4. Results and Discussion
4.1 Construction and characterization of the cwlM and lpqK knockdown mutants in
The first aim of this project was the construction of cwlM and lpqK knockdown mutants in M.
smegmatis mc2 155, using the CRISPRi-dCas9Sth1 system referred before45. The genes of interest were identified in M. smegmatis mc2 155 as MSMEG_6935 and MSMEG_4455, respectively. In M.
29 smegmatis, MSMEG_6935 encodes a N-acetylmuramoyl-L-alanine amidase and MSMEG_4455 is annotated as encoding a β-lactamase, with a gene length of 1191 bp and 1245 bp, respectively. In Mtb H37Rv, cwlM (Rv3915) encodes a probable PG hydrolase and lpqK (Rv0399c) encodes a possible conserved lipoprotein, with a gene length of 1221 bp and 1230 bp, respectively. In Mtb, cwlM is an essential gene and lpqK is not essential36. Moreover, the CwlM and LpqK proteins have similar sizes and predicted functions in both species.
4.1.1 Construction of the cwlM and lpqK knockdown mutants in M. smegmatis mc2155 using CRISPRi
The first step in the construction of the knockdown mutants was the design of suitable sgRNAs targeting a chosen PAM according to the essentiality classification of the genes of interest provided by the available data for Mtb, following the methods adopted by Rock et al.45. The CRISPRi system used in this work is ATc-inducible and the resulting gene repression is described as determined by diverse factors, namely the sgRNA-DNA target complementarity and the PAM strength. For essential genes, a stronger PAM is predicted to cause higher gene fold-repression and thus, higher growth inhibition45. For this reason, a weaker PAM strength was chosen for the knockdown of cwlM in M. smegmatis. All sgRNAs base pairing regions were confirmed as non-complementary to any other sequence in the corresponding genome, allowing a future application of these experiments. The sgRNAs were cloned into the PLJR962 plasmid (Figure 7.1A in Annexes) and E. coli chemically competent cells were transformed with the recombinant plasmids. The plasmid DNA was extracted, quantified and its sequence was confirmed by Sanger sequencing (Sequencing results of the sgRNAs in Annexes).
Lastly, M. smegmatis mc2 155 ECC were transformed with the corresponding recombinant plasmids by electroporation to obtain mycobacteria harbouring the CRISPRi system. The mycobacterial species were also transformed with a non-targeting PLJR962 plasmid to serve as a control. To induce the CRISPRi system, mycobacteria were grown in the presence of 100 ng mL-1 of ATc.
4.1.2 Phenotypic characterization of the cwlM and lpqK knockdown mutants in M. smegmatis mc2155
To investigate the effects of CRISPRi-mediated cwlM and lpqK silencing on mycobacterial growth and viability, spotting assays were performed. The results of the spotting assays of M. smegmatis mc2 155 WT and PLJR962 control strains, and cwlM and lpqK knockdown mutants (mut), in the presence and absence of 100 ng mL-1 ATc, are shown in Figure 4.1.
Figure 4.1 – Spotting assays of M. smegmatis mc2155 WT, PLJR962, knockdown mutants (cwlMand lpqK mut) in the presence (+) and absence (–) of 100 ng mL-1 ATc. M. smegmatis WT and PLJR962 were used as negative controls. The PLJR962 mutant is a non-targeting control. The first spot corresponds approximately to 0.001 OD600 and each subsequent spot
- ATc + ATc
WT PLJR962 cwlM mut lpqK mut
30
is a two-fold dilution. Each spot corresponds to the plating of 5 μL of culture volume. These data are representative of three independent experiments.
In the absence of ATc, no growth defects were observed for the controls or for the cwlM and lpqK knockdown mutants. The growth of the negative controls was identical when ATc was added, confirming the absence of toxic effects of 100 ng mL-1 ATc. In the presence of ATc, the cwlM knockdown mutant exhibited severe growth defects, which is in agreement with the predicted essentiality of cwlM in mycobacteria45. On the contrary, the induced lpqK knockdown mutant did not show observable growth defects. The results of the spotting assays support the essentiality of cwlM and the non-essentiality of lpqK in M. smegmatis and show that the growth effects obtained with CRISPRi-mediated targeted gene knockdown match the anticipated viability phenotypes for the employed sgRNAs.
4.1.3 Assessment of CRISPRi-mediated gene repression by qRT-PCR
Subsequently, the achievement of CRISPRi-mediated cwlM and lpqK repression was investigated by qRT-PCR in M. smegmatis control strains WT and PLJR962, and in both knockdown mutants, in the presence and absence of ATc (Figure 4.2). Since polarity effects were previously reported using this system45, upstream and downstream genes were investigated but not found in the same operon. Therefore, no polarity effects are expected.
Figure 4.2 – Relative cwlM and lpqK mRNA expression levels in M. smegmatis mc2 155 WT, PLJR962 and knockdown mutants (cwlM and lpqK mut) with (striped bars) and without (monocolour bars) 100 ng mL-1 ATc treatment for 6 h.
The left graph levels are for the cwlM gene, the right graph is for the lpqK gene. “ATc” after the name means the mutants were grown in the presence of ATc. M. smegmatis mc2 155 WT and PLJR962 were used as controls. The PLJR962 mutant is a non-targeting control. The data were quantified using the ΔΔCT method and normalized to the M. smegmatis housekeeping gene sigA, using M. smegmatis WT as a calibrator sample. The bars represent the mean of two independent experiments with two technical replicates each and the error bars correspond to the standard error of the mean (SEM). Asterisks indicate the significance of the difference in relative mRNA expression calculated using the Ordinary One-way ANOVA with Sidak’s multi comparisons test (* P < 0.05, ** P < 0.01, *** P < 0.001).
The CRISPRi system reaches maximal gene knockdown between one and two cell divisions after the addition of ATc45. As so, bacterial cultures for total RNA extraction were collected 6 h after ATc addition. The mRNA expression levels were quantified using the ΔΔCT method and normalized to the M. smegmatis housekeeping gene sigA, which encodes for a sigma factor that initiates and promotes attachment of the RNA polymerase to specific initiation sites and its release, found in all mycobacterial species. M. smegmatis WT was used as a calibrator sample. When comparing the mRNA expression levels of the induced cwlM knockdown mutant with the induced WT strain, induced PLJR962 strain and the respective uninduced mutant, there was a repression of ~3.77 fold, ~2.48 fold and ~2.72, respectively. This is much lower than the repression fold of 38.5 of the system described when a PAM 14 is used (Figure 7.1B in Annexes). When comparing the mRNA expression levels of the induced
31 lpqK knockdown mutant with the induced WT strain, induced PLJR962 strain and the respective uninduced mutant, there was a repression of ~9.6, ~7.85 and ~7.5 fold, respectively. Again, this is much lower than the repression fold of 145.2 expected for a PAM 4 (Figure 7.1B in Annexes). This could be explained by the lack of passages in ATc-supplemented medium and small induction time. Although the obtained fold decrease in the mRNA expression of both genes was below the previously described for the selected PAMs45, CRISPRi was able to achieve a considerable inhibition of the target genes in M.
smegmatis. These results, together with the previous ones of the phenotype characterization experiments, show that CRISPRi-mediated repression is enough to cause defects in bacterial growth.