Emb proteins in Mycobacterium smegmatis, The

Mycobacterium tuberculosis, the causative agent of tuberculosis, was once thought to be a ghost of the past. However, the emergence of drug resistant strains has brought this bacterium back into the spot light. Ethambutol, a compound with structural similarity to D-arabinose, has been shown to have many inhibitory effects on M. tuberculosis and other mycobacterial species. Some of these activities include: inhibition of synthesis of the arabinan constituent of the cell wall arabinogalactan (AG), inhibition of RNA metabolism, and phospholipid synthesis. Likewise, this inhibition of arabinan synthesis extends to that of lipoarabinomannan (LAM). One of the most significant components of all mycobacterial cell walls is LAM; it has been implicated as a central molecule involved in the virulence and immunopathogenesis of tuberculosis. The arabinan of LAM is attached to a mannan backbone which extends from a phosphatidylinositol mannoside anchor at the reducing end. Recent research efforts have been directed towards showing that the Emb proteins are involved in arabinan synthesis. These proteins are conserved throughout all mycobacterial species, and through the use of computer program algorithms (such as SOSUI, TMPRED), it has been predicted that there are 11-13 transmembrane domains in the N-terminal region of the proteins. This correlates to approximately 670 amino acids. Also, these programs showed a soluble globular C-terminal domain accounting for approximately 430 amino acids. Specifically, it was found that EmbC targets the arabinan of LAM. This was made possible by the inactivation of the embA, embB, and embC genes individually, and subsequently looking at the resulting structural alterations on the arabinan component of cell wall AG and LAM. With this knowledge, the establishment of the catalytic site of the embC gene that specifically controls the arabinosylation of lipomannan to give mature lipoarabinomannan was necessary. Generation of hybrids with variation in the N-terminus of the EmbC protein is the current strategy being used to pursue this information. An EmbC/B hybrid was created by the fusion of the N-terminus of EmbC and the C-terminal domain of EmbB. This included 668 amino acids from the N-terminal of EmbC, fused with the last 407 amino acids of EmbB. This gene fusion was cloned into the pVV16 shuttle vector and electroporated into M. smegmatis ΔembC. LAM was then extracted from these hybrid cells, and analysis showed that the EmbC/B hybrid resulted in a shortened form of LAM. After further biochemical studies on this truncated LAM such as glycosyl compositional analysis, endoarabinase digestion followed by HPAEC (High-pH anion-exchange chromatography) profiling, and mass spectrometry analysis, it was shown that LAM was not only truncated, but had a structural alteration where the nonreducing end resembled that of the arabinan of AG. After such exciting results, current work continues with the generation of hybrids. The formation of the 50:50 EmbC/B hybrid containing approximately 580 amino acids of each of the EmbC and EmbB genes has been completed. This final construct has been transformed into the ΔembC mutant by electroporation and biochemical analysis is being performed. Results pertaining to the hybrids capacity to complement the LAM defect will be discussed in this poster presentation. This new 50:50 fusion will be essential in giving us the knowledge on the contribution of the first eight transmembrane domains of the EmbC protein in LAM biosynthesis.College Honors

Estudio de resistencia a la rifampicina y la dapsona en tres pacientes con recurrencia de lepra Study of rifampin and dapsone resistance in three patients with recurring leprosy

OBJETIVO: Detectar la presencia de cepas de Mycobacterium leprae resistentes a la rifampicina y la dapsona en tres pacientes con recurrencia de lepra y sospecha clínica de resistencia antimicrobiana, mediante la aplicación de técnicas moleculares. MÉTODOS: Se realizó un estudio descriptivo retrospectivo en tres pacientes multibacilares del Sanatorio de Agua de Dios, Cundinamarca, Colombia, que habían presentado recidivas de lepra documentadas por su historia clínica, baciloscopia y biopsia. Se obtuvieron biopsias de lesiones cutáneas que se procesaron para la extracción y purificación del ADN bacilar. Se amplificaron regiones de los genes rpoB y folP1 asociadas con la resistencia antimicrobiana, mediante la reacción en cadena de la polimerasa "touch-down" y se secuenciaron los productos amplificados mediante el método de Sanger. RESULTADOS: Se detectó una mutación puntual en el nucleótido 1367 del gen rpoB en dos de las muestras estudiadas. No se encontró la mutación estudiada en el gen folP1 en ninguno de los tres pacientes. CONCLUSIONES: La mutación identificada demostró la presencia de bacilos de M. leprae resistentes a la rifampicina en dos de los tres pacientes estudiados con recurrencia de la enfermedad. No se detectó la mutación indicadora de resistencia a la dapsona en ninguno de los tres pacientes.<br>OBJECTIVE: To detect the presence of rifampin- and dapsone-resistant strains of Mycobacterium leprae in three patients with recurring leprosy and clinically-suspected antimicrobial resistance through molecular techniques. METHODS: A retrospective, descriptive study was conducted of three multibacillary patients at the "Agua de Dios" Sanitarium in Cundinamarca, Colombia, that presented leprosy relapses that were documented by medical history, bacilloscopy, and biopsy. Biopsies were taken of the skin lesions and the bacteria were subject to DNA extraction and purification. Regions of the rpoB and folP1 genes associated with antimicrobial resistance were amplified and subjected to touch-down polymerase chain reaction and the amplified products were sequenced using the Sanger method. RESULTS: A punctual mutation was identified in nucleotide 1367 of the rpoB gene in two of the samples studied. This mutation was not found in the folP1 gene of any of the three patients. CONCLUSIONS: The mutation identified showed strains of rifampin-resistant M. leprae in two of the three patients with recurring leprosy. Mutations that indicate dapsone-resistance were not detected in any of the three patients

Study of rifampin and dapsone resistance in three patients with recurring leprosy

5 páginasOBJECTIVE: To detect the presence of rifampin- and dapsone-resistant strains of Mycobacterium leprae in three patients with recurring leprosy and clinically-suspected antimicrobial resistance through molecular techniques. METHODS: A retrospective, descriptive study was conducted of three multibacillary patients at the "Agua de Dios" Sanitarium in Cundinamarca, Colombia, that presented leprosy relapses that were documented by medical history, bacilloscopy, and biopsy. Biopsies were taken of the skin lesions and the bacteria were subject to DNA extraction and purification. Regions of the rpoB and folP1 genes associated with antimicrobial resistance were amplified and subjected to touch-down polymerase chain reaction and the amplified products were sequenced using the Sanger method. RESULTS: A punctual mutation was identified in nucleotide 1367 of the rpoB gene in two of the samples studied. This mutation was not found in the folP1 gene of any of the three patients. CONCLUSIONS: The mutation identified showed strains of rifampin-resistant M. leprae in two of the three patients with recurring leprosy. Mutations that indicate dapsone-resistance were not detected in any of the three patients.OBJETIVO: Detectar la presencia de cepas de Mycobacterium leprae resistentes a la rifampicina y la dapsona en tres pacientes con recurrencia de lepra y sospecha clínica de resistencia antimicrobiana, mediante la aplicación de técnicas moleculares. MÉTODOS: Se realizó un estudio descriptivo retrospectivo en tres pacientes multibacilares del Sanatorio de Agua de Dios, Cundinamarca, Colombia, que habían presentado recidivas de lepra documentadas por su historia clínica, baciloscopia y biopsia. Se obtuvieron biopsias de lesiones cutáneas que se procesaron para la extracción y purificación del ADN bacilar. Se amplificaron regiones de los genes rpoB y folP1 asociadas con la resistencia antimicrobiana, mediante la reacción en cadena de la polimerasa "touch-down" y se secuenciaron los productos amplificados mediante el método de Sanger. RESULTADOS: Se detectó una mutación puntual en el nucleótido 1367 del gen rpoB en dos de las muestras estudiadas. No se encontró la mutación estudiada en el gen folP1 en ninguno de los tres pacientes. CONCLUSIONES: La mutación identificada demostró la presencia de bacilos de M. leprae resistentes a la rifampicina en dos de los tres pacientes estudiados con recurrencia de la enfermedad. No se detectó la mutación indicadora de resistencia a la dapsona en ninguno de los tres pacientes

Initiation of methylglucose lipopolysaccharide biosynthesis in mycobacteria.

Mycobacteria produce two unique families of cytoplasmic polymethylated polysaccharides -- the methylglucose lipopolysaccharides (MGLPs) and the methylmannose polysaccharides (MMPs) -- the physiological functions of which are still poorly defined. Towards defining the roles of these polysaccharides in mycobacterial physiology, we generated knock-out mutations of genes in their putative biosynthetic pathways.We report here on the characterization of the Rv1208 protein of Mycobacterium tuberculosis and its ortholog in Mycobacterium smegmatis (MSMEG_5084) as the enzymes responsible for the transfer of the first glucose residue of MGLPs. Disruption of MSMEG_5084 in M. smegmatis resulted in a dramatic decrease in MGLP synthesis directly attributable to the almost complete abolition of glucosyl-3-phosphoglycerate synthase activity in this strain. Synthesis of MGLPs in the mutant was restored upon complementation with wild-type copies of the Rv1208 gene from M. tuberculosis or MSMEG_5084 from M. smegmatis.This is the first evidence linking Rv1208 to MGLP biosynthesis. Thus, the first step in the initiation of MGLP biosynthesis in mycobacteria has been defined, and subsequent steps can be inferred