Alexis Apostolos

Bacteria were first detected in human tumors more than 100 years ago, but the characterization of the tumor microbiome has remained challenging because of its low biomass. We undertook a comprehensive analysis of the tumor microbiome, studying 1526 tumors and their adjacent normal tissues across seven cancer types, including breast, lung, ovary, pancreas, melanoma, bone, and brain tumors. We found that each tumor type has a distinct microbiome composition and that breast cancer has a particularly rich and diverse microbiome. The intratumor bacteria are mostly intracellular and are present in both cancer and immune cells. We also noted correlations between intratumor bacteria or their predicted functions with tumor types and subtypes, patients’ smoking status, and the response to immunotherapy.

Nature Microbiology, Published online: 18 May 2020; doi:10.1038/s41564-020-0723-z

Antibiotic adjuvant peptide SLAP-S25 binds to the inner and outer membranes of Gram-negative bacteria to cause membrane damage and boost efficacy of all major antibiotic classes.

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AsnB is responsible for peptidoglycan precursor amidation in Clostridium difficile in the presence of vancomycin.

Microbiology. 2020 Apr 30;:

Authors: Ammam F, Patin D, Coullon H, Blanot D, Lambert T, Mengin-Lecreulx D, Candela T

Abstract
Clostridium difficile 630 possesses a cryptic but functional gene cluster vanG Cd homologous to the vanG operon of Enterococcus faecalis. Expression of vanG Cd in the presence of subinhibitory concentrations of vancomycin is accompanied by peptidoglycan amidation on the meso-DAP residue. In this paper, we report the presence of two potential asparagine synthetase genes named asnB and asnB2 in the C. difficile genome whose products were potentially involved in this peptidoglycan structure modification. We found that asnB expression was only induced when C. difficile was grown in the presence of vancomycin, yet independently from the vanG Cd resistance and regulation operons. In addition, peptidoglycan precursors were not amidated when asnB was inactivated. No change in vancomycin MIC was observed in the asnB mutant strain. In contrast, overexpression of asnB resulted in the amidation of most of the C. difficile peptidoglycan precursors and in a weak increase of vancomycin susceptibility. AsnB activity was confirmed in E. coli. In contrast, the expression of the second asparagine synthetase, AsnB2, was not induced in the presence of vancomycin. In summary, our results demonstrate that AsnB is responsible for peptidoglycan amidation of C. difficile in the presence of vancomycin.

PMID: 32375990 [PubMed - as supplied by publisher]

Cell‐specific cytosolic delivery of proteins is highly challenging. Herein, we demonstrate that mutants of anthrax‐derived protective antigen (mPA) can be expressed as fusions to scFvs for targeted delivery of potent protein therapeutics to the cellular cytosol. Targeting of EGFR or carcinoembryonic antigen with this strategy led to selective delivery to and death of pancreatic cancer cells.

Abstract

The nontoxic, anthrax protective antigen/lethal factor N‐terminal domain (PA/LF_N) complex is an effective platform for translocating proteins into the cytosol of cells. Mutant PA (mPA) was recently fused to epidermal growth factor (EGF) to retarget delivery of LF_N to cells bearing EGF receptors (EGFR), but the requirement for a known cognate ligand limits the applicability of this approach. Here, we render practical protective antigen retargeting to a variety of receptors with mPA single‐chain variable fragment (scFv) fusion constructs. Our design enables the targeting of two pancreatic cancer‐relevant receptors, EGFR and carcinoembryonic antigen. We demonstrate that fusion to scFvs does not disturb the basic functions of mPA. Moreover, mPA−scFv fusions enable cell‐specific delivery of diphtheria toxin catalytic domain and Ras/Rap1‐specific endopeptidase to pancreatic cancer cells. Importantly, mPA−scFv fusion‐based treatments display potent cell‐specific toxicity in vitro, opening fundamentally new routes toward engineered immunotoxins and providing a potential solution to the challenge of targeted protein delivery to the cytosol of cancer cells.

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Listeria monocytogenes Wall Teichoic Acid Glycosylation Promotes Surface Anchoring of Virulence Factors, Resistance to Antimicrobial Peptides, and Decreased Susceptibility to Antibiotics.

Pathogens. 2020 Apr 16;9(4):

Authors: Meireles D, Pombinho R, Carvalho F, Sousa S, Cabanes D

Abstract
The cell wall of Listeria monocytogenes (Lm), a major intracellular foodborne bacterial pathogen, comprises a thick peptidoglycan layer that serves as a scaffold for glycopolymers such as wall teichoic acids (WTAs). WTAs contain non-essential sugar substituents whose absence prevents bacteriophage binding and impacts antigenicity, sensitivity to antimicrobials, and virulence. Here, we demonstrated, for the first time, the triple function of Lm WTA glycosylations in the following: (1) supporting the correct anchoring of major Lm virulence factors at the bacterial surface, namely Ami and InlB; (2) promoting Lm resistance to antimicrobial peptides (AMPs); and (3) decreasing Lm sensitivity to some antibiotics. We showed that while the decoration of WTAs by rhamnose in Lm serovar 1/2a and by galactose in serovar 4b are important for the surface anchoring of Ami and InlB, N-acetylglucosamine in serovar 1/2a and glucose in serovar 4b are dispensable for the surface association of InlB or InlB/Ami. We found that the absence of a single glycosylation only had a slight impact on the sensibility of Lm to AMPs and antibiotics, however the concomitant deficiency of both glycosylations (rhamnose and N-acetylglucosamine in serovar 1/2a, and galactose and glucose in serovar 4b) significantly impaired the Lm capacity to overcome the action of antimicrobials. We propose WTA glycosylation as a broad mechanism used by Lm, not only to properly anchor surface virulence factors, but also to resist AMPs and antibiotics. WTA glycosyltransferases thus emerge as promising drug targets to attenuate the virulence of bacterial pathogens, while increasing their susceptibility to host immune defenses and potentiating the action of antibiotics.

PMID: 32316182 [PubMed]

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Lipoprotein Lpp regulates the mechanical properties of the E. coli cell envelope.

Nat Commun. 2020 Apr 14;11(1):1789

Authors: Mathelié-Guinlet M, Asmar AT, Collet JF, Dufrêne YF

Abstract
The mechanical properties of the cell envelope in Gram-negative bacteria are controlled by the peptidoglycan, the outer membrane, and the proteins interacting with both layers. In Escherichia coli, the lipoprotein Lpp provides the only covalent crosslink between the outer membrane and the peptidoglycan. Here, we use single-cell atomic force microscopy and genetically engineered strains to study the contribution of Lpp to cell envelope mechanics. We show that Lpp contributes to cell envelope stiffness in two ways: by covalently connecting the outer membrane to the peptidoglycan, and by controlling the width of the periplasmic space. Furthermore, mutations affecting Lpp function substantially increase bacterial susceptibility to the antibiotic vancomycin, indicating that Lpp-dependent effects can affect antibacterial drug efficacy.

PMID: 32286264 [PubMed - as supplied by publisher]

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Fluorescence anisotropy assays for high throughput screening of compounds binding to lipid II, PBP1b, FtsW and MurJ.

Sci Rep. 2020 Apr 14;10(1):6280

Authors: Boes A, Olatunji S, Mohammadi T, Breukink E, Terrak M

Abstract
Lipid II precursor and its processing by a flippase and peptidoglycan polymerases are considered key hot spot targets for antibiotics. We have developed a fluorescent anisotropy (FA) assay using a unique and versatile probe (fluorescent lipid II) and monitored direct binding between lipid II and interacting proteins (PBP1b, FtsW and MurJ), as well as between lipid II and interacting antibiotics (vancomycin, nisin, ramoplanin and a small molecule). Competition experiments performed using unlabelled lipid II, four lipid II-binding antibiotics and moenomycin demonstrate that the assay can detect compounds interacting with lipid II or the proteins. These results provide a proof-of-concept for the use of this assay in a high-throughput screening of compounds against all these targets. In addition, the assay constitutes a powerful tool in the study of the mode of action of compounds that interfere with these processes. Interestingly, FA assay with lipid II probe has the advantage over moenomycin based probe to potentially identify compounds that interfere with both donor and acceptor sites of the aPBPs GTase as well as compounds that bind to lipid II. In addition, this assay would allow the screening of compounds against SEDS proteins and MurJ which do not interact with moenomycin.

PMID: 32286439 [PubMed - as supplied by publisher]

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NOD2 influences trajectories of intestinal microbiota recovery after antibiotic perturbation.

Cell Mol Gastroenterol Hepatol. 2020 Apr 11;:

Authors: Anderson JM, Lipinski S, Sommer F, Pan WH, Boulard O, Rehman A, Falk-Paulsen M, Stengel ST, Aden K, Häsler R, Bharti R, Künzel S, Baines JF, Chamaillard M, Rosenstiel P

Abstract
BACKGROUND & AIMS: Loss-of-function variants in nucleotide-binding oligomerization domain-containing protein 2 (NOD2) impair the recognition of the bacterial cell wall component muramyl-dipeptide and are associated with an increased risk for developing Crohn's disease (CD). Likewise, exposure to antibiotics increases the individual risk for developing inflammatory bowel disease (IBD). Here, we studied the long-term impact of NOD2 on the ability of the gut bacterial and fungal microbiota to recover after antibiotic treatment.
METHODS: Two cohorts of 20-week- and 52-week-old wild-type (WT) C57BL/6J and NOD2 knock-out (Nod2-KO) mice were treated with broad-spectrum antibiotics and fecal samples were collected to investigate temporal dynamics of the intestinal microbiota (bacteria and fungi) using 16S rRNA and ITS1 sequencing. In addition, two sets of germ-free (GF) WT mice were colonized with either WT or Nod2-KO post-antibiotic donor microbiota and the severity of intestinal inflammation was monitored in the colonized mice.
RESULTS: Antibiotics exposure caused long-term shifts in the bacterial and fungal community composition. Genetic ablation of NOD2 was associated with delayed body weight gain after antibiotic treatment and an impaired recovery of the bacterial gut microbiota. Transfer of the post-antibiotic fecal microbiota of Nod2-KO mice induced an intestinal inflammatory response in the colon of GF recipient mice compared to respective microbiota from WT controls based on histopathology and gene expression analyses.
CONCLUSIONS: Our data illustrate that the bacterial sensor NOD2 contributes to intestinal microbial community composition after antibiotic treatment and may add to the explanation of how defects in the NOD2 signaling pathway are involved in the etiology of CD.

PMID: 32289499 [PubMed - as supplied by publisher]

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Tools for probing host-bacteria interactions in the gut microenvironment: From molecular to cellular levels.

Bioorg Med Chem Lett. 2020 Mar 18;:127116

Authors: Wodzanowski KA, Cassel SE, Grimes CL, Kloxin AM

Abstract
Healthy function of the gut microenvironment is dependent on complex interactions between the bacteria of the microbiome, epithelial and immune (host) cells, and the surrounding tissue. Misregulation of these interactions is implicated in disease. A range of tools have been developed to study these interactions, from mechanistic studies to therapeutic evaluation. In this Digest, we highlight select tools at the cellular and molecular level for probing specific cell-microenvironment interactions. Approaches are overviewed for controlling and probing cell-cell interactions, from transwell and microfluidic devices to engineered bacterial peptidoglycan fragments, and cell-matrix interactions, from three-dimensional scaffolds to chemical handles for in situ modifications.

PMID: 32223923 [PubMed - as supplied by publisher]

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Microbiome-derived carnitine mimics as previously unknown mediators of gut-brain axis communication.

Sci Adv. 2020 03;6(11):eaax6328

Authors: Hulme H, Meikle LM, Strittmatter N, van der Hooft JJJ, Swales J, Bragg RA, Villar VH, Ormsby MJ, Barnes S, Brown SL, Dexter A, Kamat MT, Komen JC, Walker D, Milling S, Osterweil EK, MacDonald AS, Schofield CJ, Tardito S, Bunch J, Douce G, Edgar JM, Edrada-Ebel R, Goodwin RJA, Burchmore R, Wall DM

Abstract
Alterations to the gut microbiome are associated with various neurological diseases, yet evidence of causality and identity of microbiome-derived compounds that mediate gut-brain axis interaction remain elusive. Here, we identify two previously unknown bacterial metabolites 3-methyl-4-(trimethylammonio)butanoate and 4-(trimethylammonio)pentanoate, structural analogs of carnitine that are present in both gut and brain of specific pathogen-free mice but absent in germ-free mice. We demonstrate that these compounds are produced by anaerobic commensal bacteria from the family Lachnospiraceae (Clostridiales) family, colocalize with carnitine in brain white matter, and inhibit carnitine-mediated fatty acid oxidation in a murine cell culture model of central nervous system white matter. This is the first description of direct molecular inter-kingdom exchange between gut prokaryotes and mammalian brain cells, leading to inhibition of brain cell function.

PMID: 32195337 [PubMed - indexed for MEDLINE]

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PE/PPE proteins mediate nutrient transport across the outer membrane of Mycobacterium tuberculosis.

Science. 2020 03 06;367(6482):1147-1151

Authors: Wang Q, Boshoff HIM, Harrison JR, Ray PC, Green SR, Wyatt PG, Barry CE

Abstract
Mycobacterium tuberculosis has an unusual outer membrane that lacks canonical porin proteins for the transport of small solutes to the periplasm. We discovered that 3,3-bis-di(methylsulfonyl)propionamide (3bMP1) inhibits the growth of M. tuberculosis, and resistance to this compound is conferred by mutation within a member of the proline-proline-glutamate (PPE) family, PPE51. Deletion of PPE51 rendered M. tuberculosis cells unable to replicate on propionamide, glucose, or glycerol. Growth was restored upon loss of the mycobacterial cell wall component phthiocerol dimycocerosate. Mutants in other proline-glutamate (PE)/PPE clusters, responsive to magnesium and phosphate, also showed a phthiocerol dimycocerosate-dependent growth compromise upon limitation of the corresponding substrate. Phthiocerol dimycocerosate determined the low permeability of the mycobacterial outer membrane, and the PE/PPE proteins apparently act as solute-specific channels.

PMID: 32139546 [PubMed - indexed for MEDLINE]

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Harnessing β-Lactam Antibiotics for Illumination of the Activity of Penicillin-Binding Proteins in Bacillus subtilis.

ACS Chem Biol. 2020 Mar 10;:

Authors: Sharifzadeh S, Dempwolff F, Kearns DB, Carlson EE

Abstract
Selective chemical probes enable individual investigation of penicillin-binding proteins (PBPs) and provide critical information about their enzymatic activity with spatial and temporal resolution. To identify scaffolds for novel probes to study peptidoglycan biosynthesis in Bacillus subtilis, we evaluated the PBP inhibition profiles of 21 β-lactam antibiotics from different structural subclasses using a fluorescence-based assay. Most compounds readily labeled PBP1, PBP2a, PBP2b or PBP4. Almost all penicillin scaffolds were co-selective for all or combinations of PBP2a, 2b and 4. Cephalosporins, on the other hand, possessed the lowest IC50 values for PBP1 alone or along with PBP4 (ceftriaxone, cefoxitin), 2b (cefotaxime) or 2a, 2b and 4 (cephalothin). Overall, five selective inhibitors for PBP1 (aztreonam, faropenem, piperacillin, cefuroxime and cefsulodin), one selective inhibitor for PBP5 (6-aminopenicillanic acid) and various co-selective inhibitors for other PBPs in B. subtilis were discovered. Surprisingly, carbapenems strongly inhibited PBP3, formerly shown to have low affinity for β-lactams and speculated to be involved in resistance in B. subtilis. To investigate the specific roles of PBP3, we developed activity-based probes based on the meropenem core and utilized them to monitor the activity of PBP3 in living cells. We showed that PBP3 activity localizes as patches in single cells and concentrates as a ring at the septum and the division site during the cell growth cycle. Our activity-based approach enabled spatial resolution of the transpeptidation activity of individual PBPs in this model microorganism, which was not possible with previous chemical and biological approaches.

PMID: 32155044 [PubMed - as supplied by publisher]

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Protease-dependent excitation of nodose ganglion neurons by commensal gut bacteria.

J Physiol. 2020 Mar 05;:

Authors: Pradhananga S, Tashtush AA, Allen-Vercoe E, Petrof EO, Lomax AE

Abstract
KEY POINTS: The vagus nerve has been implicated in mediating behavioral effects of the gut microbiota on the central nervous system. This study examined whether the secretory products of commensal gut bacteria can modulate the excitability of vagal afferent neurons with cell bodies in nodose ganglia. Cysteine proteases from commensal bacteria increased the excitability of vagal afferent neurons via activation of protease-activated receptor 2 and modulation of the voltage-dependence of Na+ conductance activation. Lipopolysaccharide, a component of the cell wall of Gram-negative bacteria, increased the excitability of nodose ganglia neurons via TLR4-dependent activation of nuclear factor kappa B. Our study identified potential mechanisms by which gut microbiota influences the activity of vagal afferent pathways, which may in turn impact autonomic reflexes and behaviour.
ABSTRACT: Behavioural studies have implicated vagal afferent neurons as an important component of the microbiota-gut-brain axis. However, the mechanisms underlying the ability of the gut microbiota to affect vagal afferent pathways are unclear. We examined the effect of supernatant from a community of 33 commensal gastrointestinal bacterial derived from a healthy human donor (microbial ecosystem therapeutics; MET-1) on the excitability of mouse vagal afferent neurons. Perforated patch clamp electrophysiology was used to measure the excitability of dissociated nodose ganglion (NG) neurons. NG neuronal excitability was assayed by measuring the amount of current required to elicit an action potential, the rheobase. MET-1 supernatant increased the excitability of NG neurons by hyperpolarizing the voltage-dependence of activation of Na+ conductance. The increase in excitability elicited by MET-1 supernatant was blocked by the cysteine protease inhibitor E-64 (30 nM). The protease activated receptor-2 (PAR2 ) antagonist (GB 83, 10 μM) also blocked the effect of MET-1 supernatant on NG neurons. Supernatant from Lactobacillus paracasei 6MRS, a component of MET-1, recapitulated the effect of MET-1 supernatant on NG neurons. Lastly, we compared the effects of MET-1 supernatant and lipopolysaccharide (LPS) from Escherichia coli 05:B5 on NG neuron excitability. LPS increased the excitability of NG neurons in a toll like receptor 4 (TLR4 )-dependent and PAR2 -independent manner, whereas the excitatory effects of MET-1 supernatant were independent of TLR4 activation. Together, our findings suggest that cysteine proteases from commensal bacteria increase the excitability of vagal afferent neurons by the activation of PAR2 . This article is protected by copyright. All rights reserved.

PMID: 32134496 [PubMed - as supplied by publisher]

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Synthesis of a meso-Oxa-Diaminopimelic Acid Containing Peptidoglycan Pentapeptide and Coupling to the GlcNAc-anhydro-MurNAc Disaccharide.

Org Lett. 2020 Mar 05;:

Authors: Soni AS, Vacariu CM, Chen JY, Tanner ME

Abstract
The syntheses of peptidoglycan (PG)-derived peptides containing meso-diaminopimelic acid (meso-Dap) are typically quite lengthy due to the need to prepare orthogonally protected meso-Dap. In this work, the preparation of the PG pentapeptide containing the isosteric analog meso-oxa-Dap is described. The synthesis relies on the ring opening of a peptide embedded aziridine via the attack of a serine residue. The pentapeptide was attached to a GlcNAc-anhydro-MurNAc disaccharide, to produce a putative substrate for the AmpG pore protein.

PMID: 32133861 [PubMed - as supplied by publisher]

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Involvement of penicillin-binding proteins in the metabolism of a bacterial peptidoglycan containing a non-canonical D-amino acid.

Amino Acids. 2020 Feb 27;:

Authors: Miyamoto T, Katane M, Saitoh Y, Sekine M, Homma H

Abstract
Bacteria produce various D-amino acids, including non-canonical D-amino acids, to adapt to environmental changes and overcome a variety of threats. These D-amino acids are largely utilized as components of peptidoglycan, and they promote peptidoglycan remodeling and biofilm disassembly. The biosynthesis, maturation, and recycling of peptidoglycan are catalyzed by penicillin-binding proteins (PBPs). However, although non-canonical D-amino acids are known to be incorporated into peptidoglycan, the maturation and recycling of peptidoglycan containing such residues remain uncharacterized. Therefore, we investigated whether PBP4 and PBP5, low molecular mass (LMM) PBPs from Escherichia coli and Bacillus subtilis, are involved in these events of peptidoglycan metabolism. Enzyme assays using p-nitroaniline (pNA)-derivatized D-amino acids and peptidoglycan-mimicking peptides revealed that PBP4 and PBP5 from both species have peptidase activity toward substrates containing D-Asn, D-His, or D-Trp. These D-amino acids slowed the growth of dacA- or dacB-deficient E. coli (∆dacA or ∆dacB) relative to the wild-type strain. Additionally, these D-amino acids affected biofilm formation by the ∆dacB strain. Collectively, PBP4 and PBP5 are involved in the cleavage of peptidoglycan containing non-canonical D-amino acids, and these properties affect growth and biofilm formation.

PMID: 32108264 [PubMed - as supplied by publisher]

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Substrate and stereochemical control of peptidoglycan crosslinking by transpeptidation by Escherichia coli PBP1B.

J Am Chem Soc. 2020 Feb 12;:

Authors: Catherwood AC, Lloyd AJ, Tod JA, Chauhan S, Slade SE, Walkowiak G, Galley NF, Punekar A, Smart K, Rea D, Evans ND, Chappell MJ, Roper DI, Dowson CG

Abstract
Penicillin binding proteins (PBPs) catalysing transpeptidation reactions that stabilize the peptidoglycan component of the bacterial cell wall are the targets of beta-lactams, the most clinically successful antibiotics to date. However, PBP-transpeptidation enzymology has evaded detailed analysis, because of the historical unavailability of kinetically competent assays with physiologically relevant substrates and the previously unappreciated contribution of protein cofactors to PBP activity. By re-engineering peptidoglycan synthesis, we have constructed a continuous spectrophotometric assay for transpeptidation of native or near native peptidoglycan precursors and fragments by Escherichia coli PBP1B, allowing us to (a) identify recognition elements of transpeptidase substrates, (b), reveal a novel mechanism of stereochemical editing within peptidoglycan transpeptidation, (c) assess the impact of peptidoglycan substrates on beta-lactam targeting of transpeptidation and (d) demonstrate both substrates have to be bound before transpeptidation occurs. The results allow characterization of high molecular weight PBPs as enzymes and not merely the targets of beta-lactam acylation.

PMID: 32048840 [PubMed - as supplied by publisher]

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Fluorescence Assessment of the AmpR-Signaling Network of Pseudomonas aeruginosa to Exposure to β-Lactam Antibiotics.

ACS Chem Biol. 2020 Jan 28;:

Authors: Dik DA, Kim C, Madukoma CS, Fisher JF, Shrout JD, Mobashery S

Abstract
Gram-negative bacteria have evolved an elaborate pathway to sense and respond to exposure to β-actam antibiotics. The β-actam antibiotics inhibit penicillin-binding proteins, whereby the loss of their activities alters/damages the cell-wall peptidoglycan. Bacteria sense this damage and remove the affected peptidoglycan into complex recycling pathways. As an offshoot of these pathways, muropeptide chemical signals generated from the cell-wall recycling manifest the production of a class C β-actamase, which hydrolytically degrades the β-lactam antibiotic as a resistance mechanism. We disclose the use of a fluorescence probe that detects the activation of the recycling system by the formation of the key muropeptides involved in signaling. This same probe additionally detects natural-product cell-wall-active antibiotics that are produced in situ by cohabitating bacteria.

PMID: 31990176 [PubMed - as supplied by publisher]

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Peptidoglycan reshaping by a noncanonical peptidase for helical cell shape in Campylobacter jejuni.

Nat Commun. 2020 Jan 23;11(1):458

Authors: Min K, An DR, Yoon HJ, Rana N, Park JS, Kim J, Lee M, Hesek D, Ryu S, Kim BM, Mobashery S, Suh SW, Lee HH

Abstract
Assembly of the peptidoglycan is crucial in maintaining viability of bacteria and in defining bacterial cell shapes, both of which are important for existence in the ecological niche that the organism occupies. Here, eight crystal structures for a member of the cell-shape-determining class of Campylobacter jejuni, the peptidoglycan peptidase 3 (Pgp3), are reported. Characterization of the turnover chemistry of Pgp3 reveals cell wall D,D-endopeptidase and D,D-carboxypeptidase activities. Catalysis is accompanied by large conformational changes upon peptidoglycan binding, whereby a loop regulates access to the active site. Furthermore, prior hydrolysis of the crosslinked peptide stem from the saccharide backbone of the peptidoglycan on one side is a pre-requisite for its recognition and turnover by Pgp3. These analyses reveal the noncanonical nature of the transformations at the core of the events that define the morphological shape for C. jejuni as an intestinal pathogen.

PMID: 31974386 [PubMed - in process]

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Structure and function of Listeria teichoic acids and their implications.

Mol Microbiol. 2020 Jan 23;:

Authors: Sumrall ET, Keller AP, Shen Y, Loessner MJ

Abstract
Teichoic acids (TAs) are the most abundant glycopolymers in the cell wall of Listeria, an opportunistic Gram-positive pathogen that causes severe foodborne infections. Two different structural classes of Listeria TA exist: the polyribitolphosphate-based wall-teichoic acid (WTA) that is covalently anchored to the peptidoglycan, and the polyglycerolphosphate-based lipoteichoic acid (LTA) that is tethered to the cytoplasmic membrane. While TA polymers govern many important physiological processes, the diverse glycosylation patterns of WTA result in a high degree of surface variation across the species and serovars of Listeria, which in turn bestows varying effects on fitness, biofilm formation, bacteriophage susceptibility, and virulence. We review the advances made over the past two decades, and our current understanding of the relationship between TA structure and function. We describe the various types of TA that have been structurally determined to date, and discuss the genetic determinants known to be involved in TA glycosylation. We elaborate on surface proteins functionally related to TA decoration, as well as the molecular and analytical tools used to probe TAs. We anticipate that the growing knowledge of Listeria surface chemistry will also be exploited to develop novel diagnostic and therapeutic strategies for this pathogen.

PMID: 31972870 [PubMed - as supplied by publisher]

Distinct cytoskeletal proteins define zones of enhanced cell wall synthesis in Helicobacter pylori.

Elife. 2020 Jan 09;9:

Authors: Taylor JA, Bratton BP, Sichel SR, Blair KM, Jacobs HM, DeMeester KE, Kuru E, Gray J, Biboy J, VanNieuwenhze MS, Vollmer W, Grimes CL, Shaevitz JW, Salama NR

Abstract
Helical cell shape is necessary for efficient stomach colonization by Helicobacter pylori, but the molecular mechanisms for generating helical shape remain unclear. The helical centerline pitch and radius of wild-type H. pylori cells dictate surface curvatures of considerably higher positive and negative Gaussian curvatures than those present in straight- or curved-rod H. pylori. Quantitative 3D microscopy analysis of short pulses with either N-acetylmuramic acid or D-alanine metabolic probes showed that cell wall growth is enhanced at both sidewall curvature extremes. Immunofluorescence revealed MreB is most abundant at negative Gaussian curvature, while the bactofilin CcmA is most abundant at positive Gaussian curvature. Strains expressing CcmA variants with altered polymerization properties lose helical shape and associated positive Gaussian curvatures. We thus propose a model where CcmA and MreB promote PG synthesis at positive and negative Gaussian curvatures, respectively, and that this patterning is one mechanism necessary for maintaining helical shape.

PMID: 31916938 [PubMed - as supplied by publisher]

Nature Communications, Published online: 22 January 2020; doi:10.1038/s41467-020-14321-0

Selectively inhibiting N-Methyl-D-aspartate receptors (NMDARs) containing the GluN2C/2D subunits has been challenging. Here, using electrophysiology and X-ray crystallography, authors show that compounds UBP791 and UBP1700 show over 40- and 50-fold selectivity for GluN2C/2D compared to GluN2A.

Pseudomonas aeruginosa rapidly adapts to altered conditions by quorum sensing (QS), a communication system that it uses to collectively modify its behavior through the production, release, and detection of signaling molecules. QS molecules can also be sensed by hosts, although the respective receptors and signaling pathways are poorly understood. We describe a pattern of regulation in the host by the aryl hydrocarbon receptor (AhR) that is critically dependent on qualitative and quantitative sensing of P. aeruginosa quorum. QS molecules bind to AhR and distinctly modulate its activity. This is mirrored upon infection with P. aeruginosa collected from diverse growth stages and with QS mutants. We propose that by spying on bacterial quorum, AhR acts as a major sensor of infection dynamics, capable of orchestrating host defense according to the status quo of infection.

Nature Reviews Microbiology, Published online: 14 November 2019; doi:10.1038/s41579-019-0273-7

Mycobacterium tuberculosis has a distinctive cell envelope that contributes to its resistance against the human immune system and antibiotic therapy. In this Review, Dulberger, Rubin and Boutte discuss mycobacterial cell envelope dynamics and their relevance for infection and drug treatment.

Cysteine‐selective fluorogenic probes enable the fluorescence‐based screening of inhibitors of Ldt_Mt2, a transpeptidase antibiotic target in M. tuberculosis. The assay, which is amenable to high‐throughput screening, demonstrates the efficacy of the penem and carbapenem classes of β‐lactam antibiotics.

Abstract

Mycobacterium tuberculosis l,d‐transpeptidases (Ldts), which are involved in cell‐wall biosynthesis, have emerged as promising targets for the treatment of tuberculosis. However, an efficient method for testing inhibition of these enzymes is not currently available. We present a fluorescence‐based assay for Ldt_Mt2, which is suitable for high‐throughput screening. Two fluorogenic probes were identified that release a fluorophore upon reaction with Ldt_Mt2, thus making it possible to assess the availability of the catalytic site in the presence of inhibitors. The assay was applied to a panel of β‐lactam antibiotics and related inhibitors; the results validate observations that the (carba)penem subclass of β‐lactams are more potent Ldt inhibitors than other β‐lactam classes, though unexpected variations in potency were observed. The method will enable systematic structure–activity relationship studies on Ldts, thereby facilitating the identification of new antibiotics active against M. tuberculosis.

The nucleotide oligomerization domain (NOD)–like receptors 1 and 2 (NOD1/2) are intracellular pattern-recognition proteins that activate immune signaling pathways in response to peptidoglycans associated with microorganisms. Recruitment to bacteria-containing endosomes and other intracellular membranes is required for NOD1/2 signaling, and NOD1/2 mutations that disrupt membrane localization are associated with inflammatory bowel disease and other inflammatory conditions. However, little is known about this recruitment process. We found that NOD1/2 S-palmitoylation is required for membrane recruitment and immune signaling. ZDHHC5 was identified as the palmitoyltransferase responsible for this critical posttranslational modification, and several disease-associated mutations in NOD2 were found to be associated with defective S-palmitoylation. Thus, ZDHHC5-mediated S-palmitoylation of NOD1/2 is critical for their ability to respond to peptidoglycans and to mount an effective immune response.