Characterization of the CwlM and LpqK proteins

After successfully obtaining the pET-29b:cwlM and pET-29b:lpqK plasmids in E. coli BL21(DE3), inductions were tested with different conditions and, ultimately, 2 L inductions of the proteins were conducted with IPTG induction at 16ºC O.N, resulting in a non-purified mixture containing the target protein. The expression of both proteins was induced and the cells collected, but only the purification of CwlM was pursued because of time constraints.

4.4.1 Production of the CwlM and LpqK proteins

The next step was to test several temperature/conditions to express the CwlM (43.9 kDa) and LpqK (44.3 kDa) proteins. Inductions were conducted at 37ºC or 16ºC and fractions from both conditions were run in an SDS-PAGE gel to ascertain the presence of these proteins. In the gel shown in Figure 4.8, we can observe additional bands on the fourth (CwlM at 16 ºC), sixth (LpqK at 37 ºC), and seventh (LpqK at 16 ºC) lanes, when compared with the non-induced (N.I) controls (second and fifth lanes). These bands have the approximate size of the target proteins. In sum, we can observe that under the tested induction conditions, CwlM is only effectively produced at 16 ºC, while LpqK seems to be expressed at both temperatures, but with a stronger production at 16 ºC.

Figure 4.8 – SDS polyacrylamide gel of the supernatant of the non-induced and induced cell lysates of E. coli BL21 (DE3) expressing either CwlM or LpqK at different conditions. Order of the lanes from left to right: ladder (NZYColour Protein Marker II – NZYTech); E. coli BL21(DE3):pET-29b:cwlM (non-induced (N.I); induced at 37ºC; induced at 16ºC); E.

39

coli BL21(DE3):pET-29b:lpqK (non-induced; induced at 37ºC; induced at 16ºC). The red arrows indicate the presence of the proteins.

After ascertaining the ideal induction conditions of temperature and incubation time, the conditions chosen for a scale-up of 2 L of culture were 16 ºC O.N. Fractions were also collected to ascertain the presence of the proteins of interest in the gel. In Figure 4.9, we can observe that when compared to the non-induced cultures, the CwlM protein appears in the third lane with the expected size, but LpqK does not appear as it was in the small-scale inductions. We hypothesize that the LpqK band may be merged with the immediately above band, as it is also observed that the respective bands are closely placed in the small-scale induction gel. These expression inconsistencies also contributed to choosing CwlM over LpqK for the following purification assays.

Figure 4.9 – SDS polyacrylamide gel of the supernatant of the non-induced and induced cell lysates of E. coli BL21 (DE3) expressing either CwlM or LpqK at 16 ºC O.N. Order of the lanes from left to right: ladder (NZYColour Protein Marker II – NZYTech); E. coli 29b:cwlM (non-induced (N.I); induced at 16ºC); E. coli BL21(DE3):pET-29b:lpqK (non-induced; induced at 16ºC). The red arrows indicate the presence of the proteins.

4.4.2 Purification of the CwlM protein

In order to obtain the CwlM protein to use in the characterization assays, the cell pellet was sonicated, centrifuged and filtered to proceed to the purification of the protein. Expression of a recombinant protein with affinity tags is a common strategy to aid purification. Histidine-tagged proteins are commonly purified using Immobilized Metal Affinity Chromatography (IMAC), which was used in this study. IMAC is based on the interaction between amino acid residues and divalent metal ions immobilized on resins. The string of histidine residues binds to several types of immobilized metal ions, including nickel⁶⁷. The ÄKTAprime plus system was used to purify the His6-tagged protein from

40 the obtained supernatant. With this tag, the protein is trapped in the column and eluted with a solution of imidazole that is utilized as a competitive agent of histidine-tagged proteins⁶⁷.

The results of the measured absorbance at 280 nm during the elution process were plotted (Figure 4.10). In this ÄKTA plot, we can observe an absorbance increase between fractions 7 and 11, with a peak of ~250 mAU, which likely corresponds to our desired protein.

Figure 4.10 – Absorbance at 280nm during the elution step of the affinity purification of CwlM from an induced filtrate of E. coli BL21(DE3):pET-29b:cwlM in the ÄKTAprime plus system. 1 through 11 are the fractions collected and the last dot is waste.

After the purification, the CwlM protein fractions were run in an SDS-PAGE gel to see which were the most concentrated fractions (Figure 4.11). This gel verified that the most concentrated fractions were: 6, 7, 8 and 9, with the most noticeable bands at ~43.9 kDa, corresponding to the CwlM protein size.

Figure 4.11 – Fractions of the CwlM protein after purification in the AKTA (A) and final purified protein (B). Panel image A order: ladder (NZYColour Protein Marker II – NZYTech), 3, 4, 5, 6, 7, 8, 9 and 10. Panel image B order: ladder (NZYColour Protein Marker II – NZYTech), CwlM.

41 The four fractions were mixed and dialysis proceeded to remove imidazole and to exchange the buffer. After centrifugation and filtration, a final purified protein solution with a concentration of ~800 µg µL^-1 was obtained.

4.4.3 Enzymatic activity characterization

The CwlM protein is annotated as an N-acetylmuramoyl-L-alanine amidase of the AmiA/LytC family, hereafter classified as a PG amidase³². More specifically, CwlM belongs to the second class of PG hydrolases by being a specific amidohydrolase that cleaves a critical amide bond between the glycan moiety (MurNAc) and the peptide moiety (L-alanine) of the PG⁶⁸.

4.4.3.1 Zymogram

To test this possible hydrolytic activity, a zymogram protocol was conducted with PG from M.

luteus, which is a convenient and standardizable substrate for the detection of PG hydrolase activity over Gram-positive bacteria cell walls. In this particular case, it enables the evaluation of this activity without the difficult hindrance provided by the mycolic acid-arabinogalactan CW.

The zymogram (Figure 4.12B), containing M. luteus as the substrate, revealed one translucent zone, corresponding to the lytic activity of the lysozyme over the PG (positive control - Lys lane) which is supposed to have ~14 kDa. As observed, the band of Lys has a molecular weight between 17-25 kDa, meaning there was a shift in this band in relation to its predicted size. This can happen because the quantity of SDS and buffers added to zymogram gels is smaller when compared to normal SDS-PAGE gels. In addition, the zymogram gels are subjected to a longer run, heating the loaded samples, which can cause a renaturation of the Lys protein, significantly altering its size. Thus, in this case, the Lys band should be interpreted as a qualitative result rather than a quantitative one. The opposite happens in the negative control (BSA lane, 66 kDa), where no visible activity occurs because BSA has no PG hydrolytic activity. When compared to the positive control, CwlM does not appear to have any hydrolase activity against M. luteus PG. A plate assay on the right side of the image confirmed these results, with no hydrolysis halo for CwlM when compared with the Lys control. This is concordant with the hypothesis raised by Boutte et al. that the essential function of CwlM might not be enzymatic but rather regulatory³². On the contrary, CwlMMtb has been shown, by Deng et al., to have PG amidase activity by being able to release PG from the CW of M. luteus and M. smegmatis³³. However, Boutte et al. showed that key residues that coordinate PG hydrolysis are not conserved in CwlM from both Mtb and M. smegmatis, supporting the obtained results implying that CwlM is inefficient as an enzyme³².

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Figure 4.12 – SDS-PAGE (A), Zymogram (B) and plate (C) of Micrococcus luteus with the CwlM protein. Lane order of both gels: CwlM, ladder (NZYColour Protein Marker II – NZYTech), BSA (negative control), Lysozyme (Lys – positive control).

4.4.3.2 Nitrocefin hydrolysis assay

Through analysis with the MOTIF tool (https://www.genome.jp/tools/motif/) against PROSITE, NCBI and Pfam databases, we found that LpqKMtb (409 aa) is a possible conserved lipoprotein with several hits for β-lactamase motifs, in which the hit from Pfam has the best e-value (6.6e^-57 at 42-388 aa) (Figure 7.6 in Annexes). CwlMMtb (406 aa) is annotated as an N-acetylmuramoyl-L-alanine amidase with several hits for amidase and PG binding domains in which the AmiC, N-acetylmuramoyl-L-alanine amidase motif has the best e-value (2e^-55 at 156-376 aa) (Figure 7.7 in Annexes). Following these results, we decided to characterize the CwlM and LpqK proteins in terms of β-lactamase activity against a chromogenic cephalosporin, nitrocefin. A nitrocefin hydrolysis assay was carried out with the strains of E. coli BL21(DE3) producing each protein respectively. In this protocol, we used filtered cell lysates obtained from IPTG-induced cultures as a preliminary substrate to more promptly identify possibly increased β-lactamase activity due to high expression of the study proteins.

Through this experiment, we obtained a rate of µg of hydrolysed nitrocefin per unit of time and per mg of total protein. We can observe that the rates increase from lower to higher (Figure 4.13) in the following order: pET-29b (control), CwlM and LpqK. Albeit not significantly, the lysate obtained from the culture expressing LpqK possessed the highest β-lactamase activity, almost doubling the value obtained for the control. Since E. coli strains produce multiple proteins besides the protein under study, some of these may potentially interfere with the readings. Thus, to better clarify a possible β-lactamase activity for this enzyme, assays with the purified protein should be attempted. Since LpqK in M.

smegmatis was reported as a group 2a penicillinase⁶¹, other iodometric assays should be done for LpqKMtb with various β-lactam antibiotics as substrates, especially penicillins. In addition, different reaction buffers should be experimented with, given that each β-lactamase may have its optimal reaction

43 buffer. Phosphate-based buffers like the one used in this assay, PBS, have poor buffering ability in alkaline conditions and can inhibit some metal ion-dependent biochemical reactions. In fact, HEPES buffer is ideal for these assays because it has a superior ability to maintain the solution’s pH and because it cannot be complexed with metal ions⁶⁹, on which some β-lactamases might depend.

Figure 4.13 – Nitrocefin assay with activity expressed as µg of hydrolysed nitrocefin min^-1 by mg of total protein^-1 for each protein (pET-29b – control, CwlM and LpqK).Ordinary one-way ANOVA Dunnett's multiple comparisons test was applied for relative statistical differences.

4.4.4 Synergy of the CwlM protein with antibiotics

To evaluate potential synergistic effects between CwlM and different antibiotics on the growth of M. smegmatis, we applied the checkerboard method⁷⁰. The following antibiotics were tested: EMB, MER, CTX and CTX+CLAV. The absorbances were measured and a growth rate was calculated and plotted with the correspondent CwlM and antibiotic concentrations to evaluate differences (Figure 4.14).

In the CTX graph, we can see that compared to the control (0 µg ml^-1), increasing protein concentrations were consistently associated with increases in the growth rate of M. smegmatis. In the absence of the protein, the growth rate evolves from approximately 100 % to 30 % (with 32 µg ml^-1 of CTX), to 10 % (with 64 µg ml^-1 of CTX), to ~0 % (with 128 µg ml^-1 of CTX), and finally to 0 % (with 256 µg ml^-1 of CTX), the usual MIC. The same antibiotic concentrations combined with 50 µg ml^-1 of CwlM lead to a growth rate of 70%, 40%, 10%, and finally ~0 %, respectively. The presence of lower concentrations of the CwlM protein also delayed the fall in growth rate to a similar extent. Interestingly, a similar effect happens in the CTX+CLAV graph, although not so accentuated. In this case, 50 µg ml

-1 of CwlM has a positive effect on the growth at 128 µg ml^-1, when compared to the control, which is the usual MIC for the WT in this case.

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Figure 4.14 – Synergy graphs with M. smegmatis mc² 155 growth rate by antibiotic concentration (µg ml^-1) for each CwlM protein concentration (µg ml^-1).

With MER, the effect is not as clear as with CTX. Nonetheless, with 2 µg ml^-1 of the carbapenem, a clear increase in growth is observed from 10 % without CwlM to almost 50 % with 50 µg ml^-1 of the protein, although at 4 µg ml^-1 the values return to uniformity. This happens also for the other two protein concentrations (12.5 µg ml^-1 and 6.25 µg ml^-1), but in a less obvious manner. The addition of increasing protein concentrations of CwlM in combination with EMB produces small growth fluctuations and, overall, does not seem to impact the growth rate of M. smegmatis since the growth rate values between different protein concentrations do not have clear divergences as in the case of CTX.

This delay effect seems to be more pronounced with the tested β-lactams in contrast with EMB.

Normally, the addition of the CLAV to CTX reduces and stabilizes the MIC. However, that was not observed with this assay. In general, this delay effect is more pronounced with antibiotic concentrations close to or equal to the MIC values and with a protein concentration of 50 µg ml^-1. The possible effect of protein absorption was considered and removed from the plotted values, so it does not affect the observed patterns.

As said before, the CwlM protein is an amidohydrolase that cleaves a critical amide bond between MurNAc and L-alanine of the PG. The β-lactams have an amide bond between the carbonyl carbon and the nitrogen in the β-lactam ring that is normally cleaved by β-lactamases (Figure 4.15). In addition, there are other amide bonds that are not present in the β-lactam ring. Although the protein showed no PG hydrolysis against M. luteus in the zymogram assay, CwlM could be hydrolysing some

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.