Insertional mutagenesis was performed using the pAUL-A vector as described (11). Primers contained a 5′-end specific tag carrying the sequences previously described (8). Gene deletions were performed using pMAD vector as described (12, 13). Insertions and deletions were verified by PCR and DNA sequencing.Preparation of input pools
Individual overnight cultures of mutants were subcultured in 50ml of BHI (or BHI supplemented with erythromycin for insertion mutants), grown until OD600nm = 0.6, and mixed in equal volume. Equal CFU numbers of the individual cultures were confirmed by plating. The mixed culture was centrifuged at 4°C, washed in cold PBS, and re-suspended in 3ml PBS.In vivo infection
Oral infections were performed as described (14). 5×109CFU in PBS with 150mg/mL CaCO3 were delivered intragastrically to six-week-old specific-pathogen-free female BALB/c mice, which were starved for 12h (5 mice/group). At 72h post-infection, livers and spleens were recovered and CFU numbers were determined by plating organ homogenates.
Animal experiments were performed in strict accordance with the IBMC guidelines for laboratory animal husbandry. The protocol was approved by the department for animal health and protection of the Portuguese Agriculture Ministry (Permit Number: 0420/000/000/2007).Mutant screening by PCR
PCR screening was performed using the primers shown in Table 1. For input pools, PCR reactions were performed using a 1/10 dilution of the pool suspension as the template. For output pools, mouse organs were recovered 72h post-infection, homogenized, and plated on BHI. After overnight incubation, total colonies were re-suspended in 1ml sterile PBS and PCR was performed using a 1/10 dilution as the template. PCR amplifications were performed according to a protocol that comprised an initial denaturation step at 94°C for 5 min, followed by 25 cycles of 94°C (30 s), 55°C (30 s), and 72°C (30 s), and a final step at 72°C for 5 min.
These conditions were determined to allow a semi-quantitative measure of relative differences in template between samples by endpoint PCR, ensuring that saturation had not been reached. To ensure that the reaction was within the exponential phase of amplification, samples were amplified with increasing numbers of cycles and template concentrations. For insertional mutants, each tag-specific primer was used in combination with a primer specific to the gene in which the tag was inserted to generate ╛400bp products. For deletion mutants, pairs of gene-specific primers were designed to amplify a ╛400bp fragment mapping the scar resulting from the deletion of each gene. PCR products were analyzed on 1% agarose gel. Band quantification was performed using QuantityOne software (Bio-Rad, Hercules, CA, USA). The relative band intensity was calculated using the formula: %band intensity = ((SO/SI)/(CO/CI))×100. (SO = Sample band intensity Output; SI = Sample band intensity Input; CO = Positive control band intensity Output; CI = Positive control band intensity Input). Results represent means of at least three independent experiments.Statistics
Student's t-test was used and P-values were considered as: P > 0.05 = Not significant; P < 0.05 = significant(*);P < 0.01 = Very significant(**);P < 0.001 = Extremely significant(***).Results and discussion Generation of a pool of tagged insertion mutants
We first tested the feasibility of our approach using tagged insertion mutants. To create an input pool of targeted tagged insertion mutants, a collection of six unique 21-mers was synthesized (pTMF primers, Table 1) and used to tag selected genes by insertional mutagenesis. The tags were previously designed to have an invariable region for PCR optimization and a variable region for specific amplification while preserving the same melting temperature (8). A tagged mutant for a major Lm virulence factor was constructed (TM-) and used as strongly attenuated mutant (negative control). A tagged mutant for a gene previously shown to be irrelevant to virulence (TM+) was used as a non-attenuated strain (positive control). In addition, four other genes with unknown function in virulence were mutated by insertional mutagenesis to complete the input pool (TM1-TM4). To have every mutant equally represented in the input pool, individual cultures were grown until exponential phase and mixed in equal quantities as verified by CFU determination. This strategy overcomes possible growth effects of specific mutants in mixed cultures that would result in their underrepresentation in the pool. The equal representation of each mutant in the input pool was analyzed by PCR using tag-specific primers in combination with target gene-specific primers designed as described above (Table 1). After PCR and band quantification, the relative band intensity was calculated, verifying the equal abundance of each mutant in the pool (Figure 2A). In addition, the absence of cross-amplification from different tagged mutants was confirmed by sequencing the unique DNA amplification product observed for each tag.