A detailed protocol is presented for preparing the bacteriostatic diamide masarimycin, a small molecule probe that inhibits the growth of Bacillus subtilis and Streptococcus pneumoniae by targeting cell wall degradation. Its application as a chemical probe is demonstrated in synergy/antagonism assays and morphological studies with B. subtilis and S. pneumoniae.
Peptidoglycan (PG) in the cell wall of bacteria is a unique macromolecular structure that confers shape, and protection from the surrounding environment. Central to understanding cell growth and division is the knowledge of how PG degradation influences biosynthesis and cell wall assembly. Recently, the metabolic labeling of PG through the introduction of modified sugars or amino acids has been reported. While chemical interrogation of biosynthetic steps with small molecule inhibitors is possible, chemical biology tools to study PG degradation by autolysins are underdeveloped. Bacterial autolysins are a broad class of enzymes that are involved in the tightly coordinated degradation of PG. Here, a detailed protocol is presented for preparing a small molecule probe, masarimycin, which is an inhibitor of N-acetylglucosaminidase LytG in Bacillus subtilis, and cell wall metabolism in Streptococcus pneumoniae. Preparation of the inhibitor via microwave-assisted and classical organic synthesis is provided. Its applicability as a tool to study Gram-positive physiology in biological assays is presented.
Peptidoglycan (PG) is a mesh-like polymer that delineates cell shape and structure in both Gram-positive and Gram-negative bacteria1,2. This heteropolymer is a matrix of amino sugars cross-linked by short peptides3,4,5,6 with a backbone composed of β-(1,4)-linked alternating N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) residues (Figure 1)1. Attached to the C-3 lactyl moiety of MurNAc is the stem peptide. The metabolism of PG involves a tightly coordinated system of biosynthetic and degradative enzymes to incorporate new material into the cell wall7,8. Degradation of PG is carried out by enzymes collectively referred to as autolysins9 and further classified based on the specificity of the bond cleaved. Autolysins participate in many cellular processes including cell growth, cell division, motility, PG maturation, chemotaxis, protein secretion, genetic competence, differentiation, and pathogenicity10,11. Unraveling the specific biological functions of individual autolysins can be daunting, due in part to functional redundancy. However, recent biophysical8,12,13 and computational studies12 have provided new insight into their roles in PG metabolism. In addition, recent reports have provided further insight into the synthesis14 and membrane-mediated15,16,17 steps in PG metabolism. A thorough understanding of the relationship between degradative and synthetic pathways of PG metabolism could give rise to previously untapped antibiotic targets.
While there have been significant advances in methodology to study glycobiology in eukaryotes, bacterial glycobiology and, in particular, PG metabolism has not advanced at a similar rate. Current chemical approaches to study PG metabolism include fluorescently labeled antibiotics18, fluorescent probes19,20, and metabolic labeling21,22,23,24. These new approaches are providing new ways to interrogate bacterial cell wall metabolism. While some of these strategies are capable of labeling PG in vivo, they can be species-specific19, or only work in strains lacking a particular autolysin25. Many PG labeling strategies are intended for use with isolated cell walls26 or with in vitro reconstituted PG biosynthesis pathways20,27,28. The use of fluorescently labeled antibiotics is currently limited to biosynthetic steps and transpeptidation18.
The current knowledge of bacterial autolysins and their role in cell wall metabolism comes from genetic and in vitro biochemical analysis11,29,30,31,32. While these approaches have provided a wealth of information on this important class of enzymes, deciphering their biological role can be challenging. For instance, due to functional redundancy33, deletion of an autolysin in most cases does not result in halting bacterial growth. This is despite their implied role in cell growth and division7,12. Another complication is that genetic deletion of bacterial autolysins can give rise to meta-phenotypes34. Meta-phenotypes arise from the complex interplay between the pathway affected by the genetic deletion and other interconnected pathways. For instance, a meta-phenotype can arise via a direct effect such as the lack of an enzyme, or an indirect effect such as a disruption of regulators.
Currently, there are only a few inhibitors of glycosidase autolysins such as N-acetylglucosaminidases (GlcNAcase) and N-acetylmuramidases, which can be used as chemical probes to study the degradation of PG. To address this, the diamide masarimycin (previously termed as fgkc) has been identified and characterized35 as a bacteriostatic inhibitor of Bacillus subtilis growth that targets the GlcNAcase LytG32 (Figure 1). LytG is an exo-acting GlcNAcase36, a member of cluster 2 within glycosyl hydrolase family 73 (GH73). It is the major active GlcNAcase during vegetative growth32. To our knowledge, masarimycin is the first inhibitor of a PG-acting GlcNAcase that inhibits cellular growth. Additional studies of masarimycin with Streptococcus pneumoniae found that masarimycin likely inhibits cell wall metabolism in this organism37. Here, the preparation of masarimycin is reported for use as a chemical biology probe to study physiology in the Gram-positive organisms B. subtilis, and S. pneumoniae. Examples of morphological analysis of sub-minimum inhibitory concentration treatment with masarimycin, as well as a synergy/antagonism assay are presented. Synergy and antagonism assays using antibiotics with well-defined modes of action can be a useful way to explore connections between cellular processes38,39,40.
1. General methods
NOTE: All compounds were purchased from standard suppliers and used without further purification.
2. General procedure for preparation of masarimycin
NOTE: Perform the below steps in a fume hood.
3. Microwave procedure for preparation of masarimycin
4. Synergy and antagonism assay
5. Morphological study
Masarimycin is a small molecule bacteriostatic inhibitor of B. subtilis and S. pneumoniae and has been shown to inhibit the exo-acting GlcNAcase LytG in B. subtilis35,37 and target the cell wall in S. pneumoniae37. Masarimycin can be efficiently prepared either by the classical or microwave-assisted organic synthesis with yields in the 55%-70% range. Microwave-assisted synthesis has the advantage of a significant reduction in time to synthesize the compound. Microwave-assisted synthesis shortens the synthesis from 5-6 h (traditional synthesis) to 2-3 h while maintaining comparable yields. Flash chromatography provides a rapid purification of masarimycin in high purity (Supplementary Figures 1–2). Structural assignments from 1H and 13C NMR spectra along with representative spectra are provided in Supplementary Figures 3–4.
Synergy and antagonism screens can be a useful tool to reveal functional connections among cellular components (synergy) and to investigate genetic networks and mechanisms of drug action (antagonism)40. Evaluation of synergy/antagonism with the ATPase inhibitor optochin in S. pneumoniae is presented in Figure 2. Resazurin microtitre plate assay41 provides an easy readout of the growth/non-growth of the organism. The lowest concentration of compound to inhibit bacterial growth (blue color) is taken as the MIC value in the presence of a co-drug. Wells with bacterial growth will be pink in color. The relationship between masarimycin and optochin was determined by calculating the fractional inhibitor concentration index (FICI) using equations in protocol step 4.7. The FICI value for the masarimycin-optochin interaction is calculated to be 1.5, indicating an indifferent relationship based on published standards42. Phenotypic assays using masarimycin in B. subtilis at sub-MIC concentrations presented a sausage-like phenotype (Figure 3B) which differs from reported phenotypes of the ΔlytG mutant in the literature32 and more closely resembles multiple autolysin knockouts29. Phenotypic analysis of S. pneumoniae with masarimycin at sub-MIC concentrations presented a clumping phenotype (Figure 3D). This clumping phenotype is distinct from those reported for S. pneumoniae cell-wall-acting GlcNAcases43,44,45.
Figure 1: Structure of peptidoglycan showing the cleavage site of the exo-acting N-acetyl glucosaminidase LytG from Bacillus subtilis. Inset shows the structure of the LytG inhibitor masarimycin. Please click here to view a larger version of this figure.
Figure 2: Synergy/Antagonism assay to explore antagonistic/synergistic relationships with masarimycin and optochin in S. pneumoniae. Blue or purple color indicates no bacterial growth, while pink color indicates bacterial growth. The MIC in the presence of co-drug is taken as the lowest concentration that shows no bacterial growth (blue color). Please click here to view a larger version of this figure.
Figure 3: Morphological analysis. Morphological changes to B. subtilis (A,B) and S. pneumoniae (C,D) when treated with 0.75x MIC (MICB.subtilis = 3.8 µM and MICS.pneumoniae = 7.8 µM) masarimycin. Cells were fixed and stained with 0.1% (m/v) methylene blue and visualized by bright field microscopy under oil immersion at 1000x magnification. This figure has been modified from 35. Please click here to view a larger version of this figure.
Supplementary Figure 1: Representative thin layer chromatography of masarimycin post aqueous workup. The mobile phase is 90:10 hexane: isopropanol and iodine vapor is used for staining spots. Rf = 0.3 for masarimycin. Please click here to download this File.
Supplementary Figure 2: Representative flash chromatogram for the purification of masarimycin. The peak at approximately 1.2 column volumes contains masarimycin. Please click here to download this File.
Supplementary Figure 3: Representative 1H NMR of masarimycin dissolved in CDCl3 and recorded on a 400 MHz NMR spectrometer. Spectrum is referenced to residual CHCl3 solvent peak at δ = 7.26. Numbers in green above chemical shifts indicate proton assignments at the corresponding positions in the structure of masarimycin (see inset). Please click here to download this File.
Supplementary Figure 4: Representative 13C NMR spectrum of masarimycin dissolved in CDCl3 at 100 MHz. Spectrum referenced to residual CHCl3 solvent peak at δ = 77.36. Numbers in green above chemical shifts indicate carbon atom assignments at the positions in the structure of masarimycin (see inset). Please click here to download this File.
Supplementary Figure 5: Representative resazurin MIC assay of masarimycin against B. subtilis. Please click here to download this File.
Masarimycin is a single micromolar bacteriostatic inhibitor of B. subtilis35 and S. pneumoniae37 growth. In B. subtilis, masarimycin has been shown to inhibit the GlcNAcase LytG35, while the precise molecular target in the cell wall of S. pneumoniae has not been identified37. Synthesis of masarimycin using either the classical organic synthesis or microwave procedure provides the inhibitor in good yield and high purity. Low yields of masarimycin can typically be attributed to the oxidation of the cyclohexyl carboxaldehyde. To overcome this, it is recommended to store cyclohexyl carboxaldehyde under an inert atmosphere in a desiccator. Oxidation of the aldehyde to the corresponding carboxylic acid can be seen as a white solid in the bottle. Purchasing small quantities of cyclohexyl carboxaldehyde without storing it for extended periods greatly reduces this problem.
NMR assignment of masarimycin structure is complicated by the presence of a mixture of cis and trans forms of the amide bond as well as atropisomers around the o-iodophenyl ring that results in multiple peaks. This can result in a proton chemical shift spread over 1 ppm thereby complicating assignments35. As a result, partial assignment of NMR chemical shifts for both 1H and 13C NMR spectra along with representative spectra are provided in Supplementary Figures 3–4. If there is difficulty in assigning 1H and 13C chemical shifts for masarimycin due to the mixture of isomers, 2-dimensional NMR experiments can be used. Correlated spectroscopy (COSY) can be used to identify proton spin systems, while heteronuclear single quantum coherence spectroscopy (HSQC) NMR experiments can be used to identify proton-carbon single bond correlations. Once purified, masarimycin can be stored at -20 °C as oil or dissolved in DMSO to a concentration of 25 mM until needed. It is recommended to store in small aliquots to reduce the number of freeze-thaw cycles. After repeated freeze-thaw cycles of the compound, the masarimycin stock solution should be checked by TLC to monitor for any degradation.
Synergy and antagonism screens can be an effective strategy to identify pathway interactions and can be used to understand the mode of action of small molecules. Figure 2 shows an example of a synergy/antagonism assay with S. pneumonia R6 using masarimycin and the ATPase inhibitor optochin (note that the synergy/antagonism screening in B. subtilis is still an ongoing investigation). For reproducibility, second passage cells were used and grown to an OD600nm of no more than 0.4. A FICI of 1.5 was observed for the interaction between masarimycin and optochin, indicating an indifferent relationship between the antibiotic pair. The indifferent relationship between masarimycin and optochin indicates no apparent interaction between the pathways these antibiotics target. While these assays can provide useful information about drug interactions, it is important to note that synergy/antagonism assays should be run with biological replicates and use of the more conservative cutoffs as described by Odds42. This helps to prevent the over-interpretation of observed minor synergistic or antagonistic relationships.
Phenotypic analysis of B. subtilis cells treated with sub-MIC masarimycin (Figure 3B) indicates a phenotype that differs from phenotypes reported for genetic deletion of lytG32 and more closely resembles phenotypes of B. subtilis strains with multiple autolysin deletions29. This discrepancy in phenotype is intriguing because while in vitro inhibition of LytG has been demontrated35, a ΔlytG mutant has no observable phenotype32. This discrepancy can in part be explained by differences in genetic and chemical inactivation46,47. The observed differences in the chemical or genetic inactivation of LytG is an intriguing question that is currently under investigation. S. pneumoniae cells treated with masarimycin presented a phenotype (Figure 3D) distinct from the genetic deletion of the corresponding GlcNAcase (GH73, cluster 2) LytB37,43,44,48. This morphological discrepancy highlights the challenges in assigning the mode of action or attributing the biological target of small molecule inhibitors. Morphological phenotypes can arise from a more complex set of interactions other than a single genetic deletion or chemical inactivation of a cell-wall acting enzyme. These meta-phenotypes34 can arise from complex interactions via direct (lack of an enzyme(s)) or indirect (loss of regulators) mechanisms.
To the best of our knowledge, masarimycin is the first inhibitor of a bacterial autolysin that demonstrates inhibition of bacterial growth (Supplementary Figure 5). It is a narrow-spectrum bacteriostatic inhibitor of growth in B. subtilis and S.pneumoniae. This narrow spectrum is a limitation for multi-species comparative studies of cell-wall metabolism between Gram-positive and Gram-negative organisms. This narrow spectrum is in part due to differences in some of the glycosyl hydrolase autolysins used during vegetative growth between Gram-positive (GlcNAcase) and Gram-negative (lytic transglycosylase) organisms. Using small-molecule inhibitors such as masarimycin for inhibiting PG autolysins, in particular, GlcNAcases can provide an orthogonal approach to traditional genetics for elucidating autolysin function. Masarimycin has a distinct advantage over some chemical biology methods, in that it can be used in more than one species (B. subtilis and S. pneumoniae). It can allow for comparative studies of cell wall metabolism between rod-shaped (B. subtilis) and coccoid (S. pneumoniae) species. The less coregulated cell wall metabolism and division in S. pneumoniae provides a counter-point in the more tightly regulated system of rod-shaped species49,50. Future applications of this technique will be to identify the molecular target in S.pneumoniae and explore the differences between genetic and chemical inactivation of autolysins in S.pneumoniae and B. subtilis.
Critical Steps in the Protocol
It is important to pay attention to the effective concentration of masarimycin in biological and biochemical assays. Due to its hydrophobic nature, concentrations above 250 µM (65x MIC in B. subtilis) can result in solubility and aggregation issues that can impact the interpretation of biological data. Properly controlling for the effect of vehicle (i.e., DMSO) in all experiments is essential.
The authors have nothing to disclose.
Research was supported by the National Science Foundation under grant number 2009522. NMR analysis of masarimycin was supported by the National Science Foundation major research instrumentation program award under grant number 1919644. Any opinions, findings, and conclusions, or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the National Science Foundation.
2-Iodobenzoic acid | SIGMA-ALDRICH | I7675-25G | corrosive, irritant, light yellow to orange-brown powder |
2-Propanol | SIGMA-ALDRICH | 109827-4L | flammable, irritant, colorless liquid |
Acetonitrile | SIGMA-ALDRICH | 34851-4L | flammable, irritant, colorless liquid |
Aluminum backed silica plates | Sorbtech | 4434126 | silica gel XG F254 on aluminum backed plates |
chloroform-d | SIGMA-ALDRICH | 151823-50G | solvent for NMR |
Compact Mass Spectrometer | Advion-Interchim | Advion CMS | compact mass spectrometer equiped with APCI source and atmospheric solids analysis probe |
Corning Costar 96 well flat bottom plates-sterile | fisher chemical | 07-200-90 | for synergy/antagonism assays |
cover slips | fisher chemical | 12-547 | for microscopy |
Cyclohexanecarboxaldehyde | CHEM-IMPEX INT'L INC. | 24451 | flammable, irritant, colorless to pink liquid |
Cyclohexyl isocyanide | SIGMA-ALDRICH | 133302-5G | irritant, colorless liquid, extremly unpleasant odor |
Cyclohexylamine | SIGMA-ALDRICH | 240648-100ML | corrosive, flammable, irritant, colorless liquid unless contaminated |
Ethyl acetate | SIGMA-ALDRICH | 537446-4L | flammable, irritant, colorless liquid |
flash silica cartridge (12g) | Advion-Interchim | PF-50SIHP-F0012 | pack of flash silica columns (12g) for purification of masarimycin |
formaldehyde | SIGMA-ALDRICH | F8775-25ML | fixing agent for microscopy |
HEPES | SIGMA-ALDRICH | H8651-25G | buffer for microscopy fixing solution |
Hexane, mixture of isomers | SIGMA-ALDRICH | 178918-4L | environmentally damaging, flammable, irritant, health hazard, colorless liquid |
High performance compact mass spectrometer | Advion | expression | Atmospheric Solids Analysis Probe (ASAP), low resolution |
High Vac | eppendorf | Vacufuge plus | vacuum aided by centrifugal force and temperature |
Hydrochloric acid | SIGMA-ALDRICH | 258148-2.5L | corrosive, irritant, colorless liquid |
hydrochloric acid | SIGMA-ALDRICH | 320331-2.5L | strong acid |
immersion oil | fisher chemical | 12-365-19 | for microscopy |
Iodine, resublimed crystals | Alfa Aesar | 41955 | environmentally damaging, irritant, health hazard, dark grey/purple crystals |
Mestre Mnova | MestreLab Research | software for processing NMR spectra | |
Methanol | SIGMA-ALDRICH | 439193-4L | flammable, toxic, health hazard, colorless liquid |
methylene blue | SIGMA-ALDRICH | M9140-25G | microscopy stain for staining cell walls |
meuller-hinton agar plates + 5% sheep blood | fisher chemical | B21176X | growth media for Streptococcus pneumoniae |
meuller-hinton broth | fisher chemical | DF0757-17-6 | growth media for Streptococcus pneumoniae |
microscope slides | fisher chemical | 22-310397 | for microscopy |
Microwave Synthesis Labstation | MILESTONE | START SYNTH | device that requires the ventilation of a fume hood, equipped with synthesis carousel |
NMR tubes | SIGMA-ALDRICH | Z562769-5EA | 5mm NMR tubes 600 MHz |
Nuclear Magnetic Resonance (NMR) | Bruker | Ascend 400 | large superconducting magnet (400MHz) |
optochin | fisher chemical | AAB21627MC | ethylhydrocupreine hydrochloride |
petrie plates | Celltreat | 229695 | for preparing agar plates for bacterial growth |
Primo Star Bright field/Phase contrast Microscope with ERc5s camera | Zeiss | for morphology studies | |
puriFlash | interchim | XS520plus | flash chromatography purification system |
resazurin | SIGMA-ALDRICH | R7017-1G | for synergy/antagonism assays |
Rotary Evaporator | Heidolph | Hei-VAP Value "The Collegiate" | solvent evaporator |
Sodium bicarbonate | SIGMA-ALDRICH | S6014-500G | irritant, white powder |
Sodium chloride | fisher chemical | S271-1 | crystalline, colorless |
Sodium chloride | SIGMA-ALDRICH | S5886-500G | for growth of B.subtilis and preparation of LB media |
Sodium sulfate | SIGMA-ALDRICH | 7985592-500G | anhydrous, granular, white |
tryptone | fisher chemical | BP1421-500 | for growth of B.subtilis and preparation of LB media |
Whitney DG250 Workstation | Microbiology International | DG250 | anaerobic workstation. Anaerobic gas mixture used: 5% hydrogen, 10% carbon dioxide, balance nitrogen |
yeast extract | fisher chemical | BP1422-500 | for growth of B.subtilis and preparation of LB media |
Zen Lite (blue) software | Zeiss | for acquiring micrographs |