scholarly journals Compartmentalized Cell Envelope Biosynthesis in Mycobacterium tuberculosis

2022 ◽  
Author(s):  
Julia Puffal ◽  
Ian L. Sparks ◽  
James R. Brenner ◽  
Xuni Li ◽  
John D. Leszyk ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Proteomic Analysis ◽  
Fluorescent Protein ◽  
Cell Envelope ◽  
Membrane Domain ◽  
Protein Fusion ◽  
Final Maturation ◽  
Associated Proteins ◽  
Gradient Fractionation ◽  
Membrane Compartmentalization

The intracellular membrane domain (IMD) is a metabolically active and laterally discrete membrane domain initially discovered in Mycobacterium smegmatis. The IMD correlates both temporally and spatially with the polar cell envelope elongation in M. smegmatis. Whether or not a similar membrane domain exists in pathogenic species remains unknown. Here we show that the IMD is a conserved membrane structure found in Mycobacterium tuberculosis. We used two independent approaches, density gradient fractionation of membrane domains and visualization of IMD-associated proteins through fluorescence microscopy, to determine the characteristics of the plasma membrane compartmentalization in M. tuberculosis. Proteomic analysis revealed that the IMD is enriched in metabolic enzymes that are involved in the synthesis of conserved cell envelope components such as peptidoglycan, arabinogalactan, and phosphatidylinositol mannosides. Using a fluorescent protein fusion of IMD-associated proteins, we demonstrated that this domain is concentrated in the polar region of the rod-shaped cells, where active cell envelope biosynthesis is taking place. Proteomic analysis further revealed the enrichment of enzymes involved in synthesis of phthiocerol dimycocerosates and phenolic glycolipids in the IMD. We validated the IMD association of two enzymes, α1,3-fucosyltransferase and fucosyl 4-O-methyltransferase, which are involved in the final maturation steps of phenolic glycolipid biosynthesis. Taken together, these data indicate that functional compartmentalization of membrane is an evolutionarily conserved feature found in both M. tuberculosis and M. smegmatis, and M. tuberculosis utilizes this membrane location for the synthesis of its surface-exposed lipid virulence factors.

scholarly journals Fluorescence Imaging-Based Discovery of Membrane Domain-Associated Proteins in Mycobacterium smegmatis

2021 ◽  
Author(s):  
Corelle A. Z. Rokicki ◽  
James R. Brenner ◽  
Alexander H. Dills ◽  
Julius J. Judd ◽  
Jemila C. Kester ◽  
...  
Keyword(s):  
Mycobacterium Smegmatis ◽  
Fluorescent Protein ◽  
Cell Envelope ◽  
Protein Localization ◽  
Subcellular Fractionation ◽  
Polar Regions ◽  
Intracellular Membrane ◽  
Membrane Domain ◽  
Associated Proteins ◽  
Proteomic Analyses

Mycobacteria spatially organize their plasma membrane, and many enzymes involved in envelope biosynthesis associate with a membrane compartment termed the intracellular membrane domain (IMD). The IMD is concentrated in the polar regions of growing cells and becomes less polarized under non-growing conditions. Because mycobacteria elongate from the poles, the observed polar localization of the IMD during growth likely supports the localized biosynthesis of envelope components. While we have identified more than 300 IMD-associated proteins by proteomic analyses, only a handful of these have been verified by independent experimental methods. Furthermore, some IMD-associated proteins may have escaped proteomic identification and remain to be identified. Here, we visually screened an arrayed library of 523 Mycobacterium smegmatis strains, each producing a Dendra2-FLAG-tagged recombinant protein. We identified 29 fusion proteins that showed polar fluorescence patterns characteristic of IMD proteins. Twenty of these had previously been suggested to localize to the IMD based on proteomic data. Of the nine remaining IMD candidate proteins, three were confirmed by biochemical methods to be associated with the IMD. Taken together, this new co-localization strategy is effective in verifying the IMD association of proteins found by proteomic analyses, while facilitating the discovery of additional IMD-associated proteins. Importance The intracellular membrane domain (IMD) is a membrane subcompartment found in Mycobacterium smegmatis cells. Proteomic analysis of purified IMD identified more than 300 proteins, including enzymes involved in cell envelope biosynthesis. However, proteomics on its own is unlikely to detect every IMD-associated protein because of technical and biological limitations. Here, we describe fluorescent protein co-localization as an alternative, independent approach. Using a combination of fluorescence microscopy, proteomics, and subcellular fractionation, we identified three new proteins associated with the IMD. Such a robust method to rigorously define IMD proteins will benefit future investigations to decipher the synthesis, maintenance and functions of this membrane domain, and help delineate a more general mechanisms of subcellular protein localization in mycobacteria.

scholarly journals Identification of Genes Encoding Exported Mycobacterium tuberculosis Proteins Using a Tn552′phoA In Vitro Transposition System

2000 ◽  
Vol 182 (10) ◽  
pp. 2732-2740 ◽  
Author(s):  
Miriam Braunstein ◽  
Thomas J. Griffin ◽  
Jordan I. Kriakov ◽  
Sarah T. Friedman ◽  
Nigel D. F. Grindley ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Protective Immunity ◽  
Cell Envelope ◽  
Open Reading Frames ◽  
Signal Peptides ◽  
Genomic Libraries ◽  
Target Dna ◽  
Genes Encoding ◽  
Associated Proteins

ABSTRACT Secreted and cell envelope-associated proteins are important to both Mycobacterium tuberculosis pathogenesis and the generation of protective immunity to M. tuberculosis. We used an in vitro Tn552′phoA transposition system to identify exported proteins of M. tuberculosis. The system is simple and efficient, and the transposon inserts randomly into target DNA. M. tuberculosis genomic libraries were targeted with Tn552′phoA transposons, and these libraries were screened in M. smegmatis for active PhoA translational fusions. Thirty-two different M. tuberculosis open reading frames were identified; eight contain standard signal peptides, six contain lipoprotein signal peptides, and seventeen contain one or more transmembrane domains. Four of these proteins had not yet been assigned as exported proteins in the M. tuberculosisdatabases. This collection of exported proteins includes factors that are known to participate in the immune response of M. tuberculosis and proteins with homologies, suggesting a role in pathogenesis. Nine of the proteins appear to be unique to mycobacteria and represent promising candidates for factors that participate in protective immunity and virulence. This technology of creating comprehensive fusion libraries should be applicable to other organisms.

scholarly journals Definition of novel cell envelope associated proteins in Triton X-114 extracts of Mycobacterium tuberculosis H37Rv

2010 ◽  
Vol 10 (1) ◽  
pp. 132 ◽  
Author(s):  
Hiwa Målen ◽  
Sharad Pathak ◽  
Tina Søfteland ◽  
Gustavo A de Souza ◽  
Harald G Wiker
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Cell Envelope ◽  
Tuberculosis H37rv ◽  
Mycobacterium Tuberculosis H37rv ◽  
Associated Proteins ◽  
Definition Of ◽  
Triton X

scholarly journals Uptake of the ATP-Binding Cassette (ABC) Transporter Ste6 into the Yeast Vacuole Is Blocked in the doa4 Mutant

2001 ◽  
Vol 12 (4) ◽  
pp. 1047-1059 ◽  
Author(s):  
Sascha Losko ◽  
Frank Kopp ◽  
Andreas Kranz ◽  
Ralf Kölling
Keyword(s):  
Fluorescent Protein ◽  
Intracellular Localization ◽  
Sucrose Density Gradient ◽  
Multivesicular Bodies ◽  
Green Fluorescent Protein Fusion ◽  
Protein Fusion ◽  
Yeast Vacuole ◽  
Trafficking Pathway ◽  
Green Fluorescent ◽  
Gradient Fractionation

Previous experiments suggested that trafficking of thea-factor transporter Ste6 of Saccharomyces cerevisiae to the yeast vacuole is regulated by ubiquitination. To define the ubiquitination-dependent step in the trafficking pathway, we examined the intracellular localization of Ste6 in the ubiquitination-deficient doa4 mutant by immunofluorescence experiments, with a Ste6-green fluorescent protein fusion protein and by sucrose density gradient fractionation. We found that Ste6 accumulated at the vacuolar membrane in the doa4 mutant and not at the cell surface. Experiments with a doa4 pep4double mutant showed that Ste6 uptake into the lumen of the vacuole is inhibited in the doa4 mutant. The uptake defect could be suppressed by expression of additional ubiquitin, indicating that it is primarily the result of a lowered ubiquitin level (and thus of reduced ubiquitination) and not the result of a deubiquitination defect. Based on our findings, we propose that ubiquitination of Ste6 or of a trafficking factor is required for Ste6 sorting into the multivesicular bodies pathway. In addition, we obtained evidence suggesting that Ste6 recycles between an internal compartment and the plasma membrane.

scholarly journals A Family of acr-Coregulated Mycobacterium tuberculosis Genes Shares a Common DNA Motif and Requires Rv3133c (dosR or devR) for Expression

2003 ◽  
Vol 71 (9) ◽  
pp. 5332-5343 ◽  
Author(s):  
Matthew A. Florczyk ◽  
Lee Ann McCue ◽  
Anjan Purkayastha ◽  
Egidio Currenti ◽  
Meyer J. Wolin ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Fluorescent Protein ◽  
Culture Conditions ◽  
Sequence Motif ◽  
Low Oxygen ◽  
Protein Fusion ◽  
Dna Motif ◽  
Oxygen Conditions ◽  
Green Fluorescent

ABSTRACT Previous work has shown that the divergently transcribed Mycobacterium tuberculosis genes acr (hspX, Rv2031c) and acg (Rv2032) are induced under conditions of shallow standing culture and low oxygen and intracellularly within macrophages. We used a combination of computational and experimental methods to identify promoters for eight additional genes that are regulated in a similar manner and that comprise an acr-coregulated promoter (ACP) family. Transcriptional regulation of these ACP family members was evaluated by using a plasmid-based promoter-green fluorescent protein fusion system and flow cytometry. All promoters showed increased expression in shallow standing versus shaking cultures, in low- versus high-oxygen conditions, and intracellularly within macrophages versus extracellularly in tissue culture medium. However, there were quantitative differences in expression among promoters and among conditions for each promoter. A conserved 18-bp palindromic sequence motif was identified in all ACPs by Gibbs sampling-based computational analyses. Two such motifs overlap regions in the acr and acg promoters that were previously shown to be required for their expression. In addition, we found that 5% carbon dioxide was required for growth of Mycobacterium bovis BCG under microaerophilic (1.3% O2) culture conditions and fully prevented the growth cessation typically associated with rapid removal of oxygen. These findings are likely to be relevant to the in vivo environment and will contribute to our understanding of the pathogenesis of tuberculosis infection.

scholarly journals Fluorescence Imaging-Based Discovery of Membrane Domain-Associated Proteins in Mycobacterium smegmatis

2021 ◽  
Author(s):  
Corelle A. Z. Rokicki ◽  
James R. Brenner ◽  
Alexander Dills ◽  
Julius J. Judd ◽  
Jemila C. Kester ◽  
...  
Keyword(s):  
Mycobacterium Smegmatis ◽  
Cell Envelope ◽  
Subcellular Fractionation ◽  
Polar Regions ◽  
Intracellular Membrane ◽  
Membrane Domain ◽  
Membrane Compartment ◽  
Associated Proteins ◽  
Proteomic Analyses ◽  
Growing Conditions

AbstractMycobacteria spatially organize their plasma membrane, and many enzymes involved in envelope biosynthesis associate with a membrane compartment termed the intracellular membrane domain (IMD). The IMD is concentrated in the polar regions of growing cells and becomes less polarized under non-growing conditions. Because mycobacteria elongate from the poles, the observed polar localization of the IMD during growth likely supports the localized envelope biosynthesis. While we have identified more than 300 IMD-associated proteins by proteomic analyses, only a handful of these have been verified by other experimental methods. Furthermore, we speculate that some IMD-associated proteins may have escaped proteomic identification and remain to be identified. Here, we visually screened an arrayed library of 523 Mycobacterium smegmatis strains each expressing a Dendra2-FLAG-tagged recombinant protein. We identified 29 fusion proteins that showed fluorescence patterns similar to those of IMD proteins and, consistent with this co-localization, we had previously identified 20 of these using a proteomics approach. Of the nine remaining IMD candidate proteins, three were confirmed to be associated with the IMD while some others appear to be lipid droplet-associated. Taken together, our newly devised strategy is effective in verifying the IMD association of proteins found by proteomic analyses, while facilitating the discovery of additional IMD-associated proteins.ImportanceThe intracellular membrane domain (IMD) is a membrane subcompartment found in Mycobacterium smegmatis cells. Proteomic analysis of purified IMD identified more than 300 proteins, including enzymes involved in cell envelope biosynthesis, that likely contribute to the function of the IMD. How can we find more IMD-associated proteins that escaped proteomic detection? Here, as an alternative approach, fluorescence microscope images of 523 proteins were screened to identify IMD-associated proteins. We confirmed the IMD association of previously identified proteins and discovered three additional proteins associated with the IMD. Together, subcellular fractionation, proteomics, and fluorescence microscopy form a robust combination to more rigorously define IMD proteins, which will aid future investigations to decipher the synthesis, maintenance and functions of this membrane domain.

scholarly journals Demethylmenaquinone methyl transferase is a membrane domain-associated protein essential for menaquinone homeostasis in Mycobacterium smegmatis

2018 ◽  
Author(s):  
Julia Puffal ◽  
Jacob A. Mayfield ◽  
D. Branch Moody ◽  
Yasu S. Morita
Keyword(s):  
Plasma Membrane ◽  
Spatial Organization ◽  
Fluorescent Protein ◽  
Essential Gene ◽  
Sucrose Density Gradient ◽  
Cellular Level ◽  
Distinct Region ◽  
Membrane Domain ◽  
Protein Fusion ◽  
Extra Copy

AbstractThe intracellular membrane domain (IMD) in mycobacteria is a spatially distinct region of the plasma membrane with diverse functions. Previous comparative proteomic analysis of the IMD suggested that menaquinone biosynthetic enzymes are associated with this domain. In the present study, we determined the subcellular site of these enzymes using sucrose density gradient fractionation. We found that the last two enzymes, the methyltransferase MenG, and the reductase MenJ, are associated with the IMD. MenA, the prenyltransferase that mediates the first membrane-associated step of the menaquinone biosynthesis, is associated with the conventional plasma membrane. For MenG, we additionally showed the polar enrichment of the fluorescent protein fusion colocalizing with an IMD marker protein in situ. To start dissecting the roles of IMD-associated enzymes, we further tested the physiological significance of MenG. The deletion of menG at the endogenous genomic loci was possible only when an extra copy of the gene was present, indicating that it is an essential gene in M. smegmatis. Using a tetracycline-inducible switch, we achieved gradual and partial depletion of MenG over three consecutive 24 hour subcultures. This partial MenG depletion resulted in progressive slowing of growth, which corroborated the observation that menG is an essential gene. Upon MenG depletion, there was a significant accumulation of MenG substrate, demethylmenaquinone, even though the cellular level of menaquinone, the reaction product, was unaffected. Furthermore, the growth retardation was coincided with a lower oxygen consumption rate and ATP accumulation. These results imply a previously unappreciated role of MenG in regulating menaquinone homeostasis within the complex spatial organization of mycobacterial plasma membrane.

Exploration of Ion Channels in Mycobacterium tuberculosis: Implication on Drug Discovery and Potent Drug Targets Against Tuberculosis

2020 ◽  
Vol 14 (1) ◽  
pp. 14-29
Author(s):  
Manish Dwivedi
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Drug Targets ◽  
Cell Envelope ◽  
Intracellular Pathogen ◽  
Host Immune System ◽  
Transporter Proteins ◽  
Channel Proteins ◽  
Medical Importance ◽  
Potent Drug ◽  
Emergence Of Resistance

Scientific interest in mycobacteria has been sparked by the medical importance of Mycobacterium tuberculosis (Mtb) that is known to cause severe diseases in mammals, i.e. tuberculosis and by properties that distinguish them from other microorganisms which are notoriously difficult to treat. The treatment of their infections is difficult because mycobacteria fortify themselves with a thick impermeable cell envelope. Channel and transporter proteins are among the crucial adaptations of Mycobacterium that facilitate their strength to combat against host immune system and anti-tuberculosis drugs. In previous studies, it was investigated that some of the channel proteins contribute to the overall antibiotic resistance in Mtb. Moreover, in some of the cases, membrane proteins were found responsible for virulence of these pathogens. Given the ability of M. tuberculosis to survive as an intracellular pathogen and its inclination to develop resistance to the prevailing anti-tuberculosis drugs, its treatment requires new approaches and optimization of anti-TB drugs and investigation of new targets are needed for their potential in clinical usage. Therefore, it is imperative to investigate the survival of Mtb. in stressed conditions with different behavior of particular channel/ transporter proteins. Comprehensive understanding of channel proteins and their mechanism will provide us direction to find out preventive measures against the emergence of resistance and reduce the duration of the treatment, eventually leading to plausible eradication of tuberculosis.

scholarly journals Proteomic analysis of drug-susceptible and multidrug-resistant nonreplicating Beijing strains of Mycobacterium tuberculosis cultured in vitro

2021 ◽  
Vol 26 ◽  
pp. 100960
Author(s):  
Bhanubong Saiboonjan ◽  
Sittiruk Roytrakul ◽  
Arunnee Sangka ◽  
Viraphong Lulitanond ◽  
Kiatichai Faksri ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Proteomic Analysis ◽  
Multidrug Resistant

scholarly journals Angiotensin II stimulates trafficking of NHE3, NaPi2, and associated proteins into the proximal tubule microvilli

2010 ◽  
Vol 298 (1) ◽  
pp. F177-F186 ◽  
Author(s):  
Anne D. M. Riquier-Brison ◽  
Patrick K. K. Leong ◽  
Kaarina Pihakaski-Maunsbach ◽  
Alicia A. McDonough
Keyword(s):  
Angiotensin Ii ◽  
Proximal Tubule ◽  
Flow Rate ◽  
Enzyme Inhibitor ◽  
Volume Flow Rate ◽  
Ang Ii ◽  
Sprague Dawley ◽  
Water Reabsorption ◽  
Associated Proteins ◽  
Gradient Fractionation

Angiotensin II (ANG II) stimulates proximal tubule (PT) sodium and water reabsorption. We showed that treating rats acutely with the angiotensin-converting enzyme inhibitor captopril decreases PT salt and water reabsorption and provokes rapid redistribution of the Na+/H+ exchanger isoform 3 (NHE3), Na+/Pi cotransporter 2 (NaPi2), and associated proteins out of the microvilli. The aim of the present study was to determine whether acute ANG II infusion increases the abundance of PT NHE3, NaPi2, and associated proteins in the microvilli available for reabsorbing NaCl. Male Sprague-Dawley rats were infused with a dose of captopril (12 μg/min for 20 min) that increased PT flow rate ∼20% with no change in blood pressure (BP) or glomerular filtration rate (GFR). When ANG II (20 ng·kg−1·min−1 for 20 min) was added to the captopril infusate, PT volume flow rate returned to baseline without changing BP or GFR. After captopril, NHE3 was localized to the base of the microvilli and NaPi2 to subapical cytoplasmic vesicles; after 20 min ANG II, both NHE3 and NaPi2 redistributed into the microvilli, assayed by confocal microscopy and density gradient fractionation. Additional PT proteins that redistributed into low-density microvilli-enriched membranes in response to ANG II included myosin VI, DPPIV, NHERF-1, ezrin, megalin, vacuolar H+-ATPase, aminopeptidase N, and clathrin. In summary, in response to 20 min ANG II in the absence of a change in BP or GFR, multiple proteins traffic into the PT brush-border microvilli where they likely contribute to the rapid increase in PT salt and water reabsorption.

Sign in / Sign up

Export Citation Format

Share Document