sep413

Related Articles

β-lactam resistance: The role of low molecular weight penicillin binding proteins, β-lactamases and ld-transpeptidases in bacteria associated with respiratory tract infections.

IUBMB Life. 2018 May 02;:

Authors: Ealand CS, Machowski EE, Kana BD

Abstract
Disruption of peptidoglycan (PG) biosynthesis in the bacterial cell wall by β-lactam antibiotics has transformed therapeutic options for bacterial infections. These antibiotics target the transpeptidase domains in penicillin binding proteins (PBPs), which can be classified into high and low molecular weight (LMW) counterparts. While the essentiality of the former has been extensively demonstrated, the physiological roles of LMW PBPs remain poorly understood. Herein, we review the function of LMW PBPs, β-lactamases and ld-transpeptidases (Ldts) in pathogens associated with respiratory tract infections. More specifically, we explore their roles in mediating β-lactam resistance. Using a comparative genomics approach, we identified a high degree of genetic redundancy for LMW PBPs which retain the motifs, SxxN, SxN and KTG required for catalytic activity. Differences in domain architecture suggest distinct physiological roles, possibly related to bacterial cell cycle and/or adaptation to various environmental conditions. Many of the LMW PBPs play an important role in β-lactam resistance either through mutation or variation in abundance. In all of the bacterial genomes assessed, at least one β-lactamase homologue is present, suggesting that enzymatic degradation of β-lactams is a highly conserved resistance mechanism. Furthermore, the presence of Ldt homologues in the majority of species surveyed suggests that alternative PG crosslinking may further mediate β-lactam drug resistance. A deeper understanding of the interplay between these different mechanisms of β-lactam resistance will provide a framework for new therapeutics, which are urgently required given the rapid emergence of antimicrobial resistance. © 2018 IUBMB Life, 2018.

PMID: 29717815 [PubMed - as supplied by publisher]

Why does the microbiome affect behaviour?

Why does the microbiome affect behaviour?, Published online: 24 April 2018; doi:10.1038/s41579-018-0014-3

The microbiota can influence host behaviour through the gut–brain axis. In this Opinion, Johnson and Foster explore the evolution of this relationship and propose that adaptations of competing gut microorganisms may affect behaviour as a by‑product, leading to host dependence.

Antibiotic resistance has become a global public health priority. Polymyxins, a family of cationic polypeptide antibiotics, act as a final line of refuge against severe infections by Gram-negative pathogens with pan-drug resistance. Unfortunately, this last-resort antibiotic has been challenged by the emergence and global spread of mobilized colistin resistance determinants (mcr). Given the fact that it has triggered extensive concerns worldwide, we present here an updated view of MCR-like colistin resistance.

Related Articles

Mode of Action and Heterologous Expression of the Natural Product Antibiotic Vancoresmycin.

ACS Chem Biol. 2018 01 19;13(1):207-214

Authors: Kepplinger B, Morton-Laing S, Seistrup KH, Marrs ECL, Hopkins AP, Perry JD, Strahl H, Hall MJ, Errington J, Ellis Allenby NE

Abstract
Antibiotics that interfere with the bacterial cytoplasmic membrane have long-term potential for the treatment of infectious diseases as this mode of action is anticipated to result in low resistance frequency. Vancoresmycin is an understudied natural product antibiotic consisting of a terminal tetramic acid moiety fused to a linear, highly oxygenated, stereochemically complex polyketide chain. Vancoresmycin shows minimum inhibitory concentrations (MICs) from 0.125 to 2 μg/mL against a range of clinically relevant, antibiotic-resistant Gram-positive bacteria. Through a comprehensive mode-of-action study, utilizing Bacillus subtilis reporter strains, DiSC3(5) depolarization assays, and fluorescence microscopy, we have shown that vancoresmycin selectively targets the cytoplasmic membrane of Gram-positive bacteria via a non-pore-forming, concentration-dependent depolarization mechanism. Whole genome sequencing of the producing strain allowed identification of the 141 kbp gene cluster encoding for vancoresmycin biosynthesis and a preliminary model for its biosynthesis. The size and complex structure of vancoresmycin could confound attempts to generate synthetic analogues. To overcome this problem and facilitate future studies, we identified, cloned, and expressed the 141 kbp biosynthetic gene cluster in Streptomyces coelicolor M1152. Elucidation of the mode-of-action of vancoresmycin, together with the heterologous expression system, will greatly facilitate further studies of this and related molecules.

PMID: 29185696 [PubMed - indexed for MEDLINE]

Related Articles

Small Molecule Chelators Reveal That Iron Starvation Inhibits Late Stages of Bacterial Cytokinesis.

ACS Chem Biol. 2018 01 19;13(1):235-246

Authors: Santos TMA, Lammers MG, Zhou M, Sparks IL, Rajendran M, Fang D, De Jesus CLY, Carneiro GFR, Cui Q, Weibel DB

Abstract
Bacterial cell division requires identification of the division site, assembly of the division machinery, and constriction of the cell envelope. These processes are regulated in response to several cellular and environmental signals. Here, we use small molecule iron chelators to characterize the surprising connections between bacterial iron homeostasis and cell division. We demonstrate that iron starvation downregulates the transcription of genes encoding proteins involved in cell division, reduces protein biosynthesis, and prevents correct positioning of the division machinery at the division site. These combined events arrest the constriction of the cell during late stages of cytokinesis in a manner distinct from known mechanisms of inhibiting cell division. Overexpression of genes encoding cell division proteins or iron transporters partially suppresses the biological activity of iron chelators and restores growth and division. We propose a model demonstrating the effect of iron availability on the regulatory mechanisms coordinating division in response to the nutritional state of the cell.

PMID: 29227619 [PubMed - indexed for MEDLINE]

Related Articles

The ng_ζ1 toxin of the gonococcal epsilon/zeta toxin/antitoxin system drains precursors for cell wall synthesis.

Nat Commun. 2018 Apr 27;9(1):1686

Authors: Rocker A, Peschke M, Kittilä T, Sakson R, Brieke C, Meinhart A

Abstract
Bacterial toxin-antitoxin complexes are emerging as key players modulating bacterial physiology as activation of toxins induces stasis or programmed cell death by interference with vital cellular processes. Zeta toxins, which are prevalent in many bacterial genomes, were shown to interfere with cell wall formation by perturbing peptidoglycan synthesis in Gram-positive bacteria. Here, we characterize the epsilon/zeta toxin-antitoxin (TA) homologue from the Gram-negative pathogen Neisseria gonorrhoeae termed ng_ɛ1 / ng_ζ1. Contrary to previously studied streptococcal epsilon/zeta TA systems, ng_ɛ1 has an epsilon-unrelated fold and ng_ζ1 displays broader substrate specificity and phosphorylates multiple UDP-activated sugars that are precursors of peptidoglycan and lipopolysaccharide synthesis. Moreover, the phosphorylation site is different from the streptococcal zeta toxins, resulting in a different interference with cell wall synthesis. This difference most likely reflects adaptation to the individual cell wall composition of Gram-negative and Gram-positive organisms but also the distinct involvement of cell wall components in virulence.

PMID: 29703974 [PubMed - in process]

Scientists work out the specific genes and biochemical steps required for digesting the very drugs designed to kill microbes.

zinc can modulate the virulence of Escherichia coli bacteria, a pathogen that causes urinary infections in humans, according to a new study published in the journal Scientific Reports by biologist Carlos Balsalobre, from the University of Barcelona (UB). The new study reveals for the first time that zinc can regulate the expression of bacterial virulence factors, in particular, alfa-hemolysin, an exotoxin produced by some Escherichia coli pathogenic strains.

Related Articles

D-Ala-D-Ala ligase as a broad host-range counterselection marker in vancomycin-resistant lactic acid bacteria.

J Bacteriol. 2018 Apr 23;:

Authors: Zhang S, Oh JH, Alexander LM, Özçam M, Van Pijkeren JP

Abstract
The peptidoglycan composition in lactic acid bacteria dictates vancomycin resistance. Vancomycin binds relatively poor to peptidoglycan ending in D-alanyl-D-lactate while it binds with high-affinity to peptidoglycan ending in D-alanyl-D-alanine, which results in vancomycin resistance or sensitivity, respectively. The enzyme responsible for generating these peptidoglycan precursors is Ddl. A single amino acid in the Ddl active site, phenylalanine or tyrosine, determines depsipeptide or dipeptide activity, respectively. Here, we established that heterologous expression of dipeptide ligase in vancomycin-resistant lactobacilli increases their sensitivity to vancomycin in a dose-dependent manner, and overcomes the effects of a native D-Ala-D-Ala dipeptidase. We incorporated the dipeptide ligase gene on a suicide vector and demonstrated it functions as a counterselection marker (CSM) in lactobacilli: vancomycin selection only allows cells to grow in which the suicide vector has been lost from the cell. Subsequently, we developed a liquid-based approach to identify recombinants in only 5 days, which is approximately in half the time of conventional approaches. Phylogenetic analysis revealed that Ddl serves as a marker to predict vancomycin resistance, and consequently predicts broad applicability of Ddl as a counterselection marker in the genus Lactobacillus Finally, our system represents the first 'plug and play' counterselection system in lactic acid bacteria that does not require prior genome editing and/or synthetic medium.IMPORTANCE The genus Lactobacillus contains more than 200 species, many of which are exploited in the food and biotechnology industries, and medicine. Prediction of intrinsic vancomycin resistance has so-far been limited to select Lactobacillus species. Here, we show that heterologous expression of the enzyme Ddl dipeptide ligase-an essential enzyme involved in peptidoglycan synthesis-increases the sensitivity to vancomycin in a dose-dependent manner. We exploited this to develop a counterselection marker for use in vancomycin-resistant lactobacilli, thereby expanding the poorly developed genome editing toolbox that is currently available for most strains. Also, we show that Ddl is a phylogenetic marker to predict vancomycin-resistance in Lactobacillus: 81% of Lactobacillus species are intrinsically resistant to vancomycin, which makes our tool broadly applicable.

PMID: 29686137 [PubMed - as supplied by publisher]

Metabolic principles of persistence and pathogenicity in Mycobacterium tuberculosis

Metabolic principles of persistence and pathogenicity in <i>Mycobacterium tuberculosis</i>, Published online: 24 April 2018; doi:10.1038/s41579-018-0013-4

As an obligate human pathogen, Mycobacterium tuberculosis has evolved to survive and thrive in biochemically challenging niches in its host. Ehrt, Schnappinger and Rhee review the unique metabolic features that enable M. tuberculosis pathogenesis and persistence but also represent drug targets.

Related Articles

Redefining the essential trafficking pathway for outer membrane lipoproteins.

Proc Natl Acad Sci U S A. 2017 05 02;114(18):4769-4774

Authors: Grabowicz M, Silhavy TJ

Abstract
The outer membrane (OM) of Gram-negative bacteria is a permeability barrier and an intrinsic antibiotic resistance factor. Lipoproteins are OM components that function in cell wall synthesis, diverse secretion systems, and antibiotic efflux pumps. Moreover, each of the essential OM machines that assemble the barrier requires one or more lipoproteins. This dependence is thought to explain the essentiality of the periplasmic chaperone LolA and its OM receptor LolB that traffic lipoproteins to the OM. However, we show that in strains lacking substrates that are toxic when mislocalized, both LolA and LolB can be completely bypassed by activating an envelope stress response without compromising trafficking of essential lipoproteins. We identify the Cpx stress response as a monitor of lipoprotein trafficking tasked with protecting the cell from mislocalized lipoproteins. Moreover, our findings reveal that an alternate trafficking pathway exists that can, under certain conditions, bypass the functions of LolA and LolB, implying that these proteins do not perform any truly essential mechanistic steps in lipoprotein trafficking. Instead, these proteins' key function is to prevent lethal accumulation of mislocalized lipoproteins.

PMID: 28416660 [PubMed - indexed for MEDLINE]

E. coli outbreaks hospitalize people and cause food recalls pretty much annually in the United States. This year is no different.

Related Articles

On the catalytic mechanism of acetyl transfer catalysed by Mycobacterium tuberculosis GlmU.

Biochemistry. 2018 Apr 23;:

Authors: Craggs PD, Mouilleron S, Rejzek M, Chiara C, Young RJ, Field RA, Argyrou A, de Carvalho LPS

Abstract
The biosynthetic pathway of peptidoglycan is essential for Mycobacterium tuberculosis. We report here the acetyltransferase substrate specificity and catalytic mechanism of the bifunctional N-acetyltransferase/uridyltransferase from M. tuberculosis (GlmU). This enzyme is responsible for the final two steps of the synthesis of UDP-N-acetylglucosamine, which is an essential precursor of peptidoglycan, from glucosamine-1-phosphate, acetyl coenzyme A and uridine-5'-triphosphate. GlmU utilizes requires ternary complex formation to transfer an acetyl from acetyl coenzyme A to glucosamine-1-phosphate to form N-acetylglucosmaine-1-phosphate. Steady-state kinetic studies and equilibrium binding experiments indicate that GlmU follows a steady-state ordered kinetic mechanism, with acetyl coenzyme A binding first, which triggers a conformational change on GlmU, followed by glucosamine-1-phosphate binding. Coenzyme A is the last product to dissociate. Chemistry is partially rate-limiting as indicated by pH-rate studies and solvent kinetic isotope effects. A novel crystal structure of a mimic of the Michaelis complex, with glucose-1-phosphate and acetyl-coenzyme A, helps us to propose the residues involved in deprotonation of glucosamine-1-phosphate and the loop movement that likely generates the active site required for glucosamine-1-phosphate to bind. Together, these results pave the way for the rational discovery of improved inhibitors against M. tuberculosis GlmU, some of which might become candidates for antibiotic discovery programs.

PMID: 29684272 [PubMed - as supplied by publisher]

Microbiota-accessible carbohydrates suppress Clostridium difficile infection in a murine model

Microbiota-accessible carbohydrates suppress <i>Clostridium difficile</i> infection in a murine model, Published online: 23 April 2018; doi:10.1038/s41564-018-0150-6

A murine diet high in microbiota-accessible carbohydrates (MACs) reduces Clostridium difficile colonization compared to a low-MAC diet, which is associated with changes in microbiota composition, short-chain fatty acid concentrations and inflammation.

Angewandte Chemie International Edition, EarlyView.

Glycointeractions in bacterial pathogenesis

Glycointeractions in bacterial pathogenesis, Published online: 19 April 2018; doi:10.1038/s41579-018-0007-2

In this Review, Jennings and colleagues discuss interactions involving host and bacterial glycans that have a role in bacterial pathogenesis. They also highlight recent technological advances that have illuminated the glycoscience of microbial pathogenesis.

Related Articles

Unusual Escherichia coli PBP 3 Insertion Sequence Identified from a Collection of Carbapenem-Resistant Enterobacteriaceae Tested In Vitro with a Combination of Ceftazidime-, Ceftaroline-, or Aztreonam-Avibactam.

Antimicrob Agents Chemother. 2017 Aug;61(8):

Authors: Zhang Y, Kashikar A, Brown CA, Denys G, Bush K

Abstract
Carbapenemase-producing Enterobacteriaceae isolates (n = 110) from health care centers in central Indiana (from 2010 to 2013) were tested for susceptibility to combinations of avibactam (4 μg/ml) with ceftazidime, ceftaroline, or aztreonam. MIC50/MIC90 values were 1/2 μg/ml (ceftazidime-avibactam), 0.5/2 μg/ml (ceftaroline-avibactam), and 0.25/0.5 μg/ml (aztreonam-avibactam.) A β-lactam MIC of 8 μg/ml was reported for the three combinations against one Escherichia coli isolate with an unusual TIPY insertion following Tyr344 in penicillin-binding protein 3 (PBP 3) as the result of gene duplication.

PMID: 28559260 [PubMed - indexed for MEDLINE]

Related Articles

Function of alanine racemase in the physiological activity and cariogenicity of Streptococcus mutans.

Sci Rep. 2018 Apr 13;8(1):5984

Authors: Liu S, Wei Y, Zhou X, Zhang K, Peng X, Ren B, Chen V, Cheng L, Li M

Abstract
The enzyme alanine racemase (Alr) has been a new target for the development of antibacterial drugs based on the involvement of D-Ala in bacterial cell wall biosynthesis. Our previous study noted that Alr is essential for the growth and interspecies competitiveness of S. mutans, the major causative organism of dental caries. However, physiological activity and cariogenicity of S. mutans affected by Alr remains unknown. The current study examined the biofilm biomass, biofilm structure, extracellular polysaccharide (EPS) synthesis, glucosyltransferase (gtf) gene expression, acid production and acid tolerance in the alr-mutant strain. We found that biofilm formation, biofilm structure, and EPS synthesis was in a D-Ala dose-dependent manner. Biofilm structure was loose in alr-mutant group and the ratio of EPS/bacteria was also elevated. Additionally, the expression levels of multiple gtfs were up-regulated, and acid tolerance was decreased. We also established in vivo models of dental caries and found that the incidence and severity of the caries were decreased in the alr-mutant group in comparison to the parental S. mutans group. Our in vivo and in vitro experiments demonstrate that Alr is essential for the cariogenicity of S. mutans and that Alr might be a potential target for the prevention and treatment of caries.

PMID: 29654290 [PubMed - in process]

Related Articles

Atomic Force Microscopy of Side Wall and Septa Peptidoglycan From Bacillus subtilis Reveals an Architectural Remodeling During Growth.

Front Microbiol. 2018;9:620

Authors: Li K, Yuan XX, Sun HM, Zhao LS, Tang R, Chen ZH, Qin QL, Chen XL, Zhang YZ, Su HN

Abstract
Peptidoglycan is the fundamental structural constituent of the bacterial cell wall. Despite many years of research, the architecture of peptidoglycan is still largely elusive. Here, we report the high-resolution architecture of peptidoglycan from the model Gram-positive bacterium Bacillus subtilis. We provide high-resolution evidence of peptidoglycan architecture remodeling at different growth stages. Side wall peptidoglycan from B. subtilis strain AS1.398 changed from an irregular architecture in exponential growth phase to an ordered cable-like architecture in stationary phase. Thickness of side wall peptidoglycan was found to be related with growth stages, with a slight increase after transition to stationary phase. Septal disks were synthesized progressively toward the center, while the surface features were less clear than those imaged with side walls. Compared with previous studies, our results revealed slight differences in architecture of peptidoglycan from different B. subtilis strains, expanding our knowledge about the architectural features of B. subtilis peptidoglycan.

PMID: 29651285 [PubMed]

Related Articles

Bacterial Cell Wall Precursor Phosphatase Assays Using Thin-layer Chromatography (TLC) and High Pressure Liquid Chromatography (HPLC).

Bio Protoc. 2018 Mar 20;8(6):e2761

Authors: Pazos M, Otten C, Vollmer W

Abstract
Peptidoglycan encases the bacterial cytoplasmic membrane to protect the cell from lysis due to the turgor. The final steps of peptidoglycan synthesis require a membrane-anchored substrate called lipid II, in which the peptidoglycan subunit is linked to the carrier lipid undecaprenol via a pyrophosphate moiety. Lipid II is the target of glycopeptide antibiotics and several antimicrobial peptides, and is degraded by 'attacking' enzymes involved in bacterial competition to induce lysis. Here we describe two protocols using thin-layer chromatography (TLC) and high pressure liquid chromatography (HPLC), respectively, to assay the digestion of lipid II by phosphatases such as Colicin M or the LXG toxin protein TelC from Streptococcus intermedius. The TLC method can also monitor the digestion of undecaprenyl (pyro)phosphate, whereas the HPLC method allows to separate the di-, mono- or unphosphorylated disaccharide pentapeptide products of lipid II.

PMID: 29651453 [PubMed]

Related Articles

Exolytic and endolytic turnover of peptidoglycan by lytic transglycosylase Slt of Pseudomonas aeruginosa.

Proc Natl Acad Sci U S A. 2018 Apr 09;:

Authors: Lee M, Batuecas MT, Tomoshige S, Domínguez-Gil T, Mahasenan KV, Dik DA, Hesek D, Millán C, Usón I, Lastochkin E, Hermoso JA, Mobashery S

Abstract
β-Lactam antibiotics inhibit cell-wall transpeptidases, preventing the peptidoglycan, the major constituent of the bacterial cell wall, from cross-linking. This causes accumulation of long non-cross-linked strands of peptidoglycan, which leads to bacterial death. Pseudomonas aeruginosa, a nefarious bacterial pathogen, attempts to repair this aberrantly formed peptidoglycan by the function of the lytic transglycosylase Slt. We document in this report that Slt turns over the peptidoglycan by both exolytic and endolytic reactions, which cause glycosidic bond scission from a terminus or in the middle of the peptidoglycan, respectively. These reactions were characterized with complex synthetic peptidoglycan fragments that ranged in size from tetrasaccharides to octasaccharides. The X-ray structure of the wild-type apo Slt revealed it to be a doughnut-shaped protein. In a series of six additional X-ray crystal structures, we provide insights with authentic substrates into how Slt is enabled for catalysis for both the endolytic and exolytic reactions. The substrate for the exolytic reaction binds Slt in a canonical arrangement and reveals how both the glycan chain and the peptide stems are recognized by the Slt. We document that the apo enzyme does not have a fully formed active site for the endolytic reaction. However, binding of the peptidoglycan at the existing subsites within the catalytic domain causes a conformational change in the protein that assembles the surface for binding of a more expansive peptidoglycan between the catalytic domain and an adjacent domain. The complexes of Slt with synthetic peptidoglycan substrates provide an unprecedented snapshot of the endolytic reaction.

PMID: 29632171 [PubMed - as supplied by publisher]