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Appl Environ Microbiol. Feb 2013; 79(3): 768–773.
PMCID: PMC3568546
Mycobacteriophage Ms6 LysA: a Peptidoglycan Amidase and a Useful Analytical Tool
Sebabrata Mahapatra,a Charles Piechota,a Filipa Gil,b Yufang Ma,ac Hairong Huang,ad Michael S. Scherman,a Victoria Jones,a Martin S. Pavelka, Jr.,e Jose Moniz-Pereira,b Madalena Pimentel,b Michael R. McNeil,a and Dean C. Crickcorresponding authora
aDepartment of Microbiology Immunology and Pathology, Colorado State University, Fort Collins, Colorado, USA
bCentro de Patogénese Molecula, Unidade dos Retrovirus e Infecções Associadas, Faculdade de Farmácia, Universidade de Lisboa, Lisbon, Portugal
cDepartment of Biochemistry and Molecular Biology, Dalian Medical University, Dalian, People's Republic of China
dBeijing Tuberculosis and Thoracic Tumor Institute, Beijing, People's Republic of China
eDepartment of Microbiology and Immunology, University of Rochester Medical Center, Rochester, New York, USA
corresponding authorCorresponding author.
Address correspondence to Dean C. Crick, dcrick/at/colostate.edu.
Received July 18, 2012; Accepted November 9, 2012.
Since the peptidoglycan isolated from Mycobacterium spp. is refractory to commercially available murolytic enzymes, possibly due to the presence of various modifications found on this peptidoglycan, the utility of a mycobacteriophage-derived murolytic enzyme was assessed for an analysis of peptidoglycan from mycobacteria. We cloned, expressed, and purified the lysA gene product, a protein with homology to known peptidoglycan-degrading amidases, from bacteriophage Ms6. The recombinant protein was shown to cleave the bond between l-Ala and d-muramic acid of muramyl pentapeptide and to release up to 70% of the diaminopimelic acid present in the isolated mycobacterial cell wall. In contrast to lysozyme, which, in culture, inhibits the growth of both Mycobacterium smegmatis and Mycobacterium tuberculosis, LysA had no effect on the growth of either species. However, the enzyme is useful for solubilizing the peptide chains of isolated mycobacterial peptidoglycan for analysis. The data indicate that the stem peptides from M. smegmatis are heavily amidated, containing few free carboxylic acids, regardless of the cross-linking status.
Mycobacterium tuberculosis is a leading cause of disease-related deaths worldwide and is responsible for nearly 2 million deaths each year. Much of the pathology and general drug resistance that this pathogen demonstrates is believed to be related to its unique cell wall core, which consists of a peptidoglycan layer covalently attached to a mycolic acid layer via the polysaccharide arabinogalactan (1, 2). Although the overall structures of the peptidoglycans of M. tuberculosis and Mycobacterium smegmatis are currently reasonably well known, some important gaps in our understanding of the structure and synthesis of this macromolecule remain. For example, it was recently shown that the carboxylic acid functions of the stem pentapeptide moiety of lipid II, a peptidoglycan precursor, are substantially amidated in M. smegmatis (3) and other mycobacterial species (4). Hence, it is important to understand the nature, synthesis, and function of these modifications in mature peptidoglycan in M. smegmatis, M. tuberculosis, and other Mycobacterium species.
Typically, when analyzing the peptidoglycan structure, the peptidoglycan is chemically or enzymatically hydrolyzed to generate soluble fragments that can be further analyzed (36). However, chemical hydrolysis can result in the loss of features, and peptidoglycan isolated from Mycobacterium spp. is notoriously refractory to commercially available murolytic enzymes. This resistance to enzymatic hydrolysis is possibly due to the presence of some or all of the modifications found on mycobacterial peptidoglycan, including an N-glycolyl function on the muramic acid, the amidation of carboxylic acids, and the addition of a glycine or serine to the peptide (5). These modifications are not unique to mycobacteria, as the amidation of carboxylic acid functions (7); N-glycolylation (8); and the addition of glycine (9), serine, or alanine (10, 11) have been reported for the peptidoglycans of other bacteria. Thus, an endolytic enzyme that is adapted to the hydrolysis of modified peptidoglycan could be a useful tool in the armamentarium of the mycobacteriologist and other researchers. Since peptidoglycan hydrolases are often specific to peptidoglycan cross-linking and secondary modifications (12), the murolytic activity of a mycobacteriophage endolysin was investigated.
More than 220 mycobacteriophage genomes have been sequenced, all of which are predicted to encode a putative endolysin (lysin A) (12). The protein encoded by lysA in mycobacterial phage Ms6 causes Escherichia coli cells to lyse, after the addition of CHCl3, when expressed in the heterologous host (13, 14). In addition to Ms6 LysA, four other mycobacteriophages generate cleared zones in zymograms where lyophilized Micrococcus luteus was incorporated into the gel matrix (15, 1517), strongly suggesting that mycobacteriophage LysA proteins are murolytic. Thus, it seemed possible that LysA could be a useful reagent for generating peptidoglycan fragments from mycobacterial peptidoglycan for analytical purposes. This paper reports the expression, purification, and partial characterization of the enzymatic activity of LysA from Ms6 and the utilization of the enzyme in analyses of the amidation of mycobacterial peptidoglycan. It should be noted that the designation lysA is also used to identify an unrelated gene encoding an enzyme involved in diaminopimelic acid (DAP) synthesis; in this paper, lysA and LysA refer to the gene encoding the mycobacterial phage lysin and the lysin, respectively.
Expression of LysA.
Ms6 LysA was readily expressed in a soluble form with an N-terminal His tag by using the previously described plasmid pMG231 in E. coli M-15 pREP4 (Qiagen, Valencia, CA) cells (14). The protein was purified to near homogeneity by using His-Select HF nickel affinity gel (Sigma, St. Louis, MO).
Preparation of [3H]diaminopimelic acid-labeled peptidoglycan of M. smegmatis.
A total of 50 μCi of tritiated DAP (45 Ci/mmol; American Radiochemicals, St. Louis, MO) was added to 250 ml of Middlebrook 7H9 medium, without albumin, dextrose, and catalase (ADC) enrichment, supplemented with lysine at 40 μg/ml, which was then inoculated with M. smegmatis strain PM1482 (ept-1 ΔlysA4 rpsL6 ΔblaS ΔblaE) and incubated at 37°C to the late log phase. The cells were centrifuged for 10 min at 6,000 × g and resuspended in 20 mM Tris HCl (pH 7.9) containing 0.5 M NaCl and 20% glycerol (breaking buffer) at 4 ml/g of cell pellet. The cells were then broken via six passes through a French pressure cell at 20,000 lb/in2, and a cell wall-enriched pellet was recovered after centrifugation (40 min at 35,000 × g). The pellet was washed by resuspension in breaking buffer and repeated centrifugation. Noncovalently attached proteins and carbohydrates were removed by treatment in 2% SDS overnight at room temperature, and again, the pellet was isolated by centrifugation. The resulting pellet was resuspended in 2% sodium dodecyl sulfate (SDS), brought to 100°C for 1 h, and recovered by centrifugation. Finally, the pellet was washed twice with water and then with 80% acetone, resulting in a purified mycolylarabinogalactan-peptidoglycan complex (MAPc) containing tritium-labeled DAP. The final preparation was resuspended in water and stored at −80°C for future use.
Preparation of unlabeled and radiolabeled UDP-muramyl-pentapeptide and muramyl-pentapeptide.
UDP-muramyl-pentapeptide (UDP-Mur-pentapeptide) was enzymatically synthesized as previously described (3). Separate preparations of UDP-Mur-[14C]pentapeptide were made by incorporating l-[14C]Ala or d-[14C]Glu (PerkinElmer). Muramyl-pentapeptide (Mur-pentapeptide) and radiolabeled Mur-pentapeptides were prepared from UDP-Mur-pentapeptide and UDP-Mur-[14C]pentapeptide by hydrolysis in 0.2 M trifluoroacetic acid for 1 h at 60°C. The samples were then dried and dissolved in water.
Treatment of [3H]DAP-labeled peptidoglycan with LysA.
MAPc samples containing radiolabeled peptidoglycan (2,600 cpm) were suspended in 100 μl of 50 mM ammonium acetate buffer at pH 5.0, 6.0, or 7.0, and 40 μg of recombinant LysA was added. The mixtures were incubated overnight at room temperature with gentle rocking. Concentrated trichloroacetic acid was added to a final concentration of 10%, and the resulting mixture was incubated on ice for 30 min before centrifugation for 15 min at 14,000 × g. To determine the percentage of radioactivity solubilized from the peptidoglycan, aliquots of the resulting supernatants and pellets {after resuspension in 0.5% (wt/vol) 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate (CHAPS)} were analyzed by liquid scintillation spectrometry.
Treatment of Mur-[14C]pentapeptide with LysA.
Mur-[14C]pentapeptide samples, prepared as described above, containing radiolabeled peptide were suspended in 100 μl of 50 mM ammonium acetate buffer at pH 5.0, and 40 μg of recombinant LysA was added. The mixtures were incubated for the indicated times at room temperature with gentle rocking. Concentrated trichloroacetic acid was added to a final concentration of 10%, and the resulting mixture was chilled on ice for 30 min before centrifugation for 15 min at 14,000 × g. Aliquots of the resulting supernatants were applied onto Kieselgel 60 F254 thin-layer chromatography (TLC) plates, which were then developed in isobutyric acid–1 M ammonium hydroxide (5:30, vol/vol), and the radioactive bands were visualized by autoradiography.
Mass spectrometric analysis of peptides released from M. smegmatis cell walls.
Unlabeled MAPc was prepared and treated with LysA as described above for the radiolabeled MAPc. The LysA-digested samples were clarified by centrifugation, and supernatants containing MAPc-derived peptides were deproteinated by ultrafiltration using Millipore Ultrafree centrifugal ultrafiltration devices with a 5-kDa cutoff. The samples were then subjected to liquid chromatography-mass spectrometry (LC-MS). An aliquot was applied onto a Phenomenex HyperClone ODS reverse-phase C18 column (5 μm [2.0 by 150 mm]) connected to an Agilent 1200 series high-performance liquid chromatography (HPLC) system, and the soluble Mur-peptides were eluted with a 0 to 80% linear gradient of methanol in 0.1% formic acid at a flow rate of 320 μl/min The eluate was directly introduced into an Agilent 6250 quadrupole time-of-flight (Q-TOF) mass spectrometer equipped with an Agilent multimode source operated in the simultaneous electrospray ionization and atmospheric pressure chemical ionization mode. The positive-ion MS and tandem MS (MS2) data were collected by using Agilent MassHunter workstation software and processed with the molecular feature extractor algorithm (MFE) of Agilent MassHunter Qualitative Analysis software to find molecular features (compounds with a defined exact mass and retention time) present in each sample. A preset minimum abundance of 500 counts was used to filter out low-abundance molecular features. A custom database containing calculated monoisotopic masses of possible monomeric, dimeric, and trimeric peptides, including those with amidated carboxylic acid residues and/or Gly residues (up to a molecular mass of 2 kDa), was generated and used to identify the observed molecular features. The assigned identity of peptides was confirmed by MS2 when possible.
Expression of LysA and its activity against isolated mycobacterial cell walls.
Ms6 LysA (GenBank accession number AAG48318.1), a 43,079-Da protein with a predicted amidase 2 domain, was readily expressed with an N-terminal His tag and purified to apparent homogeneity, as assessed by SDS-polyacrylamide gel electrophoresis (data not shown), by immobilized nickel affinity chromatography. The ability of the enzyme to solubilize mycobacterial cell walls was tested by using [3H]DAP-labeled MAPc from M. smegmatis at pH 5.0, 6.0, or 7.0. Up to 70% of the radiolabeled DAP present in the MAPc could be solubilized by Ms6 LysA by digestion overnight at room temperature, which is consistent with the previously reported hydrolytic activity in zymograms (17). There was little difference in the amounts of material released at the three pH values tested, although 10 to 15% more radioactive material was consistently solubilized at pH 5.0 than at pH 7.0 (data not shown).
Determination of the mode of action of LysA.
Initial experiments indicated that Ms6 LysA did not hydrolyze UDP-Mur-pentapeptide but did hydrolyze Mur-pentapeptide derived from the UDP-Mur-pentapeptide (data not shown). Thus, Mur-[14C]pentapeptide containing radiolabeled d-Glu residues was treated with enzyme and analyzed by thin-layer chromatography (Fig. 1). Radiolabeled material was released in a time-dependent fashion and migrated slightly faster than the starting material under the chromatography conditions used. The treatment of Mur-[14C]pentapeptide containing radiolabeled l-Ala gave identical results (data not shown), suggesting that the enzyme cleaves between the l-Ala and the muramic acid residue or, although more unlikely, that the enzyme cleaves the pentapeptide at a position distal to the d-Glu residue, a result that is consistent with the presence of an amidase 2 domain, characteristic of N-acetylmuramoyl-l-alanine amidases identified by a search for conserved domains.
Fig 1
Fig 1
TLC analysis of Mur-[14C]Glu-pentapeptide treated with purified recombinant LysA for various periods of time. The radioactive material at the origin is residual UDP-Mur-[14C]Glu-pentapeptide after hydrolysis to generate Mur-[14C]Glu-pentapeptide.
To unambiguously identify the site of cleavage, nonradiolabeled Mur-pentapeptide was treated with Ms6 LysA, and the resulting sample was purified by HPLC (3), using a Superdex Peptide 10/300 GL sizing column (Amersham Biosciences, Piscataway, NJ), generating a single major peak consistent with a small peptide. Positive-ion mass spectroscopy showed a dominant feature with a retention time of 7.9 min and an m/z of 533.2, which is consistent with a [M + H]+ ion corresponding to a calculated monoisotopic mass of 532.249291 for the l-Ala–d-Glu–DAP–d-Ala–d-Ala pentapeptide. MS2 data confirmed this observation (Fig. 2; see also Fig. S1 in the supplemental material), demonstrating the presence of daughter ions with m/z values of 515.18, 462.07, 444.09, 373.05, and 333.04, as expected (3) (see Fig. S2 in the supplemental material for the inferred fragmentation pattern). These results unambiguously show that Ms6 LysA is an amidase that cleaves between the l-Ala residue and the lactyl moiety of the muramic acid residues of Mur-pentapeptide.
Fig 2
Fig 2
MS2 of the pentapeptide l-Ala–γd-Glu–DAP–d-Ala–d-Ala released from Mur-pentapeptide by LysA. Observed ions are labeled according to the inferred fragmentation pattern shown in Fig. S2 in the supplemental material. (more ...)
Effect of LysA on bacterial growth.
Phage lytic enzymes often have motifs resulting in tight and specific binding to the cell wall of their host bacterium, and exogenously applied phage-encoded endolysins have been shown to have effective antimicrobial activity against Gram-positive pathogens (1820). Previously reported observations indicated that lysozyme could inhibit the growth of Mycobacterium bovis BCG and M. tuberculosis under some conditions (21, 22), and an MIC of 16 μg/ml for lysozyme against M. smegmatis grown in 7H9 medium was reported previously (23). Thus, a point that is not widely appreciated is that the mycolic acid layer of mycobacteria, which is often described as a permeability barrier (2427), is not necessarily a barrier to lytic proteins. Therefore, the action of purified Ms6 LysA against intact mycobacteria was explored even though the mycolic acid-rich cell wall is generally thought to act as a permeability barrier. The addition of up to 500 μg/ml of the endolysin neither inhibited the growth of nor killed M. smegmatis or M. tuberculosis in 7H9 medium. In contrast, the treatment of 1.2 × 107 CFU M. smegmatis for 24 h with lysozyme at 20 μg/ml killed 98% of the bacteria (2.4 × 105 CFU remained), a result consistent with the low MIC of 16 μg/ml reported previously for M. smegmatis cells grown in 7H9 medium (23). It is not clear why an enzyme that cleaves the glycan chain of peptidoglycan, lysozyme, should kill mycobacteria when an enzyme that removes the peptide portion of peptidoglycan, Ms6 LysA, does not. It is possible that the explanation is trivial (instability in the culture medium or a lack of high specific activity, for example); however, phage lytic enzymes are generally ineffective against Gram-negative bacteria because the outer membranes of these organisms prevent access to the peptidoglycan, and the mycolic acid layer of the mycobacterial cell wall could act as a similar, although selective, barrier (12). It is possible that the considerably higher molecular mass of Ms6 LysA (43,079 Da) than that of lysozyme (16,240 Da) or other chemicophysical properties may prevent LysA from crossing the mycolate layer in intact mycobacteria. Alternatively, the polycationic properties of the lysozyme (28) may contribute to the molecule's lethal effect on M. smegmatis; however, it is known that relatively minor changes in the mycolic acid structure result in a dramatic increase in the lysozyme susceptibility of Mycobacterium marinum (29), suggesting that minor changes in the physical nature of the mycolic acid layer can result in significant changes in the permeability of this hydrophobic barrier. Recently, an intriguing idea that mycobacterial pathogens might be rendered susceptible to exogenous endolysins through cotreatment with LysA and LysB, a mycobacteriophage mycolylarabinogalactan esterase that releases mycolic acids from the mycobacterial cell wall, was proposed (17, 30).
Utilization of LysA for peptidoglycan analysis.
Since it had been demonstrated that Ms6 LysA efficiently solubilized the majority of radiolabeled DAP in MAPc preparations (see above), it seemed likely that the enzyme could be utilized as a tool for analyses of the mycobacterial peptidoglycan structure. MS analysis of Ms6 LysA-treated MAPc from M. smegmatis identified 28 features with exact masses that matched the calculated masses of predicted peptidoglycan fragments (Fig. 3). As can be seen, the solubilized peptides fell into one of three groups: those that were un-cross-linked monomers (Fig. 3) consisting of tri- and tetrapeptides; those that were cross-linked dimers of the stem peptide consisting of hexa-, hepta-, and octapeptides; and those that were cross-linked trimers of the stem peptide consisting of nona- and decapeptides. Thus, once incorporated into the cell wall, the entire repertoire of detectable stem peptides in M. smegmatis peptidoglycan had been truncated to either tripeptides (Tri) (Fig. 3) or tetrapeptides (Tetra) (Fig. 3). For example, features 7 and 9 are hexapeptides composed of dimers of two stem peptides truncated to tripeptides, with three carboxylic acid functions amidated (identified as Tri-Tri-3NH2 in Fig. 3). Figure 4 provides a partial representative MS2 spectrum of a Tri-Tri-3NH2 peptide. This analysis clearly shows ions with m/z values (743.3666, 726.3424, and 709.3167) consistent with the neutral loss of NH3, presumably representing the amide nitrogens (see Fig. S3 in the supplemental material). Although these ions indicate the presence of the amide modifications, they provide no information regarding the actual location of the solubilized peptidoglycan fragment.
Fig 3
Fig 3
LC-MS analysis of LysA-treated MAPc from M. smegmatis. The molecular feature extractor algorithm (MFE) in Agilent MassHunter Qualitative Analysis software identified 28 features with exact masses that matched the calculated masses of predicted peptidoglycan (more ...)
Fig 4
Fig 4
Partial MS2 spectrum of an ion with an m/z of 760.3965. The peak with an m/z of 760.3952 represents the parent molecular ion ([M + H]+) of a hexapeptide composed of two stem peptides truncated to tripeptides, with three amidated carboxylic acid functions (more ...)
The most abundant peptidoglycan fragment seen is a monomer with a tetrapeptide and 2 amide groups (feature 3), while the cross-linked dimer (Tri-Tri-2NH2) (feature 10) was the least abundant (Fig. 3). In terms of cross-linking, it is obvious that the Tri-Tri dimers must have DAP-DAP cross-links and that the Tri-Tri-Tri trimers must have two DAP-DAP cross-links. However, dimers and trimers containing a stem peptide with four amino acid residues (Tetra) may have either a DAP-DAP or a DAP-Ala cross-link. Interestingly, all of the trimers observed contain at least one DAP-DAP cross-link (Fig. 3). The reason for this observation is not intuitively obvious; however, the most abundant monomer is Tetra (feature 3), and the most abundant dimer is Tetra-Tetra (feature 18). Since the most abundant fragments are Tetra fragments, they may well represent the substrates that are available for l,d-transpeptidation. The overall abundance of the Tri-Tri dimers and Tri-Tri-Tri trimers suggests the presence of an undescribed l,d-carboxypeptidase in M. smegmatis. While an activity of this nature was alluded to in a recent review (31), there appear to be no empirical data describing the activity available.
Degree of amidation in peptidoglycan of M. smegmatis.
Cell walls were prepared from M. smegmatis and treated with Ms6 LysA, and the solubilized material was analyzed as described above. The observed peptides ranging from tripeptide monomers to decapeptide trimers have between 3 and 7 carboxylic acid residues that potentially could be amidated. In all cases observed, at least one of the carboxylic acid residues was amidated (Fig. 3). The tripeptide monomers had two amidated carboxylic acids residues in at least two configurations (as determined by relative retention times during chromatography), as did the tetrapeptide monomers. The hexa-, hepta-, and octapeptides had predominantly 3 modified carboxylic acid residues. In contrast, the nona- and decapeptides had primarily 4 or 5 modified carboxylic acid residues, although there were a few with only three modified acidic residues. Hence, the amidation previously found at the lipid II level (3, 4) carried over to mature peptidoglycan, which is heavily modified. The significance of this modification is unknown at present, but one could speculate that the reduction of polarity could make it more energetically favorable to move the lipid II across the plasma membrane; however, all bacteria with peptidoglycan transport lipid II across the plasma membrane, but amidation is not a feature of the peptidoglycan of all bacteria. Alternatively, the amide modifications could help regulate the degree and nature of the interpeptide cross-linking via an as-yet-unknown mechanism, they could play a role in the coordination of peptidoglycan assembly with that of other components of the cell wall, or they could provide resistance to the activity of exogenous lytic enzymes. The identification of the precise positions of carboxylic acid residues that are amidated could potentially shed light on this issue.
Conclusions.
We have shown that LysA from phage Ms6 is a peptidoglycan amidase that cleaves the bond between the d-Mur and l-Ala of the stem peptide of Mur-pentapeptide and mature peptidoglycan. Unlike lysozyme, the Ms6 LysA enzyme was unable to kill or inhibit the growth of either M. smegmatis or M. tuberculosis under the conditions tested. However, the enzyme has been shown to be useful for solubilizing the peptide chains of mycobacterial peptidoglycan for further analysis. In addition, it was shown that the mature peptidoglycan of M. smegmatis is highly amidated, similar to the amidation previously reported for the peptidoglycan precursor lipid II.
Supplementary Material
Supplemental material
ACKNOWLEDGMENTS
This work was supported by funds provided by U.S. Public Health Service grants NIH/NIAID AI-33706 (M.R.M.), NIH/NIAID AI049151 (D.C.C.), and NIH/NIAID AI073772 (M.S.P.) and FCT (Fundação para a Ciência e Tecnologia) project POCTI/ESP/47723/2002 (J.M.-P. and M.P.).
Footnotes
Published ahead of print 16 November 2012
Supplemental material for this article may be found at http://dx.doi.org/10.1128/AEM.02263-12.
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