Alexis Apostolos

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Chemical tools to characterize peptidoglycan synthases.

Curr Opin Chem Biol. 2019 Aug 26;53:44-50

Authors: Taguchi A, Kahne D, Walker S

Abstract
The peptidoglycan cell wall is a unique macromolecular structure in bacteria that defines their shape and confers protection from the surrounding environment. Decades of research has focused on understanding the peptidoglycan synthesis pathway and exploiting its essentiality for antibiotic development. Recently, a new class of peptidoglycan polymerases known as the SEDS (shape, elongation, division and sporulation) proteins were identified; these polytopic membrane proteins function together with the better-known penicillin-binding proteins (PBPs) to build the cell wall. In this review, we will highlight recent developments in chemical tools and methods to label the bacterial cell wall and discuss how these developments are leading to a better understanding of peptidoglycan synthases and their cellular roles.

PMID: 31466035 [PubMed - as supplied by publisher]

Publication date: 14 August 2019

Source: Cell Host & Microbe, Volume 26, Issue 2

Author(s): Won Jong Kim, Dustin Higashi, Maira Goytia, Maria A. Rendón, Michelle Pilligua-Lucas, Matthew Bronnimann, Jeanine A. McLean, Joseph Duncan, David Trees, Ann E. Jerse, Magdalene So

Summary

Graphical Abstract

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A Fluorescence-based Assay for Screening β-Lactams Targeting the Mycobacterium tuberculosis Transpeptidase LdtMt2.

Chembiochem. 2019 Jul 19;:

Authors: de Munnik M, Lohans CT, Langley GW, Bon C, Brem J, Schofield C

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 LdtMt2, which is suitable for high-throughput screening. Two fluorogenic probes were identified that release a fluorophore upon reaction with LdtMt2, 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, facilitating the identification of new antibiotics active against M. tuberculosis.

PMID: 31322798 [PubMed - as supplied by publisher]

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The Cell Wall Hydrolytic NlpC/P60 Endopeptidases in Mycobacterial Cytokinesis: A Structural Perspective.

Cells. 2019 Jun 18;8(6):

Authors: Squeglia F, Moreira M, Ruggiero A, Berisio R

Abstract
In preparation for division, bacteria replicate their DNA and segregate the newly formed chromosomes. A division septum then assembles between the chromosomes, and the mother cell splits into two identical daughters due to septum degradation. A major constituent of bacterial septa and of the whole cell wall is peptidoglycan (PGN), an essential cell wall polymer, formed by glycan chains of β-(1-4)-linked-N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc), cross-linked by short peptide stems. Depending on the amino acid located at the third position of the peptide stem, PGN is classified as either Lys-type or meso-diaminopimelic acid (DAP)-type. Hydrolytic enzymes play a crucial role in the degradation of bacterial septa to split the cell wall material shared by adjacent daughter cells to promote their separation. In mycobacteria, a key PGN hydrolase, belonging to the NlpC/P60 endopeptidase family and denoted as RipA, is responsible for the degradation of septa, as the deletion of the gene encoding for this enzyme generates abnormal bacteria with multiple septa. This review provides an update of structural and functional data highlighting the central role of RipA in mycobacterial cytokinesis and the fine regulation of its catalytic activity, which involves multiple molecular partners.

PMID: 31216697 [PubMed]

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The hydrolase LpqI primes mycobacterial peptidoglycan recycling.

Nat Commun. 2019 Jun 14;10(1):2647

Authors: Moynihan PJ, Cadby IT, Veerapen N, Jankute M, Crosatti M, Mukamolova GV, Lovering AL, Besra GS

Abstract
Growth and division by most bacteria requires remodelling and cleavage of their cell wall. A byproduct of this process is the generation of free peptidoglycan (PG) fragments known as muropeptides, which are recycled in many model organisms. Bacteria and hosts can harness the unique nature of muropeptides as a signal for cell wall damage and infection, respectively. Despite this critical role for muropeptides, it has long been thought that pathogenic mycobacteria such as Mycobacterium tuberculosis do not recycle their PG. Herein we show that M. tuberculosis and Mycobacterium bovis BCG are able to recycle components of their PG. We demonstrate that the core mycobacterial gene lpqI, encodes an authentic NagZ β-N-acetylglucosaminidase and that it is essential for PG-derived amino sugar recycling via an unusual pathway. Together these data provide a critical first step in understanding how mycobacteria recycle their peptidoglycan.

PMID: 31201321 [PubMed - in process]

Nature Microbiology, Published online: 20 May 2019; doi:10.1038/s41564-019-0454-1

Legionella pneumophila effector RavD is a deubiquitinase with specificity for linear ubiquitin that inhibits host inflammatory signalling during infection.

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Tryptophan fluorescence quenching in β-lactam-interacting proteins is modulated by the structure of intermediates and final products of the acylation reaction.

ACS Infect Dis. 2019 May 06;:

Authors: Triboulet S, Edoo Z, Compain F, Ourghanlian C, Dupuis A, Dubée V, Sütterlin L, Atze H, Etheve-Quelquejeu M, Hugonnet JE, Arthur M

Abstract
In most bacteria, β-lactam antibiotics inhibit the last cross-linking step of peptidoglycan synthesis by acylation of the active-site Ser of D,D-transpeptidases belonging to the penicillin-binding protein (PBP) family. In mycobacteria, cross-linking is mainly ensured by L,D-transpeptidases (LDTs), which are promising targets for the development of β-lactam-based therapies for multidrug-resistant tuberculosis. For this purpose, fluorescence spectroscopy is used to investigate the efficacy of LDT inactivation by β-lactams but the basis for fluorescence quenching during enzyme acylation remains unknown. In contrast to what has been reported for PBPs, we show here using a model L,D-transpeptidase (Ldtfm) that fluorescence quenching of Trp residues does not depend upon direct hydrophobic interaction between Trp residues and β-lactams. Rather, Trp fluorescence was quenched by the drug covalently bound to the active-site Cys residue of Ldtfm. Fluorescence quenching was not quantitatively determined by the size of the drug and was not specific of the thioester link connecting the β-lactam carbonyl to the catalytic Cys as quenching was also observed for acylation of the active-site Ser of β-lactamase BlaC from M. tuberculosis. Fluorescence quenching was extensive for reaction intermediates containing an amine anion and for acylenzymes containing an imine stabilized by mesomeric effect, but not for acylenzymes containing a protonated β-lactam nitrogen. Together, these results indicate that the extent of fluorescence quenching is determined by the status of the β-lactam nitrogen. Thus, fluorescence kinetics can provide information not only on the efficacy of enzyme inactivation but also on the structure of the covalent adducts responsible for enzyme inactivation.

PMID: 31056908 [PubMed - as supplied by publisher]

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Structural insight into YcbB-mediated beta-lactam resistance in Escherichia coli.

Nat Commun. 2019 Apr 23;10(1):1849

Authors: Caveney NA, Caballero G, Voedts H, Niciforovic A, Worrall LJ, Vuckovic M, Fonvielle M, Hugonnet JE, Arthur M, Strynadka NCJ

Abstract
The bacterial cell wall plays a crucial role in viability and is an important drug target. In Escherichia coli, the peptidoglycan crosslinking reaction to form the cell wall is primarily carried out by penicillin-binding proteins that catalyse D,D-transpeptidase activity. However, an alternate crosslinking mechanism involving the L,D-transpeptidase YcbB can lead to bypass of D,D-transpeptidation and beta-lactam resistance. Here, we show that the crystallographic structure of YcbB consists of a conserved L,D-transpeptidase catalytic domain decorated with a subdomain on the dynamic substrate capping loop, peptidoglycan-binding and large scaffolding domains. Meropenem acylation of YcbB gives insight into the mode of inhibition by carbapenems, the singular antibiotic class with significant activity against L,D-transpeptidases. We also report the structure of PBP5-meropenem to compare interactions mediating inhibition. Additionally, we probe the interaction network of this pathway and assay beta-lactam resistance in vivo. Our results provide structural insights into the mechanism of action and the inhibition of L,D-transpeptidation, and into YcbB-mediated antibiotic resistance.

PMID: 31015395 [PubMed - in process]

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Peptidoglycan hydrolase of an unusual cross-link cleavage specificity contributes to bacterial cell wall synthesis.

Proc Natl Acad Sci U S A. 2019 Apr 02;:

Authors: Chodisetti PK, Reddy M

Abstract
Bacteria are surrounded by a protective exoskeleton, peptidoglycan (PG), a cross-linked mesh-like macromolecule consisting of glycan strands interlinked by short peptides. Because PG completely encases the cytoplasmic membrane, cleavage of peptide cross-links is a prerequisite to make space for incorporation of nascent glycan strands for its successful expansion during cell growth. In most bacteria, the peptides consist of l-alanine, d-glutamate, meso-diaminopimelic acid (mDAP) and d-alanine (d-Ala) with cross-links occurring either between d-Ala and mDAP or two mDAP residues. In Escherichia coli, the d-Ala-mDAP cross-links whose cleavage by specialized endopeptidases is crucial for expansion of PG predominate. However, a small proportion of mDAP-mDAP cross-links also exist, yet their role in the context of PG expansion or the hydrolase(s) capable of catalyzing their cleavage is not known. Here, we identified an ORF of unknown function, YcbK (renamed MepK), as an mDAP-mDAP cross-link cleaving endopeptidase working in conjunction with other elongation-specific endopeptidases to make space for efficient incorporation of nascent PG strands into the sacculus. E. coli mutants lacking mepK and another d-Ala-mDAP-specific endopeptidase (mepS) were synthetic sick, and the defects were abrogated by lack of l,d-transpeptidases, enzymes catalyzing the formation of mDAP cross-links. Purified MepK was able to cleave the mDAP cross-links of soluble muropeptides and of intact PG sacculi. Overall, this study describes a PG hydrolytic enzyme with a hitherto unknown substrate specificity that contributes to expansion of the PG sacculus, emphasizing the fundamental importance of cross-link cleavage in bacterial peptidoglycan synthesis.

PMID: 30940749 [PubMed - as supplied by publisher]

Bacteriophage are abundant at sites of bacterial infection, but their effects on mammalian hosts are unclear. We have identified pathogenic roles for filamentous Pf bacteriophage produced by Pseudomonas aeruginosa (Pa) in suppression of immunity against bacterial infection. Pf promote Pa wound infection in mice and are associated with chronic human Pa wound infections. Murine and human leukocytes endocytose Pf, and internalization of this single-stranded DNA virus results in phage RNA production. This triggers Toll-like receptor 3 (TLR3)– and TIR domain–containing adapter-inducing interferon-β (TRIF)–dependent type I interferon production, inhibition of tumor necrosis factor (TNF), and the suppression of phagocytosis. Conversely, immunization of mice against Pf prevents Pa wound infection. Thus, Pf triggers maladaptive innate viral pattern-recognition responses, which impair bacterial clearance. Vaccination against phage virions represents a potential strategy to prevent bacterial infection.

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Lipoprotein Signal Peptidase Inhibitors with Antibiotic Properties Identified through Design of a Robust In Vitro HT Platform.

Cell Chem Biol. 2018 03 15;25(3):301-308.e12

Authors: Kitamura S, Owensby A, Wall D, Wolan DW

Abstract
As resistance to antibiotics increases, the exploration of new targets and strategies to combat pathogenic bacteria becomes more urgent. Ideal protein targets are required for viability across many species, are unique to prokaryotes to limit effects on the host, and have robust assays to quantitate activity and identify inhibitors. Lipoprotein signal peptidase (Lsp) is a transmembrane aspartyl protease required for lipoprotein maturation and comprehensively fits these criteria. Here, we have developed the first in vitro high-throughput assay to monitor proteolysis by Lsp. We employed our high-throughput screen assay against 646,275 compounds to discover inhibitors of Lsp and synthesized a range of analogs to generate molecules with nanomolar half maximal inhibitory concentration values. Importantly, our inhibitors are effective in preventing the growth of E. coli cultures in the presence of outer-membrane permeabilizer PMBN and should facilitate development of antibacterial agents with a novel mechanism of action to treat antibiotic-resistant bacteria.

PMID: 29337186 [PubMed - indexed for MEDLINE]

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Deficiency of D-alanyl-D-alanine ligase A attenuated cell division and greatly altered the proteome of Mycobacterium smegmatis.

Microbiologyopen. 2019 Mar 03;:e819

Authors: Chen Y, Xu Y, Yang S, Li S, Ding W, Zhang W

Abstract
D-Alanyl-D-alanine ligase A (DdlA) catalyses the dimerization of two D-alanines yielding D-alanyl-D-alanine required for mycobacterial peptidoglycan biosynthesis, and is a promising antimycobacterial drug target. To better understand the roles of DdlA in mycobacteria in vivo, we established a cell model in which DdlA expression was specifically downregulated by ddlA antisense RNA by introducing a 380 bp ddlA fragment into pMind followed by transforming the construct into nonpathogenic Mycobacterium smegmatis. The M. smegmatis cell model was verified by plotting the growth inhibition curves and quantifying endogenous DdlA expression using a polyclonal anti-DdlA antibody produced from the expressed DdlA. Scanning electron microscopy and transmission electron microscopy were used to investigate mycobacterial morphology. Bidimensional gel electrophoresis and mass spectrometry were used to analyze differentially expressed proteins. Consequently, the successful construction of the M. smegmatis cell model was verified. The morphological investigation of the model indicated that DdlA deficiency led to an increased number of Z rings and a rearrangement of intracellular content, including a clear nucleoid and visible filamentous DNA. Proteomic techniques identified six upregulated and 14 downregulated proteins that interacted with each other to permit cell survival by forming a regulatory network under DdlA deficiency. Finally, our data revealed that DdlA deficiency inhibited cell division in mycobacteria and attenuated the process of carbohydrate catabolism and the pathway of fatty acid anabolism, while maintaining active protein degradation and synthesis. N-Nitrosodimethylamine (NDMA)-dependent methanol dehydrogenase (MSMEG_6242) and fumonisin (MSMEG_1419) were identified as potential antimycobacterial drug targets.

PMID: 30828981 [PubMed - as supplied by publisher]

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Extra Sugar on Vancomycin: New Analogues for Combating Multidrug-Resistant Staphylococcus aureus and Vancomycin-Resistant Enterococci.

J Med Chem. 2018 01 11;61(1):286-304

Authors: Guan D, Chen F, Xiong L, Tang F, Faridoon, Qiu Y, Zhang N, Gong L, Li J, Lan L, Huang W

Abstract
Lipophilic substitution on vancomycin is an effective strategy for the development of novel vancomycin analogues against drug-resistant bacteria by enhancing bacterial cell wall interactions. However, hydrophobic structures usually lead to long elimination half-life and accumulative toxicity; therefore, hydrophilic fragments were also introduced to the lipo-vancomycin to regulate their pharmacokinetic/pharmacodynamic properties. Here, we synthesized a series of new vancomycin analogues carrying various sugar moieties on the seventh-amino acid phenyl ring and lipophilic substitutions on vancosamine with extensive structure-activity relationship analysis. The optimal analogues indicated 128-1024-fold higher activity against methicillin-susceptible S. aureus, vancomycin-intermediate resistant S. aureus (VISA), and vancomycin-resistant Enterococci (VRE) compared with that of vancomycin. In vivo pharmacokinetics studies demonstrated the effective regulation of extra sugar motifs, which shortened the half-life and addressed concerns of accumulative toxicity of lipo-vancomycin. This work presents an effective strategy for lipo-vancomycin derivative design by introducing extra sugars, which leads to better antibiotic-like properties of enhanced efficacy, optimal pharmacokinetics, and lower toxicity.

PMID: 29243921 [PubMed - indexed for MEDLINE]

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Recognition of Peptidoglycan Fragments by the Transpeptidase PBP4 From Staphylococcus aureus.

Front Microbiol. 2018;9:3223

Authors: Maya-Martinez R, Alexander JAN, Otten CF, Ayala I, Vollmer D, Gray J, Bougault CM, Burt A, Laguri C, Fonvielle M, Arthur M, Strynadka NCJ, Vollmer W, Simorre JP

Abstract
Peptidoglycan (PG) is an essential component of the cell envelope, maintaining bacterial cell shape and protecting it from bursting due to turgor pressure. The monoderm bacterium Staphylococcus aureus has a highly cross-linked PG, with ~90% of peptide stems participating in DD-cross-links and up to 15 peptide stems connected with each other. These cross-links are formed in transpeptidation reactions catalyzed by penicillin-binding proteins (PBPs) of classes A and B. Most S. aureus strains have three housekeeping PBPs with this function (PBP1, PBP2, and PBP3) but MRSA strains have acquired a third class B PBP, PBP2a, which is encoded by the mecA gene and required for the expression of high-level resistance to β-lactams. Another housekeeping PBP of S. aureus is PBP4, which belongs to the class C PBPs, and hence would be expected to have PG hydrolase (DD-carboxypeptidase or DD-endopeptidase) activity. However, previous works showed that, unexpectedly, PBP4 has transpeptidase activity that significantly contributes to both the high level of cross-linking in the PG of S. aureus and to the low level of β-lactam resistance in the absence of PBP2a. To gain insights into this unusual activity of PBP4, we studied by NMR spectroscopy its interaction in vitro with different substrates, including intact peptidoglycan, synthetic peptide stems, muropeptides, and long glycan chains with uncross-linked peptide stems. PBP4 showed no affinity for the complex, intact peptidoglycan or the smallest isolated peptide stems. Transpeptidase activity of PBP4 was verified with the disaccharide peptide subunits (muropeptides) in vitro, producing cyclic dimer and multimer products; these assays also showed a designed PBP4(S75C) nucleophile mutant to be inactive. Using this inactive but structurally highly similar variant, liquid-state NMR identified two interaction surfaces in close proximity to the central nucleophile position that can accommodate the potential donor and acceptor stems for the transpeptidation reaction. A PBP4:muropeptide model structure was built from these experimental restraints, which provides new mechanistic insights into mecA independent resistance to β-lactams in S. aureus.

PMID: 30713527 [PubMed]

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Zn(II) mediates vancomycin polymerization and potentiates its antibiotic activity against resistant bacteria.

Sci Rep. 2017 07 07;7(1):4893

Authors: Zarkan A, Macklyne HR, Chirgadze DY, Bond AD, Hesketh AR, Hong HJ

Abstract
Vancomycin is known to bind to Zn(II) and can induce a zinc starvation response in bacteria. Here we identify a novel polymerization of vancomycin dimers by structural analysis of vancomycin-Zn(II) crystals and fibre X-ray diffraction. Bioassays indicate that this structure is associated with an increased antibiotic activity against bacterial strains possessing high level vancomycin resistance mediated by the reprogramming of peptidoglycan biosynthesis to use precursors terminating in D-Ala-D-Lac in place of D-Ala-D-Ala. Polymerization occurs via interaction of Zn(II) with the N-terminal methylleucine group of vancomycin, and we show that the activity of other glycopeptide antibiotics with this feature can also be similarly augmented by Zn(II). Construction and analysis of a model strain predominantly using D-Ala-D-Lac precursors for peptidoglycan biosynthesis during normal growth supports the hypothesis that Zn(II) mediated vancomycin polymerization enhances the binding affinity towards these precursors.

PMID: 28687742 [PubMed - indexed for MEDLINE]

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Peptidoglycan precursor synthesis along the sidewall of pole-growing mycobacteria.

Elife. 2018 09 10;7:

Authors: García-Heredia A, Pohane AA, Melzer ES, Carr CR, Fiolek TJ, Rundell SR, Chuin Lim H, Wagner JC, Morita YS, Swarts BM, Siegrist MS

Abstract
Rod-shaped mycobacteria expand from their poles, yet d-amino acid probes label cell wall peptidoglycan in this genus at both the poles and sidewall. We sought to clarify the metabolic fates of these probes. Monopeptide incorporation was decreased by antibiotics that block peptidoglycan synthesis or l,d-transpeptidation and in an l,d-transpeptidase mutant. Dipeptides complemented defects in d-alanine synthesis or ligation and were present in lipid-linked peptidoglycan precursors. Characterizing probe uptake pathways allowed us to localize peptidoglycan metabolism with precision: monopeptide-marked l,d-transpeptidase remodeling and dipeptide-marked synthesis were coincident with mycomembrane metabolism at the poles, septum and sidewall. Fluorescent pencillin-marked d,d-transpeptidation around the cell perimeter further suggested that the mycobacterial sidewall is a site of cell wall assembly. While polar peptidoglycan synthesis was associated with cell elongation, sidewall synthesis responded to cell wall damage. Peptidoglycan editing along the sidewall may support cell wall robustness in pole-growing mycobacteria.

PMID: 30198841 [PubMed - indexed for MEDLINE]

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Differential modes of crosslinking establish spatially distinct regions of peptidoglycan in Caulobacter crescentus.

Mol Microbiol. 2019 04;111(4):995-1008

Authors: Stankeviciute G, Miguel AV, Radkov A, Chou S, Huang KC, Klein EA

Abstract
The diversity of cell shapes across the bacterial kingdom reflects evolutionary pressures that have produced physiologically important morphologies. While efforts have been made to understand the regulation of some prototypical cell morphologies such as that of rod-shaped Escherichia coli, little is known about most cell shapes. For Caulobacter crescentus, polar stalk synthesis is tied to its dimorphic life cycle, and stalk elongation is regulated by phosphate availability. Based on the previous observation that C. crescentus stalks are lysozyme-resistant, we compared the composition of the peptidoglycan cell wall of stalks and cell bodies and identified key differences in peptidoglycan crosslinking. Cell body peptidoglycan contained primarily DD-crosslinks between meso-diaminopimelic acid and D-alanine residues, whereas stalk peptidoglycan had more LD-transpeptidation (meso-diaminopimelic acid-meso-diaminopimelic acid), mediated by LdtD. We determined that ldtD is dispensable for stalk elongation; rather, stalk LD-transpeptidation reflects an aging process associated with low peptidoglycan turnover in the stalk. We also found that lysozyme resistance is a structural consequence of LD-crosslinking. Despite no obvious selection pressure for LD-crosslinking or lysozyme resistance in C. crescentus, the correlation between these two properties was maintained in other organisms, suggesting that DAP-DAP crosslinking may be a general mechanism for regulating bacterial sensitivity to lysozyme.

PMID: 30614079 [PubMed - indexed for MEDLINE]

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Cell Wall Hydrolytic Enzymes Enhance Antimicrobial Drug Activity Against Mycobacterium.

Curr Microbiol. 2019 Jan 02;:

Authors: Gustine JN, Au MB, Haserick JR, Hett EC, Rubin EJ, Gibson FC, Deng LL

Abstract
Cell wall hydrolases are enzymes that cleave bacterial cell walls by hydrolyzing specific bonds within peptidoglycan and other portions of the envelope. Two major sources of hydrolases in nature are from hosts and microbes. This study specifically investigated whether cell wall hydrolytic enzymes could be employed as exogenous reagents to augment the efficacy of antimicrobial agents against mycobacteria. Mycobacterium smegmatis cultures were treated with ten conventional antibiotics and six anti-tuberculosis drugs-alone or in combination with cell wall hydrolases. Culture turbidity, colony-forming units (CFUs), vital staining, and oxygen consumption were all monitored. The majority of antimicrobial agents tested alone only had minimal inhibitory effects on bacterial growth. However, the combination of cell wall hydrolases and most of the antimicrobial agents tested, revealed a synergistic effect that resulted in significant enhancement of bactericidal activity. Vital staining showed increased cellular damage when M. smegmatis and Mycobacterium bovis bacillus Calmette-Guérin (M. bovis BCG) were treated with both drug and lysozyme. Respiration analysis revealed stress responses when cells were treated with lysozyme and drugs individually, and an acute increase in oxygen consumption when treated with both drug and lysozyme. Similar trends were also observed for the other three enzymes (hydrolase-30, RipA-His6 and RpfE-His6) evaluated. These findings demonstrated that cell wall hydrolytic enzymes, as a group of biological agents, have the capability to improve the potency of many current antimicrobial drugs and render ineffective antibiotics effective in killing mycobacteria. This combinatorial approach may represent an important strategy to eliminate drug-resistant bacteria.

PMID: 30603964 [PubMed - as supplied by publisher]

Antibiotics stimulate vesicles formation in Staphylococcus aureus in a phage-dependent and independent fashion and via different routes.

Antimicrob Agents Chemother. 2018 Dec 03;:

Authors: Andreoni F, Toyofuku M, Menzi C, Kalawong R, Mairpady Shambat S, François P, Zinkernagel AS, Eberl L

Abstract
Bacterial membrane vesicles research has so far mainly focussed on Gram-negative bacteria. Only recently Gram-positive bacteria have been demonstrated to produce and release extracellular membrane vesicles (MVs) that contribute to bacterial virulence. Although treatment of bacteria with antibiotics is a well-established trigger of bacterial MVs formation, the underlying mechanisms are poorly understood. In this study we show that antibiotics can induce MVs through different routes in the important human pathogen Staphylococcus aureus DNA damaging agents and antibiotics inducing the SOS response triggered vesicle formation in lysogenic strains of S. aureus but not in their phage-devoid counterparts. β-lactam antibiotics flucloxacillin and ceftaroline increased vesicle formation in a prophage-independent manner by weakening the peptidoglycan layer. We present evidence that the amount of DNA associated with MVs formed by phage lysis is higher than that of MVs formed by β-lactam antibiotics-induced blebbing. The purified MVs derived from S. aureus protected the bacteria from challenge with daptomycin, a membrane-targeting antibiotic, both in vitro and ex vivo in whole blood. In addition, the MVs protected S. aureus from killing in whole blood, indicating that antibiotic-induced MVs function as a decoy and thereby contribute to the survival of the bacterium.

PMID: 30509943 [PubMed - as supplied by publisher]

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Enhancing Anti-Proliferative Activity and Selectivity of a FLT-3 Inhibitor by PROTAC Conversion.

J Am Chem Soc. 2018 Nov 14;:

Authors: Burslem GM, Song J, Chen X, Hines J, Crews CM

Abstract
The receptor tyrosine kinase FLT-3 is frequently mutated in acute myeloid leukemia, however current small molecule inhibitors suffer from limited efficacy in the clinic. Conversion of a FLT-3 inhibitor (quizartinib) into a proteolysis targeting chimera (PROTAC) results in a compound which induces degradation of FLT-3 ITD mutant at low nanomolar concentrations. Furthermore, the PROTAC is capable of inhibiting cell growth more potently than the warhead alone while inhibiting fewer off-target kinases. This enhanced anti-proliferative activity occurs, despite a slight reduction in the PROTAC's kinase inhibitory activity, via an increased level of apoptosis induction suggesting non-kinase roles for the FLT-3 ITD protein. Additionally, the PROTAC is capable of inducing FLT-3 ITD degradation in vivo. These results suggest that degradation of FLT-3 ITD may provide a useful method for therapeutic intervention.

PMID: 30427680 [PubMed - as supplied by publisher]

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The structures of penicillin binding protein 4 (PBP4) and PBP5 from Enterococci provide structural insights into β-lactam resistance.

J Biol Chem. 2018 Oct 24;:

Authors: Moon TM, D'Andréa ÉD, Lee CW, Soares A, Jakoncic J, Desbonnet C, Solache MG, Rice LB, Page R, Peti W

Abstract
The final steps of cell-wall biosynthesis in bacteria are carried out by penicillin-binding proteins (PBPs), whose transpeptidase domains form the crosslinks in peptidoglycan chains that define the bacterial cell wall. These enzymes are the targets of β-lactam antibiotics, as their inhibition reduces the structural integrity of the cell wall. Bacterial resistance to antibiotics is a rapidly growing concern; however, the structural underpinnings of PBP-derived antibiotic resistance are poorly understood. PBP4 and PBP5 are low-affinity, class B transpeptidases that confer antibiotic resistance to Enterococcus faecalis and Enterococcus faecium, respectively. Here, we report the crystal structures of PBP4 (1.8 Å) and PBP5 (2.7 Å) in their apo and acyl-enzyme complexes with the β-lactams benzylpenicillin, imipenem and ceftaroline. We found that, although these three β-lactams adopt geometries similar to those observed in other class B PBP structures, there are small, but significant differences that likely decrease antibiotic efficacy. Further, we also discovered that the N-terminal domain extensions in this class of PBPs undergo large rigid-body rotations without impacting the structure of the catalytic transpeptidase domain. Together, our findings are defining the subtle functional and structural differences in the enterococcus PBPs that allow them to support transpeptidase activity while also conferring bacterial resistance to antibiotics that function as substrate mimics.

PMID: 30355734 [PubMed - as supplied by publisher]

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Maturing Mycobacterium smegmatis peptidoglycan requires non-canonical crosslinks to maintain shape.

Elife. 2018 Oct 16;7:

Authors: Baranowski C, Welsh MA, Sham LT, Eskandarian HA, Lim HC, Kieser KJ, Wagner JC, McKinney J, Fantner GE, Ioerger TR, Walker S, Bernhardt TG, Rubin EJ, Rego EH

Abstract
In most well studied rod-shaped bacteria, peptidoglycan is primarily crosslinked by penicillin-binding proteins (PBPs). However, in mycobacteria, crosslinks formed by L,D-transpeptidases (LDTs) are highly abundant. To elucidate the role of these unusual crosslinks, we characterized Mycobacterium smegmatis cells lacking all LDTs. We find that crosslinks generate by LDTs are required for rod shape maintenance specifically at sites of aging cell wall, a byproduct of polar elongation. Asymmetric polar growth leads to a non-uniform distribution of these two types of crosslinks in a single cell. Consequently, in the absence of LDT-mediated crosslinks, PBP-catalyzed crosslinks become more important. Because of this, Mycobacterium tuberculosis (Mtb) is more rapidly killed using a combination of drugs capable of PBP- and LDT- inhibition. Thus, knowledge about the spatial and genetic relationship between drug targets can be exploited to more effectively treat this pathogen.

PMID: 30324906 [PubMed - as supplied by publisher]

Peptidoglycan editing by a specific ld-transpeptidase controls the muramidase-dependent secretion of typhoid toxin

Peptidoglycan editing by a specific <span class="small-caps">ld</span>-transpeptidase controls the muramidase-dependent secretion of typhoid toxin, Published online: 24 September 2018; doi:10.1038/s41564-018-0248-x

The secretion of Salmonella Typhi typhoid toxin requires translocation across the peptidoglycan layer through the action of a ld-transpeptidase. Subsequent outer membrane perturbation fully releases toxin for host cell intoxication.

Mycobacterium tuberculosis carrying a rifampicin drug resistance mutation reprograms macrophage metabolism through cell wall lipid changes

<i>Mycobacterium tuberculosis</i> carrying a rifampicin drug resistance mutation reprograms macrophage metabolism through cell wall lipid changes, Published online: 17 September 2018; doi:10.1038/s41564-018-0245-0

A prevalent rifampicin resistance mutation in Mycobacterium tuberculosis alters bacterial virulence lipid expression and enables bypasses of a host immune axis that is critical for the control of drug-susceptible infections.

Microbiota-derived short-chain fatty acids promote Th1 cell IL-10 production to maintain intestinal homeostasis

Microbiota-derived short-chain fatty acids promote Th1 cell IL-10 production to maintain intestinal homeostasis, Published online: 03 September 2018; doi:10.1038/s41467-018-05901-2

T cells play a critical role in intestinal homeostasis, with increasing evidence suggesting a role for the microbiome metabolome in modulating this response. Here the authors show short-chain fatty acids promote IL-10 production in Th1 cells.