Antibiotics that target the mycobacterial CW biosynthesis

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.

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Table 4.2 – Median of Minimum Inhibitory concentrations for each strain of interest. M. smegmatis WT and PLJR962 are used as controls. Knockdown mutants (cwlM and lpqK mut). CLAV – clavulanate; AMX– amoxicillin; CTX – cefotaxime;

MER – meropenem; EMB – ethambutol; INH – isoniazid; VAN – vancomycin. Presence of ATc (+), Absence of ATc (-).

Antibiotics

Strains (M. smegmatis mc² 155) AMX AMX+CLAV CTX CTX+CLAV MER MER+CLAV EMB INH VAN

WT - ATc 64 4 >256 128 4 4 0.5 8 2

+ATc 64 4 256 128 2 4 0.5 8 2

PLJR962 - ATc 128 8 >256 256 4 4 0.5 8 2

+ATc 128 4 >256 128 4 4 0.5 8 2

cwlM mut - ATc 64 8 256 256 4 4 0.5 8 2

+ATc 32 4 32 32 2 2 0.25 4 1

lpqK mut - ATc 64 4 256 128 4 4 0.5 16 2

+ATc 128 8 256 256 4 4 0.5 8 2

According to Li et al.⁵⁴, the M. smegmatis mc² 155 WT MIC of AMX is 64 μg mL^-1, similarly to what was obtained for the negative controls (M. smegmatis WT and PLJR962) and the uninduced knockdown mutants. Likewise, in the presence of ATc, the MICs of AMX for the negative controls and for the mutants only ranged between 32 and 128 μg mL^-1, either 2-fold below or above the WT MIC, indicating that the negative controls were not affected by ATc addition and that the repression of cwlM and lpqK did not influence the susceptibility of the mycobacteria to AMX. Since PG biosynthesis inhibition is more efficient when both PBPs and LDTs are blocked⁵⁵, no major differences with AMX were observed since AMX only inhibits PBPs^19,25. Based on the EUCAST breakpoints, all M. smegmatis controls and cwlM and lpqK mutants are classified as resistant to AMX. AMX+CLAV has been shown to have bactericidal activity in Mtb in vitro and in drug-resistant TB patients⁵⁶ although there is conflicting data between studies⁵⁵. A WHO report from 2020 stated that AMX+CLAV should not be used without imipenem–cilastatin or meropenem for MDR/RR-TB treatment⁵⁷. The obtained MICs for AMX+CLAV, in the absence of ATc, for both negative controls and the knockdown mutants, were 4–8 μg mL^-1. Compared to AMX alone, the values decreased mostly up to 16-fold. Similarly, the MIC of AMX+CLAV in patients with TB was reported to be 4–8 μg mL^{-1 56}and the MIC of AMX was found to be reduced by the presence of clavulanate up to 16-fold and at least 32-fold in M. smegmatis and Mtb, respectively^52,56. This is likely due to the presence of a class A β-lactamase in M. smegmatis, BlaS, for which penicillin-type β-lactams, like AMX, are a very good substrate⁵².The presence of ATc did not change the MIC of AMX+CLAV for any strain. M. smegmatis WT in both conditions, the induced PLJR962 control strain and the induced cwlM knockdown mutant are classified as having intermediate susceptibility to AMX+CLAV according to the EUCAST breakpoints. In contrast, the uninduced lpqK knockdown mutant is considered to have intermediate susceptibility to AMX+CLAV.

In M. smegmatis, the MIC of CTX is reported as 64 – 128 μg mL^-1 by Viswanathan et al.⁵⁸. In this work, the MIC of CTX for the uninduced M. smegmatis controls and mutants was ≥256 μg mL^-1, being more in conformity with the results obtained by Jeon et al.⁵⁹. Importantly, the induced cwlM knockdown mutant displayed a CTX MIC of 32 μg mL^-1, 8-fold lower than that obtained for the negative controls and for the lpqK knockdown mutant. In the absence of ATc, the MIC of CTX+CLAV was 128 μg mL^-1 for the WT and for the lpqK knockdown mutant and 256 μg mL^-1 for the control strain PLJR962 and for the cwlM knockdown mutant. The MICs of CTX+CLAV were lower than those obtained without clavulanate and in accordance with the obtained results by Viswanathan et al.⁵⁸. In the presence of ATc, the MIC of CTX+CLAV was 128 μg mL^-1 for the WT and PLJR962 strains and 256 μg mL^-1 for the

33 lpqK knockdown mutant. The induced cwlM knockdown mutant presented a CTX+CLAV MIC of 32 μg mL^-1, 4-fold lower than that obtained for M. smegmatis WT. Based on the EUCAST breakpoints, the M. smegmatis negative controls and the knockdown mutants are, in all conditions, classified as resistant to CTX and CTX+CLAV. Compared to AMX+CLAV MICs, the use of clavulanate does not seem to help the action of CTX, causing only a slight decrease in the MIC. In fact, BlaS hydrolyses penicillins such as AMX at a higher rate when compared to cephalosporins⁵², like CTX. As said before in section 1.3.1.2, cephalosporins like CTX mostly inhibit PBPs^17,19, but CTX has been shown to cause inhibition of some mycobacterial LDTs⁶⁰. Altogether, it seems to be easier for CTX to act on its targets in the PG when cwlM is repressed, with a 4-fold difference in the MIC. The role of CwlM in M. smegmatis as an N-acetylmuramoyl-L-alanine amidase involves CW processes and the repression of cwlM causes abnormal cell elongation due to uncoordinated synthesis of the various layers of the CW. Even with a downregulated PG biosynthesis, other associated biosynthetic and metabolic processes may remain active leading to a CW with poor structural integrity³².

According to Li et al.⁵⁴ and Viswanathan et al.⁵⁸, the MIC of MER is 8 and 4 μg mL^-1 for M.

smegmatis WT, respectively. In the absence of ATc, the MIC of MER and MER+CLAV was 4 μg mL

-1 for all M. smegmatis negative controls and for the knockdown mutants. In the presence of the inducer, no changes were found in the MICs of WT or PLJR962 strains and only the cwlM knockdown mutant exhibited a 2-fold MIC reduction when compared to the negative controls. Based on the EUCAST breakpoints, all controls and mutants have intermediate susceptibility to MER, in all conditions. An exception is the induced cwlM knockdown mutant, which is classified as susceptible to MER. Contrary to AMX results and to what was previously reported^19,30, the MICs of MER did not change in the presence of CLAV. Moreover, a lower concentration of MER is required to inhibit mycobacterial growth in comparison to AMX or CTX. Evidently, MER efficiently inhibits PG cross-linking, since carbapenems inhibit both class A PBPs¹⁹ and LDTs²⁷ while being poorly hydrolysed by β-lactamases.

The obtained results support the idea that MER-containing therapeutic schemes may be important alternative treatments for MDR-TB^19,27. Since the antimicrobial action of MER is so efficient, the repression of cwlM and the associated downregulated PG biosynthesis, does not have an impact on the already very low MIC of MER.

Furthermore, the repression of lpqK, which encodes a β-lactamase in M. smegmatis, did not seem to have an impact on the AMX, CTX and MER MICs. This probably happens because LpqK does not have an important role in resistance to the tested β-lactams, in these specific experimental conditions.

In a study⁶¹, MSMEG_4455 showed a β-lactam hydrolysis pattern similar to that of a group 2a penicillinase, with demonstrated significant susceptibility alterations for a group of penicillins like ampicillin, piperacillin, carbenicillin and penicillin G but not for AMX. This means that understanding the role of this β-lactamase by studying its kinetic activity and its preferred substrate is important.

In addition to lactams, the effect of the inhibition of a mycobacterial hydrolase and a β-lactamase in INH and EMB susceptibility was also evaluated, since these antibiotics comprise two crucial first-line anti-TB drugs that also target the mycobacterial CW. The MICs of EMB for the M.

smegmatis controls and the lpqK knockdown mutant, in both –ATc and +ATc conditions, were found to be 0.5 or 1 μg mL^-1, following previous experiments performed in identical conditions^62,63. For the cwlM knockdown mutant, the MIC was 0.25 μg mL^-1. These results suggest that the inhibition of these this specific hydrolase and β-lactamase did not result in altered susceptibility to EMB. According to Li et al.⁵⁴ and Viswanathan et al.⁵⁸, the MIC of INH for M. smegmatis is 8 μg m^-1. In –ATc conditions, the MICs of INH for the controls and for the mutants were 8–16 μg mL^-1. In the presence of ATc, both

34 negative controls and the lpqK knockdown mutant showed MICs within the range values obtained without ATc and the cwlM knockdown mutant displayed a MIC of 4 μg mL^-1. These differences are only of a 2-fold magnitude and thus, the lack of CwlM and LpqK also seemed to not affect the susceptibility of M. smegmatis to INH. EMB disrupts the synthesis of AG and INH inhibits the synthesis of the MA, meaning that both these antitubercular agents inhibit the formation of the mAGP complex.

Hence, a downregulated PG biosynthesis as a result of the repression of cwlM should destabilize this complex. MurA performs the first committed step in PG biosynthesis and its regulation by CwlM is an efficient point at which to regulate this pathway. The unaltered susceptibility of the induced cwlM knockdown mutant to EMB and INH could be explained by the fact that there are other factors that also regulate MurA, possibly mitigating the effects of the repression of cwlM. As expected, the repression of the lpqK does not cause any relevant changes in the MICs of EMB and INH since β-lactamases only target β-lactams and both EMB and INH are antitubercular agents.

The M. smegmatis VAN MIC is 4 μg mL^-1, in conformity with the values referenced by Lelovic et al.⁶⁴. VAN is a large hydrophilic molecule able to form a hydrogen bond with the terminal D-alanyl-D-alanine moieties during PG biosynthesis, thereby preventing bacterial CW backbone synthesis⁶⁵. At least, for the cwlM knockdown mutant, an alteration in the MIC of VAN was expected. The target of VAN, ubiquitous in bacteria, is thus only easily reachable in bacteria with thin cell walls, without an outer lipid membrane or without PDIM, which is not the case of the mycobacterial CW. The potency and activity of vancomycin could be improved by combining it with other CW-targeting drugs⁶⁵, such as EMB and INH.

After analysing the MICs results, we decided to proceed only with the cwlM knockdown mutant for the next antibiotic experiments since the lpqK knockdown mutant did not show any relevant MIC changes. CTX and MER produced the most significant changes in the MIC values of the induced cwlM knockdown mutant, so they were chosen for the following experiments. To confirm these results, spotting assays of the cwlM knockdown mutant in the presence of varying concentrations of cefotaxime and meropenem were performed (Figure 4.5).

As shown in section 4.1.2, cwlM is an essential gene since with 100 ng mL^-1 of ATc growth defects are observed in the knockdown mutant when compared to the WT control. In Figure 4.5, there are no prominent growth defects in M. smegmatis WT regardless of the concentration of CTX used, but with MER the growth defects are notable at 1 μg mL^-1. The WT and the cwlM knockdown mutant show similar growth without inducer, regardless of the concentration of antibiotic used. The induced cwlM knockdown mutant presents growth defects with meropenem or cefotaxime when compared to the WT, losing its capacity to form a spot when the highest antibiotic concentrations are applied and essentially disappearing (loss of viability) at the subsequent dilutions. The prominence of the growth defects is directly proportional to the concentration of antibiotic used. In sum, the knockdown of the cwlM gene seems to increase the susceptibility to CTX and MER when compared to the negative control, M.

smegmatis WT, reaffirming previous results.

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Figure 4.5 – Spotting assays of M. smegmatis mc² 155 and the cwlM knockdown mutant in presence of varying concentrations of cefotaxime and meropenem. A. negative control plates (no antibiotic); B. Agar plates with cefotaxime; C.

Agar plates with meropenem. Control - M. smegmatis WT. cwlM mut– cwlM knockdown mutant.

To further validate these results, disk diffusion assays were performed for the cwlM knockdown mutant with AMX+CLAV and MER (Figure 4.6). Disk diffusion assays are performed by assessing the diffusion (in halo) of a certain antibiotic through a homogeneous layer of bacteria, poured over solid media, that may or may not be supplemented with inducer. Therefore, the concentrations of antibiotic used to produce a visible halo effect were optimized. For AMX, this concentration was found to be 8-fold superior to the MIC determined in liquid medium and, for MER it was found to be 64-8-fold superior to the respective MIC in liquid medium. CTX was not used in the assay because the concentration of CTX needed to inhibit mycobacterial growth in liquid media is already very high. Thus, growth inhibition in solid media would require even higher concentrations, which defy the water solubility limits of a CTX solution.

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Figure 4.6 – Average length (cm) of the inhibition halo for amoxicillin (AMX+CLAV) and meropenem (MER) antibiotic disks for M. smegmatis mc²155 WT and for the cwlM knockdown mutant (mut), with (striped bars) and without (smooth bars) 100 ng mL^-1 ATc. M. smegmatis mc² 155 WT was used as a negative control. Measurements were made by ImageJ software tools. The bars represent the mean of three independent experiments with three biological triplicates each and the error bars correspond to the standard deviation. Asterisks indicate the significance of the difference in relative diameter length calculated using the ordinary one-way ANOVA with Sidak's multiple comparisons test (* P < 0.05).

With 256 mg L^-1 of MER, the diameter for both the WT and for the cwlM mutant were similar and no significant differences were observed regardless of the ATc conditions. With 512 mg L^-1 of MER, the induced cwlM knockdown mutant has increased susceptibility to the carbapenem and significant differences were observed in comparison with the induced WT strain and with the respective uninduced mutant. With 512 mg L^-1 of AMX, the halo diameter for the WT strain and for the cwlM mutant were similar, with and without inducer. With 1024 mg L^-1 of AMX, the halo diameter results were similar to those obtained with 512 mg L^-1 of MER, with a greater AMX susceptibility observed for the induced cwlM knockdown mutant, although no significant differences were observed. In sum, the cwlM knockdown mutant has a significantly increased susceptibility to MER when compared to the WT strain.

Moreover, the cwlM knockdown mutant also has increased susceptibility to AMX albeit without significance. These results together with the MIC and spotting assays, collectively show that the knockdown of the cwlM gene affects M. smegmatis susceptibility to β-lactams, specifically to CTX and MER.