Inhibitors Of Cell Wall Biosynthesis Research Articles

Dual RNA sequencing during Trichoderma harzianum-Phytophthora capsici interaction reveals multiple biological processes involved in the inhibition and highlights the cell wall as a potential target.

Phytophthora capsici is a destructive oomycete pathogen, causing huge economic losses for agricultural production. The genus Trichoderma represents one of the most extensively researched categories of biocontrol agents, encompassing a diverse array of effective strains. The commercial biocontrol agent Trichoderma harzianum strain T-22 exhibits pronounced biocontrol effects against many plant pathogens, but its activity against P. capsici is not known. T. harzianum T-22 significantly inhibited the growth of P. capsici mycelia and the culture filtrate of T-22 induced lysis of P. capsici zoospores. Electron microscopic analyses indicated that T-22 significantly modulated the ultrastructural composition of P. capsici, with a severe impact on the cell wall integrity. Dual RNA sequencing revealed multiple biological processes involved in the inhibition during the interaction between these two microorganisms. In particular, a marked upregulation of genes was identified in T. harzianum that are implicated in cell wall degradation or disruption. Concurrently, the presence of T. harzianum appeared to potentiate the susceptibility of P. capsici to cell wall biosynthesis inhibitors such as mandipropamid and dimethomorph. Further investigations showed that mandipropamid and dimethomorph could strongly inhibit the growth and development of P. capsici but had no impact on T. harzianum even at high concentrations, demonstrating the feasibility of combining T. harzianum and these cell wall synthesis inhibitors to combat P. capsici. These findings provided enhanced insights into the biocontrol mechanisms against P. capsici with T. harzianum and evidenced compatibility between specific biological and chemical control strategies. © 2024 Society of Chemical Industry.

The effects of daptomycin on cell wall biosynthesis in Enterococcal faecalis

Daptomycin is a cyclic lipodepsipeptide antibiotic reserved for the treatment of serious infections by multidrug-resistant Gram-positive pathogens. Its mode of action is considered to be multifaceted, encompassing the targeting and depolarization of bacterial cell membranes, alongside the inhibition of cell wall biosynthesis. To characterize the daptomycin mode of action, 15N cross-polarization at magic-angle spinning NMR measurements were performed on intact whole cells of Staphylococcus aureus grown in the presence of a sub-inhibitory concentration of daptomycin in a chemically defined media containing l-[ϵ-15N]Lys. Daptomycin-treated cells showed a reduction in the lysyl-ε-amide intensity that was consistent with cell wall thinning. However, the reduced lysyl-ε-amine intensity at 10 ppm indicated that the daptomycin-treated cells did not accumulate in Park’s nucleotide, the cytoplasmic peptidoglycan (PG) precursor. Consequently, daptomycin did not inhibit the transglycosylation step of PG biosynthesis. To further elucidate the daptomycin mode of action, the PG composition of daptomycin-susceptible Enterococcus faecalis grown in the presence of daptomycin was analyzed using liquid chromatography-mass spectrometry. Sixty-nine muropeptide ions correspond to PG with varying degrees of modifications including crosslinking, acetylation, alanylation, and 1,6-anhydrous ring formation at MurNAc were quantified. Analysis showed that the cell walls of daptomycin-treated E. faecalis had a significant reduction in PG crosslinking which was accompanied by an increase in lytic transglycosylase activities and a decrease in PG-stem modifications by the carboxypeptidases. The changes in PG composition suggest that daptomycin inhibits cell wall biosynthesis by impeding the incorporation of nascent PG into the cell walls by transpeptidases and maturation by carboxypeptidases. As a result, the newly formed cell walls become highly susceptible to degradation by the autolysins, resulting in thinning of the cell wall.

Bacillus licheniformis: A Producer of Antimicrobial Substances, including Antimycobacterials, Which Are Feasible for Medical Applications.

Bacillus licheniformis produces several classes of antimicrobial substances, including bacteriocins, which are peptides or proteins with different structural composition and molecular mass: ribosomally synthesized by bacteria (1.4-20 kDa), non-ribosomally synthesized peptides and cyclic lipopeptides (0.8-42 kDa) and exopolysaccharides (>1000 kDa). Different bacteriocins act against Gram-positive or Gram-negative bacteria, fungal pathogens and amoeba cells. The main mechanisms of bacteriocin lytic activity include interaction of peptides with membranes of target cells resulting in structural alterations, pore-forming, and inhibition of cell wall biosynthesis. DNase and RNase activity for some bacteriocines are also postulated. Non-ribosomal peptides are synthesized by special non-ribosomal multimodular peptide synthetases and contain unnatural amino acids or fatty acids. Their harmful effect is due to their ability to form pores in biological membranes, destabilize lipid packaging, and disrupt the peptidoglycan layer. Lipopeptides, as biosurfactants, are able to destroy bacterial biofilms. Secreted polysaccharides are high molecular weight compounds, composed of repeated units of sugar moieties attached to a carrier lipid. Their antagonistic action was revealed in relation to bacteria, viruses, and fungi. Exopolysaccharides also inhibit the formation of biofilms by pathogenic bacteria and prevent their colonization on various surfaces. However, mechanism of the harmful effect for many secreted antibacterial substances remains unknown. The antimicrobial activity for most substances has been studied in vitro only, but some substances have been characterized in vivo and they have found practical applications in medicine and veterinary. The cyclic lipopeptides that have surfactant properties are used in some industries. In this review, special attention is paid to the antimycobacterials produced by B. licheniformis as a possible approach to combat multidrug-resistant and latent tuberculosis. In particular, licheniformins and bacitracins have shown strong antimycobacterial activity. However, the medical application of some antibacterials with promising in vitro antimycobacterial activity has been limited by their toxicity to animals and humans. As such, similar to the enhancement in the antimycobacterial activity of natural bacteriocins achieved using genetic engineering, the reduction in toxicity using the same approach appears feasible. The unique capability of B. licheniformis to synthesize and produce a range of different antibacterial compounds means that this organism can act as a natural universal vehicle for antibiotic substances in the form of probiotic cultures and strains to combat various types of pathogens, including mycobacteria.

Evaluation of Efflux-Mediated Resistance and Biofilm formation in Virulent Pseudomonas aeruginosa Associated with Healthcare Infections

Pseudomonas aeruginosa is a significant pathogen identified with healthcare-associated infections. The present study evaluates the role of biofilm and efflux pump activities in influencing high-level resistance in virulent P. aeruginosa strains in clinical infection. Phenotypic resistance in biotyped Pseudomonas aeruginosa (n = 147) from diagnosed disease conditions was classified based on multiple antibiotic resistance (MAR) indices and analysed with logistic regression for risk factors. Efflux pump activity, biofilm formation, and virulence factors were analysed for optimal association in Pseudomonas infection using receiver operation characteristics (ROC). Age-specificity (OR [CI] = 0.986 [0.946-1.027]), gender (OR [CI] = 1.44 [0.211-9.827]) and infection sources (OR [CI] = 0.860 [0.438-1.688]) were risk variables for multidrug resistance (MDR)-P. aeruginosa infection (p < 0.05). Biofilm formers caused 48.2% and 18.5% otorrhea and wound infections (95% CI = 0.820-1.032; p = 0.001) respectively and more than 30% multidrug resistance (MDR) strains demonstrated high-level efflux pump activity (95% CI = 0.762-1.016; p = 0.001), protease (95% CI = 0.112-0.480; p = 0.003), lipase (95% CI = 0.143-0.523; p = 0.001), and hemolysin (95% CI = 1.109-1.780; p = 0.001). Resistance relatedness of more than 80% and 60% to cell wall biosynthesis inhibitors (ceftazidime, ceffproxil, augumentin, ampicillin) and, DNA translational and transcriptional inhibitors (gentamicin, ciprofloxacin, ofloxacin, nitrofurantoin) were observed (p < 0.05). Strong efflux correlation (r = 0.85, p = 0.034) with MDR strains, with high predictive performances in efflux pump activity (ROC-AUC 0.78), biofilm formation (ROC-AUC 0.520), and virulence hierarchical-clustering. Combine activities of the expressed efflux pump and biofilm formation in MDR-P. aeruginosa pose risk to clinical management and infection control.

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.

Next-generation membrane-active glycopeptide antibiotics that also inhibit bacterial cell division.

Resistance to vancomycin, a life-saving drug against Gram-positive bacterial infections necessitates developing alternative therapeutics. Herein, we report vancomycin derivatives that assimilate mechanisms beyond d-Ala-d-Ala binding. The role of hydrophobicity towards the structure and function of the membrane-active vancomycin showed that alkyl-cationic substitutions favored broad-spectrum activity. The lead molecule, VanQAmC10 delocalized the cell division protein MinD in Bacillus subtilis, implying an impact on bacterial cell division. Further examination of wild-type, GFP-FtsZ, or GFP-FtsI producing- and ΔamiAC mutants of Escherichia coli revealed filamentous phenotypes and delocalization of the FtsI protein. The findings indicate that VanQAmC10 also inhibits bacterial cell division, a property previously unknown for glycopeptide antibiotics. The conjunction of multiple mechanisms contributes to its superior efficacy against metabolically active and inactive bacteria, wherein vancomycin is ineffective. Additionally, VanQAmC10 exhibits high efficacy against methicillin-resistant Staphylococcus aureus (MRSA) and Acinetobacter baumannii in mouse models of infection.

Prospects of antimicrobial peptides as an alternative to chemical preservatives for food safety

Antimicrobial peptides (AMPs) are a potential alternative to antimicrobial agents that have got considerable research interest owing to their significant role in the inhibition of bacterial pathogens. These AMPs can essentially inhibit the growth and multiplication of microbes through multiple mechanisms including disruption of cellular membranes, inhibition of cell wall biosynthesis, or affecting intracellular components and cell division. Moreover, AMPs are biocompatible and biodegradable therefore, they can be a good alternative to antimicrobial agents and chemical preservatives. A few of their features for example thermostability and high selectivity are quite appealing for their potential use in the food industry for food preservation to prevent the spoilage caused by microorganisms and foodborne pathogens. Despite these advantages, very few AMPs are being used at an industrial scale for food preservation as these peptides are quite vulnerable to external environmental factors which deter their practical applications and commercialization. The review aims to provide an outline of the mechanism of action of AMPs and their prospects as an alternative to chemical preservatives in the food industry. Further studies related to the structure-activity relationship of AMPs will help to expand the understanding of their mechanism of action and to determine specific conditions to increase their stability and applicability in food preservation.

A Fungal Defensin Inhibiting Bacterial Cell-Wall Biosynthesis with Non-Hemolysis and Serum Stability.

Defensins are a class of cationic disulfide-bridged antimicrobial peptides (AMPs) present in many eukaryotic organisms and even in bacteria. They primarily include two distinct but evolutionarily related superfamilies (cis and trans). Defensins in fungi belong to the members of the cis-superfamily with the cysteine-stabilized α-helical and β-sheet fold. To date, many fungal defensin-like peptides (fDLPs) have been found through gene mining of the genome resource, but only a few have been experimentally characterized. Here, we report the structural and functional characterization of Pyronesin4 (abbreviated as Py4), a fDLP previously identified by genomic sequencing of the basal filamentous ascomycete Pyronema confluens. Chemically, synthetic Py4 adopts a native-like structure and exhibits activity on an array of Gram-positive bacteria including some clinical isolates of Staphylococcus and Staphylococcus warneri, a conditioned pathogen inhabiting in human skin. Py4 markedly altered the bacterial morphology and caused cytoplasmic accumulation of the cell-wall synthesis precursor through binding to the membrane-bound Lipid II, indicating that it works as an inhibitor of cell-wall biosynthesis. Py4 showed no hemolysis and high mammalian serum stability. This work identified a new fungal defensin with properties relevant to drug exploration. Intramolecular epistasis between mutational sites of fDLPs is also discussed.

Emerging Roles of Glycopeptide Antibiotics: Moving beyond Gram-Positive Bacteria.

Glycopeptides, a class of cell wall biosynthesis inhibitors, have been the antibiotics of choice against drug-resistant Gram-positive bacterial infections. Their unique mechanism of action involving binding to the substrate of cell wall biosynthesis and substantial longevity in clinics makes this class of antibiotics an attractive choice for drug repurposing and reprofiling. However, resistance to glycopeptides has been observed due to alterations in the substrate, cell wall thickening, or both. The emergence of glycopeptide resistance has resulted in the development of synthetic and semisynthetic glycopeptide analogues to target acquired resistance. Recent findings demonstrate that these derivatives, along with some of the FDA approved glycopeptides have been shown to have antimicrobial activity against Gram-negative bacteria, Mycobacteria, and viruses thus expanding their spectrum of activity across the microbial kingdom. Additional mechanisms of action and identification of novel targets have proven to be critical in broadening the spectrum of activity of glycopeptides. This review focuses on the applications of glycopeptides beyond their traditional target group of Gram-positive bacteria. This will aid in making the scientific community aware about the nontraditional activity profiles of glycopeptides, identify the existing loopholes, and further explore this antibiotic class as a potential broad-spectrum antimicrobial agent.

The Role of β-Glycosylated Wall Teichoic Acids in the Reduction of Vancomycin Susceptibility in Vancomycin-Intermediate Staphylococcus aureus.

ABSTRACTStaphylococcus aureus is an opportunistic pathogen that causes a wide range of infections. Due to the rapid evolution of antibiotic resistance that leads to treatment failure, it is important to understand the underlying mechanisms. Here, the cell wall structures of several laboratory vancomycin-intermediate S. aureus (VISA) strains were analyzed. Among the VISA strains were S. aureus VC40, which accumulated 79 mutations, including most importantly 2 exchanges in the histidine-kinase VraS, and developed full resistance against vancomycin (MIC, 64 μg/ml); a revertant S. aureus VC40R, which has an additional mutation in vraR (MIC, 4 μg/ml); and S. aureus VraS(VC40), in which the 2 vraS mutations were reconstituted into a susceptible background (MIC, 4 μg/ml). A ultraperformance liquid chromatography (UPLC) analysis showed that S. aureus VC40 had a significantly decreased cross-linking of the peptidoglycan. Both S. aureus VC40 and S. aureus VraS(VC40) displayed reduced autolysis and an altered autolysin profile in a zymogram. Most striking was the significant increase in d-alanine and N-acetyl-d-glucosamine (GlcNAc) substitution of the wall teichoic acids (WTAs) in S. aureus VC40. Nuclear magnetic resonance (NMR) analysis revealed that this strain had mostly β-glycosylated WTAs in contrast to the other strains, which showed only the α-glycosylation peak. Salt stress induced the incorporation of β-GlcNAc anomers and drastically increased the vancomycin MIC for S. aureus VC40R. In addition, β-glycosylated WTAs decreased the binding affinity of AtlA, the major autolysin of S. aureus, to the cell wall, compared with α-glycosylated WTAs. In conclusion, there is a novel connection between wall teichoic acids, autolysis, and vancomycin susceptibility in S. aureus.IMPORTANCE Infections with methicillin-resistant Staphylococcus aureus are commonly treated with vancomycin. This antibiotic inhibits cell wall biosynthesis by binding to the cell wall building block lipid II. We set out to characterize the mechanisms leading to decreased vancomycin susceptibility in a laboratory-generated strain, S. aureus VC40. This strain has an altered cell wall architecture with a thick cell wall with low cross-linking, which provides decoy binding sites for vancomycin. The low cross-linking, necessary for this resistance mechanism, decreases the stability of the cell wall against lytic enzymes, which separate the daughter cells. Protection against these enzymes is provided by another cell wall polymer, the teichoic acids, which contain an unusually high substitution with sugars in the β-conformation. By experimentally increasing the proportion of β-N-acetyl-d-glucosamine in a closely related isolate through the induction of salt stress, we could show that the β-conformation of the sugars plays a vital role in the resistance of S. aureus VC40.

Molecular dynamics simulation of Toxin-Antitoxin (TA) system in Acinetobacter baumannii to explore thenovel mechanism for inhibition of cell wall biosynthesis: Zeta Toxin as an effective therapeutic target.

The majority of bacteria and archaea contains Toxin-Antitoxin system (TA) that codes for the stable Toxin and unstable Antitoxin components forming a complex. The Antitoxin inhibits the catalytic activities of the Toxin. In general, the Antitoxin will be degraded by the proteases leading to the Toxin activation that subsequently targets essential cellular processes, including transcription, translation, replication, cell division, and cell wall biosynthesis. The Zeta Toxin-Epsilon Antitoxin system in ESKAPE pathogen stabilizes the resistance plasmid and promotes pathogenicity. The known TA system in Acinetobacter baumannii are known to be involved in the replication and translation, however, the mechanism of Zeta Toxin-Epsilon Antitoxin in cell wall biosynthesis remains unknown. In the present study, molecular docking and molecular dynamic (MD) simulations were employed to demonstrate whether Zeta Toxin can impair cell wall synthesis in A. baumannii. Further, the degradation mechanism of Antitoxin in the presence and absence of adenosine triphosphate (ATP) molecules are explained through MD simulation. The result reveals that the cleavage of Antitoxin could be possible with the presence of ATP by displaying its response from 20 ns, whereas the Zeta Toxin/Epsilon was unstable after 90 ns. The obtained results demonstrate that Zeta Toxin is "temporarily favorable" for ATP to undergo phosphorylation at UNAG kinase through the substrate tunneling process. The study further evidenced that phosphorylated UNAG prevents the binding of MurA, the enzyme that catalyzes the initial step of bacterial peptidoglycan biosynthesis. Therefore, the present study explores the binding mechanism of Zeta Toxin/Epsilon Antitoxin, which could be beneficial for preventing cell wall biosynthesis as well as for unveiling the alternative treatment options to antibiotics.

Unlocking the bacterial membrane as a therapeutic target for next-generation antimicrobial amphiphiles

Gram-positive bacteria like Enterococcus faecium and Staphylococcus aureus, and Gram-negative bacteria like Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter Spp. are responsible for most of fatal bacterial infections. Bacteria present a handful of targets like ribosome, RNA polymerase, cell wall biosynthesis, and dihydrofolate reductase. Antibiotics targeting the protein synthesis like aminoglycosides and tetracyclines, inhibitors of RNA/DNA synthesis like fluoroquinolones, inhibitors of cell wall biosynthesis like glycopeptides and β-lactams, and membrane-targeting polymyxins and lipopeptides have shown very good success in combating the bacterial infections. Ability of the bacteria to develop drug resistance is a serious public health challenge as bacteria can develop antimicrobial resistance against newly introduced antibiotics that enhances the challenge for antibiotic drug discovery. Therefore, bacterial membranes present a suitable therapeutic target for development of antimicrobials as bacteria can find it difficult to develop resistance against membrane-targeting antimicrobials. In this review, we present the recent advances in engineering of membrane-targeting antimicrobial amphiphiles that can be effective alternatives to existing antibiotics in combating bacterial infections.

Alkyl-Aryl-Vancomycins: Multimodal Glycopeptides with Weak Dependence on the Bacterial Metabolic State.

Resistance to last-resort antibiotics such as vancomycin for Gram-positive bacterial infections necessitates the development of new therapeutics. Furthermore, the ability of bacteria to survive antibiotic therapy through formation of biofilms and persister cells complicates treatment. Toward this, we report alkyl-aryl-vancomycins (AAVs), with high potency against vancomycin-resistant enterococci and staphylococci. Unlike vancomycin, the lead compound AAV-qC10 was bactericidal and weakly dependent on bacterial metabolism. This resulted in complete eradication of non-growing cells of MRSA and disruption of its biofilms. In addition to inhibiting cell wall biosynthesis like vancomycin, AAV-qC10 also depolarizes and permeabilizes the membrane. More importantly, the compound delocalized the cell division protein MinD, thereby impairing bacterial growth through multiple pathways. The potential of AAV-qC10 is exemplified by its superior efficacy against MRSA in a murine thigh infection model as compared to vancomycin. This work paves the way for structural optimization and drug development for combating drug-resistant bacterial infections.

The zinc cluster transcription factor Czf1 regulates cell wall architecture and integrity in Candida albicans.

The fungal cell wall is essential for the maintenance of cellular integrity and mediates interactions of the cells with the environment. It is a highly flexible organelle whose composition and organization is modulated in response to changing growth conditions. In the pathogenic yeast Candida albicans, a network of signaling pathways regulates the structure of the cell wall, and mutants with defects in these pathways are hypersensitive to cell wall stress. By harnessing a library of genetically activated forms of all C.albicans zinc cluster transcription factors, we found that a hyperactive Czf1 rescued the hypersensitivity to cell wall stress of different protein kinase deletion mutants. The hyperactive Czf1 induced the expression of many genes with cell wall-related functions and caused visible changes in the cell wall structure. C.albicans czf1Δ mutants were hypersensitive to the antifungal drug caspofungin, which inhibits cell wall biosynthesis. The changes in cell wall architecture caused by hyperactivity or absence of Czf1 resulted in an increased recognition of C.albicans by human neutrophils. Our results show that Czf1, which is known as a regulator of filamentous growth and white-opaque switching, controls the expression of cell wall genes and modulates the architecture of the cell wall.

Review: Modification of Nanocellulose as Conjugate of Infection-Causing Antibacterial Hydrogel

Infection is the process of entering and reproducing microorganisms such as bacteria, viruses, fungi, and parasites that cause tissue injury. Some of the common types of bacteria that play a role in wound infection are Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, and Staphylococcus epidermis. The antibacterial able to inhibit bacterial growth by inhibiting cell wall biosynthesis, increasing the permeability of the bacterial cytoplasmic membrane, and interfering with the normal bacterial protein synthesis. The aim of this review article is to conduct a study of nanocellulose as an antibacterial hydrogel conjugate. The method used is to summarize information from various recent journals related to nanocellulose, nanocellulose modification, nanocellulose-based hydrogels, and their application as antibacterial. Some journals from primary sources such as the PMC system (PubMed Central), National Library of Medicine (NIH), Science Direct, Elsevier, Nature, ACS Chemical Society, and several other sources. Nanocellulose consists of β-1, 4-glucose, and there are three hydroxyls active at the C2, C3, and C6 positions of the pyranose attachment. Nanocellulose can respond by the reaction of oxidation, esterification, or etherification, by adding a new functional group. Nanocellulose can become nanocellulose nanocrystals (CNC), cellulose nanofibers (NFC), and nanocellulose bacteria (BNC). Nanocellulose formulated in the form of hydrogels and combined with antibiotics will increase the effectiveness in reducing the risk of infection that is resistant to antibiotics.

Integrated Omics Strategy Reveals Cyclic Lipopeptides Empedopeptins from Massilia sp. YMA4 and Their Biosynthetic Pathway.

Empedopeptins—eight amino acid cyclic lipopeptides—are calcium-dependent antibiotics that act against Gram-positive bacteria such as Staphylococcus aureus by inhibiting cell wall biosynthesis. However, to date, the biosynthetic mechanism of the empedopeptins has not been well identified. Through comparative genomics and metabolomics analysis, we identified empedopeptin and its new analogs from a marine bacterium, Massilia sp. YMA4. We then unveiled the empedopeptin biosynthetic gene cluster. The core nonribosomal peptide gene null-mutant strains (ΔempC, ΔempD, and ΔempE) could not produce empedopeptin, while dioxygenase gene null-mutant strains (ΔempA and ΔempB) produced several unique empedopeptin analogs. However, the antibiotic activity of ΔempA and ΔempB was significantly reduced compared with the wild-type, demonstrating that the hydroxylated amino acid residues of empedopeptin and its analogs are important to their antibiotic activity. Furthermore, we found seven bacterial strains that could produce empedopeptin-like cyclic lipopeptides using a genome mining approach. In summary, this study demonstrated that an integrated omics strategy can facilitate the discovery of potential bioactive metabolites from microbial sources without further isolation and purification.

Recent Trends in Synthesis of Chloramphenicol New Derivatives.

Chloramphenicol (CAM), the bacteriostatic broad-spectrum antibiotic, isolated from Streptomyces venezuelae during the “golden era” of antibiotic discovery, nowadays has limited clinical potential due to adverse side effects and frequent antimicrobial resistance. Numerous CAM analogs were synthesized in order to find the derivatives with improved pharmacological properties and activity on resistant bacterial strains. This work aims to summarize the most recent achievements in obtaining new CAM analogs reported during the last five years. Current investigations are mainly focused on elucidating the molecular basis of the mode of CAM action and determining the mechanisms of resistance to this class of antibiotics or on studies of the possible use of the CAM scaffold to search for therapeutic agents with different CAM modes of action—such as selective antiproliferative agents or bacterial cell wall biosynthesis inhibitors. Hopefully, a deeper understanding of the CAM interactions with the target and its specificity will generate research ideas for developing new effective drugs.

Molecular Dynamics Simulation of Atomic Interactions in the Vancomycin Binding Site.

Vancomycin is a glycopeptide antibiotic produced by Amycolaptopsis orientalis used to treat serious infections by Gram-positive pathogens including methicillin-resistant Staphylococcus aureus. Vancomycin inhibits cell wall biosynthesis by targeting lipid II, which is the membrane-bound peptidoglycan precursor. The heptapeptide aglycon structure of vancomycin binds to the d-Ala-d-Ala of the pentapeptide stem structure in lipid II. The third residue of vancomycin aglycon is asparagine, which is not directly involved in the dipeptide binding. Nonetheless, asparagine plays a crucial role in substrate recognition, as the vancomycin analogue with asparagine substituted by aspartic acid (VD) shows a reduction in antibacterial activities. To characterize the function of asparagine, binding of vancomycin and its aspartic-acid-substituted analogue VD to l-Lys-d-Ala-d-Ala and l-Lys-d-Ala-d-Lac was investigated using molecular dynamic simulations. Binding interactions were analyzed using root-mean-square deviation (RMSD), two-dimensional (2D) contour plots, hydrogen bond analysis, and free energy calculations of the complexes. The analysis shows that the aspartate substitution introduced a negative charge to the binding cleft of VD, which altered the aglycon conformation that minimized the repulsive lone pair interaction in the binding of a depsipeptide. Our findings provide new insight for the development of novel glycopeptide antibiotics against the emerging vancomycin-resistant pathogens by chemical modification at the third residue in vancomycin to improve its binding affinity to the d-Ala-d-Lac-terminated peptidoglycan in lipid II found in vancomycin-resistant enterococci and vancomycin-resistant S. aureus.

Mutations Selected After Exposure to Bacteriocin Lcn972 Activate a Bce-Like Bacitracin Resistance Module in Lactococcus lactis.

Resistance against antimicrobial peptides (AMPs) is often mediated by detoxification modules that rely on sensing the AMP through a BceAB-like ATP-binding cassette (ABC) transporter that subsequently activates a cognate two-component system (TCS) to mount the cell response. Here, the Lactococcus lactis ABC transporter YsaDCB is shown to constitute, together with TCS-G, a detoxification module that protects L. lactis against bacitracin and the bacteriocin Lcn972, both AMPs that inhibit cell wall biosynthesis. Initially, increased expression of ysaDCB was detected by RT-qPCR in three L. lactis resistant to Lcn972, two of which were also resistant to bacitracin. These mutants shared, among others, single-point mutations in ysaB coding for the putative Bce-like permease. These results led us to investigate the function of YsaDCB ABC-transporter and study the impact of these mutations. Expression in trans of ysaDCB in L. lactis NZ9000, a strain that lacks a functional detoxification module, enhanced resistance to both AMPs, demonstrating its role as a resistance factor in L. lactis. When the three different ysaB alleles from the mutants were expressed, all of them outperformed the wild-type transporter in resistance against Lcn972 but not against bacitracin, suggesting a distinct mode of protection against each AMP. Moreover, PysaD promoter fusions, designed to measure the activation of the detoxification module, revealed that the ysaB mutations unlock transcriptional control by TCS-G, resulting in constitutive expression of the ysaDCB operon. Finally, deletion of ysaD was also performed to get an insight into the function of this gene. ysaD encodes a secreted peptide and is part of the ysaDCB operon. YsaD appears to modulate signal relay between the ABC transporter and TCS-G, based on the different response of the PysaD promoter fusions when it is not present. Altogether, the results underscore the unique features of this lactococcal detoxification module that warrant further research to advance in our overall understanding of these important resistance factors in bacteria.

Analysis of a cellulose synthase catalytic subunit from the oomycete pathogen of crops Phytophthora capsici

Phytophthora capsici Leonian is an important oomycete pathogen of crop vegetables, causing significant economic losses each year. Its cell wall, rich in cellulose, is vital for cellular integrity and for interactions with the host organisms. Predicted cellulose synthase (CesA) proteins are expected to catalyze the polymerization of cellulose, but this has not been biochemically demonstrated in an oomycete. Here, we present the properties of the four newly identified CesA proteins from P. capsici and compare their domain organization with that of CesAs from other lineages. Using a newly constructed glucosyltransferase-deficient variant of Saccharomyces cerevisiae with low residual background activity, we have achieved successful heterologous expression and biochemical characterization of a CesA protein from P. capsici (PcCesA1). Our results demonstrate that the individual PcCesA1 enzyme produces cellobiose as the major reaction product. Co-immunoprecipitation studies and activity assays revealed that several PcCesA proteins interact together to form a complex whose multiproteic nature is most likely required for cellulose microfibril formation. In addition to providing important insights into cellulose synthesis in the oomycetes, our data may assist the longer term identification of cell wall biosynthesis inhibitors to control infection by pathogenic oomycetes.