gibco), 1% sodium pyruvate (100×, Gibco), 1% L-glutamine (200 mM, Gibco), 1,5% MEM nonessential amino acids (100×, Gibco), 1% penicillin/streptomycin (100×, Gibco), 1% fungizon (250 μg/ml, Gibco) and 0,1% gentamicin [21]. Cells were grown at 37°C in 5% CO2. Despite its name, the SJPL cell line was recently shown to be from monkey origin [22].
Cytotoxicity detection assay
For the cytotoxicity detection assay, 105 epithelial cells in complete DMEM without antibiotics were seeded into wells of 24-well tissue culture plates (Sarstedt, Numbrecht, Germany) and incubated O/N. Cells were infected with A. pleuropneumoniae strain MBHPP147. Bacteria from an overnight culture grown at an OD600nm of 0.6 were diluted in complete DMEM cell culture medium without antibiotics and supplemented with NAD (5 μg/mL) to a concentration of 106 CFU/ml. One ml of this suspension was added to each well at a multiplicity of infection (MOI) of 10:1, and the plates were incubated for up to 96 hours. The cellular cytotoxicity was determined using the lactate dehydrogenase (LDH)-measuring CytoTox 96 nonradioactive cytotoxicity assay (Promega, Madison, WI) as prescribed by the manufacturer. Non-infected cells were used as a negative control, while total lysis of cells by a treatment with the lysis solution represented the 100%-cytotoxicity positive control. Optical densities were measured at 490 nm with a microplate reader and percentage of cytotoxicity was calculated using the following formula: (OD490 treated well/OD490 positive lysis control well) × 100.
Biofilm assay with an abiotic surface
A static microtiter plate biofilm assay was used as described by Tremblay et al. [10]. The wells of a sterile 96-well polystyrene microtiter plate (Costar® 3599, Corning, NY, USA) were filled in triplicate with a dilution (1/100) of an overnight bacterial culture. Following an incubation of 4 to 24 h at 37°C, the medium was removed by aspiration and the wells were then washed by immersion in water. The water was then removed by aspiration and the excess water was removed by inverting plates onto a paper towel. The wells were then filled with 100 μL of crystal violet (0.1%) and the plate was incubated for 2 min at room temperature. After removal of the crystal violet solution, the plate was washed and dried in 37°C for 30 min and 100 μL of ethanol (70%) were added to the wells. Absorbance was measured at 590 nm using a spectrophotometer (Powerwave, BioTek Instruments, Winooski, VT, USA).
Biofilm assay with a biotic surface
Bacteria (500 μl) from an overnight culture were transferred in 5 ml of fresh BHI-NAD and were grown at 37°C with agitation (200 rpm) until an OD600nm of 0.6. Bacteria were then diluted to a concentration of 2.5 × 106 CFU/ml in complete DMEM cell culture medium without antibiotics and supplemented with NAD. The medium was removed from wells of 96 well-microtiter plates containing confluent monolayers of SJPL cells and 100 μL of the bacterial dilution in DMEM-NAD were added (MOI of 10:1). Plates were incubated at 37°C in 5% CO2 for 3, 6, 24, or 48 hr. To disperse the biofilms, 100 μL of a Dispersin B solution (0.4 μg/mL in Dubelcco’s phosphate-buffered saline [DPBS]; Kane Biotech Inc., Winnipeg, MB, Canada) was added to the well and the plate was incubated for 5 min at 37°C. After the desired incubation period, culture medium was removed and the wells washed 3× with PBS.
Quantification of A. pleuropneumoniaeattached to SJPL cells
Adherent bacteria were quantified as described previously [13]. Briefly, SJPL cells were seeded into 24-well tissue culture plates as described in the cytotoxicity assay. Bacteria were prepared as described above and 1 ml of the bacterial suspension was added to each well. The plates were incubated for 3, 6, 24 and 48 hours. When necessary, bacteria in biofilms were dispersed by adding 100 μL of a Dispersin B solution (4 μg/mL in DPBS) to the well and the plate was incubated for 5 min at 37°C. Nonadherent bacteria were removed by washing four times with DPBS. Cell with adherent bacteria were released from the wells by adding 100 μl of 1× trypsin-EDTA (Gibco) and resuspended in 900 μl DPBS buffer. The recovered suspension was serially diluted and these were plated on agar to determine the number of adherent bacteria.
Confocal laser scanning microscopy
Biofilms were grown on a biotic or an abiotic surface as described above. SJPL cells were fixed in formaldehyde (4%) for 1 hr at room temperature and wells were washed 3 more times with PBS. The wells were filled with 100 μl of WGA–Oregon green 488 or WGA-AlexaFluor 633 (Invitrogen, Eugene, OR, USA) diluted 1/100 in PBS or/and Phalloidin-AlexaFluor 594 (Invitrogen) diluted 1/40 in PBS and the plate was incubated for 30 min at room temperature in the dark. The plate was then washed with water and filled with 100 μl PBS. The plate was observed with a confocal microscope (Olympus FV1000 IX81, Markham, ON, Canada). The fluorophores were excited and detected as prescribed by the manufacturers. The images were acquired using the Fluoview software (Olympus).