RESUMEN
Glucuronosyl diacylglycerides (GlcAGroAc2) are functionally important glycolipids and membrane anchors for cell wall lipoglycans in the Corynebacteria. Here we describe the complete synthesis of distinct acyl-isoforms of GlcAGroAc2 bearing both acylation patterns of (R)-tuberculostearic acid (C19:0) and palmitic acid (C16:0) and their mass spectral characterization. Collision-induced fragmentation mass spectrometry identified characteristic fragment ions that were used to develop "rules" allowing the assignment of the acylation pattern as C19:0 (sn-1), C16:0 (sn-2) in the natural product from Mycobacterium smegmatis, and the structural assignment of related C18:1 (sn-1), C16:0 (sn-2) GlcAGroAc2 glycolipids from M. smegmatis and Corynebacterium glutamicum. A synthetic hydrophobic octyl glucuronoside was used to characterize the GDP-mannose-dependent mannosyltransferase MgtA from C. glutamicum that extends GlcAGroAc2. This enzyme is an Mg(2+)/Mn(2+)-dependent metalloenzyme that undergoes dramatic activation upon reduction with dithiothreitol.
Asunto(s)
Proteínas Bacterianas/química , Corynebacterium/química , Glicéridos/análisis , Glicéridos/síntesis química , Glucolípidos/análisis , Glucolípidos/síntesis química , Magnesio/química , Manosiltransferasas/química , Mycobacterium smegmatis/química , Mycobacterium/química , Ácidos Esteáricos/química , Vías Biosintéticas , Glicéridos/química , Glucolípidos/química , Espectrometría de MasasRESUMEN
Lipoarabinomannans (LAMs) and phosphatidylinositol mannosides (PIMs) are abundant glycolipids in the cell walls of all corynebacteria and mycobacteria, including the devastating human pathogen Mycobacterium tuberculosis. We have recently shown that M. smegmatis mutants of the lipoprotein-encoding lpqW gene have a profound defect in LAM biosynthesis. When these mutants are cultured in complex medium, spontaneous bypass mutants consistently evolve in which LAM biosynthesis is restored at the expense of polar PIM synthesis. Here we show that restoration of LAM biosynthesis in the lpqW mutant results from secondary mutations in the pimE gene. PimE is a mannosyltransferase involved in converting AcPIM4, a proposed branch point intermediate in the PIM and LAM biosynthetic pathways, to more polar PIMs. Mutations in pimE arose due to insertion of the mobile genetic element ISMsm1 and independent point mutations that were clustered in predicted extracytoplasmic loops of this polytopic membrane protein. Our findings provide the first strong evidence that LpqW is required to channel intermediates such as AcPIM4 into LAM synthesis and that loss of PimE function results in the accumulation of AcPIM4, bypassing the need for LpqW. These data highlight new mechanisms regulating the biosynthetic pathways of these essential cell wall components.
Asunto(s)
Lipopolisacáridos/biosíntesis , Lipoproteínas/genética , Manosiltransferasas/metabolismo , Manosiltransferasas/fisiología , Mycobacterium smegmatis/metabolismo , Pared Celular/metabolismo , Lipoproteínas/metabolismo , Manosiltransferasas/química , Manosiltransferasas/genética , Mutación , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/crecimiento & desarrollo , Fosfatidilinositoles/biosíntesis , Fosfatidilinositoles/metabolismoRESUMEN
PI (phosphatidylinositol) is a ubiquitous eukaryotic phospholipid which serves as a precursor for messenger molecules and GPI (glycosylphosphatidylinositol) anchors. PI is synthesized either de novo or by head group exchange by a PIS (PI synthase). The synthesis of GPI anchors has previously been validated both genetically and chemically as a drug target in Trypanosoma brucei, the causative parasite of African sleeping sickness. However, nothing is known about the synthesis of PI in this organism. Database mining revealed a putative TbPIS gene in the T. brucei genome and by recombinant expression and characterization it was shown to encode a catalytically active PIS, with a high specificity for myo-inositol. Immunofluorescence revealed that in T. brucei, PIS is found in both the endoplasmic reticulum and Golgi. We created a conditional double knockout of TbPIS in the bloodstream form of T. brucei, which when grown under non-permissive conditions, clearly showed that TbPIS is an essential gene. In vivo labelling of these conditional double knockout cells confirmed this result, showing a decrease in the amount of PI formed by the cells when grown under non-permissive conditions. Furthermore, quantitative and qualitative analysis by GLC-MS and ESI-MS/MS (electrospray ionization MS/MS) respectively showed a significant decrease (70%) in cellular PI, which appears to affect all major PI species equally. A consequence of this fall in PI level is a knock-on reduction in GPI biosynthesis which is essential for the parasite's survival. The results presented here show that PI synthesis is essential for bloodstream form T. brucei, and to our knowledge this is the first report of the dependence on PI synthesis of a protozoan parasite by genetic validation.
Asunto(s)
Fosfatidilinositoles/biosíntesis , Trypanosoma brucei brucei/metabolismo , Secuencia de Aminoácidos , Animales , CDP-Diacilglicerol-Inositol 3-Fosfatidiltransferasa/química , CDP-Diacilglicerol-Inositol 3-Fosfatidiltransferasa/genética , CDP-Diacilglicerol-Inositol 3-Fosfatidiltransferasa/metabolismo , Clonación Molecular , Retículo Endoplásmico/metabolismo , Eliminación de Gen , Aparato de Golgi/metabolismo , Datos de Secuencia Molecular , Fenotipo , Fosfatidilinositoles/sangre , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Trypanosoma brucei brucei/citología , Trypanosoma brucei brucei/enzimologíaRESUMEN
The cell walls of the Corynebacterineae, which includes the important human pathogen Mycobacterium tuberculosis, contain two major lipopolysaccharides, lipoarabinomannan (LAM) and lipomannan (LM). LAM is assembled on a subpool of phosphatidylinositol mannosides (PIMs), whereas the identity of the LM lipid anchor is less well characterized. In this study we have identified a new gene (Rv2188c in M. tuberculosis and NCgl2106 in Corynebacterium glutamicum) that encodes a mannosyltransferase involved in the synthesis of the early dimannosylated PIM species, acyl-PIM2, and LAM. Disruption of the C. glutamicum NCgl2106 gene resulted in loss of synthesis of AcPIM2 and accumulation of the monomannosylated precursor, AcPIM1. The synthesis of a structurally unrelated mannolipid, Gl-X, was unaffected. The synthesis of AcPIM2 in C. glutamicum DeltaNCgl2106 was restored by complementation with M. tuberculosis Rv2188c. In vivo labeling of the mutant with [3H]Man and in vitro labeling of membranes with GDP-[3H]Man confirmed that NCgl2106/Rv2188c catalyzed the second mannose addition in PIM biosynthesis, a function previously ascribed to PimB/Rv0557. The C. glutamicum Delta NCgl2106 mutant lacked mature LAM but unexpectedly still synthesized the major pool of LM. Biochemical analyses of the LM core indicated that this lipopolysaccharide was assembled on Gl-X. These data suggest that NCgl2106/Rv2188c and the previously studied PimB/Rv0557 transfer mannose residues to distinct mannoglycolipids that act as precursors for LAM and LM, respectively.
Asunto(s)
Corynebacterium glutamicum/metabolismo , Regulación Bacteriana de la Expresión Génica , Lipopolisacáridos/química , Manosiltransferasas/metabolismo , Mycobacterium tuberculosis/metabolismo , Fosfatidilinositoles/química , Secuencia de Aminoácidos , Pared Celular/metabolismo , Prueba de Complementación Genética , Glicosiltransferasas/metabolismo , Modelos Biológicos , Modelos Químicos , Datos de Secuencia Molecular , Mutación , Homología de Secuencia de AminoácidoRESUMEN
A gene encoding Trypanosoma brucei UDP-N-acetylglucosamine pyrophosphorylase was identified, and the recombinant protein was shown to have enzymatic activity. The parasite enzyme is unusual in having a strict substrate specificity for N-acetylglucosamine 1-phosphate and in being located inside a peroxisome-like microbody, the glycosome. A bloodstream form T. brucei conditional null mutant was constructed and shown to be unable to sustain growth in vitro or in vivo under nonpermissive conditions, demonstrating that there are no alternative metabolic or nutritional routes to UDP-N-acetylglucosamine and providing a genetic validation for the enzyme as a potential drug target. The conditional null mutant was also used to investigate the effects of N-acetylglucosamine starvation in the parasite. After 48 h under nonpermissive conditions, about 24 h before cell lysis, the status of parasite glycoprotein glycosylation was assessed. Under these conditions, UDP-N-acetylglucosamine levels were less than 5% of wild type. Lectin blotting and fluorescence microscopy with tomato lectin revealed that poly-N-acetyllactosamine structures were greatly reduced in the parasite. The principal parasite surface coat component, the variant surface glycoprotein, was also analyzed. Endoglycosidase digestions and mass spectrometry showed that, under UDP-N-acetylglucosamine starvation, the variant surface glycoprotein was specifically underglycosylated at its C-terminal Asn-428 N-glycosylation site. The significance of this finding, with respect to the hierarchy of site-specific N-glycosylation in T. brucei, is discussed.
Asunto(s)
Nucleotidiltransferasas/biosíntesis , Modificación Traduccional de las Proteínas/fisiología , Proteínas Protozoarias/biosíntesis , Trypanosoma brucei brucei/enzimología , Uridina Difosfato N-Acetilglucosamina/biosíntesis , Acetilglucosamina/análogos & derivados , Acetilglucosamina/metabolismo , Animales , Glicosilación , Nucleotidiltransferasas/química , Nucleotidiltransferasas/genética , Lectinas de Plantas/química , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Trypanosoma brucei brucei/genética , Uridina Difosfato N-Acetilglucosamina/genéticaRESUMEN
In bloodstream-form Trypanosoma brucei (the causative agent of African sleeping sickness) the glycosylphosphatidylinositol (GPI) anchor biosynthetic pathway has been validated genetically and chemically as a drug target. The conundrum that GPI anchors could not be in vivo labelled with [3H]-inositol led us to hypothesize that de novo synthesis was responsible for supplying myo-inositol for phosphatidylinositol (PI) destined for GPI synthesis. The rate-limiting step of the de novo synthesis is the isomerization of glucose 6-phosphate to 1-D-myo-inositol-3-phosphate, catalysed by a 1-D-myo-inositol-3-phosphate synthase (INO1). When grown under non-permissive conditions, a conditional double knockout demonstrated that INO1 is an essential gene in bloodstream-form T. brucei. It also showed that the de novo synthesized myo-inositol is utilized to form PI, which is preferentially used in GPI biosynthesis. We also show for the first time that extracellular myo-inositol can in fact be used in GPI formation although to a limited extent. Despite this, extracellular inositol cannot compensate for the deletion of INO1. Supporting these results, there was no change in PI levels in the conditional double knockout cells grown under non-permissive conditions, showing that perturbation of growth is due to a specific lack of de novo synthesized myo-inositol and not a general inositol-less death. These results suggest that there is a distinction between de novo synthesized myo-inositol and that from the extracellular environment.
Asunto(s)
Glicosilfosfatidilinositoles/biosíntesis , Inositol/biosíntesis , Mio-Inositol-1-Fosfato Sintasa/genética , Trypanosoma brucei brucei/genética , Secuencia de Aminoácidos , Animales , Northern Blotting , Southern Blotting , Cromatografía Líquida de Alta Presión , Clonación Molecular , Cromatografía de Gases y Espectrometría de Masas , Eliminación de Gen , Glicosilfosfatidilinositoles/metabolismo , Inositol/metabolismo , Datos de Secuencia Molecular , Mio-Inositol-1-Fosfato Sintasa/metabolismo , Fosfatidilinositoles/biosíntesis , Fosfatidilinositoles/metabolismo , Fosfatidilinositoles/fisiología , Filogenia , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Trypanosoma brucei brucei/crecimiento & desarrollo , Trypanosoma brucei brucei/metabolismoRESUMEN
Three exocellular beta-1,3-glucanases from Acremonium blochii strain C59, BGN3.2, BGN3.3 and BGN3.4, were purified. Two, BGN3.2 and BGN3.4 appeared to act as exo-enzymes against laminarin from Laminaria digitata, while BGN3.3 displayed an endo-mode of action. The N-terminal amino acid sequence data for BGN3.2 and BGN3.4 suggested these two enzymes may be encoded by different genes. The gene encoding the BGN3.2 glucanase was fully sequenced, and its deduced amino acid sequence was similar to those for all other sequenced fungal exo-beta-1,3-glucanases. This BGN3.2 gene consists of an uninterrupted ORF of 2349 bp encoding 783 amino acids possibly with two cleavage sites for the potential removal of a pre- and pro-protein, respectively. A DNA fragment encoding a portion of the BGN3.4 gene was amplified by PCR, and the nucleotide sequence of this fragment confirmed that BGN3.2 and BGN3.4 are encoded by different genes. The internal peptide sequences of BGN3.3 were not present in the amino acid sequence deduced from the BGN3.2 gene, reinforcing the view that BGN3.3 is also genetically different to BGN3.2. Genetic differences between multiple forms of fungal beta-1,3-glucanases from a single fungus have not been reported previously.