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Biochimie Dec 2022The objective of the present review is to provide an insight into modifications of microbial cell walls and membrane constituents by using the aminoacyl-tRNA as amino... (Review)
Review
The objective of the present review is to provide an insight into modifications of microbial cell walls and membrane constituents by using the aminoacyl-tRNA as amino acid donor. In bacteria, phospholipids are modified by Multiple peptide resistance Factor enzymes and peptidoglycan precursors by so called fem ligases. Although these modifications were thought to be restricted to procaryotes, we discovered enzymes that modify ergosterol (the main component of fungal membrane) with glycine and aspartate. The focus of this review is to present the molecular mechanisms underlying all these processes together with the structure of the enzymes and their substrates. This article also reviews how substrates are recognized and modified and how the products are subsequently exported in various organisms. Finally, the physiological outcome and the discoveries of each family of enzymes is also discussed.
Topics: Amino Acids; RNA, Transfer; Cell Wall; RNA, Transfer, Amino Acyl; Peptidoglycan; Amino Acyl-tRNA Synthetases
PubMed: 36184002
DOI: 10.1016/j.biochi.2022.09.017 -
Nature Communications Jan 2024Active nutrient uptake is fundamental for survival and pathogenicity of Gram-negative bacteria, which operate a multi-protein Ton system to transport essential nutrients...
Active nutrient uptake is fundamental for survival and pathogenicity of Gram-negative bacteria, which operate a multi-protein Ton system to transport essential nutrients like metals and vitamins. This system harnesses the proton motive force at the inner membrane to energize the import through the outer membrane, but the mechanism of energy transfer remains enigmatic. Here, we study the periplasmic domain of ExbD, a crucial component of the proton channel of the Ton system. We show that this domain is a dynamic dimer switching between two conformations representing the proton channel's open and closed states. By in vivo phenotypic assays we demonstrate that this conformational switch is essential for the nutrient uptake by bacteria. The open state of ExbD triggers a disorder to order transition of TonB, enabling TonB to supply energy to the nutrient transporter. We also reveal the anchoring role of the peptidoglycan layer in this mechanism. Herein, we propose a mechanistic model for the Ton system, emphasizing ExbD duality and the pivotal catalytic role of peptidoglycan. Sequence analysis suggests that this mechanism is conserved in other systems energizing gliding motility and membrane integrity. Our study fills important gaps in understanding bacterial motor mechanism and proposes novel antibacterial strategies.
Topics: Peptidoglycan; Protons; Cell Wall; Nutrients; Bacteria
PubMed: 38184686
DOI: 10.1038/s41467-023-44606-z -
Molecular Microbiology Nov 2022A Clostridioides difficile strain deficient in the ddl gene is unable to synthesize the dipeptide D-Ala-D-Ala, an essential component of peptidoglycan and the target of...
A Clostridioides difficile strain deficient in the ddl gene is unable to synthesize the dipeptide D-Ala-D-Ala, an essential component of peptidoglycan and the target of vancomycin. We isolated spontaneous suppressors of a ∆ddl mutation that allowed cell growth in the absence of D-Ala-D-Ala. The mutations caused constitutive or partly constitutive expression of the vancomycin-inducible vanG operon responsible for the synthesis of D-Ala-D-Ser, which can replace D-Ala-D-Ala in peptidoglycan. The mutations mapped to the vanS or vanR genes, which regulate expression of the vanG operon. The constitutive level of vanG expression was about 10-fold above that obtained by vancomycin induction. The incorporation of D-Ala-D-Ser into peptidoglycan due to high expression of the vanG operon conferred only low-level resistance to vancomycin, but VanG was found to synthesize D-Ala-D-Ala in addition to D-Ala-D-Ser. However, the same, low resistance to vancomycin was also observed in cells completely unable to synthesize D-Ala-D-Ala and grown in the presence of D-Ala-D-Ser. D-Ala-D-Ala presence was required for efficient vancomycin induction of the vanG operon showing that vancomycin is not by itself able to activate VanS. D-Ala-D-Ser, similar to D-Ala-D-Ala, served as an anti-activator of DdlR, the positive regulator of the ddl gene, thereby coupling vanG and ddl expression.
Topics: Vancomycin Resistance; Vancomycin; Peptidoglycan; Clostridioides difficile; Clostridioides; Bacterial Proteins; Transcription Factors; Anti-Bacterial Agents
PubMed: 36065735
DOI: 10.1111/mmi.14980 -
Journal of Virology Nov 2019Enteric viruses exploit bacterial components, including lipopolysaccharides (LPS) and peptidoglycan (PG), to facilitate infection in humans. Because of their origin in...
Enteric viruses exploit bacterial components, including lipopolysaccharides (LPS) and peptidoglycan (PG), to facilitate infection in humans. Because of their origin in the bat enteric system, we wondered if severe acute respiratory syndrome coronavirus (SARS-CoV) or Middle East respiratory syndrome CoV (MERS-CoV) also use bacterial components to modulate infectivity. To test this question, we incubated CoVs with LPS and PG and evaluated infectivity, finding no change following LPS treatment. However, PG from reduced infection >10,000-fold, while PG from other bacterial species failed to recapitulate this. Treatment with an alcohol solvent transferred inhibitory activity to the wash, and mass spectrometry revealed surfactin, a cyclic lipopeptide antibiotic, as the inhibitory compound. This antibiotic had robust dose- and temperature-dependent inhibition of CoV infectivity. Mechanistic studies indicated that surfactin disrupts CoV virion integrity, and surfactin treatment of the virus inoculum ablated infection Finally, similar cyclic lipopeptides had no effect on CoV infectivity, and the inhibitory effect of surfactin extended broadly to enveloped viruses, including influenza, Ebola, Zika, Nipah, chikungunya, Una, Mayaro, Dugbe, and Crimean-Congo hemorrhagic fever viruses. Overall, our results indicate that peptidoglycan-associated surfactin has broad viricidal activity and suggest that bacteria by-products may negatively modulate virus infection. In this article, we consider a role for bacteria in shaping coronavirus infection. Taking cues from studies of enteric viruses, we initially investigated how bacterial surface components might improve CoV infection. Instead, we found that peptidoglycan-associated surfactin is a potent viricidal compound that disrupts virion integrity with broad activity against enveloped viruses. Our results indicate that interactions with commensal bacterial may improve or disrupt viral infections, highlighting the importance of understanding these microbial interactions and their implications for viral pathogenesis and treatment.
Topics: Animals; Cell Line; Chlorocebus aethiops; Coronavirus Infections; Flaviviridae; Lipopeptides; Middle East Respiratory Syndrome Coronavirus; Peptides, Cyclic; Peptidoglycan; RNA Viruses; Severe acute respiratory syndrome-related coronavirus; Severe Acute Respiratory Syndrome; Vero Cells; Virus Diseases
PubMed: 31462558
DOI: 10.1128/JVI.01282-19 -
Frontiers in Cellular and Infection... 2023Mycobacteria assemble a complex cell wall with cross-linked peptidoglycan (PG) which plays an essential role in maintenance of cell wall integrity and tolerance to...
INTRODUCTION
Mycobacteria assemble a complex cell wall with cross-linked peptidoglycan (PG) which plays an essential role in maintenance of cell wall integrity and tolerance to osmotic pressure. We previously demonstrated that various hydrolytic enzymes are required to remodel PG during essential processes such as cell elongation and septal hydrolysis. Here, we explore the chemistry associated with PG cross-linking, specifically the requirement for amidation of the D-glutamate residue found in PG precursors.
METHODS
Synthetic fluorescent probes were used to assess PG remodelling dynamics in live bacteria. Fluorescence microscopy was used to assess protein localization in live bacteria and CRISPR-interference was used to construct targeted gene knockdown strains. Time-lapse microscopy was used to assess bacterial growth. Western blotting was used to assess protein phosphorylation.
RESULTS AND DISCUSSION
In , we confirmed the essentiality for D-glutamate amidation in PG biosynthesis by labelling cells with synthetic fluorescent PG probes carrying amidation modifications. We also used CRISPRi targeted knockdown of genes encoding the MurT-GatD complex, previously implicated in D-glutamate amidation, and demonstrated that these genes are essential for mycobacterial growth. We show that MurT-rseGFP co-localizes with mRFP-GatD at the cell poles and septum, which are the sites of cell wall synthesis in mycobacteria. Furthermore, time-lapse microscopic analysis of MurT-rseGFP localization, in fluorescent D-amino acid (FDAA)-labelled mycobacterial cells during growth, demonstrated co-localization with maturing PG, suggestive of a role for PG amidation during PG remodelling and repair. Depletion of MurT and GatD caused reduced PG cross-linking and increased sensitivity to lysozyme and β-lactam antibiotics. Cell growth inhibition was found to be the result of a shutdown of PG biosynthesis mediated by the serine/threonine protein kinase B (PknB) which senses uncross-linked PG. Collectively, these data demonstrate the essentiality of D-glutamate amidation in mycobacterial PG precursors and highlight the MurT-GatD complex as a novel drug target.
Topics: Amides; Glutamic Acid; Mycobacterium smegmatis; Cell Wall; Carbon-Nitrogen Ligases with Glutamine as Amide-N-Donor; Bacterial Proteins; Peptidoglycan
PubMed: 37692163
DOI: 10.3389/fcimb.2023.1205829 -
Zoonoses and Public Health Aug 2023Vancomycin-resistant Staphylococcus aureus (VRSA) is a zoonotic life-threatening pathogen. Vancomycin exhibits anti-bacterial activity by inhibiting peptidoglycan...
Vancomycin-resistant Staphylococcus aureus (VRSA) is a zoonotic life-threatening pathogen. Vancomycin exhibits anti-bacterial activity by inhibiting peptidoglycan synthesis by binding to the D-ala-D-ala terminus of the peptidoglycan. But in VRSA, D-ala-D-ala is replaced by D-ala-D-lactate due to the presence of vanA, vanB or vanD genes. This study was intended to identify the molecular prevalence of VRSA in 768 bovine milk samples, risk factor association, antibiogram profile and bioinformatics analysis of VRSA by targeting vanB gene. Out of a total of 248 S. aureus isolates from mastitic milk samples, the phenotypic and genotypic prevalence of VRSA was estimated to be 17.74% and 10.89%, respectively. Farm-level risk factors including use of improper milking technique, lack of milker's care during milking, unhygienic conditions during milking and no dry cow therapy were found to be significantly associated (p < 0.05). Anti-microbial susceptibility testing of VRSA isolates exhibited the highest resistance to oxytetracycline, followed by oxacillin and Trimethoprim+sulfamethoxazole. The current study sequences showed more resemblance with reported sequences from Iraq (MN747834) and Egypt (MK095504, MK087830), which belong to vanB gene from S. aureus as compared to sequences from other countries, which belong to vanB gene from the genus Enterococcus. The Genetic Algorithm for Recombination Detection (GARD) found 234 potential breakpoints, translating into a search room of 123,883,305 models with up to 4 breakpoints. The phylogenetic motif profiling method discovered evolutionarily conserved residues across target genes' homologous protein sequences. These residues were discovered to be conserved in drug-resistant target proteins over the evolutionary process and may play a key role in their function. The current study revealed a molecular prevalence of VRSA in dairy animals, along with molecular analysis of vancomycin resistance in S. aureus by targeting the vanB gene using standard bioinformatics tools. The occurrence of VRSA in animals requires serious attention because this pathogen has zoonotic potential, so it can become a greater risk to consumer health.
Topics: Female; Cattle; Animals; Anti-Bacterial Agents; Methicillin-Resistant Staphylococcus aureus; Staphylococcus aureus; Vancomycin-Resistant Staphylococcus aureus; Peptidoglycan; Milk; Phylogeny; Bacterial Proteins; Microbial Sensitivity Tests; Staphylococcal Infections; Cattle Diseases
PubMed: 37165559
DOI: 10.1111/zph.13047 -
Communications Biology Mar 2024Peptidoglycan polymerases, enterobacterial common antigen polymerases, O-antigen ligases, and other bacterial polysaccharide polymerases (BP-Pols) are...
Peptidoglycan polymerases, enterobacterial common antigen polymerases, O-antigen ligases, and other bacterial polysaccharide polymerases (BP-Pols) are glycosyltransferases (GTs) that build bacterial surface polysaccharides. These integral membrane enzymes share the particularity of using diphospholipid-activated sugars and were previously missing in the carbohydrate-active enzymes database (CAZy; www.cazy.org ). While the first three classes formed well-defined families of similar proteins, the sequences of BP-Pols were so diverse that a single family could not be built. To address this, we developed a new clustering method using a combination of a sequence similarity network and hidden Markov model comparisons. Overall, we have defined 17 new GT families including 14 of BP-Pols. We find that the reaction stereochemistry appears to be conserved in each of the defined BP-Pol families, and that the BP-Pols within the families transfer similar sugars even across Gram-negative and Gram-positive bacteria. Comparison of the new GT families reveals three clans of distantly related families, which also conserve the reaction stereochemistry.
Topics: Glycosyltransferases; Sugars; Cluster Analysis; Peptidoglycan
PubMed: 38454040
DOI: 10.1038/s42003-024-05930-2 -
STAR Protocols Mar 2022Postbiotics cooperate to influence immune and metabolic outcomes in the host. Here we describe a protocol for assessment of blood glucose control following acute...
Postbiotics cooperate to influence immune and metabolic outcomes in the host. Here we describe a protocol for assessment of blood glucose control following acute administration of lipopolysaccharide (LPS) and peptidoglycan (PGN) in mice. This protocol can be adapted for testing a broad range of microbial molecules and ligands for host immune receptors. Experience with mouse handling is required. For complete details on the use and execution of this protocol, please refer to Anhê et al. (2021) and Cavallari et al. (2017).
Topics: Animals; Cell Wall; Lipopolysaccharides; Mice; Peptidoglycan
PubMed: 35072117
DOI: 10.1016/j.xpro.2021.101098 -
Advances in Protein Chemistry and... 2022Gram-negative bacteria are surrounded by a complex multilayered cell envelope, consisting of an inner and an outer membrane, and separated by the aqueous periplasm,... (Review)
Review
Gram-negative bacteria are surrounded by a complex multilayered cell envelope, consisting of an inner and an outer membrane, and separated by the aqueous periplasm, which contains a thin peptidoglycan cell wall. These bacteria employ an arsenal of highly specialized membrane protein machineries to ensure the correct assembly and maintenance of the membranes forming the cell envelope. Here, we review the diverse protein systems, which perform these functions in Escherichia coli, such as the folding and insertion of membrane proteins, the transport of lipoproteins and lipopolysaccharide within the cell envelope, the targeting of phospholipids, and the regulation of mistargeted envelope components. Some of these protein machineries have been known for a long time, yet still hold surprises. Others have only recently been described and some are still missing pieces or yet remain to be discovered.
Topics: Cell Membrane; Cell Wall; Escherichia coli Proteins; Membrane Proteins; Peptidoglycan
PubMed: 35034716
DOI: 10.1016/bs.apcsb.2021.10.001 -
The Journal of Biological Chemistry Mar 2020Bacteria account for 1000-fold more biomass than humans. They vary widely in shape and size. The morphological diversity of bacteria is due largely to the different... (Review)
Review
Bacteria account for 1000-fold more biomass than humans. They vary widely in shape and size. The morphological diversity of bacteria is due largely to the different peptidoglycan-based cell wall structures that encase bacterial cells. Although the basic structure of peptidoglycan is highly conserved, consisting of long glycan strands that are cross-linked by short peptide chains, the mature cell wall is chemically diverse. Peptidoglycan hydrolases and cell wall-tailoring enzymes that regulate glycan strand length, the degree of cross-linking, and the addition of other modifications to peptidoglycan are central in determining the final architecture of the bacterial cell wall. Historically, it has been difficult to biochemically characterize these enzymes that act on peptidoglycan because suitable peptidoglycan substrates were inaccessible. In this review, we discuss fundamental aspects of bacterial cell wall synthesis, describe the regulation and diverse biochemical and functional activities of peptidoglycan hydrolases, and highlight recently developed methods to make and label defined peptidoglycan substrates. We also review how access to these substrates has now enabled biochemical studies that deepen our understanding of how bacterial cell wall enzymes cooperate to build a mature cell wall. Such improved understanding is critical to the development of new antibiotics that disrupt cell wall biogenesis, a process essential to the survival of bacteria.
Topics: Bacteria; Bacterial Proteins; Cell Wall; N-Acetylmuramoyl-L-alanine Amidase; Peptidoglycan; Protein Structure, Tertiary; Staphylococcus aureus; Substrate Specificity
PubMed: 31974163
DOI: 10.1074/jbc.REV119.010155