Noel

Methods Mol Biol. 2021;2365:265-282. doi: 10.1007/978-1-0716-1665-9_14.

ABSTRACT

Target engagement and cell permeation are important parameters that may limit the efficacy of proteolysis-targeting chimeras (PROTACs). Here, we present an approach that facilitates both the quantitation of PROTAC binding affinity for an E3 ligase of interest, as well as the assessment of relative intracellular availability. We present a panel of E3 ligase target engagement assays based upon the NanoBRET Target Engagement platform. Querying E3 ligase engagement under live-cell and permeabilized-cell conditions allow calculation of an availability index that can be used to rank order the intracellular availability of PROTACs. Here we present examples where the cellular availability of PROTACs and their monovalent precursors are prioritized using NanoBRET assays for CRBN or VHL E3 ligases.

PMID:34432249 | DOI:10.1007/978-1-0716-1665-9_14

Methods Enzymol. 2021;656:495-519. doi: 10.1016/bs.mie.2021.05.002. Epub 2021 Jun 2.

ABSTRACT

With few exceptions, ribosomal protein synthesis begins with methionine (or its derivative N-formyl-methionine) across all domains of life. The role of methionine as the initiating amino acid is dictated by the unique structure of its cognate tRNA known as tRNA^fMet. By mis-acylating tRNA^fMet, we and others have shown that protein synthesis can be initiated with a variety of canonical and noncanonical amino acids both in vitro and in vivo. Furthermore, because the α-amine of the initiating amino acid is not required for peptide bond formation, translation can be initiated with a variety of structurally disparate carboxylic acids that bear little resemblance to traditional α-amino acids. Herein, we provide a detailed protocol to initiate in vitro protein synthesis with substituted benzoic acid and 1,3-dicarbonyl compounds. These moieties are introduced at the N-terminus of peptides by mis-acylated tRNA^fMet, prepared by flexizyme-catalyzed tRNA acylation. In addition, we describe a protocol to initiate in vivo protein synthesis with aromatic noncanonical amino acids (ncAAs). This method relies on an engineered chimeric initiator tRNA that is acylated with ncAAs by an orthogonal aminoacyl-tRNA synthetase. Together, these systems are useful platforms for producing N-terminally modified proteins and for engineering the protein synthesis machinery of Escherichia coli to accept additional nonproteinogenic carboxylic acid monomers.

PMID:34325796 | PMC:PMC8428779 | DOI:10.1016/bs.mie.2021.05.002

mBio. 2021 Jul 27:e0134221. doi: 10.1128/mBio.01342-21. Online ahead of print.

ABSTRACT

Peptidoglycan (PG) is a highly cross-linked peptide-glycan mesh that confers structural rigidity and shape to most bacterial cells. Polymerization of new PG is usually achieved by the concerted activity of two membrane-bound machineries, class-A penicillin binding proteins (aPBPs) and class-B penicillin binding proteins (bPBPs) in complex with shape, elongation, division, and sporulation (SEDS) proteins. Here, we have identified four phylogenetically distinct groups of bacteria that lack any identifiable aPBPs. We performed experiments on a panel of species within one of these groups, the Rickettsiales, and found that bacteria lacking aPBPs build a PG-like cell wall with minimal abundance and rigidity relative to cell walls of aPBP-containing bacteria. This reduced cell wall may have evolved to minimize the activation of host responses to pathogens and endosymbionts while retaining the minimal PG-biosynthesis machinery required for cell elongation and division. We term these "peptidoglycan-intermediate" bacteria, a cohort of host-associated species that includes some human pathogens. IMPORTANCE Peptidoglycan (PG) is a large, cross-linked polymer that forms the cell wall of most bacterial species and confers shape, rigidity, and protection from osmotic shock. It is also a potent stimulator of the immune response in animals. PG is normally polymerized by two groups of enzymes, aPBPs and bPBPs working together with shape, elongation, division, and sporulation (SEDS) proteins. We have identified a diverse set of host-associated bacteria that have selectively lost aPBP genes while retaining bPBP/SEDS and show that some of these build a minimal PG-like structure. It is expected that these minimal cell walls built in the absence of aPBPs improve the evolutionary fitness of host-associated bacteria, potentially through evasion of PG-recognition by the host immune system.

PMID:34311584 | DOI:10.1128/mBio.01342-21

ACS Chem Biol. 2021 Aug 20;16(8):1344-1349. doi: 10.1021/acschembio.1c00422. Epub 2021 Jul 13.

ABSTRACT

Glycerol phosphate (GroP)-based teichoic acids (TAs) are antigenic cell-wall components found in both enterococcus and staphylococcus species. Their immunogenicity has been explored using both native and synthetic structures, but no details have yet been reported on the structural basis of their interaction with antibodies. This work represents the first case study in which a monoclonal antibody, generated against a synthetic TA, was developed and employed for molecular-level binding analysis using TA microarrays, ELISA, SPR-analyses, and STD-NMR spectroscopy. Our findings show that the number and the chirality of the GroP residues are crucial for interaction and that the sugar appendage contributes to the presentation of the backbone to the binding site of the antibody.

PMID:34255482 | PMC:PMC8389533 | DOI:10.1021/acschembio.1c00422

Microb Pathog. 2021 Jul 8;158:105078. doi: 10.1016/j.micpath.2021.105078. Online ahead of print.

ABSTRACT

Enterococcus faecalis (E. faecalis) is associated with persistent root canal infection because of its biofilm and various virulence factors. However, E. faecalis exhibits extensive drug resistance. d-Alanine (D-Ala) metabolism is essential for bacterial peptidoglycan biosynthesis. d-cycloserine (DCS), a second line drug used in the treatment of Mycobacterium tuberculosis infection, can inhibit two key enzymes in D-Ala metabolism: alanine racemase and d-alanine-d-alanine ligase. The aim of this study was to evaluate the effect of D-Ala metabolism on E. faecalis growth, cell wall integrity, biofilm formation and virulence gene expression by additional DCS with or without D-Ala. The results showed that DCS inhibited the planktonic growth and biofilm formation of E. faecalis in a dose-dependent manner. Both the minimum inhibitory concentration (MIC) and minimum biofilm inhibition concentration (MBIC) of DCS against E. faecalis were 200 μg/ml, whereas 50 μg/ml of DCS could inhibit planktonic growth and biofilm formation effectively. The addition of DCS also resulted in bacterial cell wall damage, biofilm surface roughness increase and biofilm adhesion force reduction. Moreover, the treatment of DCS downregulated the expression of asa1, esp, efaA, gelE, sprE, fsrB and ace genes. However, all of these inhibitory effects of DCS could be rescued by the addition of exogenous D-Ala. Meanwhile, DCS exhibited no toxicity to HGEs and HOKs. Therefore, D-Ala metabolic pathway in E. faecalis is a potential target for drug designing.

PMID:34245823 | DOI:10.1016/j.micpath.2021.105078

ChemMedChem. 2021 Jul 8. doi: 10.1002/cmdc.202100315. Online ahead of print.

ABSTRACT

The major obstacle in applying peptides to intracellular targets is their low inherent cell permeability. Standard approaches to attach a fluorophore (e.g. FITC, TAMRA) can change the physicochemical properties of the parent peptide and influence their ability to penetrate and localize in cells. We report a label-free strategy for evaluating the cell permeability of cyclic peptide leads. Fluorescent tryptophan analogues 4-cyanotryptophan (4CNW) and beta-(1-azulenyl)-L-alanine (AzAla) were incorporated into in vitro translated macrocyclic peptides by initiator reprogramming. We then demonstrate these efficient blue fluorescent emitters are good tools for monitoring peptide penetration into cells.

PMID:34236771 | DOI:10.1002/cmdc.202100315

ACS Chem Biol. 2021 Jul 16;16(7):1164-1171. doi: 10.1021/acschembio.1c00346. Epub 2021 Jun 29.

ABSTRACT

By catalyzing a 3-3 cross-link in peptidoglycan, l,d-transpeptidases (Ldts) can cause resistance to β-lactams in some pathogens in vitro. However, the prevalence of Ldt and Ldt-mediated responses to different β-lactams in vivo have never been explored. Here, we apply an in vivo metabolic labeling strategy to study their biodistributions and Ldt-induced bacterial responses to β-lactams in the mouse gut microbiota. A tetrapeptide-based fluorescent probe that functions as a substrate for Ldts in Gram-positive bacteria efficiently labels ∼18% of total gut bacteria after gavage, suggesting Ldts' high prevalence in gut microbiota. The cellular distributions of 3-3 cross-links on three gut bacterial species were then identified with the aid of fluorescence in situ hybridization to identify the bacterial taxa. After oral administration of two β-lactams, ampicillin and meropenem, only the latter efficiently inhibits the tetrapeptide labeling, suggesting that Ldts may be able to cause resistance to some β-lactams in the mammalian gut.

PMID:34185512 | DOI:10.1021/acschembio.1c00346

Front Mol Biosci. 2021 Jun 4;8:691569. doi: 10.3389/fmolb.2021.691569. eCollection 2021.

ABSTRACT

Staphylococcus aureus is a leading cause of bacterial infections world-wide. Staphylococcal infections are preferentially treated with β-lactam antibiotics, however, methicillin-resistant S. aureus (MRSA) strains have acquired resistance to this superior class of antibiotics. We have developed a growth-based, high-throughput screening approach that directly identifies cell wall synthesis inhibitors capable of reversing β-lactam resistance in MRSA. The screen is based on the finding that S. aureus mutants lacking the ClpX chaperone grow very poorly at 30°C unless specific steps in teichoic acid synthesis or penicillin binding protein (PBP) activity are inhibited. This property allowed us to exploit the S. aureus clpX mutant as a unique screening tool to rapidly identify biologically active compounds that target cell wall synthesis. We tested a library of ∼50,000 small chemical compounds and searched for compounds that inhibited growth of the wild type while stimulating growth of the clpX mutant. Fifty-eight compounds met these screening criteria, and preliminary tests of 10 compounds identified seven compounds that reverse β-lactam resistance of MRSA as expected for inhibitors of teichoic acid synthesis. The hit compounds are therefore promising candidates for further development as novel combination agents to restore β-lactam efficacy against MRSA.

PMID:34150853 | PMC:PMC8212132 | DOI:10.3389/fmolb.2021.691569

Org Biomol Chem. 2021 Jun 7. doi: 10.1039/d1ob00679g. Online ahead of print.

ABSTRACT

Nucleotide-binding oligomerization domain 2 (NOD2) is an intracellular receptor that recognizes the bacterial peptidoglycan fragment muramyl dipeptide (MDP). Our group has synthesized and biologically evaluated desmuramyl peptides containing adamantane and its mannose derivatives. The most active mannosylated derivative, ManAdDMP (Man-OCH2-d-(1-Ad)Gly-l-Ala-d-isoGln), is further characterized in silico in this study. We built intact model structures of the rabbit NOD2 protein, whose crystal structure lacks seven loops, and explored the binding of ManAdDMP. Two main binding sites for ManAdDMP are located within the nucleotide-binding oligomerization domain (NOD) and C-terminal leucine-rich repeat (LRR) domains. Our analysis shows that the dipeptide isoGln moiety of ManAdDMP significantly contributes to the binding, whereas the mannose moiety interacts with modelled loop 7, which is a part of the NOD helical domain 2. The presented results point to the importance of loops 2 and 7 in ligand recognition that could be useful for further investigation of NOD2 activation/inhibition.

PMID:34095941 | DOI:10.1039/d1ob00679g

Nat Commun. 2021 May 13;12(1):2775. doi: 10.1038/s41467-021-23063-6.

ABSTRACT

The pathway for the biosynthesis of the bacterial cell wall is one of the most prolific antibiotic targets, exemplified by the widespread use of β-lactam antibiotics. Despite this, our structural understanding of class A penicillin binding proteins, which perform the last two steps in this pathway, is incomplete due to the inherent difficulty in their crystallization and the complexity of their substrates. Here, we determine the near atomic resolution structure of the 83 kDa class A PBP from Escherichia coli, PBP1b, using cryogenic electron microscopy and a styrene maleic acid anhydride membrane mimetic. PBP1b, in its apo form, is seen to exhibit a distinct conformation in comparison to Moenomycin-bound crystal structures. The work herein paves the way for the use of cryoEM in structure-guided antibiotic development for this notoriously difficult to crystalize class of proteins and their complex substrates.

PMID:33986273 | PMC:PMC8119973 | DOI:10.1038/s41467-021-23063-6

Cell Surf. 2021 May 1;7:100053. doi: 10.1016/j.tcsw.2021.100053. eCollection 2021 Dec.

ABSTRACT

Bacteria encase their cytoplasmic membrane with peptidoglycan (PG) to maintain the shape of the cell and protect it from bursting. The enlargement of the PG layer is facilitated by the coordinated activities of PG synthesising and -cleaving enzymes. In Escherichia coli, the cytoplasmic membrane-bound lytic transglycosylase MltG associates with PG synthases and was suggested to terminate the polymerisation of PG glycan strands. Using pull-down and surface plasmon resonance, we detected interactions between MltG from Bacillus subtilis and two PG synthases; the class A PBP1 and the class B PBP2B. Using in vitro PG synthesis assays with radio-labelled or fluorophore-labelled B. subtilis-type and/or E. coli-type lipid II, we showed that both, BsMltG and EcMltG, are lytic tranglycosylases and that their activity is higher during ongoing glycan strand polymerisation. MltG competed with the transpeptidase activity of class A PBPs, but had no effect on their glycosyltransferase activity, and produced glycan strands with a length of 7 disaccharide units from cleavage in the nascent strands. We hypothesize that MltG cleaves the nascent strands to produce short glycan strands that are used in the cell for a yet unknown process.

PMID:34036206 | PMC:PMC8135044 | DOI:10.1016/j.tcsw.2021.100053

J Lipid Res. 2021 May 18:100086. doi: 10.1016/j.jlr.2021.100086. Online ahead of print.

ABSTRACT

Apolipoprotein E (apoE) is a well-known lipid-binding protein that plays a main role in the metabolism and transport of lipids. More recently, apoE-derived peptides have been shown to exert antimicrobial effects. Here, we investigated the anti-bacterial activity of apoE using in vitro assays, advanced imaging techniques, and in vivo mouse models . The formation of macromolecular complexes of apoE and endotoxins from Gram-negative bacteria was explored using gel shift assays, transmission electron microscopy , and circular dichroism spectroscopy followed by calculation of the α-helical content. The binding affinity of apoE to endotoxins was also confirmed by fluorescent spectroscopy detecting the quenching and shifting of tryptophan intrinsic fluorescence. We showed that apoE exhibits antibacterial activity particularly against Gram-negative bacteria such as Pseudomonas aeruginosa and Escherichia coli. ApoE protein folding was affected by binding of bacterial endotoxins components such as lipopolysaccharide (LPS) and Lipid A , yielding similar increases in apoE αhelical content. Moreover, high molecular-weight complexes of apoE were formed in the presence of LPS, but not to the same extent as with Lipid A. Together, our results demonstrate that interaction of apoE with Gram-negative bacteria and their endotoxins leads to the structural changes in apoE and the formation of aggregate-like complexes. The protein interaction provides a molecular explanation for the observed antimicrobial effects of apoE, which binds to LPS on the bacterial surface and leads to cell membrane damage and bacterial death.

PMID:34019903 | DOI:10.1016/j.jlr.2021.100086

ACS Chem Biol. 2021 May 11. doi: 10.1021/acschembio.1c00179. Online ahead of print.

ABSTRACT

The outer membrane of Gram-negative bacteria is a formidable permeability barrier which allows only a small subset of chemical matter to penetrate. This outer membrane barrier can hinder the study of cellular processes and compound mechanism of action, as many compounds including antibiotics are precluded from entry despite having intracellular targets. Consequently, outer membrane permeabilizing compounds are invaluable tools in such studies. Many existing compounds known to perturb the outer membrane also impact inner membrane integrity, such as polymyxins and their derivatives, making these probes nonspecific. We performed a screen of ∼140 000 diverse synthetic compounds, for those that antagonized the growth inhibitory activity of vancomycin at 15 °C in Escherichia coli, to enrich for chemicals capable of perturbing the outer membrane. This led to the discovery that liproxstatin-1, an inhibitor of ferroptosis in human cells, and MAC-0568743, a novel cationic amphiphile, could potentiate the activity of large-scaffold antibiotics with low permeation into Gram-negative bacteria at 37 °C. Liproxstatin-1 and MAC-0568743 were found to physically disrupt the integrity of the outer membrane through interactions with lipopolysaccharide in the outer leaflet of the outer membrane. We showed that these compounds selectively disrupt the outer membrane while minimally impacting inner membrane integrity, particularly at the concentrations needed to potentiate Gram-positive-targeting antibiotics. Further exploration of these molecules and their structural analogues is a promising avenue for the development of outer membrane specific probes.

PMID:33974796 | DOI:10.1021/acschembio.1c00179

Nat Commun. 2021 May 7;12(1):2560. doi: 10.1038/s41467-021-22845-2.

ABSTRACT

The commensal fungus Candida albicans often causes life-threatening infections in patients who are immunocompromised with high mortality. A prominent but poorly understood risk factor for the C. albicans commensal‒pathogen transition is the use of broad-spectrum antibiotics. Here, we report that β-lactam antibiotics cause bacteria to release significant quantities of peptidoglycan fragments that potently induce the invasive hyphal growth of C. albicans. We identify several active peptidoglycan subunits, including tracheal cytotoxin, a molecule produced by many Gram-negative bacteria, and fragments purified from the cell wall of Gram-positive Staphylococcus aureus. Feeding mice with β-lactam antibiotics causes a peptidoglycan storm that transforms the gut from a niche usually restraining C. albicans in the commensal state to promoting invasive growth, leading to systemic dissemination. Our findings reveal a mechanism underlying a significant risk factor for C. albicans infection, which could inform clinicians regarding future antibiotic selection to minimize this deadly disease incidence.

PMID:33963193 | PMC:PMC8105390 | DOI:10.1038/s41467-021-22845-2

Mol Microbiol. 2021 May 4. doi: 10.1111/mmi.14734. Online ahead of print.

ABSTRACT

Surface proteins of Staphylococcus aureus play vital roles in bacterial physiology and pathogenesis. Recent work suggests that surface proteins are spatially regulated by a YSIRK/GXXS signal peptide that promotes cross-wall targeting at the mid-cell, though the mechanisms remain unclear. We previously showed that protein A (SpA), a YSIRK/GXXS protein and key staphylococcal virulence factor, mis-localizes in a ltaS mutant deficient in lipoteichoic acid (LTA) production. Here, we identified that SpA contains another cross-wall targeting signal, the LysM domain, which, in addition to the YSIRK/GXXS signal peptide, significantly enhances SpA cross-wall targeting. We show that LTA synthesis, but not LtaS, is required for SpA septal anchoring and cross-wall deposition. Interestingly, LTA is predominantly found at the peripheral cell membrane and is diminished at the septum of dividing staphylococcal cells, suggesting a restriction mechanism for SpA septal localization. Finally, we show that D-alanylation of LTA abolishes SpA cross-wall deposition by disrupting SpA distribution in the peptidoglycan layer without altering SpA septal anchoring. Our study reveals that multiple factors contribute to the spatial regulation and cross-wall targeting of SpA via different mechanisms, which coordinately ensures efficient incorporation of surface proteins into the growing peptidoglycan during the cell cycle.

PMID:33949015 | DOI:10.1111/mmi.14734

Appl Microbiol Biotechnol. 2021 Apr 16. doi: 10.1007/s00253-021-11272-4. Online ahead of print.

ABSTRACT

Bacterial cell has always been an attractive target for anti-infective drug discovery. MurA (UDP-N-acetylglucosamine enolpyruvyl transferase) enzyme of Escherichia coli (E.coli) is crucial for peptidoglycan biosynthetic pathway, as it is involved in the early stages of bacterial cell wall biosynthesis. In the present study we aim to identify novel chemical structures targeting the MurA enzyme. For screening purpose, we used in silico approach (pharmacophore based strategy) for 52,026 library compounds (Chembridge, Chemdiv and in house synthetics) which resulted in identification of 50 compounds. These compounds were screened in vitro against MurA enzyme and release of inorganic phosphate (Pi) was estimated. Two compounds (IN00152 and IN00156) were found to inhibit MurA enzyme > 70% in primary screening and IC₅₀ of 14.03 to 32.30 μM respectively. These two hits were further evaluated for their mode of inhibition studies and whole-cell activity where we observed 2-4 folds increase in activity in presence of Permeabilizer EDTA (Ethylenediaminetetraacetic acid). Combination studies were also performed with known antibiotics in presence of EDTA. Hits are reported for the first time against this target and our report also support the use of OM permeabilizer in combination with antibacterial compounds to address the permeability and efficacy issue. These lead hits can be further optimized for drug discovery. KEY POINTS: • Emerging Gram negative resistant strains is a matter of concern. • Need for new screening strategies to cope with drying up antibiotics pipeline. • Outer membrane permeabilizers could be useful to improve potency of molecules to reach its target.

PMID:33860835 | DOI:10.1007/s00253-021-11272-4

J Extracell Vesicles. 2021 Apr;10(6):e12080. doi: 10.1002/jev2.12080. Epub 2021 Apr 1.

ABSTRACT

Gram-positive bacteria ubiquitously produce membrane vesicles (MVs), and although they contribute to biological functions, our knowledge regarding their composition and immunogenicity remains limited. Here we examine the morphology, contents and immunostimulatory functions of MVs produced by three Staphylococcus aureus strains; a methicillin resistant clinical isolate, a methicillin sensitive clinical isolate and a laboratory-adapted strain. We observed differences in the number and morphology of MVs produced by each strain and showed that they contain microbe-associated molecular patterns (MAMPs) including protein, nucleic acids and peptidoglycan. Analysis of MV-derived RNA indicated the presence of small RNA (sRNA). Furthermore, we detected variability in the amount and composition of protein, nucleic acid and peptidoglycan cargo carried by MVs from each S. aureus strain. S. aureus MVs activated Toll-like receptor (TLR) 2, 7, 8, 9 and nucleotide-binding oligomerization domain containing protein 2 (NOD2) signalling and promoted cytokine and chemokine release by epithelial cells, thus identifying that MV-associated MAMPs including DNA, RNA and peptidoglycan are detected by pattern recognition receptors (PRRs). Moreover, S. aureus MVs induced the formation of and colocalized with autophagosomes in epithelial cells, while inhibition of lysosomal acidification using bafilomycin A1 resulted in accumulation of autophagosomal puncta that colocalized with MVs, revealing the ability of the host to degrade MVs via autophagy. This study reveals the ability of DNA, RNA and peptidoglycan associated with MVs to activate PRRs in host epithelial cells, and their intracellular degradation via autophagy. These findings advance our understanding of the immunostimulatory roles of Gram-positive bacterial MVs in mediating pathogenesis, and their intracellular fate within the host.

PMID:33815695 | PMC:PMC8015888 | DOI:10.1002/jev2.12080