Bacterial strains and plasmids
S. suis isolates were grown in Todd-Hewitt broth (Oxoid, London, United Kingdom) and plated on Columbia blood base agar plates (Oxoid) containing 6% (vol/vol) horse blood. Escherichia coli was grown in Luria Broth and plated on Luria Broth containing 1.5% (wt/vol) agar. If required, erythomycin was added at 1 gml-1 for S. suis and at 200 gml-1 for E. coli. S. suis strain S735 complemented with a plasmid containing a 3kb genomic fragment derived from strain 10 (S735-pCOM1-V[10]) and the other S. suis strains used in this study have been previously described [11],[14] (Figure4).
Complementation of S. suis strain S735
S735 was complemented with plasmid pCOM1 containing one of the two ORFs in the V[10] operon (i.e. orf2[10], or folC[10]) preceded by the putative promoter region of the operon from strain 10 or with plasmid pCOM1 containing orf2 and the cognate upstream promoter from strain S735 (orf2[S735]) (Figure4). To construct these plasmids, primers with restriction sites were designed to amplify orf2[10] or orf2[S735] (comE1 comE2), folC[10] (comE4 comE6) or the promoter region of the operon (comE1 comE3) (Table3). The resulting PCR products orf2[10] and orf2[S735] were digested using restriction enzymes Sac I and BamH I, cloned into pKUN19 [22], digested with the same restriction enzymes and subsequently cloned into pCOM1, yielding pCOM1-orf2[10] and pCOM1-orf2[S735], respectively. The PCR amplicon of folC[10] was digested using restriction enzymes Sm aI and BamH I and cloned into pKUN19 cleaved with the same restriction enzymes. The PCR product comprising the promoter region of V[10] was cloned in front of folC[10] using restriction enzymes Sac I and Sma I. Subsequently, the complete fragment of promoter V[10] folC[10] was digested from pKUN19 using Sac I and Bam HI and cloned into pCOM1 digested with the same restriction enzymes, yielding pCOM1-folC[10]. To confirm that the fusion product of promoter folC[10] was transcribed, in vitro transcription/translation was performed using 35S-methionine. A clear band of the molecular weight of FolC (46.8kDa) was detected demonstrating that the fusion product could be expressed and translated. All plasmids were introduced into S. suis strain S735 by electroporation. In addition, pCOM1-V[10] was introduced into the avirulent serotype 2 strain T15 [3] by electroporation to yield T15-pCOM1-V[10].
Experimental infection
Experimental infection of caesarean derived germ-free piglets was performed as previously described [11]. Prior to infection, germ-free status of piglets was confirmed by plating tonsil swabs on Columbia agar plates containing 6% horse blood. Briefly, 4 or 5 one-week-old germ-free pigs were infected intravenously with 106 colony-forming units (CFU) of S. suis and then immediately orally administered 40mgkg-1 body weight of erythomycin (erythomycin-stearate, Abbott B.V., Amstelveen, The Netherlands) twice a day to keep selective pressure on S. suis isolates harbouring the pCOM plasmids. Infected pigs were monitored twice daily for clinical signs and tonsil swabs collected for bacteriological analysis. Pigs were euthanized when clinical signs of arthritis, meningitis, or sepsis were observed after infection with S. suis. Tissue specimens of CNS, serosae and joints were collected during necropsy, homogenized and bacterial cell counts were determined by plating serial dilutions on Columbia agar plates containing 6% horse blood and 1 μg ml-1 of erythomycin. To be able to compare results from different animal experiments included in this manuscript, a uniform scoring of non-specific and specific symptoms was applied to all animal experiments. Non-specific symptoms included inappetite and depression that were scored 0 (none), 0.5 (mild inapptite/depression) or 1 (severe inapetite/depression). Specific symptoms included lameness, central nervous system (CNS) symptoms (locomotive disorders like cycling, or walking in circles; opistotonus; nystagmus), as well as raised hairs, arched back (kyphosis), and shivering, since these are all symptoms of sepsis or serositis. Based on these observation clinical indices were calculated by dividing the number of observations where either specific or non-specific symptoms were observed by the total number of observations for this parameter. This represents a percentage of observations where either specific or non-specific symptoms were observed. Fever was defined as a body temperature >40C. `Mean number of days till death was used as a survival parameter. Although animals were euthanized after reaching humane end points (HEP), the time between inoculation and reaching HEPs is still indicative of severeness of infection. It is calculated by averaging the survival in days from inoculation until death.
All animal experiments were approved by the ethical committee of the Central Veterinary Institute of Wageningen UR, Lelystad, The Netherlands, in accordance with the Dutch law on animal experiments (#809.47126.04/00/01 & #870.47126.04/01/01).
Statistical analyses were performed on clinical indices of the groups (fever index, specific symptoms and non-specific symptoms) using a non-parametric KruskalWallis test as there was no homogeneity of variance among groups. In subsequent analyses all groups were compared pairwise to the control group (S735-pCOM1) on all three parameters, using MannWhitney U tests. Differences were considered statistically significant at p <0.05. Calculations were performed using SPSS 19 (IBM, New York, USA).
Isolation and stimulation of porcine peripheral blood mononuclear cells
Blood from 3 4week old specific pathogen free (SPF) pigs was aseptically collected and mixed with heparin (LEO Pharma, Breda, The Netherlands) to a final concentration of 5 international units (IU) ml-1 blood. Blood was subsequently diluted 1:1 with DPBS (Invitrogen, Carlsbad, CA, USA). Peripheral blood mononuclear cells (PBMCs) were isolated using Leucosep tubes (Greiner bio-one, Frickenhausen, Germany) according to manufacturers instructions. A cell suspension was generated by passage through a 100 μm cell strainer (BD Falcon, Bedford, MA, USA). PBMCs were washed once in DPBS containing 30 μg ml-1 penicillin, and once in RPMI 1640 (Invitrogen) supplemented with 2% of homologous serum (derived from the same donor pig as the PBMCs) and 30 μg ml-1 of penicillin. Cells (1ml) were seeded at a concentration of 5.106 cells per well in 24-well tissue culture plates. After overnight incubation, cells were stimulated with 5.106 CFU exponentially growing S. suis cells (multiplicity of infection (MOI) = 1), with 1 μg of lipopolysaccharide as a positive control, or with DPBS as a negative control. Antibiotics were not washed away to increase stimulation of PBMCs as described by Wichgers Scheur [23]. At time point 0 (before stimulation) and after 2, 4, and 6h of stimulation, the supernatant was removed from the PBMCs and RNA was isolated using Nucleospin RNA II kit (Machery Nagel, Dren, Germany) according to manufacturers instructions.
cDNA synthesis and quantitative PCR
RT-PCR
Two hundred ng of RNA was used to synthesize cDNA in a reaction containing 25 ng μl-1 random primers (Promega, Madison, WI, USA), 10mM dNTPs (Promega), 10mM DTT (Invitrogen), 40 U RNAsin (Promega) and SuperScriptII Reverse Transcriptase (Invitrogen) according to manufacturers instructions.
qPCR
cDNA was diluted 20 times for qPCR analysis. Primers were designed using PrimerExpress software (Applied Biosystems, Foster City, CA, USA) (Table3), except for the porcine reference genes that were ordered at GeNorm (Sequenom, San Diego, CA, USA). Each reaction contained 12.5 pmol forward primer, 12.5 pmol reverse primer and POWR SYBR Green PCR Master Mix (Applied Biosystems) according to manufacturers instructions. qPCR was performed using an ABI7500 (Applied Biosystems). GeNorm software (GeNorm) was used to determine the most stably expressed reference genes. Of the 5 candidate reference genes tested for porcine RNA, the transcript amounts of ppiA and gp1 were the least variable between the different samples. For S. suis recA was the least variable in expression of the 6 potential reference genes (phosphogelycerate dehydrogenase (pgd), acetyl coA acetyltransferase (aca), mutS, glutamate dehydrogenase (gdh), gyrase B) tested. Genorm combines expression data into a number, representing stability of expression, where 1 represents the most stabile gene. Stability numbers for S. suis ranged from 1.667 for gdh to 1.217 for recA. The level of expression of these reference genes was measured to control for variation in RNA-yield and RT-reaction conditions. In each qPCR run a standard curve was incorporated consisting of a vector containing a cloned PCR product of the target gene of that reaction. The standard curve consisted of seven 10-fold dilutions of the control vector. In this way both the expression level of the target gene and the expression levels of external reference genes could be calculated from a standard curve. For each reaction water was included in place of cDNA or template as a negative control. Analysis was performed using the ABI7500 Software (Applied Biosystems).
Sequence analysis
Sequence reactions were performed by Baseclear (Leiden, The Netherlands).
Site-directed mutagenesis
Site directed mutagenesis was achieved using the Quick-change II site-directed mutagenesis kit (Agilent Technologies, La Jolla, CA, USA) according to manufacturers instructions. PCR primers were designed with the accompanying software (Agilent Technologies) (Table3). Using primers t448a and t488a_antisense the plasmid pCOM-orf2[S735] was amplified, introducing the desired mutation that changed the -35 region of the putative promotor region of the orf2-folC-operon of S735 from 5’-TGGTCA-3’ to 5’-TGGACA-3’ (Figure4). The reaction mixture was digested using Dpn I to inactivate the original template vector and subsequently transformed to XL-1-blue competent cells (Invitrogen). To exclude the possibility of introducing PCR errors into the vector backbone, the insert of the plasmid (orf2[S735]) was isolated from the template vector after digestion with restriction enzymes Bam HI and Sac I and cloned into pCOM1 digested with the same restriction enzymes. The resulting plasmid was introduced into S. suis isolate S735 by electroporation and transformants were selected on Columbia agar containing 1 μg ml-1 erythomycin, yielding S735-pCOM1-orf2[S735][t488a]. Sequencing was used to exclude presence of PCR errors in the final construct.