Cell Wall Biosynthesis Research Articles

Synthesis and Testing of Analogs of the Tuberculosis Drug Candidate SQ109 against Bacteria and Protozoa: Identification of Lead Compounds against Mycobacterium abscessus and Malaria Parasites.

SQ109 is a tuberculosis drug candidate that has high potency against Mycobacterium tuberculosis and is thought to function at least in part by blocking cell wall biosynthesis by inhibiting the MmpL3 transporter. It also has activity against bacteria and protozoan parasites that lack MmpL3, where it can act as an uncoupler, targeting lipid membranes and Ca2+ homeostasis. Here, we synthesized 18 analogs of SQ109 and tested them against M. smegmatis, M. tuberculosis, M. abscessus, Bacillus subtilis, and Escherichia coli, as well as against the protozoan parasites Trypanosoma brucei, T. cruzi, Leishmania donovani, L. mexicana, and Plasmodium falciparum. Activity against the mycobacteria was generally less than with SQ109 and was reduced by increasing the size of the alkyl adduct, but two analogs were ∼4-8-fold more active than SQ109 against M. abscessus, including a highly drug-resistant strain harboring an A309P mutation in MmpL3. There was also better activity than found with SQ109 with other bacteria and protozoa. Of particular interest, we found that the adamantyl C-2 ethyl, butyl, phenyl, and benzyl analogs had 4-10× increased activity against P. falciparum asexual blood stages, together with low toxicity to a human HepG2 cell line, making them of interest as new antimalarial drug leads. We also used surface plasmon resonance to investigate the binding of inhibitors to MmpL3 and differential scanning calorimetry to investigate binding to lipid membranes. There was no correlation between MmpL3 binding and M. tuberculosis or M. smegmatis cell activity, suggesting that MmpL3 is not a major target in mycobacteria. However, some of the more active species decreased lipid phase transition temperatures, indicating increased accumulation in membranes, which is expected to lead to enhanced uncoupler activity.

β-Lactams from the Ocean.

The title of this essay is as much a question as it is a statement. The discovery of the β-lactam antibiotics-including penicillins, cephalosporins, and carbapenems-as largely (if not exclusively) secondary metabolites of terrestrial fungi and bacteria, transformed modern medicine. The antibiotic β-lactams inactivate essential enzymes of bacterial cell-wall biosynthesis. Moreover, the ability of the β-lactams to function as enzyme inhibitors is of such great medical value, that inhibitors of the enzymes which degrade hydrolytically the β-lactams, the β-lactamases, have equal value. Given this privileged status for the β-lactam ring, it is therefore a disappointment that the exemplification of this ring in marine secondary metabolites is sparse. It may be that biologically active marine β-lactams are there, and simply have yet to be encountered. In this report, we posit a second explanation: that the value of the β-lactam to secure an ecological advantage in the marine environment might be compromised by its close structural similarity to the β-lactones of quorum sensing. The steric and reactivity similarities between the β-lactams and the β-lactones represent an outside-of-the-box opportunity for correlating new structures and new enzyme targets for the discovery of compelling biological activities.

Nanosized extracellular vesicles released by Neurospora crassa hyphae

Extracellular vesicles (EVs) are nanosized structures containing proteins, lipids, and nucleic acids, released by living cells to the surrounding medium. EVs participate in diverse processes, such as intercellular communication, virulence, and disease. In pathogenic fungi, EVs carry enzymes that allow them to invade the host or undergo environmental adaptation successfully. In Neurospora crassa, a non-pathogenic filamentous fungus widely used as a model organism, the vesicle-dependent secretory mechanisms that lead to polarized growth are well studied. In contrast, biosynthesis of EVs in this fungus has been practically unexplored. In the present work, we analyzed N. crassa culture's supernatant for the presence of EVs by dynamic light scattering (DLS), transmission electron microscopy (TEM) and proteomic analysis. We identified spherical membranous structures, with a predominant subpopulation averaging a hydrodynamic diameter (dh) of 68 nm and a particle diameter (dp) of 38 nm. EV samples stained with osmium tetroxide vapors were better resolved than those stained with uranyl acetate. Mass spectrometry analysis identified 252 proteins, including enzymes involved in carbohydrate metabolic processes, oxidative stress response, cell wall organization/remodeling, and circadian clock-regulated proteins. Some of these proteins have been previously reported in exosomes from human cells or in EVs of other fungi. In view of the results, it is suggested a putative role for EVs in cell wall biosynthesis and vegetative development in N. crassa.

Nitric oxide induces S-nitrosylation of CESA1 and CESA9 and increases cellulose content in Arabidopsis hypocotyls

Nitric oxide (NO), a small signaling gas molecule, participates in several growth and developmental processes in plants. However, how NO regulates cell wall biosynthesis remains unclear. Here, we demonstrate a positive effect of NO on cellulose content that may be related to S-nitrosylation of cellulose synthase 1 (CESA1) and CESA9. Two S-nitrosylated cysteine (Cys) residues, Cys562 and Cys641, which are exposed on the surface of CESA1 and CESA9 and located in the cellulose synthase catalytic domain, were identified to be S-nitrosylated. Meanwhile, Cys641 was located on the binding surface of CESA1 and CESA9, and Cys562 was very close to the binding surface. Cellulose synthase complexes (CSCs) dynamics are closely associated with cellulose content. S-nitrosylation of CESA1 and CESA9 improved particles mobility and thus increased the accumulation of cellulose in Arabidopsis hypocotyl cells. An increase in hemicellulose content as well as an alteration in pectin content facilitated cell wall extension and contributed to cell growth, finally promoting elongation of Arabidopsis hypocotyls. Overall, our work provides a path to investigate the way NO affects the cellulose content of plants.

Exploring the Flexibility of the Glycopeptide Antibiotic Crosslinking Cascade for Extended Peptide Backbones.

The glycopeptide antibiotics (GPAs) are a clinically approved class of antimicrobial agents that classically function through the inhibition of bacterial cell-wall biosynthesis by sequestration of the precursor lipid II. The oxidative crosslinking of the core peptide by cytochrome P450 (Oxy) enzymes during GPA biosynthesis is both essential to their function and the source of their synthetic challenge. Thus, understanding the activity and selectivity of these Oxy enzymes is of key importance for the future engineering of this important compound class. Recent reports of GPAs that display an alternative mode of action and a wider range of core peptide structures compared to classic lipid II-binding GPAs raises the question of the tolerance of Oxy enzymes for larger changes in their peptide substrates. In this work, we explore the ability of Oxy enzymes from the biosynthesis pathways of lipid II-binding GPAs to accept altered peptide substrates based on a vancomycin template. Our results show that Oxy enzymes are more tolerant of changes at the N terminus of their substrates, whilst C-terminal extension of the peptide substrates is deleterious to the activity of all Oxy enzymes. Thus, future studies should prioritise the study of Oxy enzymes from atypical GPA biosynthesis pathways bearing C-terminal peptide extension to increase the substrate scope of these important cyclisation enzymes.

New insights into the Undecaprenol monophosphate recycling pathway of Streptococcus pneumoniae.

Recycling of undecaprenol pyrophosphate is critical to regenerate the pool of undecaprenol monophosphate required for cell wall biosynthesis. Undecaprenol pyrophosphate is dephosphorylated by membrane-associated undecaprenyl pyrophosphate phosphatases such as UppP or type 2 Phosphatidic Acid Phosphatases (PAP2) and then transferred across the cytoplasmic membrane by Und-P flippases such as PopT (DUF368-containing protein) or UptA (a DedA family protein). While the deletion of uppP in S. pneumoniae has been reported to increase susceptibility to bacitracin and reduce infectivity in a murine infection model, the presence of PAP2 family proteins or Und-P flippases and their potential interplay with UppP in S. pneumoniae remained unknown. In this report, we identified two PAP2 family proteins and a DUF368-containing protein and investigated their roles together with that of UppP in cell growth, cell morphology and susceptibility to bacitracin in S. pneumoniae. Our results suggest that the undecaprenol monophosphate recycling pathway in S. pneumoniae could result from a functional redundancy between UppP, the PAP2-family protein Spr0434 and the DUF368-containing protein Spr0889.

Overexpression of an actin Gh_D04G0865 gene in cotton reduced fineness of fiber

AbstractCotton fibers are the world's most important renewable source of fiber for textile industry. The cell wall of cotton fiber determines fiber quality parameters for textile industry. The thickness of secondary cell wall and the fiber perimeter define the fineness and micronaire (MIC) of cotton fiber. The fineness of cotton fiber plays an important role in affecting the yarn quality. Actin cytoskeleton impacts the cell extension and shape, however how it affects secondary cell wall is unclear. We overexpressed the actin isovariant Gh_D04G0865 (previously shown mutation in this gene impaired fiber length) under fiber‐specific promoter in cotton. We obtained two independent transgenic lines. Fiber characteristics of their first‐generation progenies were evaluated by high volume instrument (HVI), advanced fiber information system (AFIS), Cottonscope, and Fourier‐transform infrared (FT‐IR) spectroscopy. Fiber of transgenic lines with excessive expression of actin showed significant reduction in MIC and fineness, while increase in strength. Seed index was increased in transgenic lines overexpressing actin. FT‐IR analysis detected changes in vibrations associated with crystalline cellulose. Taken together, our findings revealed that actin cytoskeleton may play roles in reducing fiber wall thickening process during secondary cell wall biosynthesis.

Evidence for the role of sound on the growth and signal response in duckweed

ABSTRACT Sound vibration, an external mechanical force, has been proven to modulate plant growth and development like rain, wind, and vibration. However, the role of sound on plants, especially on signal response, has been usually neglected in research. Herein, we investigated the growth state, gene expression, and signal response in duckweed treated with soft music. The protein content in duckweed after music treatment for 7 days was about 1.6 times that in duckweed without music treatment. Additionally, the potential maximum photochemical efficiency of photosystem II (Fv/Fm) ratio in duckweed treated with music was 0.78, which was significantly higher in comparison with the control group (P < .01). Interestingly, music promoted the Glu and Ca signaling response. To further explore the global molecular mechanism, we performed transcriptome analysis and the library preparations were sequenced on an Illumina Hiseq platform. A total of 1296 differentially expressed genes (DEGs) were found for all these investigated genes in duckweed treated with music compared to the control group. Among these, up-regulation of the expression of metabolism-related genes related to glycolysis, cell wall biosynthesis, oxidative phosphorylation, and pentose phosphate pathways were found. Overall, these results provided a molecular basis to music-triggered signal response, transcriptomic, and growth changes in duckweed, which also highlighted the potential of music as an environmentally friendly stimulus to promote improved protein production in duckweed.

Transcriptomic profiling of wheat stem during meiosis in response to freezing stress.

Low temperature injury in spring has seriously destabilized the production and grain quality of common wheat. However, the molecular mechanisms underlying spring frost tolerance remain elusive. In this study, we investigated the response of a frost-tolerant wheat variety Zhongmai8444 to freezing stress at the meiotic stage. Transcriptome profiles over a time course were subsequently generated by high-throughput sequencing. Our results revealed that the prolonged freezing temperature led to the significant reductions in plant height and seed setting rate. Cell wall thickening in the vascular tissue was also observed in the stems. RNA-seq analyses demonstrated the identification of 1010 up-regulated and 230 down-regulated genes shared by all time points of freezing treatment. Enrichment analysis revealed that gene activity related to hormone signal transduction and cell wall biosynthesis was significantly modulated under freezing. In addition, among the identified differentially expressed genes, 111 transcription factors belonging to multiple gene families exhibited dynamic expression pattern. This study provided valuable gene resources beneficial for the breeding of wheat varieties with improved spring frost tolerance.

A receptor-like kinase controls the amplitude of secondary cell wall synthesis in rice

Cell wall expansion is a key element in determining plant morphology and growth, and cell wall integrity changes are relayed to the cell to fine-tune growth responses. Here, we show that variations in the ectodomain of a cell wall-associated receptor-like kinase, WAK10, in temperate Oryza japonica accessions differentially amplify fluctuations in cell wall integrity to control rice stem height. Mutation in the WAK10 gene exhibited increased cell wall thickening in stem sclerenchyma and reduced cell expansion in the stem. Two WAK10 ectodomain variants bound pectic oligosaccharides with different affinities. The pectic oligosaccharide binding regulated WAK10 phosphorylation activity, the amplitude of secondary wall deposition, and ultimately, stem height. Rice population analyses revealed active enrichment of the short-stem WAK10 ectodomain alleles in japonica subspecies during domestication. Our study outlines not only a mechanism for how variations in ligand affinities of a receptor kinase control cell wall biosynthesis and plant growth, but it also provides breeding targets for new semi-dwarf rice cultivars.

An extensively validated whole-cell biosensor for specific, sensitive and high-throughput detection of antibacterial inhibitors targeting cell-wall biosynthesis.

Whole-cell biosensor strains are powerful tools for antibacterial drug discovery, in principle allowing the identification of inhibitors acting on specific, high-value target pathways. Whilst a variety of biosensors have been described for detecting cell-wall biosynthesis inhibitors (CWBIs), these strains typically lack specificity and/or sensitivity, and have for the most part not been rigorously evaluated as primary screening tools. Here, we describe several Staphylococcus aureus CWBI biosensors and show that specific and sensitive biosensor-based discovery of CWBIs is achievable. Biosensors comprised lacZ reporter fusions with S. aureus promoters (PgltB, PilvD, PmurZ, PoppB, PORF2768, PsgtB) that are subject to up-regulation following inhibition of cell-wall biosynthesis. Induction of biosensors was detected by measuring expression of β-galactosidase using fluorogenic or luminogenic substrates. Three of the six biosensors tested (those based on PgltB, PmurZ, PsgtB) exhibited apparently specific induction of β-galactosidase expression in the presence of CWBIs. Further validation of one of these (PmurZ) using an extensive array of positive and negative control compounds and conditional mutants established that it responded appropriately and uniquely to inhibition of cell-wall biosynthesis. Using this biosensor, we established, validated and deployed a high-throughput assay that identified a potentially novel CWBI from a screen of >9000 natural product extracts. Our extensively validated PmurZ biosensor strain offers specific and sensitive detection of CWBIs, and is well-suited for high-throughput screening; it therefore represents a valuable tool for antibacterial drug discovery.

Cleavage of α-1,4-glycosidic linkages by the glycosylphosphatidylinositol-anchored α-amylase AgtA decreases the molecular weight of cell wall α-1,3-glucan in Aspergillus oryzae.

Aspergillus fungi contain α-1,3-glucan with a low proportion of α-1,4-glucan as a major cell wall polysaccharide. Glycosylphosphatidylinositol (GPI)-anchored α-amylases are conserved in Aspergillus fungi. The GPI-anchored α-amylase AmyD in Aspergillus nidulans has been reported to directly suppress the biosynthesis of cell wall α-1,3-glucan but not to degrade it in vivo. However, the detailed mechanism of cell wall α-1,3-glucan biosynthesis regulation by AmyD remains unclear. Here we focused on AoAgtA, which is encoded by the Aspergillus oryzae agtA gene, an ortholog of the A. nidulans amyD gene. Similar to findings in A. nidulans, agtA overexpression in A. oryzae grown in submerged culture decreased the amount of cell wall α-1,3-glucan and led to the formation of smaller hyphal pellets in comparison with the wild-type strain. We analyzed the enzymatic properties of recombinant (r)AoAgtA produced in Pichia pastoris and found that it degraded soluble starch, but not linear bacterial α-1,3-glucan. Furthermore, rAoAgtA cleaved 3-α-maltotetraosylglucose with a structure similar to the predicted boundary structure between the α-1,3-glucan main chain and a short spacer composed of α-1,4-linked glucose residues in cell wall α-1,3-glucan. Interestingly, rAoAgtA randomly cleaved only the α-1,4-glycosidic bonds of 3-α-maltotetraosylglucose, indicating that AoAgtA may cleave the spacer in cell wall α-1,3-glucan. Consistent with this hypothesis, heterologous overexpression of agtA in A. nidulans decreased the molecular weight of cell wall α-1,3-glucan. These in vitro and in vivo properties of AoAgtA suggest that GPI-anchored α-amylases can degrade the spacer α-1,4-glycosidic linkages in cell wall α-1,3-glucan before its insolubilization, and this spacer cleavage decreases the molecular weight of cell wall α-1,3-glucan in vivo.

Overexpression of a predicted transketolase gene and disruption of an α-1,3-glucan synthase gene in Aspergillus oryzae DGLA3 strain enhances the yield of free dihomo-γ-linolenic acid.

Free dihomo-γ-linolenic acid (DGLA), a polyunsaturated free fatty acid (FFA), can potentially be used to produce eicosanoid pharmaceuticals, such as prostaglandin E1. Previously, we constructed an Aspergillus oryzae mutant strain, named DGLA3, which produced free DGLA at an increased yield by faaA gene disruption and cooverexpression of one elongase and two desaturase genes. In this study, we achieved a further increase. Since FFA production is increased by enhancing the pentose phosphate pathway, we overexpressed a predicted transketolase gene composing the pathway in DGLA3, which consequently increased the free DGLA yield by 1.9-fold to 403mg/L. Additionally, we disrupted the α-1,3-glucan synthase gene agsB involved in cell-wall biosynthesis, which further increased it by 1.3-fold to 533mg/L. Overall, the yield increased by 2.5-fold. Free DGLA productivity and biomass increased similarly, but residual glucose concentration decreased. Increased hyphal dispersion appeared to cause additional glucose consumption, resulting in an increase in biomass and yield.

WGCNA Reveals Genes Associated with Lignification in the Secondary Stages of Wood Formation

The lignified tissue in the secondary stem is the main source of wood. In this study, we applied RNA-Seq analysis to the poplar stems in three developmental stages, including primary stem (PS), transitional stem (TS), and secondary stem (SS), to identify a total of 2028 genes that were highly expressed in the SS. Gene annotation indicated that the functions of these genes are mainly involved in cell wall biosynthesis, xylem development, and programmed cell death (PCD) processes. Subsequently, we explored the expression pattern of these genes at various developmental stages in the horizontal direction of the wood by ASPwood. The expression of these genes was modularized and correlated with the percentage of lignified xylem, using weighted gene co-expression network analysis (WGCNA). Among the genes, as many as 690 were identified as directly associated with lignification in the SS. In addition, the gene promoter cis-elements and protein interactions were predicted by PlantRegMap and STRING, respectively. The results were introduced into a co-expression network to confirm their relationship. We eventually found 54 TFs dominating this network, of which ADOF1, ATMYB3, AtbZIP44 (Potri.005G231300), ANAC043, ATWRKY40, ATEBP (Potri.010G006800), ARF5, anac075, RAP2.1, ARF16, AT- HSFB3, Potri.014G050000 (from WRKY family), HAT22, AT-HSFB2B, and AtWRKY20 had extremely high connectivity, which may play an important role in the lignification of wood formation at secondary stages.

Intestinal epithelial cell-derived components regulate transcriptome of Lactobacillus rhamnosus GG.

Intestinal epithelial cells (IECs) provide the frontline responses to the gut microbiota for maintaining intestinal homeostasis. Our previous work revealed that IEC-derived components promote the beneficial effects of a commensal and probiotic bacterium, Lactobacillus rhamnosus GG (LGG). This study aimed to elucidate the regulatory effects of IEC-derived components on LGG at the molecular level. Differential gene expression in LGG cultured with IEC-derived components at the timepoint between the exponential and stationary phase was studied by RNA sequencing and functional analysis. The transcriptomic profile of LGG cultured with IEC-derived components was significantly different from that of control LGG, with 231 genes were significantly upregulated and 235 genes significantly down regulated (FDR <0.05). The Clusters of Orthologous Groups (COGs) and Gene Ontology (GO) analysis demonstrated that the predominant genes enriched by IEC-derived components are involved in nutrient acquisition, including transporters for amino acids, metals, and sugars, biosynthesis of amino acids, and in the biosynthesis of cell membrane and cell wall, including biosynthesis of fatty acid and lipoteichoic acid. In addition, genes associated with cell division and translation are upregulated by IEC-derived components. The outcome of the increased transcription of these genes is supported by the result that IEC-derived components significantly promoted LGG growth. The main repressed genes are associated with the metabolism of amino acids, purines, carbohydrates, glycerophospholipid, and transcription, which may reflect regulation of metabolic mechanisms in response to the availability of nutrients in bacteria. These results provide mechanistic insight into the interactions between the gut microbiota and the host.

Transcriptome analysis reveals the molecular mechanisms of Phragmites australis tolerance to CuO-nanoparticles and/or flood stress induced by arbuscular mycorrhizal fungi.

The molecular mechanism of arbuscular mycorrhizal fungi (AMF) in vertical flow constructed wetlands (VFCWs) for the purification of copper oxide nanoparticles (CuO-NPs) contaminated wastewater remains unclear. In this study, transcriptome analysis was used to explore the effect of AMF inoculation on the gene expression profile of Phragmites australis roots under different concentrations of CuO-NPs and/or flood stress. 551, 429 and 2281 differentially expressed genes (DEGs) were specially regulated by AMF under combined stresses of CuO-NPs and flood, single CuO-NPs stress and single flood stress, respectively. Based on the results of DEG function annotation and enrichment analyses, AMF inoculation under CuO-NPs and/or flood stress up-regulated the expression of a number of genes involved in antioxidant defense systems, cell wall biosynthesis and transporter protein, which may contribute to plant tolerance. The expression of 30 transcription factors (TFs) was up-regulated by AMF inoculation under combined stresses of CuO-NPs and flood, and 44 and 44 TFs were up-regulated under single CuO-NPs or flood condition, respectively, which may contribute to the alleviating effect of symbiosis on CuO-NPs and/or flood stress. These results provided a theoretical basis for enhancing the ecological restoration function of wetland plants for metallic nanoparticles (MNPs) by mycorrhizal technology in the future.

Targeting membrane-bound bacterial cell wall precursors: a tried and true antibiotic strategy in nature and the clinic.

Since Fleming's discovery of penicillin nearly a century ago, a bounty of natural product antibiotics have been discovered, many of which continue to be of clinical importance today. The structural diversity encountered among nature's repertoire of antibiotics is mirrored by the varying mechanisms of action by which they selectively target and kill bacterial cells. The ability for bacteria to construct and maintain a strong cell wall is essential for their robust growth and survival under a range of conditions. However, the need to maintain the cell wall also presents a vulnerability that is exploited by many natural antibiotics. Bacterial cell wall biosynthesis involves both the construction of complex membrane-bound precursor molecules and their subsequent crosslinking by dedicated enzymes. Interestingly, many naturally occurring antibiotics function not by directly inhibiting the enzymes associated with cell wall biosynthesis, but rather by binding tightly to their membrane-bound substrates. Such substrate sequestration mechanisms are comparatively rare outside of the antibiotics space with most small-molecule drug discovery programs instead aimed at developing inhibitors of target enzymes. In this feature article we provide the reader with an overview of the unique and ever increasing family of natural product antibiotics known to specifically function by binding to membrane-anchored bacterial cell wall precursors. In doing so, we highlight both our own contributions to the field as well as those made by other researchers engaged in exploring the potential offered by antibiotics that target bacterial cell wall precursors.

Mycobacterium Tuberculosis Curli Pili (MTP) and Heparin-Binding Hemagglutinin Adhesin (HBHA) Facilitate Regulation of Central Carbon Metabolism, Enhancement of ATP Synthesis and Cell Wall Biosynthesis

Mycobacterium Tuberculosis Curli Pili (MTP) and Heparin-Binding Hemagglutinin Adhesin (HBHA) Facilitate Regulation of Central Carbon Metabolism, Enhancement of ATP Synthesis and Cell Wall Biosynthesis

Effect of potassium intake on cadmium transporters and root cell wall biosynthesis in sweet potato

Large areas of farmland soil in southern China are deficient in potassium (K) and are contaminated with cadmium (Cd). Previously, we suggested that the K supplementation could reduce Cd accumulation in sweet potatoes (Ipomoea batatas (L.) Lam). In the present study, we investigated the underlying physiological and molecular mechanisms. A hydroponic experiment with different K and Cd treatments was performed to compare the transcriptome profile and the cell wall structure in the roots of sweet potato using RNA sequencing, Fourier transform infrared spectroscopy (FTIR) and transmission electron microscopy (TEM). The results showed that K supply inhibits the expressions of IRT1 and YSL3, which are responsible for root Cd uptake under Cd exposure. Furthermore, the expressions of COPT5 and Nramp3 were downregulated by K, which increased Cd retention in the root vacuoles. The upregulation of POD, CAD, INT1 and SUS by K contributed to lignin and cellulose biosynthesis and thickening of root xylem cell wall, which further reduced Cd translocation to the shoot. In addition, K affected the expressions of LHT, ACS, TPS and TPP associated with the production of ethylene and trehalose, which involved in plant resistance to Cd toxicity. In general, K application could decrease the uptake and translocation of Cd in sweet potatoes by regulating the expression of genes associated with Cd transporters and root cell wall components.

High-throughput screen with the l,d-transpeptidase LdtMt2 of Mycobacterium tuberculosis reveals novel classes of covalently reacting inhibitors.

Disruption of bacterial cell wall biosynthesis in Mycobacterium tuberculosis is a promising target for treating tuberculosis. The l,d-transpeptidase LdtMt2, which is responsible for the formation of 3 → 3 cross-links in the cell wall peptidoglycan, has been identified as essential for M. tuberculosis virulence. We optimised a high-throughput assay for LdtMt2, and screened a targeted library of ∼10 000 electrophilic compounds. Potent inhibitor classes were identified, including established (e.g., β-lactams) and unexplored covalently reacting electrophilic groups (e.g., cyanamides). Protein-observed mass spectrometric studies reveal most classes to react covalently and irreversibly with the LdtMt2 catalytic cysteine (Cys354). Crystallographic analyses of seven representative inhibitors reveal induced fit involving a loop enclosing the LdtMt2 active site. Several of the identified compounds have a bactericidal effect on M. tuberculosis within macrophages, one with an MIC50 value of ∼1 μM. The results provide leads for the development of new covalently reaction inhibitors of LdtMt2 and other nucleophilic cysteine enzymes.