scholarly journals Lcp1 Is a Phosphotransferase Responsible for Ligating Arabinogalactan to Peptidoglycan in Mycobacterium tuberculosis

mBio ◽  
2016 ◽  
Vol 7 (4) ◽  
Author(s):  
James Harrison ◽  
Georgina Lloyd ◽  
Maju Joe ◽  
Todd L. Lowary ◽  
Edward Reynolds ◽  
...  
Keyword(s):  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
Essential Gene ◽  
Cell Envelope ◽  
New Drugs ◽  
Cell Wall Assembly ◽  
Mycobacterial Cell ◽  
Unique Cell ◽  
Mycobacterial Cell Wall ◽  
Wall Assembly

ABSTRACT Mycobacterium tuberculosis , the etiological agent of tuberculosis (TB), has a unique cell envelope which accounts for its unusual low permeability and contributes to resistance against common antibiotics. The main structural elements of the cell wall consist of a cross-linked network of peptidoglycan (PG) in which some of the muramic acid residues are covalently attached to a complex polysaccharide, arabinogalactan (AG), via a unique α- l -rhamnopyranose–(1→3)-α- d -GlcNAc-(1→P) linker unit. While the molecular genetics associated with PG and AG biosynthetic pathways have been largely delineated, the mechanism by which these two major pathways converge has remained elusive. In Gram-positive organisms, the LytR-CpsA-Psr (LCP) family of proteins are responsible for ligating cell wall teichoic acids to peptidoglycan, through a linker unit that bears a striking resemblance to that found in mycobacterial arabinogalactan. In this study, we have identified Rv3267 as a mycobacterial LCP homolog gene that encodes a phosphotransferase which we have named Lcp1. We demonstrate that lcp1 is an essential gene required for cell viability and show that recombinant Lcp1 is capable of ligating AG to PG in a cell-free radiolabeling assay. IMPORTANCE Tuberculosis is an infectious disease caused by the bacterial organism Mycobacterium tuberculosis . Survival of M. tuberculosis rests critically on the integrity of its unique cell wall; therefore, a better understanding of how the genes and enzymes involved in cell wall assembly work is fundamental for us to develop new drugs to treat this disease. In this study, we have identified Lcp1 as an essential phosphotransferase that ligates together arabinogalactan and peptidoglycan, two crucial cell wall macromolecules found within the mycobacterial cell wall. The discovery of Lcp1 sheds new light on the final stages of mycobacterial cell wall assembly and represents a key biosynthetic step that could be exploited for new anti-TB drug discovery.

Synthesis and anti-mycobacterial activity of glycosyl sulfamides of arabinofuranose

2016 ◽  
Vol 14 (5) ◽  
pp. 1748-1754 ◽  
Author(s):  
Kajitha Suthagar ◽  
Antony J. Fairbanks
Keyword(s):  
Cell Wall ◽  
Hydroxyl Group ◽  
Cell Wall Assembly ◽  
Mycobacterial Cell ◽  
Furanose Form ◽  
Mycobacterial Cell Wall ◽  
Wall Assembly

A series ofarabino N-glycosyl sulfamides, forced to adopt the furanose form by removal of the 5-hydroxyl group, were synthesised as putative isosteric mimics of decaprenolphosphoarabinose, the donor processed by arabinosyltransferases during mycobacterial cell wall assembly.

scholarly journals MmpL3 is a lipid transporter that binds trehalose monomycolate and phosphatidylethanolamine

2019 ◽  
Vol 116 (23) ◽  
pp. 11241-11246 ◽  
Author(s):  
Chih-Chia Su ◽  
Philip A. Klenotic ◽  
Jani Reddy Bolla ◽  
Georgiana E. Purdy ◽  
Carol V. Robinson ◽  
...  
Keyword(s):  
Cell Wall ◽  
Cell Envelope ◽  
Native Mass Spectrometry ◽  
Mycolic Acids ◽  
Mycobacterial Cell ◽  
Exact Function ◽  
Species Specific ◽  
Mycobacterial Cell Wall ◽  
Trehalose Monomycolate ◽  
Lipid Transporter

The cell envelope ofMycobacterium tuberculosisis notable for the abundance of mycolic acids (MAs), essential to mycobacterial viability, and of other species-specific lipids. The mycobacterial cell envelope is extremely hydrophobic, which contributes to virulence and antibiotic resistance. However, exactly how fatty acids and lipidic elements are transported across the cell envelope for cell-wall biosynthesis is unclear. Mycobacterial membrane protein Large 3 (MmpL3) is essential and required for transport of trehalose monomycolates (TMMs), precursors of MA-containing trehalose dimycolates (TDM) and mycolyl arabinogalactan peptidoglycan, but the exact function of MmpL3 remains elusive. Here, we report a crystal structure ofMycobacterium smegmatisMmpL3 at a resolution of 2.59 Å, revealing a monomeric molecule that is structurally distinct from all known bacterial membrane proteins. A previously unknown MmpL3 ligand, phosphatidylethanolamine (PE), was discovered inside this transporter. We also show, via native mass spectrometry, that MmpL3 specifically binds both TMM and PE, but not TDM, in the micromolar range. These observations provide insight into the function of MmpL3 and suggest a possible role for this protein in shuttling a variety of lipids to strengthen the mycobacterial cell wall.

Structure of the Mycobacterium tuberculosis OmpATb protein: A model of an oligomeric channel in the mycobacterial cell wall

2010 ◽  
Vol 79 (2) ◽  
pp. 645-661 ◽  
Author(s):  
Yinshan Yang ◽  
Daniel Auguin ◽  
Stéphane Delbecq ◽  
Emilie Dumas ◽  
Gérard Molle ◽  
...  
Keyword(s):  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
Protein A ◽  
Mycobacterial Cell ◽  
Mycobacterial Cell Wall

scholarly journals Antibiotic hypersensitivity signatures identify targets for attack in the Acinetobacter baumannii cell envelope

2020 ◽  
Author(s):  
Edward Geisinger ◽  
Nadav J. Mortman ◽  
Yunfei Dai ◽  
Murat Cokol ◽  
Sapna Syal ◽  
...  
Keyword(s):  
Cell Wall ◽  
Acinetobacter Baumannii ◽  
Cell Envelope ◽  
Opportunistic Pathogen ◽  
Drug Action ◽  
Antibiotic Development ◽  
Cell Wall Assembly ◽  
Combination Strategies ◽  
High Doses ◽  
Wall Assembly

AbstractAcinetobacter baumannii is an opportunistic pathogen that is a critical, high-priority target for new antibiotic development. Clearing of A. baumannii requires relatively high doses of antibiotics across the spectrum, primarily due to its protective cell envelope. Many of the proteins that support envelope integrity and modulate drug action are uncharacterized, largely because there is an absence of orthologs for several proteins that perform essential envelope-associated processes, impeding progress on this front. To identify targets that can synergize with current antibiotics, we performed an exhaustive analysis of A. baumannii mutants causing hypersensitivity to a multitude of antibiotic treatments. By examining mutants with antibiotic hypersensitivity profiles that parallel mutations in proteins of known function, we show that the function of poorly annotated proteins can be predicted and used to identify candidate missing link proteins in essential A. baumannii processes. Using this strategy, we uncovered multiple uncharacterized proteins with critical roles in cell division or cell elongation, and revealed that a predicted cell wall D,D-endopeptidase has an unappreciated function in lipooligosaccharide synthesis. Moreover, we provide a genetic strategy that uses hypersensitivity signatures to predict drug synergies, allowing the identification of β-lactams that work cooperatively based on the cell wall assembly machineries that they preferentially target. These data reveal multiple pathways critical for envelope growth in A. baumannii that can be targeted in combination strategies for attacking the pathogen.

scholarly journals A cytoplasmic peptidoglycan amidase homologue controls mycobacterial cell wall synthesis

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Cara C Boutte ◽  
Christina E Baer ◽  
Kadamba Papavinasasundaram ◽  
Weiru Liu ◽  
Michael R Chase ◽  
...  
Keyword(s):  
Cell Wall ◽  
Antibiotic Tolerance ◽  
Bacterial Survival ◽  
Cell Wall Metabolism ◽  
Cell Wall Assembly ◽  
Enzymatic Function ◽  
Precursor Synthesis ◽  
Mycobacterial Cell Wall ◽  
Wall Assembly ◽  
Multiple Antibiotics

Regulation of cell wall assembly is essential for bacterial survival and contributes to pathogenesis and antibiotic tolerance in Mycobacterium tuberculosis (Mtb). However, little is known about how the cell wall is regulated in stress. We found that CwlM, a protein homologous to peptidoglycan amidases, coordinates peptidoglycan synthesis with nutrient availability. Surprisingly, CwlM is sequestered from peptidoglycan (PG) by localization in the cytoplasm, and its enzymatic function is not essential. Rather, CwlM is phosphorylated and associates with MurA, the first enzyme in PG precursor synthesis. Phosphorylated CwlM activates MurA ~30 fold. CwlM is dephosphorylated in starvation, resulting in lower MurA activity, decreased cell wall metabolism, and increased tolerance to multiple antibiotics. A phylogenetic analysis of cwlM implies that localization in the cytoplasm drove the evolution of this factor. We describe a system that controls cell wall metabolism in response to starvation, and show that this regulation contributes to antibiotic tolerance.

scholarly journals Multiple Mutations in Mycobacterium tuberculosis MmpL3 Increase Resistance to MmpL3 Inhibitors

mSphere ◽  
2020 ◽  
Vol 5 (5) ◽  
Author(s):  
Matthew B. McNeil ◽  
Theresa O’Malley ◽  
Devon Dennison ◽  
Catherine D. Shelton ◽  
Bjorn Sunde ◽  
...  
Keyword(s):  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
Drug Targets ◽  
New Drugs ◽  
Content Type ◽  
Mechanism Of Resistance ◽  
Primary Mechanism ◽  
Resistant Mutants ◽  
Mycobacterial Cell Wall

ABSTRACT The Mycobacterium tuberculosis protein MmpL3 performs an essential role in cell wall synthesis, since it effects the transport of trehalose monomycolates across the inner membrane. Numerous structurally diverse pharmacophores have been identified as inhibitors of MmpL3 largely based on the identification of resistant isolates with mutations in MmpL3. For some compounds, it is possible there are different primary or secondary targets. Here, we have investigated resistance to the spiral amine class of compounds. Isolation and sequencing of resistant mutants demonstrated that all had mutations in MmpL3. We hypothesized that if additional targets of this pharmacophore existed, then successive rounds to generate resistant isolates might reveal mutations in other loci. Since compounds were still active against resistant isolates, albeit with reduced potency, we isolated resistant mutants in this background at higher concentrations. After a second round of isolation with the spiral amine, we found additional mutations in MmpL3. To increase our chance of finding alternative targets, we ran a third round of isolation using a different molecule scaffold (AU1235, an adamantyl urea). Surprisingly, we obtained further mutations in MmpL3. Multiple mutations in MmpL3 increased the level and spectrum of resistance to different pharmacophores but did not incur a fitness cost in vitro. These results support the hypothesis that MmpL3 is the primary mechanism of resistance and likely target for these pharmacophores. IMPORTANCE Mycobacterium tuberculosis is a major global human pathogen, and new drugs and new drug targets are urgently required. Cell wall biosynthesis is a major target of current tuberculosis drugs and of new agents under development. Several new classes of molecules appear to have the same target, MmpL3, which is involved in the export and synthesis of the mycobacterial cell wall. However, there is still debate over whether MmpL3 is the primary or only target for these classes. We wanted to confirm the mechanism of resistance for one series. We identified mutations in MmpL3 which led to resistance to the spiral amine series. High-level resistance to these compounds and two other series was conferred by multiple mutations in the same protein (MmpL3). These mutations did not reduce growth rate in culture. These results support the hypothesis that MmpL3 is the primary mechanism of resistance and likely target for these pharmacophores.

scholarly journals Mycobacterial Cell Wall: A Source of Successful Targets for Old and New Drugs

2020 ◽  
Vol 10 (7) ◽  
pp. 2278 ◽  
Author(s):  
Catherine Vilchèze
Keyword(s):  
Cell Wall ◽  
New Drugs ◽  
Wall Structure ◽  
Tb Treatment ◽  
Mycobacterial Cell ◽  
Short Course ◽  
Cell Wall Disruption ◽  
The Face ◽  
Mycobacterial Cell Wall ◽  
Wall Components

Eighty years after the introduction of the first antituberculosis (TB) drug, the treatment of drug-susceptible TB remains very cumbersome, requiring the use of four drugs (isoniazid, rifampicin, ethambutol and pyrazinamide) for two months followed by four months on isoniazid and rifampicin. Two of the drugs used in this “short”-course, six-month chemotherapy, isoniazid and ethambutol, target the mycobacterial cell wall. Disruption of the cell wall structure can enhance the entry of other TB drugs, resulting in a more potent chemotherapy. More importantly, inhibition of cell wall components can lead to mycobacterial cell death. The complexity of the mycobacterial cell wall offers numerous opportunities to develop drugs to eradicate Mycobacterium tuberculosis, the causative agent of TB. In the past 20 years, researchers from industrial and academic laboratories have tested new molecules to find the best candidates that will change the face of TB treatment: drugs that will shorten TB treatment and be efficacious against active and latent, as well as drug-resistant TB. Two of these new TB drugs block components of the mycobacterial cell wall and have reached phase 3 clinical trial. This article reviews TB drugs targeting the mycobacterial cell wall in use clinically and those in clinical development.

Identification of inhibitors targeting Mycobacterium tuberculosis cell wall biosynthesis via dynamic combinatorial chemistry

2017 ◽  
Vol 53 (77) ◽  
pp. 10632-10635 ◽  
Author(s):  
Jian Fu ◽  
Huixiao Fu ◽  
Marc Dieu ◽  
Iman Halloum ◽  
Laurent Kremer ◽  
...  
Keyword(s):  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
Combinatorial Chemistry ◽  
Cell Wall Biosynthesis ◽  
Combinatorial Approach ◽  
Dynamic Combinatorial Chemistry ◽  
Mycobacterial Cell ◽  
Essential Enzyme ◽  
Mycobacterial Cell Wall

In this study, we report a dynamic combinatorial approach along with highly efficient in situ screening to identify inhibitors of UDP-galactopyranose mutase (UGM), an essential enzyme involved in mycobacterial cell wall biosynthesis.

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