@article {1441, title = {Mycobacterial Esx-3 requires multiple components for iron acquisition.}, journal = {MBio}, volume = {5}, year = {2014}, month = {2014}, pages = {e01073-14}, abstract = {

ABSTRACT The type VII secretion systems are conserved across mycobacterial species and in many Gram-positive bacteria. While the well-characterized Esx-1 pathway is required for the virulence of pathogenic mycobacteria and conjugation in the model organism Mycobacterium smegmatis, Esx-3 contributes to mycobactin-mediated iron acquisition in these bacteria. Here we show that several Esx-3 components are individually required for function under low-iron conditions but that at least one, the membrane-bound protease MycP3 of M. smegmatis, is partially expendable. All of the esx-3 mutants tested, including the ΔmycP3ms mutant, failed to export the native Esx-3 substrates EsxHms and EsxGms to quantifiable levels, as determined by targeted mass spectrometry. Although we were able to restore low-iron growth to the esx-3 mutants by genetic complementation, we found a wide range of complementation levels for protein export. Indeed, minute quantities of extracellular EsxHms and EsxGms were sufficient for iron acquisition under our experimental conditions. The apparent separation of Esx-3 function in iron acquisition from robust EsxGms and EsxHms secretion in the ΔmycP3ms mutant and in some of the complemented esx-3 mutants compels reexamination of the structure-function relationships for type VII secretion systems. IMPORTANCE Mycobacteria have several paralogous type VII secretion systems, Esx-1 through Esx-5. Whereas Esx-1 is required for pathogenic mycobacteria to grow within an infected host, Esx-3 is essential for growth in vitro. We and others have shown that Esx-3 is required for siderophore-mediated iron acquisition. In this work, we identify individual Esx-3 components that contribute to this process. As in the Esx-1 system, most mutations that abolish Esx-3 protein export also disrupt its function. Unexpectedly, however, ultrasensitive quantitation of Esx-3 secretion by multiple-reaction-monitoring mass spectrometry (MRM-MS) revealed that very low levels of export were sufficient for iron acquisition under similar conditions. Although protein export clearly contributes to type VII function, the relationship is not absolute.

}, keywords = {Bacterial Secretion Systems, Gene Order, Genetic Loci, Iron, Mycobacterium, Oxazoles}, issn = {2150-7511}, doi = {10.1128/mBio.01073-14}, author = {Siegrist, M Sloan and Steigedal, Magnus and Ahmad, Rushdy and Mehra, Alka and Dragset, Marte S and Schuster, Brian M and Philips, Jennifer A and Carr, Steven A and Rubin, Eric J} } @article {1443, title = {Mycobacterial lipid logic.}, journal = {Cell Host Microbe}, volume = {15}, year = {2014}, month = {2014 Jan 15}, pages = {1-2}, abstract = {

During infection of the lung epithelium, Mycobacterium tuberculosis must infect and survive within macrophages long enough to be transported into deeper lung tissues. Cambier et~al. (2013) show that pathogenic mycobacteria use the coordinated action of two cell wall glycolipids to regulate macrophage recruitment to initial infection sites.

}, keywords = {Animals, Female, Immune Evasion, Macrophages, Membrane Lipids, Mycobacterium}, issn = {1934-6069}, doi = {10.1016/j.chom.2013.12.005}, author = {Siegrist, M Sloan and Bertozzi, Carolyn R} } @article {1446, title = {Probing the mycobacterial trehalome with bioorthogonal chemistry.}, journal = {J Am Chem Soc}, volume = {134}, year = {2012}, month = {2012 Oct 3}, pages = {16123-6}, abstract = {

Mycobacteria, including the pathogen Mycobacterium tuberculosis, use the non-mammalian disaccharide trehalose as a precursor for essential cell-wall glycolipids and other metabolites. Here we describe a strategy for exploiting trehalose metabolic pathways to label glycolipids in mycobacteria with azide-modified trehalose (TreAz) analogues. Subsequent bioorthogonal ligation with alkyne-functionalized probes enabled detection and visualization of cell-surface glycolipids. Characterization of the metabolic fates of four TreAz analogues revealed unique labeling routes that can be harnessed for pathway-targeted investigation of the mycobacterial trehalome.

}, keywords = {Alkynes, Azides, Fluorescent Dyes, Glycolipids, Mycobacterium, Trehalose}, issn = {1520-5126}, doi = {10.1021/ja3062419}, author = {Swarts, Benjamin M and Holsclaw, Cynthia M and Jewett, John C and Alber, Marina and Fox, Douglas M and Siegrist, M Sloan and Leary, Julie A and Kalscheuer, Rainer and Bertozzi, Carolyn R} } @article {687, title = {Inositol lipid metabolism in mycobacteria: biosynthesis and regulatory mechanisms.}, journal = {Biochim Biophys Acta}, volume = {1810}, year = {2011}, month = {2011 Jun}, pages = {630-41}, abstract = {BACKGROUND: The genus Mycobacterium includes a number of medically important pathogens. The cell walls of these bacteria have many unique features, including the abundance of various inositol lipids, such as phosphatidylinositol mannosides (PIMs), lipomannan (LM), and lipoarabinomannan (LAM). The biosynthesis of these lipids is believed to be prime drug targets, and has been clarified in detail over the past several years. SCOPE OF REVIEW: Here we summarize our current understanding of the inositol lipid metabolism in mycobacteria. We will highlight unsolved issues and future directions especially in the context of metabolic regulation. MAJOR CONCLUSIONS: Inositol is a building block of phosphatidylinositol (PI), which is further elaborated to become PIMs, LM and LAM. d-myo-inositol 3-phosphate is an intermediate of the de novo inositol synthesis, but it is also the starting substrate for mycothiol synthesis. Controlling the level of d-myo-inositol 3-phosphate appears to be important for maintaining the steady state levels of mycothiol and inositol lipids. Several additional control mechanisms must exist to control the complex biosynthetic pathways of PI, PIMs, LM and LAM. These may include regulatory proteins such as a lipoprotein LpqW, and spatial separation of enzymes, such as the amphipathic PimA mannosyltransferase and later enzymes in the PIMs/LM biosynthetic pathway. Finally, we discuss mechanisms that underlie control of LM/LAM glycan polymer elongation. GENERAL SIGNIFICANCE: Mycobacteria have evolved a complex network of inositol metabolism. Clarifying its metabolism will not only provide better understanding of bacterial pathogenesis, but also understanding of the evolution and general functions of inositol lipids in nature.}, keywords = {Inositol, Lipid Metabolism, Lipids, Models, Biological, Mycobacterium, Phosphatidylinositols}, issn = {0006-3002}, doi = {10.1016/j.bbagen.2011.03.017}, author = {Morita, Yasu S and Fukuda, Takeshi and Sena, Chubert B C and Yamaryo-Botte, Yoshiki and McConville, Malcolm J and Kinoshita, Taroh} } @article {1447, title = {Mycobacterial Esx-3 is required for mycobactin-mediated iron acquisition.}, journal = {Proc Natl Acad Sci U S A}, volume = {106}, year = {2009}, month = {2009 Nov 3}, pages = {18792-7}, abstract = {

The Esx secretion pathway is conserved across Gram-positive bacteria. Esx-1, the best-characterized system, is required for virulence of Mycobacterium tuberculosis, although its precise function during infection remains unclear. Esx-3, a paralogous system present in all mycobacterial species, is required for growth in vitro. Here, we demonstrate that mycobacteria lacking Esx-3 are defective in acquiring iron. To compete for the limited iron available in the host and the environment, these organisms use mycobactin, high-affinity iron-binding molecules. In the absence of Esx-3, mycobacteria synthesize mycobactin but are unable to use the bound iron and are impaired severely for growth during macrophage infection. Mycobacteria thus require a specialized secretion system for acquiring iron from siderophores.

}, keywords = {Animals, Bacterial Proteins, Genome, Bacterial, Iron, Macrophages, Mice, Mutation, Mycobacterium, Mycobacterium Infections, Oxazoles, Protein Binding, Secretory Pathway, Siderophores, Transcription, Genetic, Up-Regulation}, issn = {1091-6490}, doi = {10.1073/pnas.0900589106}, author = {Siegrist, M Sloan and Unnikrishnan, Meera and McConnell, Matthew J and Borowsky, Mark and Cheng, Tan-Yun and Siddiqi, Noman and Fortune, Sarah M and Moody, D Branch and Rubin, Eric J} } @article {1448, title = {Phage transposon mutagenesis.}, journal = {Methods Mol Biol}, volume = {465}, year = {2009}, month = {2009}, pages = {311-23}, abstract = {

Phage transduction is an attractive method of genetic manipulation in mycobacteria. PhiMycoMarT7 is well suited for transposon mutagenesis as it is temperature sensitive for replication and contains T7 promoters that promote transcription, a highly active transposase gene, and an Escherichia coli oriR6 K origin of replication. Mycobacterial transposon mutant libraries produced by PhiMycoMarT7 transduction are amenable to both forward and reverse genetic studies. In this protocol, we detail the preparation of PhiMycoMarT7, including a description of the phage, reconstitution of the phage, purification of plaques, preparation of phage stock, and titering of phage stock. We then describe the transduction procedure and finally outline the isolation of individual transposon mutants.

}, keywords = {Bacteriophages, DNA Transposable Elements, Mutagenesis, Mutation, Mycobacterium, Transduction, Genetic}, issn = {1940-6029}, doi = {10.1007/978-1-59745-207-6_21}, author = {Siegrist, M Sloan and Rubin, Eric J} } @article {692, title = {PimE is a polyprenol-phosphate-mannose-dependent mannosyltransferase that transfers the fifth mannose of phosphatidylinositol mannoside in mycobacteria.}, journal = {J Biol Chem}, volume = {281}, year = {2006}, month = {2006 Sep 1}, pages = {25143-55}, abstract = {Phosphatidylinositol mannosides (PIMs) are a major class of glycolipids in all mycobacteria. AcPIM2, a dimannosyl PIM, is both an end product and a precursor for polar PIMs, such as hexamannosyl PIM (AcPIM6) and the major cell wall lipoglycan, lipoarabinomannan (LAM). The mannosyltransferases that convert AcPIM2 to AcPIM6 or LAM are dependent on polyprenol-phosphate-mannose (PPM), but have not yet been characterized. Here, we identified a gene, termed pimE that is present in all mycobacteria, and is required for AcPIM6 biosynthesis. PimE was initially identified based on homology with eukaryotic PIG-M mannosyltransferases. PimE-deleted Mycobacterium smegmatis was defective in AcPIM6 synthesis, and accumulated the tetramannosyl PIM, AcPIM4. Loss of PimE had no affect on cell growth or viability, or the biosynthesis of other intracellular and cell wall glycans. However, changes in cell wall hydrophobicity and plasma membrane organization were detected, suggesting a role for AcPIM6 in the structural integrity of the cell wall and plasma membrane. These defects were corrected by ectopic expression of the pimE gene. Metabolic pulse-chase radiolabeling and cell-free PIM biosynthesis assays indicated that PimE catalyzes the alpha1,2-mannosyl transfer for the AcPIM5 synthesis. Mutation of an Asp residue in PimE that is conserved in and required for the activity of human PIG-M resulted in loss of PIM-biosynthetic activity, indicating that PimE is the catalytic component. Finally, PimE was localized to a distinct membrane fraction enriched in AcPIM4-6 biosynthesis. Taken together, PimE represents the first PPM-dependent mannosyl-transferase shown to be involved in PIM biosynthesis, where it mediates the fifth mannose transfer.}, keywords = {Amino Acid Sequence, Cell Proliferation, Cell Wall, Cell-Free System, Genome, Bacterial, Humans, Mannose, Mannosides, Mannosyltransferases, Molecular Sequence Data, Mycobacterium, Mycobacterium smegmatis, Phosphates, Phosphatidylinositols, Sequence Homology, Amino Acid}, issn = {0021-9258}, doi = {10.1074/jbc.M604214200}, author = {Morita, Yasu S and Sena, Chubert B C and Waller, Ross F and Kurokawa, Ken and Sernee, M Fleur and Nakatani, Fumiki and Haites, Ruth E and Billman-Jacobe, Helen and McConville, Malcolm J and Maeda, Yusuke and Kinoshita, Taroh} }