The emb proteins in Mycobacterium smegmatis
Date
2004
Authors
Nagy, Toni A., author
Vissa, Varalakshmi, author
Chatterjee, Delphi, author
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Abstract
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.
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Subject
Mycobacterium tuberculosis
Recombinant proteins