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PLoS Biology Nov 2020Lifeact is a short actin-binding peptide that is used to visualize filamentous actin (F-actin) structures in live eukaryotic cells using fluorescence microscopy....
Lifeact is a short actin-binding peptide that is used to visualize filamentous actin (F-actin) structures in live eukaryotic cells using fluorescence microscopy. However, this popular probe has been shown to alter cellular morphology by affecting the structure of the cytoskeleton. The molecular basis for such artefacts is poorly understood. Here, we determined the high-resolution structure of the Lifeact-F-actin complex using electron cryo-microscopy (cryo-EM). The structure reveals that Lifeact interacts with a hydrophobic binding pocket on F-actin and stretches over 2 adjacent actin subunits, stabilizing the DNase I-binding loop (D-loop) of actin in the closed conformation. Interestingly, the hydrophobic binding site is also used by actin-binding proteins, such as cofilin and myosin and actin-binding toxins, such as the hypervariable region of TccC3 (TccC3HVR) from Photorhabdus luminescens and ExoY from Pseudomonas aeruginosa. In vitro binding assays and activity measurements demonstrate that Lifeact indeed competes with these proteins, providing an explanation for the altering effects of Lifeact on cell morphology in vivo. Finally, we demonstrate that the affinity of Lifeact to F-actin can be increased by introducing mutations into the peptide, laying the foundation for designing improved actin probes for live cell imaging.
Topics: Actins; Animals; Bacterial Toxins; Binding Sites; Binding, Competitive; Cofilin 1; Cryoelectron Microscopy; Fluorescent Dyes; HEK293 Cells; Humans; Hydrophobic and Hydrophilic Interactions; In Vitro Techniques; Microfilament Proteins; Microscopy, Confocal; Models, Molecular; Myosins; Peptide Fragments; Protein Engineering; Protein Interaction Domains and Motifs; Rabbits; Recombinant Fusion Proteins; Saccharomyces cerevisiae Proteins
PubMed: 33216759
DOI: 10.1371/journal.pbio.3000925 -
MBio Jun 2022With the overmining of actinomycetes for compounds acting against Gram-negative pathogens, recent efforts to discover novel antibiotics have been focused on other groups...
With the overmining of actinomycetes for compounds acting against Gram-negative pathogens, recent efforts to discover novel antibiotics have been focused on other groups of bacteria. Teixobactin, the first antibiotic without detectable resistance that binds lipid II, comes from an uncultured , a betaproteobacterium; odilorhabdins, from , are broad-spectrum inhibitors of protein synthesis, and darobactins from target BamA, the essential chaperone of the outer membrane of Gram-negative bacteria. and are symbionts of the nematode gut microbiome and attractive producers of secondary metabolites. Only small portions of their biosynthetic gene clusters (BGC) are expressed To access their silent operons, we first separated extracts from a small library of isolates into fractions, resulting in 200-fold concentrated material, and then screened them for antimicrobial activity. This resulted in a hit with selective activity against Escherichia coli, which we identified as a novel natural product antibiotic, 3'-amino 3'-deoxyguanosine (ADG). Mutants resistant to ADG mapped to and , kinases of guanosine. Biochemical analysis shows that ADG is a prodrug that is converted into an active ADG triphosphate (ADG-TP), a mimic of GTP. ADG incorporates into a growing RNA chain, interrupting transcription, and inhibits cell division, apparently by interfering with the GTPase activity of FtsZ. Gsk of the purine salvage pathway, which is the first kinase in the sequential phosphorylation of ADG, is restricted to E. coli and closely related species, explaining the selectivity of the compound. There are probably numerous targets of ADG-TP among GTP-dependent proteins. The discovery of ADG expands our knowledge of prodrugs, which are rare among natural compounds. Drug-resistant Gram-negative bacteria have become the major problem driving the antimicrobial resistance crisis. Searching outside the overmined actinomycetes, we focused on , gut symbionts of enthomopathogenic nematodes that carry up to 40 biosynthetic gene clusters coding for secondary metabolites. Most of these are silent and do not express . To gain access to silent operons, we first fractionated supernatant from and then tested 200-fold concentrated material for activity. This resulted in the isolation of a novel antimicrobial, 3'-amino 3'-deoxyguanosine (ADG), active against E. coli. ADG is an analog of guanosine and is converted into an active ADG-TP in the cell. ADG-TP inhibits transcription and probably numerous other GTP-dependent targets, such as FtsZ. Natural product prodrugs have been uncommon; discovery of ADG broadens our knowledge of this type of antibiotic.
Topics: Animals; Anti-Bacterial Agents; Bacterial Outer Membrane Proteins; Biological Products; Deoxyguanosine; Escherichia coli; Escherichia coli Proteins; Gram-Negative Bacteria; Guanosine; Guanosine Triphosphate; Nematoda; Operon; Photorhabdus; Prodrugs; Xenorhabdus
PubMed: 35575547
DOI: 10.1128/mbio.00700-22 -
Beilstein Journal of Organic Chemistry 2020The global threat of multiresistant pathogens has to be answered by the development of novel antibiotics. Established antibiotic applications are often based on...
The global threat of multiresistant pathogens has to be answered by the development of novel antibiotics. Established antibiotic applications are often based on so-called secondary or specialized metabolites (SMs), identified in large screening approaches. To continue this successful strategy, new sources for bioactive compounds are required, such as the bacterial genera or . In these strains, fabclavines are widely distributed SMs with a broad-spectrum bioactivity. Fabclavines are hybrid SMs derived from nonribosomal peptide synthetases (NRPS), polyunsaturated fatty acid (PUFA), and polyketide synthases (PKS). Selected and mutant strains were generated applying a chemically inducible promoter in front of the suggested fabclavine () biosynthesis gene cluster (BGC), followed by the analysis of the occurring fabclavines. Subsequently, known and unknown derivatives were identified and confirmed by MALDI-MS and MALDI-MS experiments in combination with an optimized sample preparation. This led to a total number of 22 novel fabclavine derivatives in eight strains, increasing the overall number of fabclavines to 32. Together with the identification of fabclavines as major antibiotics in several entomopathogenic strains, our work lays the foundation for the rapid fabclavine identification and dereplication as the basis for future work of this widespread and bioactive SM class.
PubMed: 32461774
DOI: 10.3762/bjoc.16.84 -
AMB Express Aug 2021Azasugars are monosaccharide analogs in which the ring oxygen is replaced with a nitrogen atom. These well-known glycosidase inhibitors are of interest as therapeutics,...
Azasugars are monosaccharide analogs in which the ring oxygen is replaced with a nitrogen atom. These well-known glycosidase inhibitors are of interest as therapeutics, yet several aspects of azasugars remain unknown including their distribution, structural diversity, and chemical ecology. The hallmark signature of bacterial azasugar biosynthesis is a three gene cluster (3GC) coding for aminotransferase, phosphatase, and dehydrogenase enzymes. Using the bioinformatics platform Enzyme Similarity Tool (EST), we identified hundreds of putative three gene clusters coding for azasugar production in microbial species. In the course of this work, we also report a consensus sequence for the aminotransferase involved in azasugar biosynthesis as being: SGNXFRXXXFPNXXXXXXXLXVPXPYCXRC. Most clusters are found in Bacillus and Streptomyces species which typically inhabit soil and the rhizosphere, but some clusters are found with diverse species representation such as Photorhabdus and Xenorhabdus which are symbiotic with entomopathogenic nematodes; the human skin commensal Cutibacterium acnes, and the marine Bacillus rugosus SPB7, a symbiont to the sea sponge Spongia officinalis. This pan-taxonomic survey of the azasugar 3GC signature may lead to the identification of new azasugar producers, facilitate studies of their natural functions, and lead to new potential therapeutics.
PubMed: 34424396
DOI: 10.1186/s13568-021-01279-5 -
Frontiers in Microbiology 2022Phylum Nematoda is of great economic importance. It has been a focused area for various research activities in distinct domains across the globe. Among nematodes, there... (Review)
Review
Phylum Nematoda is of great economic importance. It has been a focused area for various research activities in distinct domains across the globe. Among nematodes, there is a group called entomopathogenic nematodes, which has two families that live in symbiotic association with bacteria of genus and , respectively. With the passing years, researchers have isolated a wide array of bioactive compounds from these symbiotically associated nematodes. In this article, we are encapsulating bioactive compounds isolated from members of the family Heterorhabditidae inhabiting in its gut. Isolated bioactive compounds have shown a wide range of biological activity against deadly pathogens to both plants as well as animals. Some compounds exhibit lethal effects against fungi, bacteria, protozoan, insects, cancerous cell lines, neuroinflammation, etc., with great potency. The main aim of this article is to collect and analyze the importance of nematode and its associated bacteria, isolated secondary metabolites, and their biomedical potential, which can serve as potential leads for further drug discovery.
PubMed: 35422783
DOI: 10.3389/fmicb.2022.790339 -
Frontiers in Microbiology 2020Temperature plays an important role in bacteria-host interactions and can be a determining factor for host switching. In this study we sought to investigate the reasons...
Temperature plays an important role in bacteria-host interactions and can be a determining factor for host switching. In this study we sought to investigate the reasons behind growth temperature restriction in the entomopathogenic enterobacterium . has a complex dual symbiotic and pathogenic life cycle. The genus consists of 19 species but only one subgroup, previously all classed together as , have been shown to cause human disease. These clinical isolates necessarily need to be able to grow at 37°C, whilst the remaining species are largely restricted to growth temperatures below 34°C and are therefore unable to infect mammalian hosts. Here, we have isolated spontaneous mutant lines of DJC that were able to grow up to 36-37°C. Following whole genome sequencing of 29 of these mutants we identified a single gene, encoding a protein with a RecG-like helicase domain that for the majority of isolates contained single nucleotide polymorphisms. Importantly, provision of the wild-type allele of this gene in restored the temperature restriction, confirming the mutations are recessive, and the dominant effect of the protein product of this gene. The gene appears to be part of a short three cistron operon, which we have termed the Temperature Restricting Locus (TRL). Transcription reporter strains revealed that this operon is induced upon the switch from 30 to 36°C, leading to replication arrest of the bacteria. TRL is absent from all of the human pathogenic species so far examined, although its presence is not uniform in different strains of the subgroup. In a wider context, the presence of this gene is not limited to , being found in phylogenetically diverse proteobacteria. We therefore suggest that this system may play a more fundamental role in temperature restriction in diverse species, relating to as yet cryptic aspects of their ecological niches and life cycle requirements.
PubMed: 33101227
DOI: 10.3389/fmicb.2020.548800 -
Frontiers in Microbiology 2022The importance of and symbionts to their respective and nematode hosts is that they not only contribute to their entomopathogenicity but also to their fecundity... (Review)
Review
The importance of and symbionts to their respective and nematode hosts is that they not only contribute to their entomopathogenicity but also to their fecundity through the production of small molecules. Thus, this mini-review gives a brief introductory overview of these nematophilic bacteria. Specifically, their type species, nematode hosts, and geographic region of isolations are tabulated. The use of nucleotide sequence-based techniques for their species delineation and how pangenomes can improve this are highlighted. Using the - association as an example, the bacterium-nematode lifecycle is visualized with an emphasis on the role of bacterial biomolecules. Those currently in drug development are discussed, and two potential antimalarial lead compounds are highlighted. Thus, this mini-review tabulates forty-eight significant nematophilic bacteria and visualizes the ecological importance of their biomolecules. It further discusses three of these biomolecules that are currently in drug development. Through it, one is introduced to and bacteria, their natural production of biomolecules in the nematode-bacterium lifecycle, and how these molecules are useful in developing novel therapies.
PubMed: 36187939
DOI: 10.3389/fmicb.2022.993688 -
Insects Aug 2020This special issue contains articles that add to the ever-expanding toolbox of insect pathogenic nematodes (entomopathogenic nematodes; EPNs) as well articles that...
This special issue contains articles that add to the ever-expanding toolbox of insect pathogenic nematodes (entomopathogenic nematodes; EPNs) as well articles that provide new insights into the mutualistic interaction between EPNs and their hosts. The study of natural infection models such as EPNs allows detailed insight into micro- and macro-evolutionary dynamics of innate immune reactions, including known but also emerging branches of innate immunity. Additional new insights into the kinetics of EPN infections are gained by increased spatiotemporal resolution of advanced transcriptome studies and live imaging.
PubMed: 32872298
DOI: 10.3390/insects11090577 -
Nature Microbiology Oct 2022Discovery of antibiotics acting against Gram-negative species is uniquely challenging due to their restrictive penetration barrier. BamA, which inserts proteins into the...
Discovery of antibiotics acting against Gram-negative species is uniquely challenging due to their restrictive penetration barrier. BamA, which inserts proteins into the outer membrane, is an attractive target due to its surface location. Darobactins produced by Photorhabdus, a nematode gut microbiome symbiont, target BamA. We reasoned that a computational search for genes only distantly related to the darobactin operon may lead to novel compounds. Following this clue, we identified dynobactin A, a novel peptide antibiotic from Photorhabdus australis containing two unlinked rings. Dynobactin is structurally unrelated to darobactins, but also targets BamA. Based on a BamA-dynobactin co-crystal structure and a BAM-complex-dynobactin cryo-EM structure, we show that dynobactin binds to the BamA lateral gate, uniquely protruding into its β-barrel lumen. Dynobactin showed efficacy in a mouse systemic Escherichia coli infection. This study demonstrates the utility of computational approaches to antibiotic discovery and suggests that dynobactin is a promising lead for drug development.
Topics: Animals; Anti-Bacterial Agents; Bacterial Outer Membrane Proteins; Escherichia coli; Escherichia coli Proteins; Gram-Negative Bacteria; Mice; Peptides; Phenylpropionates
PubMed: 36163500
DOI: 10.1038/s41564-022-01227-4 -
The Journal of Biological Chemistry May 2024Mono-O-glycosylation of target proteins by bacterial toxins or effector proteins is a well-known mechanism by which bacteria interfere with essential functions of host...
Mono-O-glycosylation of target proteins by bacterial toxins or effector proteins is a well-known mechanism by which bacteria interfere with essential functions of host cells. The respective glycosyltransferases are important virulence factors such as the Clostridioides difficile toxins A and B. Here, we describe two glycosyltransferases of Yersinia species that have a high sequence identity: YeGT from the zoonotic pathogen Yersinia enterocolitica and YkGT from the murine pathogen Yersinia kristensenii. We show that both modify Rho family proteins by attachment of GlcNAc at tyrosine residues (Tyr-34 in RhoA). Notably, the enzymes differed in their target protein specificity. While YeGT modified RhoA, B, and C, YkGT possessed a broader substrate spectrum and glycosylated not only Rho but also Rac and Cdc42 subfamily proteins. Mutagenesis studies indicated that residue 177 is important for this broader target spectrum. We determined the crystal structure of YeGT shortened by 16 residues N terminally (sYeGT) in the ligand-free state and bound to UDP, the product of substrate hydrolysis. The structure assigns sYeGT to the GT-A family. It shares high structural similarity to glycosyltransferase domains from toxins. We also demonstrated that the 16 most N-terminal residues of YeGT and YkGT are important for the mediated translocation into the host cell using the pore-forming protective antigen of anthrax toxin. Mediated introduction into HeLa cells or ectopic expression of YeGT and YkGT caused morphological changes and redistribution of the actin cytoskeleton. The data suggest that YeGT and YkGT are likely bacterial effectors belonging to the family of tyrosine glycosylating bacterial glycosyltransferases.
PubMed: 38703997
DOI: 10.1016/j.jbc.2024.107331