RESUMEN
Tuberculosis (TB), caused by Mycobacterium tuberculosis, remains a major human pandemic. Germline-encoded mycolyl lipid-reactive (GEM) T cells are donor-unrestricted and recognize CD1b-presented mycobacterial mycolates. However, the molecular requirements governing mycolate antigenicity for the GEM T cell receptor (TCR) remain poorly understood. Here, we demonstrate CD1b expression in TB granulomas and reveal a central role for meromycolate chains in influencing GEM-TCR activity. Meromycolate fine structure influences T cell responses in TB-exposed individuals, and meromycolate alterations modulate functional responses by GEM-TCRs. Computational simulations suggest that meromycolate chain dynamics regulate mycolate head group movement, thereby modulating GEM-TCR activity. Our findings have significant implications for the design of future vaccines that target GEM T cells.
Asunto(s)
Antígenos CD1/inmunología , Mycobacterium tuberculosis/inmunología , Mycobacterium tuberculosis/metabolismo , Ácidos Micólicos/inmunología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Tuberculosis/inmunología , Antígenos Bacterianos/inmunología , Antígenos Bacterianos/metabolismo , Antígenos CD1/química , Antígenos CD1/genética , Expresión Génica , Granuloma/inmunología , Granuloma/metabolismo , Granuloma/microbiología , Granuloma/patología , Humanos , Inmunohistoquímica , Activación de Linfocitos/inmunología , Modelos Moleculares , Conformación Molecular , Ácidos Micólicos/química , Ácidos Micólicos/metabolismo , Unión Proteica , Receptores de Antígenos de Linfocitos T/metabolismo , Tuberculosis/microbiologíaRESUMEN
Mycolic acids (MAs) are highly hydrophobic long-chain α-alkyl ß-hydroxy fatty acids present in the cell wall of Mycobacterium tuberculosis (Mtb) as a complex mixture of molecules with a common general structure but with variable functional groups in the meromycolate chain. In this study, we addressed the relationship between the MA molecular structure and their contribution to the development of T-cell immune responses. Hereto, we used the model antigen ovalbumin and single synthetic MAs, differing in oxygenation class and cis versus trans proximal cyclopropane configuration, as immune stimulatory agents. Subcutaneous delivery of liposome-formulated MAs with a proximal cis cyclopropane elicited antigen-specific Th1 and cytotoxic T-cell immune responses, whereas intratracheal immunization elicited pulmonary Th17 responses. These immune stimulatory activities depended not only on the cis versus trans proximal cyclopropane configuration but also on the MA oxygenation class. Our study thus shows that both the presence and nature of the functional groups in the meromycolate chain affect the immune stimulatory adjuvant activity of Mtb mycolates and suggests that Mtb bacilli may impact on the host protective immune response by modulating the cis versus trans stereochemistry of its mycolates as well as by altering the oxygenation class of the meromycolate functional group.
Asunto(s)
Adyuvantes Inmunológicos , Mycobacterium tuberculosis/inmunología , Ácidos Micólicos/inmunología , Tuberculosis/inmunología , Animales , Citocinas/biosíntesis , Femenino , Inmunización , Inmunoglobulina G/inmunología , Factores Inmunológicos , Inmunomodulación , Inyección Intratimpánica , Inyecciones Subcutáneas , Interferón gamma/biosíntesis , Liposomas/química , Activación de Linfocitos , Ratones , Mycobacterium tuberculosis/metabolismo , Ácidos Micólicos/administración & dosificación , Ácidos Micólicos/química , Fosfatidilcolinas/química , Especificidad del Receptor de Antígeno de Linfocitos T/inmunología , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/metabolismo , Células TH1/inmunología , Células TH1/metabolismo , Células Th17/inmunología , Células Th17/metabolismo , Tuberculosis/prevención & control , Vacunas contra la Tuberculosis/administración & dosificación , Vacunas contra la Tuberculosis/inmunologíaRESUMEN
THE PROBLEM: Ante-mortem diagnosis of Johne's disease, caused by Mycobacterium avium subsp. paratuberculosis (MAP), is normally achieved through faecal culture, PCR, or serological tests, but agreement as to which samples are positive for Johne's disease is often poor and sensitivities are low, particularly in early-stage infections. The potential solution: Mycobacterial cells contain very complex characteristic mixtures of mycolic acid derivatives that elicit antibodies during infection; this has been used to detect infections in humans. Here, we explore its application in providing an assay differentiating infected from vaccinated animals (DIVA assay) for Johne's disease in cattle. METHOD: Antibody responses to different classes of mycolic acid derivatives were measured using ELISA for serum from cattle positive for MAP by both faecal PCR and commercial serum ELISA, or just by PCR, and from animals from herds with no history of Johne's disease, bovine tuberculosis reactors, BCG-vaccinated, BCG-vaccinated and M. bovis-infected, and Gudair-vaccinated animals. RESULTS: The best-performing antigens, ZAM295 and ST123-the latter a molecule present in the cells of MAP but not of Mycobacterium bovis-achieved a sensitivity of 75% and 62.5%, respectively, for serum from animals positive by both faecal PCR and a commercial MAP serum ELISA, at a specificity of 94% compared to 80 no-history negatives. Combining the results of separate assays with two antigens (ST123 and JRRR121) increased the sensitivity/specificity to 75/97.5%. At the same cut-offs, animals vaccinated with Gudair or BCG vaccines and bTB reactors showed a similar specificity. The specificity in BCG-vaccinated but M. bovis-infected animals dropped to 85%. Combining the results of two antigens gave a sensitivity/specificity of 37.5/97.5% for the full set of 80 PCR-positive samples, detecting 30 positives compared 16 for IDEXX. CONCLUSION: Serum ELISA using synthetic lipids distinguishes effectively between MAP-negative cattle samples and those positive by both PCR and a commercial MAP serodiagnostic, without interference by Gudair or BCG vaccination. It identified almost twice as many PCR positives as the commercial serodiagnostic, offering the possibility of earlier detection of infection.
RESUMEN
Mycolic acids (MAs) occur in the cell wall of Mycobacterium tuberculosis as variable mixtures of different classes and chain lengths. Here, we address the relationship between the structure and its inflammatory function of this virulence factor using single synthetic MA isomers, differing in oxygenation class and cis- versus α-methyl-trans proximal cyclopropane orientation. Analysis of bronchoalveolar inflammation, lung histopathology and alveolar macrophage transcription revealed a strong dependence on these meromycolic chemistries of mouse pulmonary inflammation in response to intratracheal treatments with MAs. Whereas α-MA was inert, oxygenated methoxy- and keto-MA with cis-cyclopropane stereochemistry elicited solid to mild inflammatory responses respectively. In trans-cyclopropane orientation, methoxy-MA partially lost its inflammatory activity and keto-MA exerted anti-inflammatory alternative activation of alveolar macrophages and counteracted cis-methoxy-MA induced airway inflammation. The differential innate immune activities of MAs demonstrated here, dependent on oxygenation class and cis versus α-methyl-trans cyclopropane chemistry, identify a novel means for M. tuberculosis to steer host immune responses during infection.
Asunto(s)
Mycobacterium tuberculosis/química , Ácidos Micólicos/química , Ácidos Micólicos/inmunología , Factores de Virulencia/química , Factores de Virulencia/inmunología , Animales , Líquido del Lavado Bronquioalveolar/citología , Líquido del Lavado Bronquioalveolar/inmunología , Recuento de Células , Femenino , Expresión Génica/genética , Inmunidad Innata/inmunología , Inflamación/inducido químicamente , Inflamación/inmunología , Inflamación/patología , Liposomas , Pulmón/inmunología , Pulmón/patología , Activación de Macrófagos/efectos de los fármacos , Activación de Macrófagos/inmunología , Macrófagos Alveolares/inmunología , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/patología , Ratones , Ratones Endogámicos C57BL , Estructura Molecular , Mycobacterium tuberculosis/inmunología , Ácidos Micólicos/administración & dosificación , Ácidos Micólicos/farmacología , Neutrófilos/inmunología , Neutrófilos/patología , Estereoisomerismo , Factores de Virulencia/administración & dosificación , Factores de Virulencia/farmacologíaRESUMEN
Background: mycobacterial cells contain complex mixtures of mycolic acid esters. These can be used as antigens recognised by antibodies in the serum of individuals with active tuberculosis, caused by Mycobacterium tuberculosis. In high burden populations, a significant number of false positives are observed; possibly these antigens are also recognised by antibodies generated by other mycobacterial infections, particularly ubiquitous 'environmental mycobacteria'. This suggests similar responses may be observed in a low burden TB population, particularly in groups regularly exposed to mycobacteria. Methods: ELISA using single synthetic trehalose mycolates corresponding to major classes in many mycobacteria was used to detect antibodies in serum of individuals with no known mycobacterial infection, comprising farmers, abattoir workers, and rural and urban populations. Results: serum from four Welsh or Scottish cohorts showed lower (with some antigens significantly lower) median responses than those reported for TB negatives from high-burden TB populations, and significantly lower responses than those with active TB. A small fraction, particularly older farmers, showed strong responses. A second study examined BCG vaccinated and non-vaccinated farmers and non-farmers. Farmers gave significantly higher median responses than non-farmers with three of five antigens, while there was no significant difference between vaccinated or non-vaccinated for either farmer or non-farmer groups. Conclusions: this initial study shows that serodiagnosis with mycobacterial lipid antigens can detect antibodies in a population sub-group that is significantly exposed to mycobacteria, in an assay that is not interfered with by vaccination. Given the links between mycobacterial exposure and a range of immune system diseases, further understanding such responses may provide a new opportunity for monitoring public health and directing treatment.
RESUMEN
Mycolic acid methyl esters were extracted from Mycobacterium avium by a mild saponification protocol, designed to preserve labile components. The resulting mixture of α-, keto- and wax ester mycolates was accompanied by some degraded ω-carboxymycolic acid dimethyl esters, whose overall structures were found to support previous studies. Chromatography of the mono-carboxylic mycolates gave an inseparable mixture of keto- and wax ester mycolates and separate α-mycolates. Reduction of the ketomycolate components allowed isolation and characterisation of intact wax ester mycolates for the first time. Minor α- and ω-carboxymycolates were detected in which methyl branches were located on either the proximal or distal sides of trans-alkene groups.
Asunto(s)
Mycobacterium avium/química , Ácidos Micólicos/química , Alquenos/química , Dimerización , Ésteres/química , Ácidos Micólicos/aislamiento & purificación , EstereoisomerismoRESUMEN
Cells of Mycobacterium alvei are known to contain a unique set of mycolic acids with a (ω-1)-methoxy group; although the various enzymes in the biosynthesis of other types of mycolic acid have been widely studied, the biosynthetic route to this substituent is unclear. We now define the stereochemistry of the (ω-1)-methoxy fragment as R, and describe the synthesis of a major R-(ω-1)-methoxy-mycolic acid and its sugar esters, and of two natural M. alvei diene mycolic acids.
Asunto(s)
Mycobacteriaceae/química , Ácidos Micólicos/síntesis química , Estructura Molecular , Mycobacteriaceae/citología , Ácidos Micólicos/química , EstereoisomerismoRESUMEN
Complex mixtures of natural dimycoloyl diarabinoglycerols isolated from mycobacteria have been shown to be both potent immune signalling agents and potentially valuable antigens in the serodiagnosis of mycobacterial infections. We now report the highly stereocontrolled synthesis of diacyl l-glycerol-(1'â1)-ß-d-arabinofuranosyl-α-d-arabinofuranosides based on simple fatty acids and single defined synthetic mycolic acids. NMR analysis confirmed that the synthetic core was identical to that in natural mixtures.
Asunto(s)
Disacáridos/síntesis química , Mycobacterium/química , Ácidos Micólicos/síntesis química , Disacáridos/química , Conformación Molecular , Ácidos Micólicos/química , EstereoisomerismoRESUMEN
An efficient synthetic approach to tri-arabino di-mycolates, using structurally defined synthetic α-, keto and methoxy mycolic acids is described.
Asunto(s)
Galactanos/química , Ácidos Micólicos/química , Adyuvantes Inmunológicos/síntesis química , Adyuvantes Inmunológicos/química , Adyuvantes Inmunológicos/farmacología , Pared Celular/química , Técnicas de Química Sintética , Galactanos/síntesis química , Galactanos/farmacología , Interleucina-6/metabolismo , Mycobacterium tuberculosis/química , Estereoisomerismo , Trisacáridos/síntesis química , Trisacáridos/química , Trisacáridos/farmacología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
R- and S-Glycerol mycolates derived from single synthetic α-, keto- and methoxy-mycolic acids are described.
Asunto(s)
Glicerol/química , Ácidos Micólicos/química , Ácidos Micólicos/síntesis química , EstereoisomerismoRESUMEN
BACKGROUND: During pulmonary tuberculosis (PTB) antibodies are generated to trehalose esters of mycolic acids which are cell wall lipids of Mycobacterium tuberculosis (Mtb). Attempts have been made to use these complex natural mixtures in serological tests for PTB diagnosis. AIM: The aim of this work was to determine whether a serological test based on a panel of defined individual trehalose esters of characteristic synthetic mycolic acids has improved diagnostic accuracy in distinguishing patients with culture positive PTB from individuals who were Mtb culture negative. METHOD: One hundred serum samples from well-characterized patients with presumptive tuberculosis, and diagnosed as having pulmonary smear and culture positive TB, or being culture and smear negative were evaluated by ELISA using different combinations of synthetic antigens and secondary antibodies. Using cut-off values determined from these samples, we validated this study blind in samples from a further 249 presumptive TB patients. RESULTS: With the first 100 samples, detailed responses depended both on the precise structure of the antigen and on the secondary antibody. Using a single antigen, a sensitivity/specificity combination for smear and culture positive PTB detection of 85 and 88% respectively was achieved; this increased to 96% and 95% respectively by a statistical combination of the results with seven antigens. In the blind study a sensitivity/specificity of 87% and 83% was reached with a single antigen. With some synthetic antigens, the responses from all 349 samples were significantly better than those with the natural mixture. Combining the results for seven antigens allowed a distinction between culture positive and negative with a ROC AUC of 0.95. CONCLUSION: We have identified promising antigen candidates for serological assays that could be used to diagnose PTB and which could be the basis of a much-needed, simple, rapid diagnostic test that would bring care closer to communities.
Asunto(s)
Antígenos Bacterianos/metabolismo , Pruebas Serológicas/métodos , Tuberculosis Pulmonar/sangre , Tuberculosis Pulmonar/diagnóstico , Adulto , Algoritmos , Antígenos Bacterianos/química , Demografía , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Masculino , Persona de Mediana Edad , Ácidos Micólicos/química , Valor Predictivo de las Pruebas , Tuberculosis Pulmonar/microbiología , Adulto JovenRESUMEN
The preparation of 6-O-mycolylglucoses (GMMs) from single synthetic mycolic acids matching the overall structure of some of the major natural glucose monomycolates of Mycobacterium tuberculosis and other mycobacteria is reported.
Asunto(s)
Glucolípidos/síntesis química , Mycobacterium/química , Ácidos Micólicos/química , Glucolípidos/química , Conformación MolecularRESUMEN
The synthesis of an α'-mycolic acid of Mycobacterium smegmatis and other mycobacteria, containing a cis,cis-diene, and of the trehalose mono- and di-mycolates of this, and of a related α'-mycolic acid containing one cis-alkene, is reported.
Asunto(s)
Alquenos/química , Ácidos Micólicos/química , Trehalosa/química , Ésteres , Isomerismo , Espectroscopía de Resonancia Magnética , Mycobacterium/química , Mycobacterium/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Trehalosa/síntesis químicaRESUMEN
We report a synthesis of a single enantiomer of a dicyclopropane containing mycolic acid from Mycobacterium tuberculosis; this method can be simply varied to modify the chain lengths or the absolute stereochemistry of either cyclopropane.
Asunto(s)
Mycobacterium tuberculosis/química , Ácidos Micólicos/síntesis química , Ciclopropanos , Estructura Molecular , Ácidos Micólicos/química , EstereoisomerismoRESUMEN
A 23-26-carbon chain length range of omega-19 (1'R,2'S) cyclopropane fatty acids, related to mycobacterial mycolic acids, has been prepared. The key cyclopropyl intermediate, (1'R,2'S)-(Z)-1-formyl-2-octadecylcyclopropane, underwent Wittig chemistry with various reagents to provide vinylic precursors, which were selectively reduced to the corresponding saturated omega-19 cyclopropane fatty acids or esters. The 24-carbon omega-19 cyclopropane ester was made by chain elongation of the 23-carbon ester. Saturated and unsaturated chiral cyclopropane acids and esters were assayed, using wall extracts of Mycobacterium smegmatis; the incorporation of 14C-acetate was used to measure inhibition or stimulation of mycolic acid synthesis. Minor inhibition (2-3%) was shown by the 23- and 24-carbon saturated esters; all the other compounds were stimulants. The most effective (38-55%) stimulators of mycolate synthesis were the unsaturated esters with 23- and 26-carbons and the saturated and unsaturated 25-carbon acids.
Asunto(s)
Ciclopropanos/síntesis química , Ácidos Grasos/síntesis química , Mycobacterium smegmatis/química , Ácidos Micólicos/síntesis química , Valeratos/síntesis química , Isótopos de Carbono/química , Pared Celular/química , Cromatografía en Capa Delgada , Ciclopropanos/química , Ésteres/química , Ácidos Grasos/química , Ácidos Micólicos/química , Valeratos/químicaRESUMEN
Ozonolysis of Z,Z,Z-cylonona-1,4,7-triene leads to a 1,9-difunctionalised Z,Z-3,6-nonadiene which is readily converted into a range of polyunsaturated pheromones and fatty acids.
Asunto(s)
Carbono/química , Ácidos Grasos Insaturados/síntesis química , Oxígeno/química , Feromonas/síntesis química , Polienos/síntesis químicaRESUMEN
Both diastereomers of the tris-allene, cyclododeca-1,2,5,6,9,10-hexaene have been obtained using a triple cyclopropylidene-allene rearrangement. On the NMR timescale, one has D3 symmetry, and is the smallest hydrocarbon synthesised to have this symmetry, and the second has C2 symmetry.
Asunto(s)
Alcadienos/química , Espectroscopía de Resonancia Magnética , Conformación Molecular , Teoría Cuántica , EstereoisomerismoRESUMEN
Patient serum antibodies to mycolic acids have the potential to be surrogate markers of active tuberculosis (TB) when they can be distinguished from the ubiquitously present cross-reactive antibodies to cholesterol. Mycolic acids are known to interact more strongly with antibodies present in the serum of patients with active TB than in patients with latent TB or no TB. Examples of single stereoisomers of mycolic acids with chain lengths corresponding to major homologues of those present in Mycobacterium tuberculosis have now been synthesised with a sulfur substituent on the terminal position of the α-chain; initial studies have established that one of these binds to a gold electrode surface, offering the potential to develop second generation sensors for diagnostic patient antibody detection.
Asunto(s)
Ácidos Micólicos/química , Compuestos de Sulfhidrilo/química , Anticuerpos/inmunología , Técnicas Electroquímicas , Electrodos , Humanos , Ácidos Micólicos/síntesis química , Ácidos Micólicos/inmunología , Estereoisomerismo , Tuberculosis/inmunología , Tuberculosis/metabolismo , Tuberculosis/patologíaRESUMEN
Cell wall mycolic acids (MA) from Mycobacterium tuberculosis (M.tb) are CD1b presented antigens that can be used to detect antibodies as surrogate markers of active TB, even in HIV coinfected patients. The use of the complex mixtures of natural MA is complicated by an apparent antibody cross-reactivity with cholesterol. Here firstly we report three recombinant monoclonal scFv antibody fragments in the chicken germ-line antibody repertoire, which demonstrate the possibilities for cross-reactivity: the first recognized both cholesterol and mycolic acids, the second mycolic acids but not cholesterol, and the third cholesterol but not mycolic acids. Secondly, MA structure is experimentally interrogated to try to understand the cross-reactivity. Unique synthetic mycolic acids representative of the three main functional classes show varying antigenicity against human TB patient sera, depending on the functional groups present and on their stereochemistry. Oxygenated (methoxy- and keto-) mycolic acid was found to be more antigenic than alpha-mycolic acids. Synthetic methoxy-mycolic acids were the most antigenic, one containing a trans-cyclopropane apparently being somewhat more antigenic than the natural mixture. Trans-cyclopropane-containing keto- and hydroxy-mycolic acids were also found to be the most antigenic among each of these classes. However, none of the individual synthetic mycolic acids significantly and reproducibly distinguished the pooled serum of TB positive patients from that of TB negative patients better than the natural mixture of MA. This argues against the potential to improve the specificity of serodiagnosis of TB with a defined single synthetic mycolic acid antigen from this set, although sensitivity may be facilitated by using a synthetic methoxy-mycolic acid.