47 results found
Dortet L, Broda A, bernabeu S, et al., Optimization of the MALDIxin test for the rapid identification of colistin resistance in Klebsiella pneumoniae using MALDI-TOF-MS, Journal of Antimicrobial Chemotherapy, ISSN: 0305-7453
Background. With the dissemination of carbapenemase producers, a revival of colistin was observed for the treatment of infections caused by multidrug-resistant Gram-negatives. Unfortunately, the increasing usage of colistin led to the emergence of resistance. In Klebsiella pneumoniae, colistin resistance arises through addition of L-arabinose-4N (L-Ara4N) or phosphoethanolamine (pEtN) on the native lipid A. The underlying mechanisms involve numerous chromosome-encoded genes or the plasmid-encoded phosphoethanolamine transferase MCR. Currently, detection of colistin resistance is time consuming since it still relies on MIC determination by broth microdilution. Recently, a rapid diagnostic test based on MALDI-TOF detection of modified lipid A was developed (the MALDIxin test) and tested on Escherichia coli and Acinetobacter baumannii.Objectives. Optimize the MALDIxin test for the rapid detection of colistin resistance in Klebsiella pneumoniae.Methods. This optimization consists on an additional mild-acid hydrolysis of 15 min in 1% acetic acid. The optimized method was tested on a collection of 81 clinical K. pneumoniae isolates including 49 colistin resistant strains among which 45 correspond to chromosome-encoded resistance, 3 MCR-related resistance and one isolate harbouring both mechanisms.Results. The optimized method allowed the rapid (< 30 min) identification of L-Ara4N and pEtN modified lipid A of K. pneumoniae which are known to be the real triggers of polymyxin resistance. In the same time, it discriminates between chromosome-encoded and MCR-related polymyxin resistance.Conclusions. The MALDIxin test has the potential to become an accurate tool for the rapid diagnostic of colistin resistance in clinically-relevant Gram negative bacteria.
Tang W, Ranganathan N, Shahrezaei V, et al., 2019, MALDI-TOF mass spectrometry on intact bacteria combined with a refined analysis framework allows accurate classification of MSSA and MRSA., PLoS ONE, Vol: 14, Pages: 1-16, ISSN: 1932-6203
Fast and reliable detection coupled with accurate data-processing and analysis of antibiotic-resistant bacteria is essential in clinical settings. In this study, we use MALDI-TOF on intact cells combined with a refined analysis framework to demonstrate discrimination between methicillin-susceptible (MSSA) and methicillin-resistant (MRSA) Staphylococcus aureus. By combining supervised and unsupervised machine learning methods, we firstly show that the mass spectroscopy data contains strong signal for the clustering of MSSA and MRSA. Then we concentrate on applying supervised learning to extract and verify the important features. A new workflow is proposed that allows for extracting a fixed set of reference peaks so that any new data can be aligned to it and hence consistent feature matrices can be obtained. Also note that by doing so we are able to examine the robustness of the important features that have been found. We also show that appropriate size of the benchmark data, appropriate alignment of the testing data and use of an optimal set of features via feature selection results in prediction accuracy over 90%. In summary, as proof-of-principle, our integrated experimental and bioinformatics study suggests a novel intact cell MALDI-TOF to be of great promise for fast and reliable detection of MRSA strains.
Potron A, Vuillemenot J-B, Puja H, et al., 2019, ISAba1-dependent overexpression of eptA in clinical strains of Acinetobacter baumannii resistant to colistin., J Antimicrob Chemother
BACKGROUND: Colistin resistance in Acinetobacter baumannii often results from mutational activation of the two-component system PmrAB and subsequent addition of phospho-ethanolamine (pEtN) to lipooligosaccharide by up-regulated pEtN transferase PmrC. OBJECTIVES: To characterize mechanisms of colistin resistance independent of PmrCAB in A. baumannii. METHODS: Twenty-seven colistin-resistant A. baumannii were collected from 2012 to 2018. Analysis of operon pmrCAB was performed by PCR and sequencing. Seven strains were investigated further by WGS and whole-genome MLST (wgMLST). RESULTS: Seven out of the 27 selected isolates were found to overexpress eptA, a gene homologous to pmrC, likely as a consequence of upstream insertion of an ISAba1 element. Insertion sites of ISAba1 were mapped 13, 18 and 156 bp ahead of the start codon of eptA in five strains, one strain and one strain, respectively. The finding that the isolates did not cluster together when compared by wgMLST analysis supports the notion that distinct insertion events occurred in close, but different, genetic backgrounds. CONCLUSIONS: Activation of eptA and subsequent addition of pEtN to the cell surface represents a novel mechanism of resistance to colistin in A. baumannii.
Rebollo-Ramirez S, Larrouy-Maumus G, 2019, NaCl triggers the CRP-dependent increase of cAMP in Mycobacterium tuberculosis, Tuberculosis, Vol: 116, Pages: 8-16, ISSN: 1472-9792
The second messenger 3′,5′-cyclic adenosine monophosphate (3′,5′-cAMP) has been shown to be involved in the regulation of many biological processes ranging from carbon catabolite repression in bacteria to cell signalling in eukaryotes. In mycobacteria, the role of cAMP and the mechanisms utilized by the bacterium to adapt to and resist immune and pharmacological sterilization remain poorly understood. Among the stresses encountered by bacteria, ionic and non-ionic osmotic stresses are among the best studied. However, in mycobacteria, the link between ionic osmotic stress, particularly sodium chloride, and cAMP has been relatively unexplored. Using a targeted metabolic analysis combined with stable isotope tracing, we show that the pathogenic Mycobacterium tuberculosis but not the opportunistic pathogen Mycobacterium marinum nor the non-pathogenic Mycobacterium smegmatis responds to NaCl stress via an increase in intracellular cAMP levels. We further showed that this increase in cAMP is dependent on the cAMP receptor protein and in part on the threonine/serine kinase PnkD, which has previously been associated with the NaCl stress response in mycobacteria.
Larrouy-Maumus GJ, 2019, Lipids as biomarkers of cancer and bacterial infections, Current Medicinal Chemistry, Vol: 26, Pages: 1924-1932, ISSN: 0929-8673
Lipids are ubiquitous molecules, known to play important roles in various cellular processes. Alterations to the lipidome can therefore be used as a read-out of the signs of disease, highlighting the importance to consider lipids as biomarkers in addition of nucleic acid and proteins. This mini-review exposes the current knowledge and limitations of the use of lipids as biomarkers of the top global killers which are cancer and bacterial infections.
Krokowski S, Lobato-Marquez D, Chastanet A, et al., 2018, Septins recognize and entrap dividing bacterial cells for delivery to lysosomes, Cell Host and Microbe, Vol: 24, Pages: 866-874, ISSN: 1931-3128
The cytoskeleton occupies a central role in cellular immunity by promoting bacterial sensing and antibacterial functions. Septins are cytoskeletal proteins implicated in various cellular processes, including cell division. Septins also assemble into cage-like structures that entrap cytosolic Shigella, yet how septins recognize bacteria is poorly understood. Here, we discover that septins are recruited to regions of micron-scale membrane curvature upon invasion and division by a variety of bacterial species. Cardiolipin, a curvature-specific phospholipid, promotes septin recruitment to highly curved membranes of Shigella, and bacterial mutants lacking cardiolipin exhibit less septin cage entrapment. Chemically inhibiting cell separation to prolong membrane curvature or reducing Shigella cell growth respectively increases and decreases septin cage formation. Once formed, septin cages inhibit Shigella cell division upon recruitment of autophagic and lysosomal machinery. Thus, recognition of dividing bacterial cells by the septin cytoskeleton is a powerful mechanism to restrict the proliferation of intracellular bacterial pathogens.
Dortet L, Bonnin RA, Pennisi I, et al., 2018, Rapid detection and discrimination of chromosome-and MCR-plasmid-mediated resistance to polymyxins by MALDI-TOF MS in Escherichia coli: the MALDIxin test, Journal of Antimicrobial Chemotherapy, Vol: 73, Pages: 3359-3367, ISSN: 0305-7453
BackgroundPolymyxins are currently considered a last-resort treatment for infections caused by MDR Gram-negative bacteria. Recently, the emergence of carbapenemase-producing Enterobacteriaceae has accelerated the use of polymyxins in the clinic, resulting in an increase in polymyxin-resistant bacteria. Polymyxin resistance arises through modification of lipid A, such as the addition of phosphoethanolamine (pETN). The underlying mechanisms involve numerous chromosome-encoded genes or, more worryingly, a plasmid-encoded pETN transferase named MCR. Currently, detection of polymyxin resistance is difficult and time consuming.ObjectivesTo develop a rapid diagnostic test that can identify polymyxin resistance and at the same time differentiate between chromosome- and plasmid-encoded resistances.MethodsWe developed a MALDI-TOF MS-based method, named the MALDIxin test, which allows the detection of polymyxin resistance-related modifications to lipid A (i.e. pETN addition), on intact bacteria, in <15 min.ResultsUsing a characterized collection of polymyxin-susceptible and -resistant Escherichia coli, we demonstrated that our method is able to identify polymyxin-resistant isolates in 15 min whilst simultaneously discriminating between chromosome- and plasmid-encoded resistance. We validated the MALDIxin test on different media, using fresh and aged colonies and show that it successfully detects all MCR-1 producers in a blindly analysed set of carbapenemase-producing E. coli strains.ConclusionsThe MALDIxin test is an accurate, rapid, cost-effective and scalable method that represents a major advance in the diagnosis of polymyxin resistance by directly assessing lipid A modifications in intact bacteria.
Dortet L, Potron A, Bonnin RA, et al., 2018, Rapid detection of colistin resistance in Acinetobacter baumannii using MALDI-TOF-based lipidomics on intact bacteria, Scientific Reports, Vol: 8, ISSN: 2045-2322
With the dissemination of extremely drug resistant bacteria, colistin is now considered as the last-resort therapy for the treatment of infection caused by Gram-negative bacilli (including carbapenemase producers). Unfortunately, the increase use of colistin has resulted in the emergence of resistance as well. In A. baumannii, colistin resistance is mostly caused by the addition of phosphoethanolamine to the lipid A through the action of a phosphoethanolamine transferase chromosomally-encoded by the pmrC gene, which is regulated by the two-component system PmrA/PmrB. In A. baumannii clinical isolate the main resistance mechanism to colistin involves mutations in pmrA, pmrB or pmrC genes leading to the overexpression of pmrC. Although, rapid detection of resistance is one of the key issues to improve the treatment of infected patient, detection of colistin resistance in A. baumannii still relies on MIC determination through microdilution, which is time-consuming (16–24 h). Here, we evaluated the performance of a recently described MALDI-TOF-based assay, the MALDIxin test, which allows the rapid detection of colistin resistance-related modifications to lipid A (i.e phosphoethanolamine addition). This test accurately detected all colistin-resistant A. baumannii isolates in less than 15 minutes, directly on intact bacteria with a very limited sample preparation prior MALDI-TOF analysis.
Decout A, Silva-Gomes S, Drocourt D, et al., 2018, Deciphering the molecular basis of mycobacteria and lipoglycan recognition by the C-type lectin Dectin-2, Scientific Reports, Vol: 8, ISSN: 2045-2322
Dectin-2 is a C-type lectin involved in the recognition of several pathogens such as Aspergillus fumigatus, Candida albicans, Schistosoma mansonii, and Mycobacterium tuberculosis that triggers Th17 immune responses. Identifying pathogen ligands and understanding the molecular basis of their recognition is one of the current challenges. Purified M. tuberculosis mannose-capped lipoarabinomannan (ManLAM) was shown to induce signaling via Dectin-2, an activity that requires the (α1 → 2)-linked mannosides forming the caps. Here, using isogenic M. tuberculosis mutant strains, we demonstrate that ManLAM is a bona fide and actually the sole ligand mediating bacilli recognition by Dectin-2, although M. tuberculosis produces a variety of cell envelope mannoconjugates, such as phosphatidyl-myo-inositol hexamannosides, lipomannan or manno(lipo)proteins, that bear (α1 → 2)-linked mannosides. In addition, we found that Dectin-2 can recognize lipoglycans from other bacterial species, such as Saccharotrix aerocolonigenes or the human opportunistic pathogen Tsukamurella paurometabola, suggesting that lipoglycans are prototypical Dectin-2 ligands. Finally, from a structure/function relationship perspective, we show, using lipoglycan variants and synthetic mannodendrimers, that dimannoside caps and multivalent interaction are required for ligand binding to and signaling via Dectin-2. Better understanding of the molecular basis of ligand recognition by Dectin-2 will pave the way for the rational design of potent adjuvants targeting this receptor.
Filloux A, Larrouy-Maumus G, Dortet L, 2018, Method of detection: Use of Lipid A and its modifications as a direct detection of antimicrobials resistance, WO2018158573
The present invention is directed to a method for detecting the presence or absence of a bacterium resistant to a cyclic cationic polypeptide antibiotic, comprising: (a) subjecting a test sample to mass spectrometry analysis and generating a mass spectrum output; wherein said test sample comprises a bacterial membrane or a fragment thereof, wherein the fragment comprises a non- Lipid A component; (b) identifying in said mass spectrum output a first defined peak indicative of the presence of Lipid A modified by phosphoethanolamine, wherein said first defined peak is a peak present in a mass spectrum output for Lipid A modified by phosphoethanolamine and wherein said first defined peak is absent from a corresponding mass spectrum output for native Lipid A; and (c) wherein the presence of said first defined peak indicates the presence of a bacterium resistant to a cyclic cationic polypeptide antibiotic, and wherein the absence of said first defined peak indicates the absence of a bacterium resistant to a cyclic cationic polypeptide antibiotic. This method is also used in a screening method to identify an inhibitor of cyclic cationic polypeptide antibiotic resistance in a bacterium. The matrix solution can contain 2,5-dihydroxybenzoic acid and aids in the selective extraction, co-crystallisation and ionisation of native Lipid A and/or modified Lipid A as an integral part of a bacterial membrane.
Larrouy-Maumus GJ, Mostowy S, Lobato-Márquez D, et al., 2018, Intact cell lipidomics reveal changes to the ratio of cardiolipins to phosphatidylinositols in response to kanamycin in HeLa and primary cells, Chemical Research in Toxicology, Vol: 31, Pages: 688-696, ISSN: 0893-228X
Antimicrobial resistance is a major threat the world is currently facing. Development of new antibiotics and the assessment of their toxicity represent important challenges. Current methods for addressing antibiotic toxicity rely on measuring mitochondrial damage using ATP and/or membrane potential as a readout. In this study, we propose an alternative readout looking at changes in the lipidome on intact and unprocessed cells by matrix-assisted laser desorption ionization mass spectrometry. As a proof of principle, we evaluated the impact of known antibiotics (levofloxacin, ethambutol, and kanamycin) on the lipidome of HeLa cells and mouse bone marrow-derived macrophages. Our methodology revealed that clinically relevant concentrations of kanamycin alter the ratio of cardiolipins to phosphatidylinositols. Unexpectedly, only kanamycin had this effect even though all antibiotics used in this study led to a decrease in the maximal mitochondrial respiratory capacity. Altogether, we report that intact cell-targeted lipidomics can be used as a qualitative method to rapidly assess the toxicity of aminoglycosides in HeLa and primary cells. Moreover, these results demonstrate there is no direct correlation between the ratio of cardiolipins to phosphatidylinositols and the maximal mitochondrial respiratory capacity.
Rebollo-Ramirez S, Thomson M, Larrouy-Maumus G, 2018, The cyclic AMP receptor protein (CRP) orchestrates the increase in intracellular levels of cAMP in response to osmotic stress in pathogenic mycobacteria, Publisher: WILEY, Pages: 96-96, ISSN: 2211-5463
Rycroft J, Gollan B, Grabe G, et al., 2018, Activity of acetyltransferase toxins involved in persister formation of Salmonella during macrophage infection, Nature Communications, Vol: 9, ISSN: 2041-1723
Non-typhoidal Salmonella strains are responsible for invasive infections associated withhigh mortality and recurrence in sub-Saharan Africa and there is strong evidence for clonalrelapse following antibiotic treatment. Persisters are non-growing bacteria that are thought tobe responsible for the recalcitrance of many infections to antibiotics. Toxin-antitoxin systemsare stress-responsive elements that are important for Salmonella persister formation,specifically during infection. Here we report analysis of persister formation of clinical invasive strains of S. Typhimurium and Enteritidis in human primary macrophages. We show that allthe invasive clinical isolates of both serovars that we tested produce high levels of persisters following internalization by human macrophages. Our genome comparison reveals that S.Enteritidis and S. Typhimurium strains contain three acetyltransferase toxins that we characterize structurally and functionally. We show that all induce the persister state byinhibiting translation through acetylation of aminoacyl-tRNAs. However, they differ in theirpotency and target partially different subsets of aminoacyl-tRNAs, potentially accounting fortheir non-redundant effect.
Lobato-Marquez D, Krokowski S, Sirianni A, et al., 2018, A requirement for septins and the autophagy receptor p62 in the proliferation of intracellular Shigella, Cytoskeleton, Vol: 76, Pages: 163-172, ISSN: 1949-3584
Shigella flexneri, a Gram‐negative enteroinvasive pathogen, causes inflammatory destruction of the human intestinal epithelium. During infection of epithelial cells, Shigella escape from the phagosome to the cytosol, where they reroute host cell glycolysis to obtain nutrients for proliferation. Septins, a poorly understood component of the cytoskeleton, can entrap cytosolic Shigella targeted to autophagy in cage‐like structures to restrict bacterial proliferation. Although bacterial entrapment by septin caging has been the subject of intense investigation, the role of septins and the autophagy machinery in the proliferation of non‐caged Shigella is mostly unknown. Here, we found that intracellular Shigella fail to efficiently proliferate in SEPT2‐, SEPT7‐ or p62/SQSTM1‐depleted cells. Consistent with a failure to proliferate, single cell analysis of bacteria not entrapped in septin cages showed that the number of metabolically active Shigella in septin‐ or p62‐depleted cells is reduced. Targeted metabolomic analysis revealed that host cell glycolysis is dysregulated in septin‐depleted cells, suggesting a key role for septins in modulation of glycolysis. Together, these results suggest that septins and the autophagy machinery may regulate metabolic pathways that promote the proliferation intracellular Shigella not entrapped in septin cages.
Berger C, Crepin V, Roumeliotis TI, et al., 2017, Citrobacter rodentium subverts ATP flux 1 and cholesterol homeostasis in 2 intestinal epithelial cell in vivo, Cell Metabolism, Vol: 26, Pages: 738-752.e6, ISSN: 1550-4131
The intestinal epithelial cells (IECs) that line the gut form a robust line of defense against ingested pathogens. We investigated the impact of infection with the enteric pathogen Citrobacter rodentium on mouse IEC metabolism using global proteomic and targeted metabolomics and lipidomics. The major signatures of the infection were upregulation of the sugar transporter Sglt4, aerobic glycolysis, and production of phosphocreatine, which mobilizes cytosolic energy. In contrast, biogenesis of mitochondrial cardiolipins, essential for ATP production, was inhibited, which coincided with increased levels of mucosal O2 and a reduction in colon-associated anaerobic commensals. In addition, IECs responded to infection by activating Srebp2 and the cholesterol biosynthetic pathway. Unexpectedly, infected IECs also upregulated the cholesterol efflux proteins AbcA1, AbcG8, and ApoA1, resulting in higher levels of fecal cholesterol and a bloom of Proteobacteria. These results suggest that C. rodentium manipulates host metabolism to evade innate immune responses and establish a favorable gut ecosystem.
Robert CB, Thomson M, Vercellone A, et al., 2017, Mass spectrometry analysis of intact Francisella bacteria identifies lipid A structure remodeling in response to acidic pH stress, BIOCHIMIE, Vol: 141, Pages: 16-20, ISSN: 0300-9084
Buchieri MV, Cimino M, Rebollo-Ramirez S, et al., 2017, Nitazoxanide Analogs Require Nitroreduction for Antimicrobial Activity in Mycobacterium smegmatis, JOURNAL OF MEDICINAL CHEMISTRY, Vol: 60, Pages: 7425-7433, ISSN: 0022-2623
Pacholarz KJ, Burnley RJ, Jowitt TA, et al., 2017, Hybrid Mass Spectrometry Approaches to Determine How L-Histidine Feedback Regulates the Enzyzme MtATP-Phosphoribosyltransferase, Structure, Vol: 25, Pages: 730-738.e4, ISSN: 1878-4186
MtATP-phosphoribosyltransferase (MtATP-PRT) is an enzyme catalyzing the first step of the biosynthesis of L-histidine in Mycobacterium tuberculosis, and proposed to be regulated via an allosteric mechanism. Native mass spectrometry (MS) reveals MtATP-PRT to exist as a hexamer. Conformational changes induced by L-histidine binding and the influence of buffer pH are determined with ion mobility MS, hydrogen deuterium exchange (HDX) MS, and analytical ultracentrifugation. The experimental collision cross-section (DTCCSHe) decreases from 76.6 to 73.5 nm2 upon ligand binding at pH 6.8, which correlates to the decrease in CCS calculated from crystal structures. No such changes in conformation were found at pH 9.0. Further detail on the regions that exhibit conformational change on L-histidine binding is obtained with HDX-MS experiments. On incubation with L-histidine, rapid changes are observed within domain III, and around the active site at longer times, indicating an allosteric effect.
Larrouy-Maumus G, Layre E, Clark S, et al., 2017, Protective efficacy of a lipid antigen vaccine in a guinea pig model of tuberculosis, VACCINE, Vol: 35, Pages: 1395-1402, ISSN: 0264-410X
Sirianni A, Krokowski S, Lobato-Márquez D, et al., 2016, Mitochondria mediate septin cage assembly to promote autophagy of Shigella, EMBO Reports, Vol: 17, ISSN: 1469-221X
Septins, cytoskeletal proteins with well-characterised roles in cytokinesis, form cage-like structures around cytosolic Shigella flexneri and promote their targeting to autophagosomes. However, the processes underlying septin cage assembly, and whether they influence S. flexneri proliferation, remain to be established. Using single cell analysis, we show that septin cages inhibit S. flexneri proliferation. To study mechanisms of septin cage assembly, we used proteomics and found mitochondrial proteins associate with septins in S. flexneriinfected cells. Strikingly, mitochondria associated with S. flexneri promote septin assembly into the cages that entrap bacteria for autophagy. We demonstrate that the cytosolic GTPase dynamin-related protein 1 (Drp1) interacts with septins to enhance mitochondrial fission. To avoid autophagy, actin-polymerising Shigella fragment mitochondria to escape from septin caging. Our results have demonstrated a role for mitochondria in anti-Shigella autophagy, and uncovered a fundamental link between septin assembly and mitochondria.
Larrouy-Maumus GJ, Leonardo B Marino, Ashoka V R Madduri, et al., 2016, Cell-Envelope Remodeling as a Determinant of Phenotypic Antibacterial Tolerance in Mycobacterium tuberculosis, ACS Infectious Diseases, Vol: 2, Pages: 352-360, ISSN: 2373-8227
The mechanisms that lead to phenotypic antibacterial tolerance in bacteria remain poorly understood. We investigate whether changes in NaCl concentration toward physiologically higher values affect antibacterial efficacy against Mycobacterium tuberculosis (Mtb), the causal agent of human tuberculosis. Indeed, multiclass phenotypic antibacterial tolerance is observed during Mtb growth in physiologic saline. This includes changes in sensitivity to ethionamide, ethambutol, d-cycloserine, several aminoglycosides, and quinolones. By employing organism-wide metabolomic and lipidomic approaches combined with phenotypic tests, we identified a time-dependent biphasic adaptive response after exposure of Mtb to physiological levels of NaCl. A first rapid, extensive, and reversible phase was associated with changes in core and amino acid metabolism. In a second phase, Mtb responded with a substantial remodelling of plasma membrane and outer lipid membrane composition. We demonstrate that phenotypic tolerance at physiological concentrations of NaCl is the result of changes in plasma and outer membrane lipid remodeling and not changes in core metabolism. Altogether, these results indicate that physiologic saline-induced antibacterial tolerance is kinetically coupled to cell envelope changes and demonstrate that metabolic changes and growth arrest are not the cause of phenotypic tolerance observed in Mtb exposed to physiologic concentrations of NaCl. Importantly, this work uncovers a role for bacterial cell envelope remodeling in antibacterial tolerance, alongside well-documented allterations in respiration, metabolism, and growth rate.
Larrouy-Maumus GJ, Abigail Clements, Alain Filloux, et al., 2015, Direct detection of lipid A on intact Gram-negative bacteria byMALDI-TOF mass spectrometry, Journal of Microbiological Methods, Vol: 120, Pages: 68-71, ISSN: 1872-8359
The purification and characterization of Gram-negative bacterial lipid A is tedious and time-consuming. Herein we report a rapid and sensitive method to identify lipid A directly on intact bacteria without any chemical treatment or purification, using an atypical solvent system to solubilize the matrix combined with MALDI-TOF mass spectrometry.
Larrouy-Maumus GJ, Gilleron M, Skovierova H, et al., 2015, A glycomic approach reveals a new mycobacterial polysaccharide, Glycobiology, Vol: 25, Pages: 1163-1171, ISSN: 1460-2423
Mycobacterium tuberculosis lipoarabinomannan (LAM) and biosynthetically related lipoglycans and glycans play an important role in host–pathogen interactions. Therefore, the elucidation of the complete biosynthetic pathways of these important molecules is expected to afford novel therapeutic targets. The characterization of biosynthetic enzymes and transporters involved in the formation and localization of these complex macromolecules in the bacterial cell envelope largely relies on genetic manipulation of mycobacteria and subsequent analyses of lipoglycan structural alterations. However, lipoglycans are present in relatively low amounts. Their purification to homogeneity remains tedious and time-consuming. To overcome these issues and to reduce the biomass and time required for lipoglycan purification, we report here the development of a methodology to efficiently purify lipoglycans by sodium deoxycholate–polyacrylamide gel electrophoresis. This faster purification method can be applied on a small amount of mycobacterial cells biomass (10–50 mg), resulting in tens of micrograms of purified lipoglycans. This amount of purified products was found to be sufficient to undertake structural analyses of lipoglycans and glycans carbohydrate domains by a combination of highly sensitive analytical procedures, involving cryoprobe NMR analysis of intact macromolecules and chemical degradations monitored by gas chromatography and capillary electrophoresis. This glycomic approach was successfully applied to the purification and structural characterization of a newly identified polysaccharide, structurally related to LAM, in the model fast-growing species Mycobacterium smegmatis.
Wheat WH, Dhouib R, Angala SK, et al., 2015, The presence of a galactosamine substituent on the arabinogalactan of Mycobacterium tuberculosis abrogates full maturation of human peripheral blood monocyte-derived dendritic cells and increases secretion of IL-10, TUBERCULOSIS, Vol: 95, Pages: 476-489, ISSN: 1472-9792
Larrouy-Maumus GJ, Cholesterol acquisition by Mycobacterium tuberculosis, Virulence, ISSN: 2150-5608
Larrouy-Maumus G, Puzo G, 2015, Mycobacterial envelope lipids fingerprint from direct MALDI-TOF MS analysis of intact bacilli, TUBERCULOSIS, Vol: 95, Pages: 75-85, ISSN: 1472-9792
Belardinelli JM, Larrouy-Maumus G, Jones V, et al., 2014, Biosynthesis and Translocation of Unsulfated Acyltrehaloses in Mycobacterium tuberculosis, Journal of Biological Chemistry, Vol: 289, Pages: 27952-27965, ISSN: 1083-351X
Prosser GA, Larrouy-Maumus G, de Carvalho LPS, 2014, Metabolomic strategies for the identification of new enzyme functions and metabolic pathways, EMBO REPORTS, Vol: 15, Pages: 657-669, ISSN: 1469-221X
Gouzy A, Larrouy-Maumus G, Bottai D, et al., 2014, Mycobacterium tuberculosis Exploits Asparagine to Assimilate Nitrogen and Resist Acid Stress during Infection, PLOS PATHOGENS, Vol: 10, ISSN: 1553-7366
Gouzy A, Larrouy-Maumus G, Wu T-D, et al., 2014, Mycobacterium tuberculosis nitrogen assimilation and host colonization require aspartate (vol 9, pg 674, 2013), NATURE CHEMICAL BIOLOGY, Vol: 10, Pages: 164-164, ISSN: 1552-4450
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