Bacterial Cell Wall Peptidoglycan Research Articles

Crystal Structure of Human Lysozyme Complexed with N-Acetyl-α-d-glucosamine

Human lysozyme is a natural non-specific immune protein that participates in the immune response of infants against bacterial and viral infections. Lysozyme is a well-known hydrolase that cleaves peptidoglycan in bacterial cell walls. Several crystal structures of human lysozyme have been reported, but little is known regarding how it recognizes sugar molecules. In this study, the crystal structures of human lysozyme in its native and two N-acetyl-α-d-glucosamine (α-D-NAG)-bound forms were determined at 1.3 Å and 1.55/1.60 Å resolution, respectively. Human lysozyme formed a typical c-type lysozyme fold and the α-D-NAG molecule was bound to the middle of subsites C and D. The N-acetyl and glucosamine groups of α-D-NAG were stabilized by hydrophobic interactions (Val117, Ala126, and Trp127), hydrogen bonds (Asn64, Asn78, Ala126, and Val128), and water bridges. Conformational changes of Arg80, Tyr81, Val128, and Arg131 of human lysozyme were observed due to the interactions of α-D-NAG with the active-site cleft. The binding configuration of α-D-NAG in human lysozyme was distinct compared with that of other sugar-bound lysozymes. Findings from this structural analysis provide a better understanding of the sugar recognition of human lysozyme during the immune response to microbial pathogens.

Antibacterial Effects of Recombinant Endolysins in Disinfecting Medical Equipment: A Pilot Study.

Nosocomial infections caused by multidrug-resistant (MDR) bacteria are severe life-threatening factors. Endolysins (lysins) degrade the bacterial cell wall peptidoglycan and may help control pathogens, especially MDR bacteria prevalent in hospital settings. This study was conducted to verify the potential of lysin as disinfectant to kill bacteria contaminating medical devices that cause hospital infections. Eight catheters removed from hospitalized patients were collected and tested for their ability to kill bacteria contaminating the catheters using two lysins, LysSS and CHAP-161. Catheter-contaminating bacterial species were isolated and identified by 16s rRNA sequencing. From the eight catheters, bacteria were cultured from seven catheters, and five bacterial species (Bacillus megaterium, Bacillus muralis, Corynebacterium striatum, Enterococcus faecium, and Staphylococcus epidermidis) were identified. LysSS could inhibit catheter-contaminating bacteria, including C. striatum and S. epidermidis, compared with untreated controls but could not inhibit the growth of E. faecium. CHAP-161 showed more bactericidal effects than LysSS, but could not inhibit the growth of S. epidermidis. This study showed the potential of lysin as an alternative disinfectant for hazardous chemical disinfectants used in hospitals.

Penicillin Binding Proteins and β-Lactamases of Mycobacterium tuberculosis: Reexamination of the Historical Paradigm.

ABSTRACTPenicillin binding proteins (PBPs) have been extensively studied due to their importance to the physiology of bacterial cell wall peptidoglycan and as targets of the most widely used class of antibiotics, the β-lactams. The existing paradigm asserts that PBPs catalyze the final step of peptidoglycan biosynthesis, and β-lactams inhibit their activities. According to this paradigm, a distinct enzyme class, β-lactamases, exists to inactivate β-lactams. This paradigm has been the basis for how bacterial diseases are treated with β-lactams. We tested whether this historical view accurately reflects the relationship between β-lactams and the PBPs and the β-lactamase, BlaC, of Mycobacterium tuberculosis. BlaC was the major inactivator of the cephalosporin subclass of β-lactams. However, the PBPs PonA1 and PonA2 inactivated penicillins and carbapenems more effectively than BlaC. These findings demonstrate that select M. tuberculosis PBPs are effective at inactivating several β-lactams. Lesser-known PBPs, DacB, DacB1, DacB2, and Rv2864c, a putative PBP, were comparably more resistant to inhibition by all β-lactam subclasses. Additionally, Rv1730c exhibited low affinity to most β-lactams. Based on these findings, we conclude that in M. tuberculosis, BlaC is not the only source of inactivation of β-lactams. Therefore, the historical paradigm does not accurately describe the relationship between β-lactams and M. tuberculosis.IMPORTANCE M. tuberculosis, the causative agent of tuberculosis, kills more humans than any other bacterium. β-lactams are the most widely used class of antibiotics to treat bacterial infections. Unlike in the historical model that describes the relationship between β-lactams and M. tuberculosis, we find that M. tuberculosis penicillin binding proteins are able to inactivate select β-lactams with high efficiency.

Multiscale Invasion Assay for Probing Macrophage Response to Gram-Negative Bacteria.

The immune system is a complex network of various cellular components that must differentiate between pathogenic bacteria and the commensal bacteria of the human microbiome, where misrecognition is linked to inflammatory disorders. Fragments of bacterial cell wall peptidoglycan bind to pattern recognition receptors within macrophages, leading to immune activation. To study this complex process, a methodology to remodel and label the bacterial cell wall of two different species of bacteria was established using copper (I) catalyzed azide-alkyne cycloaddition (CuAAC) and strain-promoted azide-alkyne cycloaddition (SPAAC). Additionally, an approach for three-dimensional (3D) culture of human macrophages and their invasion with relevant bacteria in a well-defined hydrogel-based synthetic matrix inspired by the microenvironment of the gut was established. Workflows were developed for human monocyte encapsulation and differentiation into macrophages in 3D culture with high viability. Bacteria invaded into macrophages permitted in situ peptidoglycan labeling. Macrophages exhibited biologically-relevant cytokine release in response to bacteria. This molecularly engineered, multi-dimensional bacteria-macrophage co-culture system will prove useful in future studies to observe immunostimulatory, bacterial fragment production and localization in the cell at the carbohydrate level for insights into how the immune system properly senses bacteria.

Peptidoglycan Deacetylases in Bacterial Cell Wall Remodeling and Pathogenesis.

The bacterial cell wall peptidoglycan (PG) is a dynamic structure that is constantly synthesized, re-modeled and degraded during bacterial division and growth. Postsynthetic modifications modulate the action of endogenous autolysis during PG lysis and remodeling for growth and sporulation, but also they are a mechanism used by pathogenic bacteria to evade the host innate immune system. Modifications of the glycan backbone are limited to the C-2 amine and C-6 hydroxyl moieties of either GlcNAc or MurNAc residues. This paper reviews the functional roles and properties of peptidoglycan de-Nacetylases (distinct PG GlcNAc and MurNAc deacetylases) and recent progress through genetic studies and biochemical characterization to elucidate their mechanism of action, 3D structures, substrate specificities and biological functions. Since they are virulence factors in pathogenic bacteria, peptidoglycan deacetylases are potential targets for the design of novel antimicrobial agents.

Genome-wide identification and characteristic analysis of PGRP gene family in Tegillarca granosa reveals distinct immune response of the invasive pathogen

The peptidoglycan recognition proteins (PGRPs) are conserved innate immune molecular in invertebrates and vertebrates, which play important roles in immune system by recognize the peptidoglycans of bacterial cell walls. Although PGRPs have been extensively characterized in insects, a systematic analysis of PGRPs in bivalves is lacking. In the present study, the phylogenic relationships, gene structures and expression profiles of PGRPs in marine bivalves were analyzed. The results indicated that the most PGRPs of bivalves were predicted to degrade the peptidoglycans and prevent excessive immunostimulation of bacteria. In addition, the results of the present study showed that the protein diversity of PGRPs in most marine bivalves was mainly generated by the alternative splicing of genes, however the alternative splicing of PGRP gene family was absent in Tegillarca granosa. The differences of PGRPs might be related to the genetic and environmental differences of marine bivalves. Spatiotemporal expression profiling in T. granosa suggested that PGRPs play important roles in the immune response of invasive pathogens. The present study describes a comprehensive view of PGRPs in the blood clam T. granosa and provides a foundation for functional characterization of this gene family in innate immune of marine bivalves.

Recent Insights Into the Prognostic and Therapeutic Applications of Lysozymes.

Lysozymes are naturally occurring enzymes present in a variety of biological organisms, such as bacteria, fungi, and animal bodily secretions and tissues. It is also the main ingredient of many ethnomedicines. It is well known that lysozymes and lysozyme-like enzymes can be used as anti-bacterial agents by degrading bacterial cell wall peptidoglycan that leads to cell death, and can also inhibit fungi, yeasts, and viruses. In addition to its direct antimicrobial activity, lysozyme is also an important component of the innate immune system in most mammals. Increasing evidence has shown the immune-modulatory effects of lysozymes against infection and inflammation. More recently, studies have revealed the anti-cancer activities of lysozyme in multiple types of tumors, potentially through its immune-modulatory activities. In this review, we summarized the major functions and underlying mechanisms of lysozymes derived from animal and plant sources. We highlighted the therapeutic applications and recent advances of lysozymes in cancers, hypertension, and viral diseases, aiming toseeking alternative therapies for standard medical treatment bypassing side effects. We also evaluated the role of lysozyme as a promising cancer marker for prognosis to indicate the outcomes recurrence for patients.

Peptidoglycan of Bacterial Cell Wall Affects Competitive Properties of Microorganisms.

We studied the effect of bacterial wall peptidoglycan of 7 bacterial species on the competitive properties of human-associated microorganisms. Addition of peptidoglycan to the culture medium did not change the growth characteristics of the test cultures; however, an increase in the antagonism and hydrophobicity of Bifidobacterium sp. and Enterococcus sp. was observed, while the effect on enterobacteria was predominantly indifferent or inhibitory. The effect did not depend much on the source of peptidoglycan and was equally manifested on both indigenous and probiotic strains. The observed new property of peptidoglycan indicates its participation in the formation and functioning of microbiota. The obtained data on the regulation of the properties of microorganisms provide new possibilities for the correction and maintenance of host homeostasis through host-associated microbiota.

Demonstration of the role of cell wall homeostasis in Staphylococcus aureus growth and the action of bactericidal antibiotics

Bacterial cell wall peptidoglycan is essential, maintaining both cellular integrity and morphology, in the face of internal turgor pressure. Peptidoglycan synthesis is important, as it is targeted by cell wall antibiotics, including methicillin and vancomycin. Here, we have used the major human pathogen Staphylococcus aureus to elucidate both the cell wall dynamic processes essential for growth (life) and the bactericidal effects of cell wall antibiotics (death) based on the principle of coordinated peptidoglycan synthesis and hydrolysis. The death of S. aureus due to depletion of the essential, two-component and positive regulatory system for peptidoglycan hydrolase activity (WalKR) is prevented by addition of otherwise bactericidal cell wall antibiotics, resulting in stasis. In contrast, cell wall antibiotics kill via the activity of peptidoglycan hydrolases in the absence of concomitant synthesis. Both methicillin and vancomycin treatment lead to the appearance of perforating holes throughout the cell wall due to peptidoglycan hydrolases. Methicillin alone also results in plasmolysis and misshapen septa with the involvement of the major peptidoglycan hydrolase Atl, a process that is inhibited by vancomycin. The bactericidal effect of vancomycin involves the peptidoglycan hydrolase SagB. In the presence of cell wall antibiotics, the inhibition of peptidoglycan hydrolase activity using the inhibitor complestatin results in reduced killing, while, conversely, the deregulation of hydrolase activity via loss of wall teichoic acids increases the death rate. For S. aureus, the independent regulation of cell wall synthesis and hydrolysis can lead to cell growth, death, or stasis, with implications for the development of new control regimes for this important pathogen.

Correlative Super-Resolution Optical and Atomic Force Microscopy Reveals Relationships Between Bacterial Cell Wall Architecture and Synthesis in Bacillus subtilis.

Understanding how bacteria grow and divide requires insight into both the molecular-level dynamics of ultrastructure and the chemistry of the constituent components. Atomic force microscopy (AFM) can provide near molecular resolution images of biological systems but typically provides limited chemical information. Conversely, while super-resolution optical microscopy allows localization of particular molecules and chemistries, information on the molecular context is difficult to obtain. Here, we combine these approaches into STORMForce (stochastic optical reconstruction with atomic force microscopy) and the complementary SIMForce (structured illumination with atomic force microscopy), to map the synthesis of the bacterial cell wall structural macromolecule, peptidoglycan, during growth and division in the rod-shaped bacterium Bacillus subtilis. Using “clickable” d-amino acid incorporation, we fluorescently label and spatially localize a short and controlled period of peptidoglycan synthesis and correlate this information with high-resolution AFM of the resulting architecture. During division, septal synthesis occurs across its developing surface, suggesting a two-stage process with incorporation at the leading edge and with considerable in-filling behind. During growth, the elongation of the rod occurs through bands of synthesis, spaced by ∼300 nm, and corresponds to denser regions of the internal cell wall as revealed by AFM. Combining super-resolution optics and AFM can provide insights into the synthesis processes that produce the complex architectures of bacterial structural biopolymers.

Characterization of PGRP-LB and immune deficiency in the white-backed planthopper Sogatella furcifera (Hemiptera: Delphacidae)

Peptidoglycan recognition proteins (PGRPs) participate in insect defense against bacterial pathogens by recognizing bacterial cell wall peptidoglycans (PGNs). Here, we identified the PGRP-LB gene in the white-backed planthopper Sogatella furcifera (SfPGRP-LB). SfPGRP-LB is a secreted protein with a typical PGN-binding domain and five conserved amino acid (aa) residues required for amidase activity. Expression analysis showed that the SfPGRP-LB transcript levels were significantly higher in the midgut than in other tissues. Silencing SfPGRP-LB with dsRNA significantly downregulated the expression of Toll pathway genes Toll and Dorsal and Imd pathway genes Imd and Relish after Escherichia coli challenge. However, only Toll and Dorsal expressions were downregulated after Staphylococcus aureus challenge. E. coli and S. aureus challenges rapidly and strongly upregulated SfPGRP-LB expression. Recombinantly expressed SfPGRP-LB (rSfPGRP-LB) had strong affinities for E. coli Dap-type PGN and S. aureus Lys-type PGN and agglutinated the bacteria. However, rSfPGRP-LB inhibited S. aureus but not E. coli growth. Furthermore, rSfPGRP-LB had amidase activity, degraded Lys-type PGN, and destroyed S. aureus cell walls but had no such effects on E. coli Dap-type PGN. Thus, SfPGRP-LB recognizes and binds various bacterial PGNs but only has amidase activity against Lys-type PGN.

Substrate-dependent effects of quaternary structure on RNase E activity.

RNase E is an essential, multifunctional ribonuclease encoded in E. coli by the rne gene. Structural analysis indicates that the ribonucleolytic activity of this enzyme is conferred by rne-encoded polypeptide chains that (1) dimerize to form a catalytic site at the protein-protein interface, and (2) multimerize further to generate a tetrameric quaternary structure consisting of two dimerized Rne-peptide chains. We identify here a mutation in the Rne protein's catalytic region (E429G), as well as a bacterial cell wall peptidoglycan hydrolase (Amidase C [AmiC]), that selectively affect the specific activity of the RNase E enzyme on long RNA substrates, but not on short synthetic oligonucleotides, by enhancing enzyme multimerization. Unlike the increase in specific activity that accompanies concentration-induced multimerization, enhanced multimerization associated with either the E429G mutation or interaction of the Rne protein with AmiC is independent of the substrate's 5' terminus phosphorylation state. Our findings reveal a previously unsuspected substrate length-dependent regulatory role for RNase E quaternary structure and identify cis-acting and trans-acting factors that mediate such regulation.

Peptidoglycan Salvage Enables the Periodontal Pathogen Tannerella forsythia to Survive within the Oral Microbial Community

Tannerella forsythia is an anaerobic, fusiform Gram-negative oral pathogen strongly associated with periodontitis, a multibacterial inflammatory disease that leads to the destruction of the teeth-supporting tissue, ultimately causing tooth loss. To survive in the oral habitat, T. forsythia depends on cohabiting bacteria for the provision of nutrients. For axenic growth under laboratory conditions, it specifically relies on the external supply of N-acetylmuramic acid (MurNAc), which is an essential constituent of the peptidoglycan (PGN) of bacterial cell walls. T. forsythia comprises a typical Gram-negative PGN; however, as evidenced by genome sequence analysis, the organism lacks common enzymes required for the de novo synthesis of precursors of PGN, which rationalizes its MurNAc auxotrophy. Only recently insights were obtained into how T. forsythia gains access to MurNAc in its oral habitat, enabling synthesis of the own PGN cell wall. This report summarizes T. forsythia’s strategies to survive in the oral habitat by means of PGN salvage pathways, including recovery of exogenous MurNAc and PGN-derived fragments but also polymeric PGN, which are all derived from cohabiting bacteria either via cell wall turnover or decay of cells. Salvage of polymeric PGN presumably requires the removal of peptides from PGN by an unknown amidase, concomitantly with the translocation of the polymer across the outer membrane. Two recently identified exo-lytic N-acetylmuramidases (Tf_NamZ1 and Tf_NamZ2) specifically cleave the peptide-free, exogenous (nutrition source) PGN in the periplasm and release the MurNAc and disaccharide substrates for the transporters Tf_MurT and Tf_AmpG, respectively, whereas the peptide-containing, endogenous (the self-cell wall) PGN stays unattached. This review also outlines how T. forsythia synthesises the PGN precursors UDP-MurNAc and UDP-N-acetylglucosamine (UDP-GlcNAc), involving homologs of the Pseudomonas sp. recycling enzymes AmgK/MurU and a monofunctional uridylyl transferase (named Tf_GlmU*), respectively.

Cloning and functional characterization of two peptidoglycan recognition protein isoforms (PGRP-LC) in Bactrocera dorsalis (Diptera: Tephritidae)

The innate immune system of insects is the front line of self-defense against pathogen invasion. Peptidoglycan recognition proteins (PGRPs) are important components and play key roles in insect immune systems by recognizing peptidoglycan (PGN) in bacterial cell walls. We characterized two isoforms of the PGRP-LC gene, BdPGRP-LCa and BdPGRP-LCb, from Bactrocera dorsalis (Hendel), an important fruit and vegetable pest worldwide. These two isoforms contain an open reading frames of 1 668 bp and 1 731 bp, encoding a protein of 555 and 576 amino acids, respectively. Quantitative real-time PCR results showed that both transcripts were prominently expressed in midgut and fat body of B. dorsalis adult. Inoculation of pathogens showed that both isoforms actively responded to Escherichia coli PGN. We also observed a light response to Staphylococcus aureus PGN. Upon Beauveria bassiana inoculation, the expression of BdPGRP-LCa was enhanced, but the expression of BdPGRP-LCb was suppressed. Suppression of both transcripts by RNA interference led to increased mortality of flies challenged by E. coli, indicating that the two isoforms are involved in sensing Gram-negative bacterial infections.

Supported lysozyme for improved antimicrobial surface protection

Surface protection against biofilms is still an open challenge. Current strategies rely on coatings that are meant to guarantee antiadhesive or antimicrobial effects. While it seems difficult to ensure antiadhesion in complex media and against all the adhesive arsenal of microbes, strategies based on antimicrobials lack from sustainable functionalization methodologies to allow the perfect efficiency of the grafted molecules. Here we used the high affinity ligand-receptor interaction between biotin and streptavidin to functionalize surfaces with lysozyme, an enzyme that degrades the bacterial peptidoglycan cell wall. Biotinylated lysozyme was grafted on surfaces coated with streptavidin receptors. Using atomic force microscopy (AFM)-based single molecule force spectroscopy, we showed that grafting through ligand-receptor interaction allows the correct orientation of the enzyme on the substrate for enhanced activity towards the microbial target. The antibacterial efficiency was tested against Micrococcus luteus and revealed that surface protection was improved when lysozyme was grafted through the ligand-receptor interaction. These results suggest that bio-molecular interactions are promising for a sustainable grafting of antimicrobial agents on surfaces.

Potential Fatty Acid as Antibacterial Agent Against Oral Bacteria of Streptococcus mutans and Streptococcus sanguinis from Basil (Ocimum americanum): In vitro and In silico Studies.

Streptococcus mutans and Streptococcus sanguinis are Gram-positive bacteria that cause dental caries. MurA enzyme acts as a catalyst in the formation of peptidoglycan in bacterial cell walls, making it ideal as an antibacterial target. Basil (Ocimum americanum) is an edible plant that is diverse and has been used as a herbal medicine for a long time. It has been reported that basil has a pharmacological effect as well as antibacterial activity. The purpose of this study was to identify antibacterial compounds in O. americanum and analyze their inhibition activity on MurA enzyme. Fresh leaves from O. americanum were extracted with n-hexane and purified by a combination of column chromatography on normal and reverse phases together with in vitro bioactivity assay against S. mutans ATCC 25175 and S. sanguinis ATCC 10556, respectively, while in silico molecular docking simulation of lauric acid (1) was conducted using PyRx 0.8. The structure determination of antibacterial compound by spectroscopic methods resulted in an active compound lauric acid (1). The in vitro evaluation of antibacterial activity in compound 1 showed Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) values of 78.13 and 156.3 ppm and 1250 and 2500 ppm against S. sanguinis and S. mutans, respectively. Further analysis and in silico evaluation determined lauric acid (1) as MurA Enzyme inhibitor. Lauric acid (1) showed a binding affinity of -5.2 Kcal/mol, which was higher than fosfomycin. Lauric acid showed the potential as a new natural antibacterial agent through MurA inhibition in bacterial cell wall biosynthesis.

Designing 3D Synthetic Matrices for Studying Interactions Between Bacterial Peptidoglycan and Macrophages in the Context of Immune Misrecognition Diseases

The immune system is a complex network comprised of various cellular components that work together to protect the body from damage, with a key function of differentiating between pathogenic bacteria and the 39 trillion commensal bacteria that constitute our microbiome. Misrecognition of commensal bacteria has been linked to many inflammatory bowel disorders in humans yet remains not fully understood and difficult to study. Enabled by recent advances in molecular tools, fragments of the bacterial cell wall peptidoglycan have been identified that bind to pattern recognition receptors within macrophages, which activate macrophages to start a signaling cascade that turns on the immune response. Typically, these interactions are studied using two‐dimensional invasion assays on tissue culture plastic plates; however, the dimensionality and ‘stiffness’ of these traditional culture systems may not be representative of the cell‐microenvironment interactions that exist in vivo. In order to better simulate the native cellular environment, three‐dimensional (3D) cell culture systems can be used to control key biochemical and mechanical properties of the cell microenvironment. Here, we established an approach for the encapsulation and culture of human macrophages in 3D within well‐defined, biomimetic hydrogels decorated with integrin binding peptides that mimic the natural in vivo environment and linked with cell‐degradable sites for cell‐driven migration and remodeling to probe macrophage response to bacterial invasion. With this approach, we have shown invasion with bacteria whose glycans are remodeled and their engulfment through immunostaining and assessed activation of macrophages using RT‐qPCR and ELISA. These studies provide new insights into the interactions between bacteria and immune cells and this new method affords unique opportunities for examining how our immune system recognizes and misrecognizes bacteria toward a better understanding of the mechanisms of immune misrecognition diseases.Support or Funding InformationNIH CBI Training Grant‐5T32GM008550NIH U01 Common Fund program‐U01CA221230‐01

Influence of Carbohydrate Residues on Antibacterial Activity of Vancomycin

This paper presents synthesis of vancomycin derivatives modified with selected 1- and 2-aminoalditols to carboxylic function and 2,5-anhydro-D-mannose and D-talose to amino function of vancosamine via reductive alkylation. MIC and MBC of these derivatives were determined for reference strains of bacteria: Staphylococcus aureus ATCC 25923, ATCC 6538, ATCC 6538/P, S. epidemidis ATCC 14490, E. faecium PCM 1859, E. faecalis PCM 2673, S. pyogenes PCM 465, and S. pneumonia ATCC 49619 and compared with the activity of vancomycin and its aglycone. Our findings confirm that sugar fragments can play an important role in the mechanism of interaction of vancomycin with bacterial cell wall peptidoglycan.

Comparative Molecular Dynamics Simulations Provide Insight Into Antibiotic Interactions: A Case Study Using the Enzyme L,L-Diaminopimelate Aminotransferase (DapL).

The L,L-diaminopimelate aminotransferase (DapL) pathway, a recently discovered variant of the lysine biosynthetic pathway, is an attractive pipeline to identify targets for the development of novel antibiotic compounds. DapL is a homodimer that catalyzes the conversion of tetrahydrodipicolinate to L,L-diaminopimelate in a single transamination reaction. The penultimate and ultimate products of the lysine biosynthesis pathway, meso-diaminopimelate and lysine, are key components of the Gram-negative and Gram-positive bacterial peptidoglycan cell wall. Humans are not able to synthesize lysine, and DapL has been identified in 13% of bacteria whose genomes have been sequenced and annotated to date, thus it is an attractive target for the development of narrow spectrum antibiotics through the prevention of both lysine biosynthesis and peptidoglycan crosslinking. To address the common lack of structural information when conducting compound screening experiments and provide support for the use of modeled structures, our analyses utilized inferred structures from related homologous enzymes. Using a comprehensive and comparative molecular dynamics (MD) software package—DROIDS (Detecting Relative Outlier Impacts in Dynamic Simulations) 2.0, we investigated the binding dynamics of four previously identified antagonistic ligands of DapL from Verrucomicrobium spinosum, a non-pathogenic relative of Chlamydia trachomatis. Here, we present putative docking positions of the four ligands and provide confirmatory comparative molecular dynamics simulations supporting the conformations. The simulations performed in this study can be applied to evaluate putative targets to predict compound effectiveness prior to in vivo and in vitro experimentation. Moreover, this approach has the potential to streamline the process of antibiotic development.

Mass spectrometry study of noncovalent complexes formation of antibiotic cycloserine with N-acetyl-D-glucosamine and ascorbic acid

Background: While antibiotic cycloserine (CYS) is widely applied in the treatment of tuberculosis, our knowledge of the drug intermolecular interactions with targeting biomolecules and other drugs remains incomplete. It is believed that the CYS antibacterial activity is related to inhibiting the bacterial cell wall biosynthesis. On the other hand, intermolecular interactions of CYS with ascorbic acid (ASC) molecules is worth of studying taking into account that ASC can be used as supporting vitamin preparation or can be affiliated with the patients nutrition. Objectives: The purpose of the current model study are to examine biologically significant intermolecular interactions of CYS with N-acetyl-D-glucosamine (NAG) as one of the main component of peptidoglycan of bacterial cell wall and to verify the possibility of noncovalent complexes formation between CYS and ASC molecules using electrospray ionization mass spectrometry (ESI MS) technique. Materials and methods: The objects of the study are model systems composed of CYS and NAG or CYS and ASC prepared in a polar methanol solvent for the ESI MS probing. ESI mass spectra are obtained using the approach earlier developed by us for investigation of the noncovalent complexation of drugs with targeting biomolecules. Results: The experiments reveal that the ESI mass spectrum of (CYS–NAG) model system contains peaks of protonated molecular clusters of CYS with NAG: [CYS•NAG•H]+ and [CYS•2NAG•H]+. Existing of such peaks in the spectrum testifies to formation of stable noncovalent complexes between CYS and NAG in the studied solution. ESI MS examining of (CYS–ASC) system reveals the noncovalent pair complexation of CYS and ASC molecules confirmed by the recording of intensive peak of [CYS•ASC•H]+ cluster in the spectrum. Conclusions: The ESI MS findings point to the possibility of noncovalent complexation of CYS with NAG in the polar media including biological systems. Such complexation between the antibiotic and NAG as component of peptidoglycan of bacterial cell wall is considered to be biologically significant for the process of the cell wall biosynthesis inhibiting by CYS. Stable noncovalent complexes formation between the CYS and ASC molecules is suggested as a potential molecular mechanism of the drugs activity modulation under their joint usage.