Agarose-based 3D culture improved the developmental competence of oocyte-granulosa complex isolated from porcine preantral follicle.

Various models have been established to culture whole follicles of the Preantral stage; however, the process remains inefficient and is an ongoing challenge formation. It is reported that oocyte-cumulus-granulosa complexes (OCGCs) isolated from Early Antral follicles (EAFs) undergo in vitro growth (IVG) and acquire meiotic competence in some animals. However, IVG for the oocyte-granulosa complexes (OGCs) from Preantral Follicles (PAFs) has not been firmly established. The present study indicated that the use of a modified medium with Ascorbic Acid (50 µM) facilitated granulosa cell proliferation, promoted cumulus cell differentiations, and increased antrum formation for the OGCs isolated from PAFs (0.3-0.4 mm). However, the two-dimensional 96-well plate system (2D) experienced smaller size follicles and could not prolong more than 10 days of IVG. Another method is to use an Agarose matrix 3D system to provide a soft, non-adhesive base that supports the IVG of OGCs isolated from PAFs and promotes cell proliferation, antrum formation, and maintenance for 14 days. OGCs that were grown using this method retained their spherical morphology, which in turn helped to attain healthy granulosa cells and maintain their connection with oocytes, in addition, these oocytes significantly increased diameter and lipid content, indicating developmental competence. Our result indicated that the OGCs from PAFs after IVG undergo a change in chromatin morphology and expression of acetylation of histone H3 at lysine 9 (Ac-H3-K9) and methylation of histone H3 at lysine 4 (Me-H3-K4), similar to the in vivo oocytes isolated from the ovary. Likewise, IVG oocytes cultured for maturation showed full cumulus expansion and reached mature oocytes. Furthermore, after in vitro maturation, IVG oocytes underwent the first cleavage following parthenogenetic activation. In conclusion, while most studies used whole follicles from the Preantral stage for IVG, our research finding was the first to reveal that oocytes isolated from the final stage of PAFs can migrate out of the follicle and undergo IVG under suitable conditions.

Antimicrobial Lasso Peptide Cloacaenodin Utilizes a Unique TonB-Dependent Transporter to Access Susceptible Bacteria.

The development of new antimicrobial agents effective against Gram-negative bacteria remains a major challenge in drug discovery. The lasso peptide cloacaenodin has potent antimicrobial activity against multiple strains in the Enterobacter genus, one of the ESKAPE pathogens. Here, we show that cloacaenodin uses a previously uncharacterized TonB-dependent transporter, which we name CloU, to cross the outer membrane (OM) of susceptible bacteria. Inner membrane transport is mediated by the protein SbmA. CloU is distinct from the known OM transporters (FhuA and PupB) utilized by other antimicrobial lasso peptides and thus offers important insight into the spectrum of activity of cloacaenodin. Using knowledge of the transport pathway to predict other cloacaenodin-susceptible strains, we demonstrate the activity of cloacaenodin against clinical isolates of Enterobacter and of a Kluyvera strain. Further, we use molecular dynamics simulations and mutagenesis of CloU to explain the variation in cloacaenodin susceptibility observed across different strains of Enterobacter. This work expands the currently limited understanding of lasso peptide uptake and advances the potential of cloacaenodin as an antibiotic.

Genome Mining and Discovery of Imiditides, a Family of RiPPs with a Class-Defining Aspartimide Modification.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a large and diverse class of natural products of ribosomal origin. In the past decade, various sophisticated machine-learning-based software packages have been established to discover novel RiPPs that do not resemble the known families. Here, we show that tailoring enzymes that cluster with various RiPP families can serve as effective bioinformatic seeds, providing a complementary approach for novel RiPP discovery. Leveraging the fact that O-methyltransferases homologous to protein isoaspartyl methyltransferases (PIMTs) are associated with lasso peptide, graspetide, and lanthipeptide biosynthetic gene clusters (BGCs), we utilized a C-terminal motif unique to RiPP-associated O-methyltransferases as the search query to discover a novel family of RiPPs, the imiditides. Our genome-mining algorithm reveals a total of 670 imiditide BGCs, distributed across Gram-positive bacterial genomes. In addition, we demonstrate the heterologous production of the founding member of the imiditide family, mNmaAM, encoded in the genome of Nonomuraea maritima. In contrast to other RiPP-associated PIMTs that recognize constrained peptides as substrates, the PIMT homologue in the mNmaAM BGC, NmaM, methylates a specific Asp residue on the linear precursor peptide, NmaA. The methyl ester is then turned into an aspartimide spontaneously. Substrate specificity is achieved by extensive charge-charge interactions between the precursor NmaA and the modifying enzyme NmaM suggested by both experiments and an AlphaFold model prediction. Our study shows that PIMT-mediated aspartimide formation is an emerging backbone modification strategy in the biosynthesis of multiple RiPP families.

Genome Mining and Discovery of Imiditides, a Novel Family of RiPPs with a Class-defining Aspartimide Modification.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a fascinating class of natural products of ribosomal origins. In the past decade, various sophisticated machine learning-based software packages have been established to discover novel RiPPs that do not resemble the known families. Instead, we argue that tailoring enzymes that cluster with various RiPP families can serve as effective bioinformatic seeds for novel RiPP discovery. Leveraging that O -methyltransferases homologous to protein isoaspartyl methyltransferases (PIMTs) are associated with lasso peptide, graspetide, and lanthipeptide biosynthetic gene clusters (BGCs), we utilized the C-terminal motif unique to RiPP-associated O -methyltransferases as the search query to discover a novel family of RiPPs, imiditides. Our genome-mining algorithm reveals a total of 670 imiditide BGCs, widely distributed in Gram-positive bacterial genomes. In addition, we demonstrate the heterologous production of the founding member of the imiditide family, mNmaA M , encoded in the genome of Nonomuraea maritima . In contrast to other RiPP associated PIMTs that recognize constrained peptides as substrates, the PIMT homolog in mNmaA M BGC, NmaM, methylates a specific Asp residue on the linear precursor peptide, NmaA. The methyl ester is then turned into an aspartimide spontaneously. The aspartimide moiety formed is unusually stable, leading to the accumulation of the aspartimidylated product in vivo . The substrate specificity is achieved by extensive charge-charge interactions between the precursor NmaA and the modifying enzyme NmaM suggested by both experimental validations as well as an AlphaFold model prediction. Our study suggests that PIMT-mediated aspartimide formation is an underappreciated backbone modification strategy in RiPP biosynthesis, compared to the well-studied backbone rigidification chemistries, such as thiazol(in)e and oxazol(in)e formations. Additionally, our findings suggest that aspartimide formation in Gram-positive bacterial proteomes are not limited to spontaneous protein aging and degradation.

High-Throughput Screen Reveals the Structure-Activity Relationship of the Antimicrobial Lasso Peptide Ubonodin.

The Burkholderia cepacia complex (Bcc) is a group of bacteria including opportunistic human pathogens. Immunocompromised individuals and cystic fibrosis patients are especially vulnerable to serious infections by these bacteria, motivating the search for compounds with antimicrobial activity against the Bcc. Ubonodin is a lasso peptide with promising activity against Bcc species, working by inhibiting RNA polymerase in susceptible bacteria. We constructed a library of over 90 000 ubonodin variants with 2 amino acid substitutions and used a high-throughput screen and next-generation sequencing to examine the fitness of the entire library, generating the most comprehensive data set on lasso peptide activity so far. This screen revealed information regarding the structure-activity relationship of ubonodin over a large sequence space. Remarkably, the screen identified one variant with not only improved activity compared to wild-type ubonodin but also a submicromolar minimum inhibitory concentration (MIC) against a clinical isolate of the Bcc member Burkholderia cenocepacia. Ubonodin and several of the variants identified in this study had lower MICs against certain Bcc strains than those of many clinically approved antibiotics. Finally, the large library size enabled us to develop DeepLasso, a deep learning model that can predict the RNAP inhibitory activity of an ubonodin variant.

Protein Engineering in Ribosomally Synthesized and Post-translationally Modified Peptides (RiPPs).

Ribosomally synthesized and post-translationally modified peptides (RiPPs) make up a rapidly growing superfamily of natural products. RiPPs exhibit an extraordinary range of structures, but they all begin as gene-encoded precursor peptides that are linear chains of amino acids produced by ribosomes. Given the gene-encoded nature of RiPP precursor peptides, the toolbox of protein engineering can be directly applied to these precursors. This Perspective will discuss examples of site-directed mutagenesis, noncanonical amino acid mutagenesis, and the construction and screening of combinatorial libraries as applied to RiPPs. These studies have led to important insights into the biosynthesis and bioactivity of RiPPs and the reengineering of RiPPs for entirely new functions.

Photocurable acrylate epoxy/ZnO-Ag nanocomposite coating: fabrication, mechanical and antibacterial properties.

In this study, a UV-curable acrylate epoxy nanocomposite coating has been prepared by incorporation of ZnO-Ag hybrid nanoparticles. For this purpose, firstly ZnO-Ag hybrid nanoparticles were fabricated by a seed-mediated growth method. Then, these ZnO-Ag hybrid nanoparticles (2 wt%) were added into the UV-curable acrylate resin matrices. The photocuring process of nanocomposite was evaluated by various factors, such as the conversion of acrylate double bonds, pendulum hardness and gel fraction. Under the 4.8 s UV-exposure time for full crosslinking, the obtained data indicated that incorporation of ZnO-Ag nanohybrids into the coating matrix changed the crosslinking process of coating significantly. A mechanical teat indicated that the presence of nanohybrids in photocurable coating matrix enhanced its abrasion resistance from 98.7 to 131.6 L per mil (33.3%). The antibacterial test against E. coli over 7 h indicated that E. coli bacteria were killed totally by nanocomposite coating, whereas it was 2.6 × 104 CFU mL-1 for the neat coating without nanoparticles.

Phenotype-Guided Comparative Genomics Identifies the Complete Transport Pathway of the Antimicrobial Lasso Peptide Ubonodin in Burkholderia.

New antibiotics are needed as bacterial infections continue to be a leading cause of death, but efforts to develop compounds with promising antibacterial activity are hindered by a poor understanding of─and limited strategies for elucidating─their modes of action. We recently discovered a novel lasso peptide, ubonodin, that is active against opportunistic human lung pathogens from the Burkholderia cepacia complex (Bcc). Ubonodin inhibits RNA polymerase, but only select strains were susceptible, indicating that having a conserved cellular target does not guarantee activity. Given the cytoplasmic target, we hypothesized that cellular uptake of ubonodin determines susceptibility. Although Bcc strains harbor numerous nutrient uptake systems, these organisms lack close homologues of the single known lasso peptide membrane receptor, FhuA. Thus, a straightforward homology-driven approach failed to uncover the identity of the ubonodin transporter(s). Here, we used phenotype-guided comparative genomics to identify genes uniquely associated with ubonodin-susceptible Bcc strains, leading to the identification of PupB as the ubonodin outer membrane (OM) receptor in Burkholderia. The loss of PupB renders B. cepacia resistant to ubonodin, whereas expressing PupB sensitizes a resistant strain. We also examine how a conserved iron-regulated transcriptional pathway controls PupB to further tune ubonodin susceptibility. PupB is only the second lasso peptide OM receptor to be uncovered and the first outside of enterobacteria. Finally, we elucidate the full transport pathway for ubonodin by identifying its inner membrane receptor YddA in Burkholderia. Our work provides a complete picture of the mode of action of ubonodin and establishes a general framework for deciphering the transport pathways of other natural products with cytoplasmic targets.

Metal cofactor stabilization by a partner protein is a widespread strategy employed for amidase activation.

Construction and remodeling of the bacterial peptidoglycan (PG) cell wall must be carefully coordinated with cell growth and division. Central to cell wall construction are hydrolases that cleave bonds in peptidoglycan. These enzymes also represent potential new antibiotic targets. One such hydrolase, the amidase LytH in Staphylococcus aureus, acts to remove stem peptides from PG, controlling where substrates are available for insertion of new PG strands and consequently regulating cell size. When it is absent, cells grow excessively large and have division defects. For activity, LytH requires a protein partner, ActH, that consists of an intracellular domain, a large rhomboid protease domain, and three extracellular tetratricopeptide repeats (TPRs). Here, we demonstrate that the amidase-activating function of ActH is entirely contained in its extracellular TPRs. We show that ActH binding stabilizes metals in the LytH active site and that LytH metal binding in turn is needed for stable complexation with ActH. We further present a structure of a complex of the extracellular domains of LytH and ActH. Our findings suggest that metal cofactor stabilization is a general strategy used by amidase activators and that ActH houses multiple functions within a single protein.

Mechanisms of action of ribosomally synthesized and posttranslationally modified peptides (RiPPs).

Natural products remain a critical source of medicines and drug leads. One of the most rapidly growing superclasses of natural products is RiPPs: ribosomally synthesized and posttranslationally modified peptides. RiPPs have rich and diverse bioactivities. This review highlights examples of the molecular mechanisms of action that underly those bioactivities. Particular emphasis is placed on RiPP/target interactions for which there is structural information. This detailed mechanism of action work is critical toward the development of RiPPs as therapeutics and can also be used to prioritize hits in RiPP genome mining studies.

The length of lipoteichoic acid polymers controls Staphylococcus aureus cell size and envelope integrity.

The opportunistic pathogen Staphylococcus aureus is protected by a cell envelope that is crucial for viability. In addition to peptidoglycan, lipoteichoic acid (LTA) is an especially important component of the S. aureus cell envelope. LTA is an anionic polymer anchored to a glycolipid in the outer leaflet of the cell membrane. It was known that deleting the gene for UgtP, the enzyme that makes this glycolipid anchor, causes cell growth and division defects. In Bacillus subtilis, growth abnormalities from the loss of ugtP have been attributed to both the absence of the encoded protein and to the loss of its products. Here, we show that growth defects in S. aureus ugtP deletion mutants are due to the long, abnormal LTA polymer that is produced when the glycolipid anchor is missing from the outer leaflet of the membrane. Dysregulated cell growth leads to defective cell division, and these phenotypes are corrected by mutations in the LTA polymerase, ltaS, that reduce polymer length. We also show that S. aureus mutants with long LTA are sensitized to cell wall hydrolases, beta-lactam antibiotics, and compounds that target other cell envelope pathways. We conclude that control of LTA polymer length is important for S. aureus physiology and promotes survival under stressful conditions, including antibiotic stress.IMPORTANCE Methicillin-resistant Staphylococcus aureus (MRSA) is a common cause of community- and hospital-acquired infections and is responsible for a large fraction of deaths caused by antibiotic-resistant bacteria. S. aureus is surrounded by a complex cell envelope that protects it from antimicrobial compounds and other stresses. Here we show that controlling the length of an essential cell envelope polymer, lipoteichoic acid, is critical for controlling S. aureus cell size and cell envelope integrity. We also show that genes involved in LTA length regulation are required for resistance to beta-lactam antibiotics in MRSA. The proteins encoded by these genes may be targets for combination therapy with an appropriate beta-lactam.

Uncovering the activities, biological roles, and regulation of bacterial cell wall hydrolases and tailoring enzymes.

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.

Staphylococcus aureus cell growth and division are regulated by an amidase that trims peptides from uncrosslinked peptidoglycan.

Bacteria are protected by a polymer of peptidoglycan that serves as an exoskeleton1. In Staphylococcus aureus, the peptidoglycan assembly enzymes relocate during the cell cycle from the periphery, where they are active during growth, to the division site where they build the partition between daughter cells2-4. But how peptidoglycan synthesis is regulated throughout the cell cycle is poorly understood5,6. Here, we used a transposon screen to identify a membrane protein complex that spatially regulates S. aureus peptidoglycan synthesis. This complex consists of an amidase that removes stem peptides from uncrosslinked peptidoglycan and a partner protein that controls its activity. Amidases typically hydrolyse crosslinked peptidoglycan between daughter cells so that they can separate7. However, this amidase controls cell growth. In its absence, peptidoglycan synthesis becomes spatially dysregulated, which causes cells to grow so large that cell division is defective. We show that the cell growth and division defects due to loss of this amidase can be mitigated by attenuating the polymerase activity of the major S. aureus peptidoglycan synthase. Our findings lead to a model wherein the amidase complex regulates the density of peptidoglycan assembly sites to control peptidoglycan synthase activity at a given subcellular location. Removal of stem peptides from peptidoglycan at the cell periphery promotes peptidoglycan synthase relocation to midcell during cell division. This mechanism ensures that cell expansion is properly coordinated with cell division.

Genome-wide mutant profiling predicts the mechanism of a Lipid II binding antibiotic.

Identifying targets of antibacterial compounds remains a challenging step in the development of antibiotics. We have developed a two-pronged functional genomics approach to predict mechanism of action that uses mutant fitness data from antibiotic-treated transposon libraries containing both upregulation and inactivation mutants. We treated a Staphylococcus aureus transposon library containing 690,000 unique insertions with 32 antibiotics. Upregulation signatures identified from directional biases in insertions revealed known molecular targets and resistance mechanisms for the majority of these. Because single-gene upregulation does not always confer resistance, we used a complementary machine-learning approach to predict the mechanism from inactivation mutant fitness profiles. This approach suggested the cell wall precursor Lipid II as the molecular target of the lysocins, a mechanism we have confirmed. We conclude that docking to membrane-anchored Lipid II precedes the selective bacteriolysis that distinguishes these lytic natural products, showing the utility of our approach for nominating the antibiotic mechanism of action.

Antibiotic Combinations That Enable One-Step, Targeted Mutagenesis of Chromosomal Genes.

Targeted modification of bacterial chromosomes is necessary to understand new drug targets, investigate virulence factors, elucidate cell physiology, and validate results of -omics-based approaches. For some bacteria, reverse genetics remains a major bottleneck to progress in research. Here, we describe a compound-centric strategy that combines new negative selection markers with known positive selection markers to achieve simple, efficient one-step genome engineering of bacterial chromosomes. The method was inspired by the observation that certain nonessential metabolic pathways contain essential late steps, suggesting that antibiotics targeting a late step can be used to select for the absence of genes that control flux into the pathway. Guided by this hypothesis, we have identified antibiotic/counterselectable markers to accelerate reverse engineering of two increasingly antibiotic-resistant pathogens, Staphylococcus aureus and Acinetobacter baumannii. For S. aureus, we used wall teichoic acid biosynthesis inhibitors to select for the absence of tarO and for A. baumannii, we used colistin to select for the absence of lpxC. We have obtained desired gene deletions, gene fusions, and promoter swaps in a single plating step with perfect efficiency. Our method can also be adapted to generate markerless deletions of genes using FLP recombinase. The tools described here will accelerate research on two important pathogens, and the concept we outline can be readily adapted to any organism for which a suitable target pathway can be identified.

Crystal structure of 4-amino-3-(thio-phen-3-ylmeth-yl)-1H-1,2,4-triazole-5(4H)-thione.

In the title compound, C7H8N4S2, the thio-phene ring shows rotational disorder over two orientations in a 0.6957 (15):0.3043 (15) ratio. The plane of the 1,2,4-triazole ring makes a dihedral angle of 75.02 (17)° with the major-disorder component of the thiophene ring. In the crystal, two types of inversion dimers, described by the graph-set motifs R22(8) and R22(10), are formed by N-H⋯S inter-actions. Chains of mol-ecules running in the [101] direction are linked by weaker N-H⋯N inter-actions. The thio-phene ring is involved in π-π and C-H⋯π inter-actions.