Noel

by Joshua A. F. Sutton, Oliver T. Carnell, Lucia Lafage, Joe Gray, Jacob Biboy, Josie F. Gibson, Eric J. G. Pollitt, Simone C. Tazoll, William Turnbull, Natalia H. Hajdamowicz, Bartłomiej Salamaga, Grace R. Pidwill, Alison M. Condliffe, Stephen A. Renshaw, Waldemar Vollmer, Simon J. Foster

Peptidoglycan is the major structural component of the Staphylococcus aureus cell wall, in which it maintains cellular integrity, is the interface with the host, and its synthesis is targeted by some of the most crucial antibiotics developed. Despite this importance, and the wealth of data from in vitro studies, we do not understand the structure and dynamics of peptidoglycan during infection. In this study we have developed methods to harvest bacteria from an active infection in order to purify cell walls for biochemical analysis ex vivo. Isolated ex vivo bacterial cells are smaller than those actively growing in vitro, with thickened cell walls and reduced peptidoglycan crosslinking, similar to that of stationary phase cells. These features suggested a role for specific peptidoglycan homeostatic mechanisms in disease. As S. aureus missing penicillin binding protein 4 (PBP4) has reduced peptidoglycan crosslinking in vitro its role during infection was established. Loss of PBP4 resulted in an increased recovery of S. aureus from the livers of infected mice, which coincided with enhanced fitness within murine and human macrophages. Thicker cell walls correlate with reduced activity of peptidoglycan hydrolases. S. aureus has a family of 4 putative glucosaminidases, that are collectively crucial for growth. Loss of the major enzyme SagB, led to attenuation during murine infection and reduced survival in human macrophages. However, loss of the other three enzymes Atl, SagA and ScaH resulted in clustering dependent attenuation, in a zebrafish embryo, but not a murine, model of infection. A combination of pbp4 and sagB deficiencies resulted in a restoration of parental virulence. Our results, demonstrate the importance of appropriate cell wall structure and dynamics during pathogenesis, providing new insight to the mechanisms of disease.

Tailor-made derivatives of pseudaminic acid (Pse) allow for the detection of Gram-positive and Gram-negative bacteria expressing this unique virulence-associated nonulosonic acid. The synthesis of azido-labeled Pse derivatives starting from d-glucose and respective labeling studies are described.

Abstract

Pseudaminic acid (Pse) is a significant prokaryotic monosaccharide found in important Gram-negative and Gram-positive bacteria. This unique sugar serves as a component of cell-surface-associated glycans or glycoproteins and is associated with their virulence. We report the synthesis of azidoacetamido-functionalized Pse derivatives as part of a search for Pse-derived metabolic labeling reagents. The synthesis was initiated with d-glucose (Glc), which served as a cost-effective chiral pool starting material. Key synthetic steps involve the conversion of C1 of Glc into the terminal methyl group of Pse, and inverting deoxyaminations at C3 and C5 of Glc followed by backbone elongation with a three-carbon unit using the Barbier reaction. Metabolic labeling experiments revealed that, of the four Pse derivatives, ester-protected C5 azidoacetamido-Pse successfully labeled cells of Pse-expressing Gram-positive and Gram-negative strains. No labeling was observed in cells of non-Pse-expressing strains. The ester-protected and C5 azidoacetamido-functionalized Pse is thus a useful reagent for the identification of bacteria expressing this unique virulence-associated nonulosonic acid.

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Lipoteichoic Acid Biosynthesis Inhibitors as Potent Inhibitors of S. aureus and E. faecalis Growth and Biofilm Formation.

Molecules. 2020 May 12;25(10):

Authors: Naclerio GA, Onyedibe KI, Sintim HO

Abstract
Methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecalis (VRE) have been deemed as serious threats by the CDC. Many chronic MRSA and VRE infections are due to biofilm formation. Biofilm are considered to be between 10-10,000 times more resistant to antibiotics, and therefore new chemical entities that inhibit and/or eradicate biofilm formation are needed. Teichoic acids, such as lipoteichoic acids (LTAs) and wall teichoic acids (WTAs), play pivotal roles in Gram-positive bacteria's ability to grow, replicate, and form biofilms, making the inhibition of these teichoic acids a promising approach to fight infections by biofilm forming bacteria. Here, we describe the potent biofilm inhibition activity against MRSA and VRE biofilms by two LTA biosynthesis inhibitors HSGN-94 and HSGN-189 with MBICs as low as 0.0625 µg/mL against MRSA biofilms and 0.5 µg/mL against VRE biofilms. Additionally, both HSGN-94 and HSGN-189 were shown to potently synergize with the WTA inhibitor Tunicamycin in inhibiting MRSA and VRE biofilm formation.

PMID: 32408616 [PubMed - indexed for MEDLINE]

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Detection of Bacterial Membrane Vesicles by NOD-Like Receptors.

Int J Mol Sci. 2021 Jan 20;22(3):

Authors: Johnston EL, Heras B, Kufer TA, Kaparakis-Liaskos M

Abstract
Bacterial membrane vesicles (BMVs) are nanoparticles produced by both Gram-negative and Gram-positive bacteria that can function to modulate immunity in the host. Both outer membrane vesicles (OMVs) and membrane vesicles (MVs), which are released by Gram-negative and Gram-positive bacteria, respectively, contain cargo derived from their parent bacterium, including immune stimulating molecules such as proteins, lipids and nucleic acids. Of these, peptidoglycan (PG) and lipopolysaccharide (LPS) are able to activate host innate immune pattern recognition receptors (PRRs), known as NOD-like receptors (NLRs), such as nucleotide-binding oligomerisation domain-containing protein (NOD) 1, NOD2 and NLRP3. NLR activation is a key driver of inflammation in the host, and BMVs derived from both pathogenic and commensal bacteria have been shown to package PG and LPS in order to modulate the host immune response using NLR-dependent mechanisms. Here, we discuss the packaging of immunostimulatory cargo within OMVs and MVs, their detection by NLRs and the cytokines produced by host cells in response to their detection. Additionally, commensal derived BMVs are thought to shape immunity and contribute to homeostasis in the gut, therefore we also highlight the interactions of commensal derived BMVs with NLRs and their roles in limiting inflammatory diseases.

PMID: 33498269 [PubMed - in process]

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The Novel Membrane-Associated Auxiliary Factors AuxA and AuxB Modulate β-lactam Resistance in MRSA by stabilizing Lipoteichoic Acids.

Int J Antimicrob Agents. 2021 Jan 24;:106283

Authors: Mikkelsen K, Sirisarn W, Alharbi O, Alharbi M, Liu H, Nøhr-Meldgaard K, Mayer K, Vestergaard M, Gallagher LM, Derrick JP, McBain AJ, Biboy J, Vollmer W, O'Gara JP, Grunert T, Ingmer H, Xia G

Abstract
A major determinant of β-lactam resistance in methicillin-resistant Staphylococcus aureus (MRSA) is the drug insensitive transpeptidase, PBP2a, encoded by mecA. However, full expression of the resistance phenotype requires auxiliary factors. We identified two such factors, auxiliary factor A (auxA, SAUSA300_0980) and B (auxB, SAUSA300_1003) in a screen against mutants with increased susceptibility to β-lactams in the MRSA strain JE2. auxA and auxB encode transmembrane proteins, with AuxA predicted to be a transporter. Inactivation of auxA or auxB enhanced β-lactam susceptibility in community-, hospital- and livestock associated MRSA strains without affecting PBP2a expression, peptidoglycan cross-linking or wall teichoic acid synthesis. Both mutants displayed increased susceptibility to inhibitors of lipoteichoic acid synthesis (LTA) and alanylation pathways, and released LTA even in the absence of β-lactams. The β-lactam susceptibility of the aux mutants was suppressed by mutations inactivating gdpP, which was previously found to allow growth of mutants lacking the lipoteichoic synthase enzyme, LtaS. Using the Galleria mellonella infection model, we observed enhanced survival of larvae inoculated with either auxA or auxB mutants compared to the wild type strain following treatment with amoxicillin. Collectively, our results indicate that AuxA and AuxB are central for LTA stability and potential inhibitors can be tools to re-sensitize MRSA strains to β-lactams and combat MRSA infections.

PMID: 33503451 [PubMed - as supplied by publisher]

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Mycobacterium tuberculosis Cells Surviving in the Continued Presence of Bactericidal Concentrations of Rifampicin in vitro Develop Negatively Charged Thickened Capsular Outer Layer That Restricts Permeability to the Antibiotic.

Front Microbiol. 2020;11:554795

Authors: Sebastian J, Nair RR, Swaminath S, Ajitkumar P

Abstract
Majority of the cells in the bacterial populations exposed to lethal concentrations of antibiotics for prolonged duration succumbs to the antibiotics' sterilizing activity. The remaining cells survive by diverse mechanisms that include reduced permeability of the antibiotics. However, in the cells surviving in the continued presence of lethal concentrations of antibiotics, it is not known whether any cell surface alterations occur that in turn may reduce permeability of the antibiotics. Here we report the presence of a highly negatively charged, hydrophilic, thickened capsular outer layer (TCOL) on a small proportion of the rifampicin surviving population (RSP) of Mycobacterium tuberculosis (Mtb) cells upon prolonged continuous exposure to bactericidal concentrations of rifampicin in vitro. The TCOL reduced the intracellular entry of 5-carboxyfluorescein-rifampicin (5-FAM-rifampicin), a fluorochrome-conjugated rifampicin permeability probe of negligible bacteriocidal activity but comparable properties. Gentle mechanical removal of the TCOL enabled significant increase in the 5-FAM-rifampicin permeability. Zeta potential measurements of the cells' surface charge and hexadecane assay for cell surface hydrophobicity showed that the TCOL imparted high negative charge and polar nature to the cells' surface. Flow cytometry using the MLP and RSP cells, stained with calcofluor white, which specifically binds glucose/mannose units in β (1 → 4) or β (1 → 3) linkages, revealed the presence of lower content of polysaccharides containing such residues in the TCOL. GC-MS analyses of the TCOL and the normal capsular outer layer (NCOL) of MLP cells showed elevated levels of α-D-glucopyranoside, mannose, arabinose, galactose, and their derivatives in the TCOL, indicating the presence of high content of polysaccharides with these residues. We hypothesize that the significantly high thickness and the elevated negative charge of the TCOL might have functioned as a physical barrier restricting the permeability of the relatively non-polar rifampicin. This might have reduced intracellular rifampicin concentration enabling the cells' survival in the continued presence of high doses of rifampicin. In the context of our earlier report on the de novo emergence of rifampicin-resistant genetic mutants of Mtb from the population surviving under lethal doses of the antibiotic, the present findings attain clinical significance if a subpopulation of the tubercle bacilli in tuberculosis patients possesses TCOL.

PMID: 33391194 [PubMed]

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Genome-Wide Analysis of LysM-Containing Gene Family in Wheat: Structural and Phylogenetic Analysis during Development and Defense.

Genes (Basel). 2020 Dec 29;12(1):

Authors: Chen Z, Shen Z, Zhao D, Xu L, Zhang L, Zou Q

Abstract
The lysin motif (LysM) family comprise a number of defense proteins that play important roles in plant immunity. The LysM family includes LysM-containing receptor-like proteins (LYP) and LysM-containing receptor-like kinase (LYK). LysM generally recognizes the chitin and peptidoglycan derived from bacteria and fungi. Approximately 4000 proteins with the lysin motif (Pfam PF01476) are found in prokaryotes and eukaryotes. Our study identified 57 LysM genes and 60 LysM proteins in wheat and renamed these genes and proteins based on chromosome distribution. According to the phylogenetic and gene structure of intron-exon distribution analysis, the 60 LysM proteins were classified into seven groups. Gene duplication events had occurred among the LysM family members during the evolution process, resulting in an increase in the LysM gene family. Synteny analysis suggested the characteristics of evolution of the LysM family in wheat and other species. Systematic analysis of these species provided a foundation of LysM genes in crop defense. A comprehensive analysis of the expression and cis-elements of LysM gene family members suggested that they play an essential role in defending against plant pathogens. The present study provides an overview of the LysM family in the wheat genome as well as information on systematic, phylogenetic, gene duplication, and intron-exon distribution analyses that will be helpful for future functional analysis of this important protein family, especially in Gramineae species.

PMID: 33383636 [PubMed - in process]

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MreC and MreD balance the interaction between the elongasome proteins PBP2 and RodA.

PLoS Genet. 2020 Dec 28;16(12):e1009276

Authors: Liu X, Biboy J, Consoli E, Vollmer W, den Blaauwen T

Abstract
Rod-shape of most bacteria is maintained by the elongasome, which mediates the synthesis and insertion of peptidoglycan into the cylindrical part of the cell wall. The elongasome contains several essential proteins, such as RodA, PBP2, and the MreBCD proteins, but how its activities are regulated remains poorly understood. Using E. coli as a model system, we investigated the interactions between core elongasome proteins in vivo. Our results show that PBP2 and RodA form a complex mediated by their transmembrane and periplasmic parts and independent of their catalytic activity. MreC and MreD also interact directly with PBP2. MreC elicits a change in the interaction between PBP2 and RodA, which is suppressed by MreD. The cytoplasmic domain of PBP2 is required for this suppression. We hypothesize that the in vivo measured PBP2-RodA interaction change induced by MreC corresponds to the conformational change in PBP2 as observed in the MreC-PBP2 crystal structure, which was suggested to be the "on state" of PBP2. Our results indicate that the balance between MreC and MreD determines the activity of PBP2, which could open new strategies for antibiotic drug development.

PMID: 33370261 [PubMed - as supplied by publisher]

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Unravelling the mechanism of action of "de novo" designed peptide P1 with model membranes and gram-positive and gram-negative bacteria.

Arch Biochem Biophys. 2020 10 30;693:108549

Authors: Espeche JC, Martínez M, Maturana P, Cutró A, Semorile L, Maffia PC, Hollmann A

Abstract
In the last years, the decreasing effectiveness of conventional antimicrobial-drugs has caused serious problems due to the rapid emergence of multidrug-resistant pathogens. This situation has brought attention to other antimicrobial agents like antimicrobial peptides (AMPs), for being considered an alternative to conventional drugs. These compounds target bacterial membranes for their activity, which gives them a broad spectrum of action and less probable resistance development. That is why the peptide-membrane interaction is a crucial aspect to consider in the study of AMPs. The aim of this work was the characterization of the "de novo" designed peptide P1, studying its interactions with model membranes (i.e. liposomes of DMPC:DMPG 5:1) in order to evaluate the final position of the peptide upon interacting with the membrane. Also, we tested the effects of the peptide in gram-positive and gram-negative bacteria. Later, by spectroscopic methods, the ability of the peptide to permeabilize the inner and outer membrane of E. coli and plasmatic membrane of S. aureus was assessed. The results obtained confirmed that P1 can disrupt both membranes, showing some difference in its activity as a function of the nature of each bacterial cell wall, confirming higher effects on gram-positive S. aureus. Finally, we also showed the ability of P1 to inhibit biofilms of that gram-positive bacterium. All data obtained in this work allowed us to propose a model, where the first interactions of the peptide with the bacterial envelope, seem to depend on the gram-negative and gram-positive cell wall structure. After that first interaction, the peptide is stabilized by Trp residues depth inserted into the hydrocarbon region, promoting several changes in the organization of the lipid bilayer, following a carpet-like mechanism, which results in permeabilization of the membrane, triggering the antimicrobial activity.

PMID: 32828795 [PubMed - indexed for MEDLINE]

We previously identified a small-molecule inhibitor of capsule biogenesis (designated DU011) and identified its target as MprA, a MarR family transcriptional repressor of multidrug efflux pumps. Unlike other proposed MprA ligands, such as salicylate and 2,4-dinitrophenol (DNP), DU011 does not alter Escherichia coli antibiotic resistance and has significantly enhanced inhibition of capsule expression. We hypothesized that the potency and the unique action of DU011 are due to novel interactions with the MprA binding pocket and the conformation assumed by MprA upon binding DU011 relative to other ligands. To understand the dynamics of MprA-DU011 interaction, we performed hydrogen-deuterium exchange mass spectrometry (HDX-MS); this suggested that four peptide regions undergo conformational changes upon binding DU011. We conducted isothermal calorimetric titration (ITC) to quantitatively characterize MprA binding to DU011 and canonical ligands and observed a distinct two-site binding isotherm associated with the binding reaction of MprA to DU011; however, salicylate and DNP showed a one-site binding isotherm with lower affinity. To elucidate the binding pocket(s) of MprA, we selected single point mutants of MprA that included mutated residues predicted to be within the putative binding pocket (Q51A, F58A, and E65D) as well as on or near the DNA-binding domain (L81A, S83T, and T86A). Our ITC studies suggest that two of the tested MprA mutants had lower affinity for DU011: Q51A and F58A. In addition to elucidating the MprA binding pocket for DU011, we studied the binding of these mutants to salicylate and DNP to reveal the binding pockets of these canonical ligands.

Nature Chemical Biology, Published online: 16 November 2020; doi:10.1038/s41589-020-00674-6

The difficulty of antibiotic discovery posed by the double-membrane cell envelope of Gram-negative bacteria and active drug efflux requires better understanding of bacterial permeability and compound accumulation, and more diverse chemical libraries.

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Systematic Investigation of the Permeability of Androgen Receptor PROTACs.

ACS Med Chem Lett. 2020 Aug 13;11(8):1539-1547

Authors: Scott DE, Rooney TPC, Bayle ED, Mirza T, Willems HMG, Clarke JH, Andrews SP, Skidmore J

Abstract
Bifunctional molecules known as PROTACs simultaneously bind an E3 ligase and a protein of interest to direct ubiquitination and clearance of that protein, and they have emerged in the past decade as an exciting new paradigm in drug discovery. In order to investigate the permeability and properties of these large molecules, we synthesized two panels of PROTAC molecules, constructed from a range of protein-target ligands, linkers, and E3 ligase ligands. The androgen receptor, which is a well-studied protein in the PROTAC field was used as a model system. The physicochemical properties and permeability of PROTACs are discussed.

PMID: 32832021 [PubMed - as supplied by publisher]

Chemical Biology Tools for Examining the Bacterial Cell Wall.

Cell Chem Biol. 2020 Aug 20;27(8):1052-1062

Authors: Brown AR, Gordon RA, Hyland SN, Siegrist MS, Grimes CL

Abstract
Bacteria surround themselves with cell walls to maintain cell rigidity and protect against environmental insults. Here we review chemical and biochemical techniques employed to study bacterial cell wall biogenesis. Recent advances including the ability to isolate critical intermediates, metabolic approaches for probe incorporation, and isotopic labeling techniques have provided critical insight into the biochemistry of cell walls. Fundamental manuscripts that have used these techniques to discover cell wall-interacting proteins, flippases, and cell wall stoichiometry are discussed in detail. The review highlights that these powerful methods and techniques have exciting potential to identify and characterize new targets for antibiotic development.

PMID: 32822617 [PubMed - as supplied by publisher]

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Vancomycin C-Terminus Guanidine Modifications and Further Insights into an Added Mechanism of Action Imparted by a Peripheral Structural Modification.

ACS Infect Dis. 2020 Jun 29;:

Authors: Wu ZC, Cameron MD, Boger DL

Abstract
A series of vancomycin C-terminus guanidine modifications are disclosed that improve antimicrobial activity, enhance the durability of antimicrobial action against selection or induction of resistance, and introduce a synergistic mechanism of action independent of D-Ala-D-Ala binding and inhibition of cell wall biosynthesis. The added mechanism of action results in induced bacterial cell permeability, which we show may involve interaction with cell envelope teichoic acid. Significantly, the compounds examined that contain two combined peripheral modifications, a (4-chlorobiphenyl)methyl (CBP) and C-terminus guanidinium modification, offer opportunities for new treatments against not only vancomycin-sensitive, but especially vancomycin-resistant bacteria where they act by two synergistic and now durable mechanisms of action independent of D-Ala-D-Ala/D-Lac binding and display superb antimicrobial potencies (MIC 0.6-0.15 μg/mL, VanA VRE). For the first time, we demonstrate that the synergistic behavior of the peripheral modifications examined requires the presence of both the CBP and guanidine modifications in a single molecule versus their combined use as an equimolar mixture of singly modified compounds. Finally, we show that a prototypical member of the series, G3-CBP-vancomycin (15), exhibits no hemolytic activity, displays no mammalian cell growth inhibition, and possesses improved and especially attractive in vivo pharmacokinetic (PK) properties.

PMID: 32598127 [PubMed - as supplied by publisher]

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The Protein A-mediated binding of Staphylococcus to antibodies in flow cytometric assays and its reduction using FcR blocking reagent.

Appl Environ Microbiol. 2020 Jun 26;:

Authors: Cronin UP, Girardeaux L, O'Meara E, Wilkinson M

Abstract
Staphylococcus aureus and other coagulase-positive Staphylococcus spp. bind the Fc region of IgG antibodies through expression of protein A (SpA). These species have consequently been a source of false positive signals in antibody-based assays designed to detect other target bacteria. In this work, flow cytometry was used to study the influence of a number of factors on the SpA-mediated binding of single cells to an anti-human IgG antibody, including: strain; heat killing; overnight storage; growth phase; cell physiology; surface adhesion; and growth in model food systems. Through the co-staining of antibody-stained cells with the permeability dye, propidium iodide and Calcein Violet AM, cell physiological status was related to SpA-mediated antibody binding. Generally, permeabilised cells lacking esterase activity did not strongly bind antibody. The binding of a number of commercially available polyclonal IgG antibodies to non-Staphylococcus spp. was also characterised. Not all SpA-expressing species showed strong binding of mouse IgG, and one species not known to express SpA showed strong binding. Most SpA-expressing strains bound rabbit IgG antibodies to some extent, whereas only one strain bound goat IgG. To reduce or eliminate SpA-mediated IgG binding, the following products were evaluated as blocking reagents and applied prior to staining with primary or secondary antibody: normal rabbit serum, mouse IgG isotype control, goat IgG and a commercial FcR blocking reagent. Only the FcR blocking reagent consistently reduced SpA-mediated binding of Staphylococcus spp. to antibodies against other species and could be recommended as a blocking reagent in immunoassays designed to detect non-Staphylococcus species.Importance This study characterises a widespread, but little-studied problem associated with the antibody-based detection of microbes - the Staphylococcus Protein A (SpA)-mediated binding of IgG antibodies - and offers a solution: the use of commercial FcR blocking reagent. A common source of false-positive signals in the detection of microbes in clinical, food or environmental samples can be eliminated by following the authors' findings. Using flow cytometry, the authors demonstrate the extent of heterogeneity in a culture's SpA-mediated binding of antibodies and that the degree of SpA-mediated antibody binding is strain-, growth-phase- and food matrix-dependent and influenced by simulated food processing treatments and cell adherence. Additionally, studies of SpA-mediated binding of Staphylococcus spp. to antibodies against other bacterial species produced a very nuanced picture, leading the authors to recommend testing against multiple strains of S. aureus and S. hyicus of all antibodies to be incorporated into any immunoassay designed to detect a non-Staphylococcus spp.

PMID: 32591386 [PubMed - as supplied by publisher]

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Breaching the Barrier: Quantifying Antibiotic Permeability across Gram-negative Bacterial Membranes.

J Mol Biol. 2019 08 23;431(18):3531-3546

Authors: Cama J, Henney AM, Winterhalter M

Abstract
The double-membrane cell envelope of Gram-negative bacteria is a sophisticated barrier that facilitates the uptake of nutrients and protects the organism from toxic compounds. An antibiotic molecule must find its way through the negatively charged lipopolysaccharide layer on the outer surface, pass through either a porin or the hydrophobic layer of the outer membrane, then traverse the hydrophilic peptidoglycan layer only to find another hydrophobic lipid bilayer before it finally enters the cytoplasm, where it typically finds its target. This complex uptake pathway with very different physico-chemical properties is one reason that Gram-negative are intrinsically protected against multiple classes of antibiotic-like molecules, and is likely the main reason that in vitro target-based screening programs have failed to deliver novel antibiotics for these organisms. Due to the lack of general methods available for quantifying the flux of drugs into the cell, little is known about permeation rates, transport pathways and accumulation at the target sites for particular molecules. Here we summarize the current tools available for measuring antibiotic uptake across the different compartments of Gram-negative bacteria.

PMID: 30959052 [PubMed - indexed for MEDLINE]

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Gut dysbiosis is associated with primary hypothyroidism with interaction on gut-thyroid axis.

Clin Sci (Lond). 2020 Jun 10;:

Authors: Su X, Zhao Y, Li Y, Ma S, Wang Z

Abstract
BACKGROUND: Previous studies have shown that the gut microbiome is associated with thyroid diseases, including Graves' disease, Hashimoto's disease, thyroid nodules, and thyroid cancer. However, the association between intestinal flora and primary hypothyroidism remains elusive. We aimed to characterize gut microbiome in primary hypothyroidism patients.
METHODS: Fifty-two primary hypothyroidism patients and 40 healthy controls were recruited. The differences in gut microbiota between the two groups were analyzed by 16S rRNA sequencing technology. Fecal microbiota transplantation (FMT) was performed in mice using flora from both groups; changes in thyroid function were then assessed in the mice.
RESULTS: There were significant differences in α and β diversities of gut microbiota between primary hypothyroidism patients and healthy individuals. The random forest analysis indicated that four intestinal bacteria (Veillonella, Paraprevotella, Neisseria, and Rheinheimera) could distinguish untreated primary hypothyroidism patients from healthy individuals with the highest accuracy; this was confirmed by receiver operator characteristic curve analysis. The short chain fatty acid producing ability of the primary hypothyroidism patients' gut was significantly decreased, which resulted in the increased serum lipopolysaccharides (LPS) levels. The FMT showed that mice receiving the transplant from primary hypothyroidism patients displayed decreased total thyroxine levels.
CONCLUSIONS: Our study suggests that primary hypothyroidism causes changes in gut microbiome. In turn, an altered flora can affect thyroid function in mice. These findings could help understand the development of primary hypothyroidism and might be further used to develop potential probiotics to facilitate the adjuvant treatment of this disease.

PMID: 32519746 [PubMed - as supplied by publisher]

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Bacterial Peptidoglycans from Microbiota in Neurodevelopment and Behavior.

Trends Mol Med. 2020 Jun 04;:

Authors: Gonzalez-Santana A, Diaz Heijtz R

Abstract
It is increasingly recognized that the gut microbiota profoundly influences many aspects of host development and physiology, including the modulation of brain development and behavior. However, the precise molecular mechanisms and signaling pathways involved in communication between the microbiota and the developing brain remain to be fully elucidated. Germline-encoded pattern-recognition receptors (PRRs) that recognize conserved microbial molecular signatures such as bacterial surface molecules (e.g., peptidoglycans, PGNs) have emerged as potential key regulators of gut microbiota-brain interactions. We highlight current evidence supporting multiple and essential roles for PGNs and their sensing molecules beyond innate immunity, extending to neurodevelopment and behavior. In addition, the possible implications of the PGN signaling pathway for the pathogenesis of neurodevelopmental disorders such as autism spectrum disorder (ASD) are considered.

PMID: 32507655 [PubMed - as supplied by publisher]

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Opioid system influences gut-brain axis: Dysbiosis and related alterations.

Pharmacol Res. 2020 Jun 03;:104928

Authors: Rueda-Ruzafa L, Cruz F, Cardona D, Hone AJ, Molina-Torres G, Sánchez-Labraca N, Roman P

Abstract
Opiod drugs are widely used to treat chronic pain, but their misuse can lead to tolerance, dependence, and addiction and have created a significant public health problem. In addition, food-derived opioid peptides, known as exorphins, like gluten exorphins have been shown to have harmful effects in certain pathologies like celiac disease, for example. Several studies support the involvement of the opioid system in the development of disorders such as autism spectrum syndrome. Moreover, bidirectional communication between the intestine and brain has been shown to be altered in various neurodegenerative diseases including Alzheimer´s and Parkinson´s. The presence of opiod receptors in both the digestive tract and the central nervous system (CNS) suggests that opioid drugs and exorphins may modulate the gut-brain axis. Morphine, for example, has shown a dysbiotic effect on the bacterial microbiota in addition to inducing an increase in intestinal permeability facilitating bacterial translocation. Furthermore, certain components of bacteria can modify the expression of opioid receptors at the central level increasing sensitivity to pain. Strategies based on use of probiotics have resulted in improvements in symptoms of autism and Parkinson´s disease. In this manuscript, we review the role of the opioid system in disorders and CNS pathologies and the involvement of the gut-brain axis.

PMID: 32504837 [PubMed - as supplied by publisher]

Molecules from symbiotic microorganisms pervasively infiltrate almost every organ system of a mammalian host, marking the initiation of microbial–host mutualism in utero, long before the newborn acquires its own microbiota. Starting from in utero development, when maternal microbial molecules can penetrate the placental barrier, we follow the different phases of adaptation through the life events of birth, lactation, and weaning, as the young mammal adapts to the microbes that colonize its body surfaces. The vulnerability of early-life mammals is mitigated by maternal detoxification and excretion mechanisms, the protective effects of maternal milk, and modulation of neonatal receptor systems. Host adaptations to microbial exposure during specific developmental windows are critical to ensure organ function for development, growth, and immunity.

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Opposite effect of vancomycin and D-Cycloserine combination in both vancomycin resistant Staphylococcus aureus and enterococci.

FEMS Microbiol Lett. 2020 Apr 11;:

Authors: Boudrioua A, Li Y, Hartke A, Giraud C

Abstract
The increasing spread of antibiotic resistant bacteria is a major human health concern. The challenging development of new effective antibiotics has led to focus on seeking synergistic antibiotic combinations. Vancomycin (VAN) is a glycopeptide antibiotic used to treat Staphylococcus aureus and enterococci infections. It is targeting D-Alanyl-D-Alanine dimers during peptidoglycan biosynthesis. D-cycloserine (DCS) is a D-Alanine analogue that targets peptidoglycan biosynthesis by inhibiting D-Alanine:D-Alanine ligase (Ddl). The VAN-DCS combination was found to be synergistic in VAN resistant S. aureus strains lacking van genes cluster. We hypothesize that this combination leads to opposite effects in S. aureus and enterococci strains harboring van genes cluster where VAN resistance is conferred by the synthesis of modified peptidoglycan precursors ending in D-Alanyl-D-Lactate. The calculated Fractional Inhibitory Concentration of VAN-DCS combination in a van- vancomycin-intermediate, VanA type, and VanB type strains were 0.5, 5 and 3 respectively. As a result, VAN-DCS combination leads to synergism in van-lacking strains, and to antagonism in strains harboring van genes cluster. The VAN-DCS antagonism is due to a mechanism that we named van-mediated Ddl inhibition bypass. Our results show that antibiotic combinations can lead to opposite effects depending on the genetic backgrounds.

PMID: 32277698 [PubMed - as supplied by publisher]

Gut sensing : The indigenous metabolic status of mouse gut microbiota is quantified by flow cytometry using the signal from fluorescent d ‐amino acid‐based in vivo labeling as the indicator of bacterial metabolic activities, and signals from fluorescence in situ hybridization to indicate their taxonomic identification.

Abstract

Herein, we propose a metabolic d ‐amino acid‐based labeling and in situ hybridization‐facilitated (MeDabLISH) strategy for the quantitative analysis of the indigenous metabolic status of gut bacteria. The fluorescent d ‐amino acid (FDAA)‐based labeling intensities of bacteria were found to highly correlate with their temporal and steady‐state metabolic status. Then, after taxonomic identification of bacterial genera in the in vivo FDAA‐labeled mouse gut microbiota, by corresponding fluorescence in situ hybridization (FISH) probes, the metabolic activities of different gut bacterial genera are quantified by flow cytometry, using FISH signals to differentiate genera and FDAA signals to indicate their basal metabolic levels. It was found that Gram‐negative genera in the mouse microbiota have stronger metabolic activities during the daytime, and Gram‐positive genera have higher activities at the night. Our strategy will be instrumental in deepening our understanding of the highly complex microbiota.

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Synthesis of the New Cyanine-Labeled Bacterial Lipooligosaccharides for Intracellular Imaging and in Vitro Microscopy Studies.

Bioconjug Chem. 2019 06 19;30(6):1649-1657

Authors: Wang TC, Cochet F, Facchini FA, Zaffaroni L, Serba C, Pascal S, Andraud C, Sala A, Di Lorenzo F, Maury O, Huser T, Peri F

Abstract
Endotoxin (lipooligosaccharide, LOS, and lipopolysaccharide, LPS) is the major molecular component of Gram-negative bacteria outer membrane, and very potent pro-inflammatory substance. Visualizing and tracking the distribution of the circulating endotoxin is one of the fundamental approaches to understand the molecular aspects of infection with subsequent inflammatory and immune responses, LPS also being a key player in the molecular dialogue between microbiota and host. While fluorescently labeled LPS has previously been used to track its subcellular localization and colocalization with TLR4 receptor and downstream effectors, our knowledge on lipopolysaccharide (LOS) localization and cellular activity remains almost unexplored. In this study, LOS was labeled with a novel fluorophore, Cy7N, featuring a large Stokes-shifted emission in the deep-red spectrum resulting in lower light scattering and better imaging contrast. The LOS-Cy7N chemical identity was determined by mass spectrometry, and immunoreactivity of the conjugate was evaluated. Interestingly, its application to microscopic imaging showed a faster cell internalization compared to LPS-Alexa488, despite that it is also CD14-dependent and undergoes the same endocytic pathway as LPS toward lysosomal detoxification. Our results suggest the use of the new infrared fluorophore Cy7N for cell imaging of labeled LOS by confocal fluorescence microscopy, and propose that LOS is imported in the cells by mechanisms different from those responsible for LPS uptake.

PMID: 31136151 [PubMed - indexed for MEDLINE]

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A Critical Role of Formyl Peptide Receptors in Host Defense against Escherichia coli.

J Immunol. 2020 Mar 27;:

Authors: Zhang M, Gao JL, Chen K, Yoshimura T, Liang W, Gong W, Li X, Huang J, McDermott DH, Murphy PM, Wang X, Wang JM

Abstract
Formyl peptide receptors (FPRs, mouse Fprs) belong to the G protein-coupled receptor superfamily and mediate phagocyte migration in response to bacteria- and host-derived chemoattractants; however, knowledge about their in vivo roles in bacterial pathogenesis is limited. In this study, we investigated the role of Fpr1 and Fpr2 in host defense against Escherichia coli infection. In vitro, we found that supernatants from E. coli cultures induced chemotaxis of wild-type (WT) mouse bone marrow-derived neutrophils and that the activity was significantly reduced in cells genetically deficient in either Fpr1 or Fpr2 and was almost absent in cells lacking both receptors. Consistent with this, E. coli supernatants induced chemotaxis and MAPK phosphorylation in HEK293 cells expressing either recombinant Fpr1 or Fpr2 but not untransfected parental cells. WT bone marrow -derived neutrophils could actively phagocytose and kill E. coli, whereas both activities were diminished in cells lacking Fpr1 or Fpr2; again, an additive effect was observed in cells lacking both receptors. In vivo, Fpr1 and Fpr2 deficiency resulted in reduced recruitment of neutrophils in the liver and peritoneal cavity of mice infected with inactivated E. coli Moreover, Fpr1-/- and Fpr2-/- mice had significantly increased mortality compared with WT mice after i.p. challenge with a virulent E. coli clinical isolate. These results indicate a critical role of Fprs in host defense against E. coli infection.

PMID: 32221037 [PubMed - as supplied by publisher]

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Remodeling of Crossbridges Controls Peptidoglycan Crosslinking Levels in Bacterial Cell Walls.

ACS Chem Biol. 2020 Mar 13;:

Authors: Apostolos AJ, Pidgeon SE, Pires MM

Abstract
Cell walls are barriers found in almost all known bacterial cells. These structures establish a controlled interface be-tween the external environment and vital cellular compo-nents. A primary component of cell wall is a highly cross-linked matrix called peptidoglycan (PG). PG crosslinking, car-ried out by transglycosylases and transpeptidases, is neces-sary for proper cell wall assembly. Transpeptidases, targets of -lactam antibiotics, stitch together two neighboring PG stem peptides (acyl-donor and acyl-acceptor strands). We recently described a novel class of cellular PG probes that were pro-cessed exclusively as acyl-donor strands. Herein, we have accessed the other half of the transpeptidase reaction by de-veloping probes that are processed exclusively as acyl-acceptor strands. The critical nature of the crossbridge on the PG peptide was demonstrated in live bacterial cells and sur-prising promiscuity in crossbridge primary sequence was found in various bacterial species. Additionally, acyl-acceptor probes provided insight into how chemical remodeling of the PG crossbridge (e.g., amidation) can modulate crosslinking levels, thus establishing a physiological role of PG structural variations. Together, the acyl-donor and -acceptor probes will provide a versatile platform to interrogate PG crosslinking in physiologically relevant settings.

PMID: 32167281 [PubMed - as supplied by publisher]