45 of these amide bonds reducing the quantity of β-lactam available to produce a bactericidal effect against M. smegmatis. This could happen since the zymogram and synergy assays were performed in different conditions that could influence CwlM activity. Also, in the nitrocefin assay, CwlM has some increased β-lactamase activity when compared to the control, which may support this hypothesis, albeit without significance.
The difference between CTX and MER could be due to the spatial orientation of the amide bond or due to substrate affinity of the CwlM protein to these antibiotics. In contrast, EMB does not have amide bonds and as observed on the graph, CwlM does not influence its bactericidal activity against M. smegmatis. To better access this, a β-lactamase assay with CwlM against the same antibiotics would enlighten about this possible hydrolysis of the amide bonds and understand how CwlM contributes to M. smegmatis survival. Altogether, CwlM seems to have an influence in cefotaxime resistance.
Figure 4.15 – Scaffold of lactam antibiotics (cefotaxime and meropenem) and ethambutol. The amide bond in the β-lactam ring is highlighted in red.
46 is critical to avoiding the acquisition of additional drug resistance. An improved TB vaccine would be the most effective means of preventing the spread of all forms of the disease. The evaluation of available antibiotics has gained importance in recent years for the treatment of drug-resistant TB. β-lactams are the oldest and most widely used class of antibiotics but the presence of a chromosomally encoded β-lactamase, known to inactivate beta-lactams, is considered the main reason for the ineffectiveness of penicillins and cephalosporins against Mtb. However, several studies support the pairing of β-lactams with a β-lactamase inhibitor to combat drug-resistant TB. For instance, although amoxicillin-clavulanate is occasionally used against XDR-TB, there is not enough data to support its routine use. Another possible pair is meropenem-clavulanate, which has been used in conjunction with anti-tubercular drugs but has not yet been tested on its own for its bactericidal activity against TB in humans. A lot of doctors that have resorted to all possible antibiotics commonly used against TB have turned to β-lactams to find a treatment for their patients. There is a lot of potential for testing several different β-lactam-β-lactamase combinations against MDR-TB. Therefore, it is imperative to enlighten the mechanism of TB pathogenesis and uncover the role of genes that could be related with antibiotic resistance.
In this study, we chose to characterize two genes to uncover their role in antibiotic resistance, especially to β-lactams, since both were found to be associated with differential β-lactam phenotypes.
Therefore, the main objective of this study was to assess how the repression of the expression of the cwlM and lpqK orthologs in M. smegmatis affects the susceptibility of bacteria to β-lactams and then characterize the corresponding Mtb proteins to better enlighten their function. The first gene, cwlM, is an essential gene encoding a protein involved in PG biosynthesis regulation with a conserved regulatory pathway: PknB-CwlM-MurA32. The second gene, a non-essential gene, lpqK, encodes a lipoprotein with a β-lactamase domain. First, the construction of knockdown mutants of cwlM and lpqK was successful.
The characterization of both knockdown mutants was carried out by spotting assays, with and without inducer. A qRT-PCR assay confirmed the successful repression of both target genes in the presence of an inducer. Subsequently, to evaluate the role of both genes in antibiotic susceptibility, MIC assays with the WT, PLJR962, and the knockdown mutant strains, with and without ATc, were performed. In general, we found that amoxicillin and meropenem, with and without clavulanate, were considered more effective at inhibiting mycobacterial growth when compared to cefotaxime. In addition, ethambutol, isoniazid, and vancomycin were highly effective in inhibiting mycobacterial growth. For the cwlM knockdown mutant, differences were observed in the MIC of cefotaxime and meropenem. No differences were observed in the MICs of the lpqK knockdown mutant when compared to the control strains WT and PLJR962, suggesting that the lpqK ortholog gene in M. smegmatis does not affect susceptibility to β-lactams. After the MIC assays, spotting assays with cefotaxime and meropenem and disk diffusion assays with meropenem and amoxicillin-clavulanate were performed with the cwlM knockdown mutant, with and without inducer. The obtained results, together with those from the MIC assays, collectively show that the knockdown of the cwlM gene affects the susceptibility of M.
smegmatis to β-lactams, specifically to cefotaxime and meropenem. Since CwlM is annotated as an N-acetylmuramoyl-L-alanine amidase and was demonstrated to be involved in a conserved regulatory pathway of PG biosynthesis32, a phenotype of susceptibility to β-lactams was expected. Nevertheless, substantial differences were only observed for cefotaxime and meropenem. PG biosynthesis has numerous regulatory pathways that can compensate for this repression. On the contrary, the repression of lpqK did not seem to affect susceptibility to β-lactams.
To characterize both proteins from Mtb, a zymogram was performed with CwlM since this protein is reported to have PG hydrolytic activity. A nitrocefin assay was also conducted with both CwlM and LpqK producing strains since LpqK has a β-lactamase domain. Due to time constraints, the
47 LpqK protein was not purified. The zymogram revealed that CwlM does not have PG-hydrolytic activity, supporting the hypothesis that CwlM acts more like a regulator of its conserved pathway instead of possessing an enzymatic role32. Moreover, the nitrocefin assays revealed that the LpqK protein has the highest β-lactamase activity when compared to the control, although without significance, which was expected since LpqK has a β-lactamase domain and was reported as a group 2a penicillinase in M.
smegmatis 61. To uncover the role of CwlM even further in antibiotic resistance/susceptibility, a synergy assay with β-lactams was performed in which CwlM was revealed to hinder M. smegmatis antibiotic killing with cefotaxime. Since lactams have an amidase bond that normally is hydrolysed by β-lactamases, CwlM could be delaying the effect of cefotaxime because this protein is indeed an amidase.
In view of the results obtained in this study, future perspectives should focus on:
• Constructing and characterizing cwlM and lpqK knockdown mutants in Mtb, by performing growth curves and spotting assays. The knockdown of cwlM and lpqK should then be confirmed through qRT-PCR.
• Performing MIC, spotting assays with antibiotic and disk diffusion assays to evaluate how the knockdown of cwlM and lpqK in Mtb affects β-lactams susceptibility and compare the obtained results with those observed with M. smegmatis.
• Extract and purify the CwlM and LpqK proteins to conduct new β-lactamase assays and other classification assays with kinetic studies to better understand the role of these proteins in Mtb antibiotic resistance.
In this project, the implemented CRISPRi technique, allowed us to show that cwlM contributes to β-lactam resistance in M. smegmatis. On the contrary, the full extent of the impact of lpqK on the susceptibility of M. smegmatis to β-lactams is not yet known. Additionally, in the protein studies we found that CwlM role might be important to β-lactam resistance. In conclusion, these findings may give an important contribution to the future understanding of mycobacterial pathogenesis and to the future development of alternative TB therapeutics with or without β-lactams.
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