sep413

New insight on how antibiotics affect the gut microbiome—the community of microbes that live inside us—has been published in the journal eLife.

Related Articles

Design and synthesis of pyrophosphate-targeting vancomycin derivatives for combating vancomycin-resistant Enterococci.

ChemMedChem. 2018 Jun 19;:

Authors: Guan D, Chen F, Faridoon F, Liu J, Li J, Lan L, Huang W

Abstract
Vancomycin, as the last resort for intractable Gram-positive bacterial infections, is losing the efficacy with the emergence of vancomycin-resistant bacteria especially vancomycin-resistant Enterococci (VRE). To combat this threat, we rationally designed and synthesized 39 novel vancomycin derivatives, via respective or combined modifications with metal-chelating, lipophilic, and galactose-attached strategies, for extensive SAR analysis. In a proposed mechanism, the conjugation of dipicolylamine (DPA) on 7th amino acid resorcinol position or C-terminus endued the vancomycin backbone with the binding activity to pyrophosphate moiety in lipid II while keeping the intrinsic binding affinity to the dipeptide terminus of the bacterial cell wall peptidoglycan precursor. The in vitro antibacterial activities were evaluated and the optimal compounds indicated 16-1024 fold higher activity against VRE compared with vancomycin. It was also found compound 11b showed the synergistic effect combining two peripheral modification and mechanism especially towards VRE.

PMID: 29920964 [PubMed - as supplied by publisher]

Publication date: September 2018
Source:Biochimie, Volume 152
Author(s): Ismahene Dahmane, Caroline Montagner, André Matagne, Shrinivas Dumbre, Piet Herdewijn, Mohammed Terrak
Peptidoglycan glycosyltransferases (GTase) of family 51 are essential enzymes for the synthesis of the glycan chains of the bacterial cell wall. They are considered potential antibacterial target, but discovery of inhibitors was hampered so far by the lack of efficient and affordable screening assay. Here we used Staphylococcus aureus MtgA to introduce a single tryptophan reporter residue in selected positions flanking the substrates binding cavity of the protein. We selected a mutant (Y181W) that shows strong fluorescence quenching in the presence of moenomycin A and two lipid II analogs inhibitors. The assay provides a simple method to study GTase-ligand interactions and can be used as primary high throughput screening of GTase inhibitors without the need for lipid II substrate or reporter ligands.

Graphical abstract

Interactions of commensal and pathogenic microorganisms with the intestinal mucosal barrier

Interactions of commensal and pathogenic microorganisms with the intestinal mucosal barrier, Published online: 14 June 2018; doi:10.1038/s41579-018-0036-x

The first line of host defence against both encroaching commensal bacteria and invading enteric pathogens is the intestinal mucosal barrier, which is composed of epithelial cells and a host-secreted mucous layer. In this Review, Martens and colleagues discuss the complex interactions of commensal and pathogenic microorganisms with the intestinal mucosal barrier.

Related Articles

IL-1R and MyD88 Contribute to the Absence of a Bacterial Microbiome on the Healthy Murine Cornea.

Front Microbiol. 2018;9:1117

Authors: Wan SJ, Sullivan AB, Shieh P, Metruccio MME, Evans DJ, Bertozzi CR, Fleiszig SMJ

Abstract
Microbial communities are important for the health of mucosal tissues. Traditional culture and gene sequencing have demonstrated bacterial populations on the conjunctiva. However, it remains unclear if the cornea, a transparent tissue critical for vision, also hosts a microbiome. Corneas of wild-type, IL-1R (-/-) and MyD88 (-/-) C57BL/6 mice were imaged after labeling with alkyne-functionalized D-alanine (alkDala), a probe that only incorporates into the peptidoglycan of metabolically active bacteria. Fluorescence in situ hybridization (FISH) was also used to detect viable bacteria. AlkDala labeling was rarely observed on healthy corneas. In contrast, adjacent conjunctivae harbored filamentous alkDala-positive forms, that also labeled with DMN-Tre, a Corynebacterineae-specific probe. FISH confirmed the absence of viable bacteria on healthy corneas, which also cleared deliberately inoculated bacteria within 24 h. Differing from wild-type, both IL-1R (-/-) and MyD88 (-/-) corneas harbored numerous alkDala-labeled bacteria, a result abrogated by topical antibiotics. IL-1R (-/-) corneas were impermeable to fluorescein suggesting that bacterial colonization did not reflect decreased epithelial integrity. Thus, in contrast to the conjunctiva and other mucosal surfaces, healthy murine corneas host very few viable bacteria, and this constitutive state requires the IL-1R and MyD88. While this study cannot exclude the presence of fungi, viruses, or non-viable or dormant bacteria, the data suggest that healthy murine corneas do not host a resident viable bacterial community, or microbiome, the absence of which could have important implications for understanding the homeostasis of this tissue.

PMID: 29896179 [PubMed]

Related Articles

Structure and mutagenic analysis of the lipid II flippase MurJ from Escherichia coli.

Proc Natl Acad Sci U S A. 2018 Jun 11;:

Authors: Zheng S, Sham LT, Rubino FA, Brock KP, Robins WP, Mekalanos JJ, Marks DS, Bernhardt TG, Kruse AC

Abstract
The peptidoglycan cell wall provides an essential protective barrier in almost all bacteria, defining cellular morphology and conferring resistance to osmotic stress and other environmental hazards. The precursor to peptidoglycan, lipid II, is assembled on the inner leaflet of the plasma membrane. However, peptidoglycan polymerization occurs on the outer face of the plasma membrane, and lipid II must be flipped across the membrane by the MurJ protein before its use in peptidoglycan synthesis. Due to its central role in cell wall assembly, MurJ is of fundamental importance in microbial cell biology and is a prime target for novel antibiotic development. However, relatively little is known regarding the mechanisms of MurJ function, and structural data for MurJ are available only from the extremophile Thermosipho africanus Here, we report the crystal structure of substrate-free MurJ from the gram-negative model organism Escherichia coli, revealing an inward-open conformation. Taking advantage of the genetic tractability of E. coli, we performed high-throughput mutagenesis and next-generation sequencing to assess mutational tolerance at every amino acid in the protein, providing a detailed functional and structural map for the enzyme and identifying sites for inhibitor development. Lastly, through the use of sequence coevolution analysis, we identify functionally important interactions in the outward-open state of the protein, supporting a rocker-switch model for lipid II transport.

PMID: 29891673 [PubMed - as supplied by publisher]

Staphylococcus aureus modulation of innate immune responses through Toll-like (TLRs), (NOD)-like (NLRs) and C-type lectin (CLRs) receptors.

FEMS Microbiol Rev. 2018 Jun 08;:

Authors: Askarian F, Wagner T, Johannessen M, Nizet V

Abstract
Early recognition of pathogens by the innate immune system is crucial for bacterial clearance. Many pattern recognition receptors (PRRs) such as Toll-like (TLRs) and (NOD)-like (NLRs) receptors have been implicated in initial sensing of bacterial components. The intracellular signaling cascades triggered by these receptors result in transcriptional upregulation of inflammatory pathways. Although this step is crucial for bacterial elimination, it is also associated with the potential for substantial immunopathology, which underscores the need for tight control of inflammatory responses. The leading human bacterial pathogen Staphylococcus aureus expresses over 100 virulence factors that exert numerous effects upon host cells. In this manner, the pathogen seeks to avoid host recognition or perturb PRR-induced innate immune responses to allow optimal survival in the host. These immune system interactions may result in enhanced bacterial proliferation but also provoke systemic cytokine responses associated with sepsis. This review summarizes recent findings on the various mechanisms applied by S. aureus to modulate or interfere with inflammatory responses through PRRs. Detailed understanding of these complex interactions can provide new insights toward future immune-stimulatory therapeutics against infection or immunomodulatory therapeutics to suppress or correct dysregulated inflammation.

PMID: 29893825 [PubMed - as supplied by publisher]

Chemistry – A European Journal, Volume 24, Issue 32, June 7, 2018.

Overcoming resistance: A new fluorogenic sensor for the detection of metallo‐β‐lactamase activities has been disclosed. With carbapenem as an enzymatic recognition moiety, this reagent shows excellent specificity to metallo‐β‐lactamase over other serine‐β‐lactamases, including serine carbapenemases, and allows the rapid identification of metallo‐β‐lactamase‐expressing pathogenic bacteria with high selectivity.

Abstract

Metallo‐β‐lactamase is one of the major clinical threats because this β‐lactam‐hydrolyzing enzyme confers significant resistance to most β‐lactam antibiotics, including carbapenems, among bacterial pathogens. Reported herein is a novel fluorogenic sensor for the specific detection of metallo‐β‐lactamase activities. This carbapenem‐based reagent exhibits excellent selectivity to metallo‐β‐lactamase over other serine‐β‐lactamases, including serine carbapenemases. The usefulness of this probe was further demonstrated in the rapid identification of metallo‐β‐lactamase‐expressing pathogenic bacteria.

Monoclonal antibodies are considered promising therapeutic alternatives to fight antibiotic-resistant bacteria. Upon binding to their targets, they either act alone (e.g., by neutralizing bacterial toxins) or in concert with the host’s immune system (with complement or phagocytes). Storek et al. have described a unique, directly bactericidal antibody against Escherichia coli.

Antibiotics as food for bacteria

Antibiotics as food for bacteria, Published online: 11 June 2018; doi:10.1038/s41564-018-0181-z

Antibiotic resistance against β-lactams is of paramount therapeutic importance. Although such resistance is known to involve degradation by hydrolysis, the molecular details of what happens next remain unclear. A new study reveals how soil microbes perform β-lactam catabolism and how this process can support bacterial growth.

Immunological mechanisms of human resistance to persistent Mycobacterium tuberculosis infection

Immunological mechanisms of human resistance to persistent <i>Mycobacterium tuberculosis</i> infection, Published online: 12 June 2018; doi:10.1038/s41577-018-0025-3

Resisters are individuals who show resistance to infection despite long-term, high exposure to Mycobacterium tuberculosis. In this Review, Simmons and colleagues discuss potential mechanisms underlying this resistance, such as those mediated by macrophages, T cells and B cells, and how an understanding of these mechanisms might aid in the development of therapies for tuberculosis.

The peptidoglycan composition in lactic acid bacteria dictates vancomycin resistance. Vancomycin binds relatively poorly to peptidoglycan ending in d-alanyl-d-lactate and binds with high affinity to peptidoglycan ending in d-alanyl-d-alanine (d-Ala-d-Ala), which results in vancomycin resistance and sensitivity, respectively. The enzyme responsible for generating these peptidoglycan precursors is dipeptide ligase (Ddl). A single amino acid in the Ddl active site, phenylalanine or tyrosine, determines depsipeptide or dipeptide activity, respectively. Here, we established that heterologous expression of dipeptide ligase in vancomycin-resistant lactobacilli increases their sensitivity to vancomycin in a dose-dependent manner and overcomes the effects of the presence of a native d-Ala-d-Ala dipeptidase. We incorporated the dipeptide ligase gene on a suicide vector and demonstrated that it functions as a counterselection marker (CSM) in lactobacilli; vancomycin selection allows only those cells to grow in which the suicide vector has been lost. Subsequently, we developed a liquid-based approach to identify recombinants in only 5 days, which is approximately half the time required by conventional approaches. Phylogenetic analysis revealed that Ddl serves as a marker to predict vancomycin resistance and consequently indicated the broad applicability of the use of Ddl as a counterselection marker in the genus Lactobacillus. Finally, our system represents the first "plug and play" counterselection system in lactic acid bacteria that does not require prior genome editing and/or synthetic medium.

IMPORTANCE The genus Lactobacillus contains more than 200 species, many of which are exploited in the food and biotechnology industries and in medicine. Prediction of intrinsic vancomycin resistance has thus far been limited to selected Lactobacillus species. Here, we show that heterologous expression of the enzyme Ddl (dipeptide ligase)—an essential enzyme involved in peptidoglycan synthesis—increases sensitivity to vancomycin in a dose-dependent manner. We exploited this to develop a counterselection marker for use in vancomycin-resistant lactobacilli, thereby expanding the poorly developed genome editing toolbox that is currently available for most strains. Also, we showed that Ddl is a phylogenetic marker that can be used to predict vancomycin resistance in Lactobacillus; 81% of Lactobacillus species are intrinsically resistant to vancomycin, which makes our tool broadly applicable.

Related Articles

Enzyme structures of the bacterial peptidoglycan and wall teichoic acid biogenesis pathways.

Curr Opin Struct Biol. 2018 Jun 06;53:45-58

Authors: Caveney NA, Li FK, Strynadka NC

Abstract
The bacterial cell wall is a complex polymeric structure with essential roles in defence, survival and pathogenesis. Common to both Gram-positive and Gram-negative bacteria is the mesh-like peptidoglycan sacculus that surrounds the outer leaflet of the cytoplasmic membrane. Recent crystallographic studies of enzymes that comprise the peptidoglycan biosynthetic pathway have led to significant new understanding of all stages. These include initial multi-step cytosolic formation of sugar-pentapeptide precursors, transfer of the precursors to activated polyprenyl lipids at the membrane inner leaflet and flippase mediated relocalization of the resulting lipid II precursors to the outer leaflet where glycopolymerization and subsequent peptide crosslinking are finalized. Additional, species-specific enzymes allow customized peptidoglycan modifications and biosynthetic regulation that are important to bacterial virulence and survival. These studies have reinforced the unique and specific catalytic mechanisms at play in cell wall biogenesis and expanded the atomic foundation to develop novel, structure guided, antibacterial agents.

PMID: 29885610 [PubMed - as supplied by publisher]

Related Articles

Molecular insight on the non-covalent interactions between carbapenems and L,D-transpeptidase 2 from Mycobacterium tuberculosis: ONIOM study.

J Comput Aided Mol Des. 2018 May 29;:

Authors: Ntombela T, Fakhar Z, Ibeji CU, Govender T, Maguire GEM, Lamichhane G, Kruger HG, Honarparvar B

Abstract
Tuberculosis remains a dreadful disease that has claimed many human lives worldwide and elimination of the causative agent Mycobacterium tuberculosis also remains elusive. Multidrug-resistant TB is rapidly increasing worldwide; therefore, there is an urgent need for improving the current antibiotics and novel drug targets to successfully curb the TB burden. L,D-Transpeptidase 2 is an essential protein in Mtb that is responsible for virulence and growth during the chronic stage of the disease. Both D,D- and L,D-transpeptidases are inhibited concurrently to eradicate the bacterium. It was recently discovered that classic penicillins only inhibit D,D-transpeptidases, while L,D-transpeptidases are blocked by carbapenems. This has contributed to drug resistance and persistence of tuberculosis. Herein, a hybrid two-layered ONIOM (B3LYP/6-31G+(d): AMBER) model was used to extensively investigate the binding interactions of LdtMt2 complexed with four carbapenems (biapenem, imipenem, meropenem, and tebipenem) to ascertain molecular insight of the drug-enzyme complexation event. In the studied complexes, the carbapenems together with catalytic triad active site residues of LdtMt2 (His187, Ser188 and Cys205) were treated at with QM [B3LYP/6-31+G(d)], while the remaining part of the complexes were treated at MM level (AMBER force field). The resulting Gibbs free energy (ΔG), enthalpy (ΔH) and entropy (ΔS) for all complexes showed that the carbapenems exhibit reasonable binding interactions towards LdtMt2. Increasing the number of amino acid residues that form hydrogen bond interactions in the QM layer showed significant impact in binding interaction energy differences and the stabilities of the carbapenems inside the active pocket of LdtMt2. The theoretical binding free energies obtained in this study reflect the same trend of the experimental  observations. The electrostatic, hydrogen bonding and Van der Waals interactions between the carbapenems and LdtMt2 were also assessed. To further examine the nature of intermolecular interactions for carbapenem-LdtMt2 complexes, AIM and NBO analysis were performed for the QM region (carbapenems and the active residues of LdtMt2) of the complexes. These analyses revealed that the hydrogen bond interactions and charge transfer from the bonding to anti-bonding orbitals between catalytic residues of the enzyme and selected ligands enhances the binding and stability of carbapenem-LdtMt2 complexes. The two-layered ONIOM (B3LYP/6-31+G(d): Amber) model was used to evaluate the efficacy of FDA approved carbapenems antibiotics towards LdtMt2.

PMID: 29845435 [PubMed - as supplied by publisher]

Related Articles

Elucidating the inhibition of peptidoglycan biosynthesis in Staphylococcus aureus by albocycline, a macrolactone isolated from Streptomyces maizeus.

Bioorg Med Chem. 2018 07 23;26(12):3453-3460

Authors: Liang H, Zhou G, Ge Y, D'Ambrosio EA, Eidem TM, Blanchard C, Shehatou C, Chatare VK, Dunman PM, Valentine AM, Voelz VA, Grimes CL, Andrade RB

Abstract
Antibiotic resistance is a serious threat to global public health, and methicillin-resistant Staphylococcus aureus (MRSA) is a poignant example. The macrolactone natural product albocycline, derived from various Streptomyces strains, was recently identified as a promising antibiotic candidate for the treatment of both MRSA and vancomycin-resistant S. aureus (VRSA), which is another clinically relevant and antibiotic resistant strain. Moreover, it was hypothesized that albocycline's antimicrobial activity was derived from the inhibition of peptidoglycan (i.e., bacterial cell wall) biosynthesis. Herein, preliminary mechanistic studies are performed to test the hypothesis that albocycline inhibits MurA, the enzyme that catalyzes the first step of peptidoglycan biosynthesis, using a combination of biological assays alongside molecular modeling and simulation studies. Computational modeling suggests albocycline exists as two conformations in solution, and computational docking of these conformations to an ensemble of simulated receptor structures correctly predicted preferential binding to S. aureus MurA-the enzyme that catalyzes the first step of peptidoglycan biosynthesis-over Escherichia coli (E. coli) MurA. Albocycline isolated from the producing organism (Streptomyces maizeus) weakly inhibited S. aureus MurA (IC50 of 480 μM) but did not inhibit E. coli MurA. The antimicrobial activity of albocycline against resistant S. aureus strains was superior to that of vancomycin, preferentially inhibiting Gram-positive organisms. Albocycline was not toxic to human HepG2 cells in MTT assays. While these studies demonstrate that albocycline is a promising lead candidate against resistant S. aureus, taken together they suggest that MurA is not the primary target, and further work is necessary to identify the major biological target.

PMID: 29805074 [PubMed - indexed for MEDLINE]

Related Articles

Measuring the Adhesion Forces for the Multivalent Binding of Vancomycin-conjugated Dendrimer to Bacterial Cell-Wall Peptide.

Langmuir. 2018 May 24;:

Authors: Peterson E, Joseph C, Peterson H, Bouwman R, Tang S, Cannon J, Sinniah K, Choi SK

Abstract
Multivalent ligand-receptor interaction provides the fundamental basis for the hypothetical notion that high binding avidity relates to the strong force of adhesion. Despite its increasing importance in the design of targeted nanoconjugates, an understanding of the physical forces underlying the multivalent interaction remains a subject of urgent investigation. In this study, we designed three vancomycin (Van)-conjugated dendrimers G5(Van)n (n = mean valency = 0, 1, 4) for bacterial targeting with generation 5 (G5) poly(amidoamine) dendrimer as a multivalent scaffold, and evaluated both their binding avidity and physical force of adhesion to a bacterial model surface by employing surface plasmon resonance (SPR) spectroscopy and atomic force microscopy (AFM). The SPR experiment for these conjugates was performed in a biosensor chip surface immobilized with a bacterial cell-wall peptide Lys-D-Ala-D-Ala. Of these, G5(Van)4 bound most tightly with KD of 0.34 nM, which represents an increase in avidity by two or three orders of magnitude relative to a monovalent conjugate G5(Van)1 or free vancomycin, respectively. By single molecule force spectroscopy, we measured the adhesion force between G5(Van)n and the same cell-wall peptide immobilized on the surface. The distribution of adhesion forces increased in proportion to vancomycin valency with the mean force of 134 pN at n = 4 greater than 96 pN at n = 1 at a loading rate of 5200 pN/s. In summary, our results are strongly supportive of the positive correlation between the avidity and adhesion force in the multivalent interaction of vancomycin nanoconjugates.

PMID: 29792710 [PubMed - as supplied by publisher]

The micro-bio-electronic device combines bacteria that can detect certain molecules along with wifi-connected electrical outputs.

Related Articles

Blue fluorescent amino acid for biological spectroscopy and microscopy.

Proc Natl Acad Sci U S A. 2017 06 06;114(23):6005-6009

Authors: Hilaire MR, Ahmed IA, Lin CW, Jo H, DeGrado WF, Gai F

Abstract
Many fluorescent proteins are currently available for biological spectroscopy and imaging measurements, allowing a wide range of biochemical and biophysical processes and interactions to be studied at various length scales. However, in applications where a small fluorescence reporter is required or desirable, the choice of fluorophores is rather limited. As such, continued effort has been devoted to the development of amino acid-based fluorophores that do not require a specific environment and additional time to mature and have a large fluorescence quantum yield, long fluorescence lifetime, good photostability, and an emission spectrum in the visible region. Herein, we show that a tryptophan analog, 4-cyanotryptophan, which differs from tryptophan by only two atoms, is the smallest fluorescent amino acid that meets these requirements and has great potential to enable in vitro and in vivo spectroscopic and microscopic measurements of proteins.

PMID: 28533371 [PubMed - indexed for MEDLINE]

Related Articles

PBP2b plays a key role in both peripheral growth and septum positioning in Lactococcus lactis.

PLoS One. 2018;13(5):e0198014

Authors: David B, Duchêne MC, Haustenne GL, Pérez-Núñez D, Chapot-Chartier MP, De Bolle X, Guédon E, Hols P, Hallet B

Abstract
Lactococcus lactis is an ovoid bacterium that forms filaments during planktonic and biofilm lifestyles by uncoupling cell division from cell elongation. In this work, we investigate the role of the leading peptidoglycan synthase PBP2b that is dedicated to cell elongation in ovococci. We show that the localization of a fluorescent derivative of PBP2b remains associated to the septal region and superimposed with structural changes of FtsZ during both vegetative growth and filamentation indicating that PBP2b remains intimately associated to the division machinery during the whole cell cycle. In addition, we show that PBP2b-negative cells of L. lactis are not only defective in peripheral growth; they are also affected in septum positioning. This septation defect does not simply result from the absence of the protein in the cell growth machinery since it is also observed when PBP2b-deficient cells are complemented by a catalytically inactive variant of PBP2b. Finally, we show that round cells resulting from β-lactam treatment are not altered in septation, suggesting that shape elongation as such is not a major determinant for selection of the division site. Altogether, we propose that the specific PBP2b transpeptidase activity at the septum plays an important role for tagging future division sites during L. lactis cell cycle.

PMID: 29791496 [PubMed - in process]