Determination of the sensitivity and reliance of the PCR screening
To further confirm that the PCR band intensity can be directly related to the quantity of the corresponding mutant in a pool and to determine the detection limit of PCR screening, we assessed the sensitivity of our method. The quantity of one tagged mutant (TM3) was artificially decreased (from 1/6 to 1/6000) in the pool while maintaining other mutants at the 1/6 proportion. After PCR, we observed a dose-dependent decrease in the intensity of the band corresponding to TM3. Each 10-fold dilution resulted in a ╛30% reduction of the corresponding band intensity (Figure 2B). In addition, the variation of the proportion of TM3 did not affect the band intensity corresponding to another mutant (TM+) in the pool (Figure 2C).
Results indicate that the band intensity can be directly related to the amount of the corresponding strain and showed that the variation of the quantity of one mutant did not affect the relative quantities of other strains in the pool. However, quantifications based on PCR as performed here are only semiquantitative. Also, results obtained using this approach are an indication of a mutant being attenuated rather than an exact quantification of its growth attenuation.PCR-based screening of targeted mutants: detection of new virulence genes
To refine the in vivo identification of bacterial virulence factors, we developed an approach for the simultaneous analysis of multiple targeted mutants (Figure 1). The pool of insertion mutants described above was used for in vivo challenge of mice. The relative quantity of each mutant in mouse liver and spleen was assessed after oral infection. Three days post-infection, a time point that—under these experimental conditions—corresponded to the peak of the infection, organs were recovered, homogenized, and plated on BHI. Total colonies were re-suspended in PBS to constitute the output pool. The proportion of each tagged mutant in the output pool was estimated by PCR screening and band intensity quantification. The relative quantity of each mutant corresponded to the ratio of band intensities obtained from the output vs. input pools, relative to the positive control. The formula used (see Materials and methods) allowed the correction of intensity variations between mutant bands resulting from slight differences in oligonucleotide efficiencies. As expected, both in livers and spleens, TM- was nearly undetected as compared with TM+ (Figure 3A). These results validate our approach and confirm that PCR screening easily identifies virulence mutants. Interestingly, we also observed that, after oral inoculation, TM2 appeared underrepresented (decrease in band intensity >30%) in both organs (Figure 3A). These results indicate a new gene implicated in Listeria pathogenicity and show the potential of the PCR-based screening of targeted mutants.
To definitively demonstrate the direct correlation between PCR detection and colony forming units (CFUs) of a specific mutant within a pool, we performed mixed infections using pools composed of the wild type (WT) strain and an insertion mutant carrying an antibiotic resistance for differential selection. Two different pools were produced: (i) a pool containing the WT strain and an insertion mutant for a major Lm virulence factor highly attenuated in virulence (TM-) and (ii) a pool containing the WT strain and an insertion mutant moderately attenuated in virulence (TM2). The presence of equal quantities of both strains in each pool was verified by PCR and by plating on BHI and BHI with antibiotics. Results obtained by both techniques confirmed the equal quantity of each strain in input pools (Figure 2D). These two input pools were used for oral inoculation of BALB/c mice. Three days post-infection organs were recovered, homogenized, and plated on BHI. Total colonies were re-suspended in PBS to constitute the output pool. The proportion of each tagged mutant in the output pool was estimated by PCR screening and band intensity quantification. In parallel, serial dilution of organ homogenates were plated on BHI and BHI with antibiotics to determine CFUs. Results clearly confirmed a direct correlation between the intensity of the bands observed by PCR screening and the real number of bacteria in infected mouse organs (Figure 2E).