RESUMO
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 nongrowing 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 few 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 colocalization 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 colocalization 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 mechanism of subcellular protein localization in mycobacteria.
Assuntos
Proteínas de Bactérias/metabolismo , Mycobacterium smegmatis/metabolismo , Imagem Óptica/métodos , Proteínas de Bactérias/genética , Membrana Celular , Regulação Bacteriana da Expressão Gênica/fisiologia , Mycobacterium smegmatis/genética , Domínios ProteicosRESUMO
Ferret Systemic Coronaviral Disease (FSCD) is a systemic disease caused by ferret systemic coronavirus, which is considered lethal in most of the ferrets that are affected by it. To our knowledge, no treatment has been shown to be effective against FSCD in vivo, and most of the ferrets are euthanized or die after the development of clinical disease. GS-441524 has been shown to be effective in successfully treating cats with Feline Infectious Peritonitis (FIP), a disease that shares similarities with FSCD. However, to our knowledge, treatment with GS-441524 has not been reported for the treatment of FSCD in ferrets. Here, we describe three cases of ferrets diagnosed with FSCD successfully cured utilizing oral GS-441524. FSCD may be effectively treated following similar protocols utilized for feline infectious peritonitis in cats.
RESUMO
Toxin-antitoxin modules are present in many bacterial pathogens. The VapBC family is particularly abundant in members of the Mycobacterium tuberculosis complex, with 50 modules present in the M. tuberculosis genome. In type IIA modules the VapB antitoxin protein binds to and inhibits the activity of the co-expressed cognate VapC toxin protein. VapB proteins also bind to promoter region sequences and repress expression of the vapB-vapC operon. Though VapB-VapC interactions can control the amount of free VapC toxin in the bacterial cell, the mechanisms that affect this interaction are poorly understood. Based on our recent finding of Ser/Thr phosphorylation of VapB proteins in M. tuberculosis, we substituted phosphomimetic or phosphoablative amino acids at the phosphorylation sites of two VapB proteins. We found that phosphomimetic substitution of VapB27 and VapB46 resulted in decreased interaction with their respective cognate VapC proteins, whereas phosphoablative substitution did not alter binding. Similarly, we determined that phosphomimetic substitution interfered with VapB binding to promoter region DNA sequences. Both decreased VapB-VapC interaction and decreased VapB repression of vapB-vapC operon transcription would result in increased free VapC in the M. tuberculosis cell. M. tuberculosis strains expressing vapB46-vapC46 constructs containing a phosphoablative vapB mutation resulted in lower toxicity compared to a strain expressing native vapB46, whereas similar or greater toxicity was observed in the strain expressing the phosphomimetic vapB mutation. These results identify a novel mechanism by which VapC toxicity activity can be regulated by VapB phosphorylation, potentially in response to extracytoplasmic as well as intracellular signals.
RESUMO
Many antibiotics target the assembly of cell wall peptidoglycan, an essential, heteropolymeric mesh that encases most bacteria. In rod-shaped bacteria, cell wall elongation is spatially precise yet relies on limited pools of lipid-linked precursors that generate and are attracted to membrane disorder. By tracking enzymes, substrates, and products of peptidoglycan biosynthesis in Mycobacterium smegmatis, we show that precursors are made in plasma membrane domains that are laterally and biochemically distinct from sites of cell wall assembly. Membrane partitioning likely contributes to robust, orderly peptidoglycan synthesis, suggesting that these domains help template peptidoglycan synthesis. The cell wall-organizing protein DivIVA and the cell wall itself promote domain homeostasis. These data support a model in which the peptidoglycan polymer feeds back on its membrane template to maintain an environment conducive to directional synthesis. Our findings are applicable to rod-shaped bacteria that are phylogenetically distant from M. smegmatis, indicating that horizontal compartmentalization of precursors may be a general feature of bacillary cell wall biogenesis.
Assuntos
Parede Celular/metabolismo , Mycobacterium smegmatis/metabolismo , Peptidoglicano/metabolismo , Ciclo Celular , Membrana Celular/metabolismoRESUMO
Mycobacteria and related bacteria in the Actinobacteria phylum are unusual in that they produce phosphatidylinositol (PI) as a major phospholipid species. PI can be further modified by glycan polymers, leading to the synthesis of PI mannosides (PIMs), lipomannan (LM), and lipoarabinomannan (LAM). Small lipids such as PI and PIMs are extracted with a mixture of chloroform, methanol, and water and analyzed by thin layer chromatography. For larger glycolipids, such as LM and LAM, more hydrophilic solvent is needed for the extraction, and SDS-PAGE is better suited for the analysis. For LM, further structural characterization can be performed by MALDI-TOF mass spectrometry. Precise quantification of PIMs, LM, and LAM can be performed by quantification of glycan staining using analytical software. The metabolic radiolabeling protocol is also described.
Assuntos
Fracionamento Químico/métodos , Cromatografia em Camada Fina/métodos , Lipopolissacarídeos/análise , Mycobacterium/química , Fosfatidilinositóis/análise , Clorofórmio/química , Humanos , Lipopolissacarídeos/isolamento & purificação , Metanol/química , Infecções por Mycobacterium/microbiologia , Mycobacterium smegmatis/química , Fosfatidilinositóis/isolamento & purificação , Solventes/químicaRESUMO
The 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 in Mycobacterium smegmatis. 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 h sub-cultures. 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.
RESUMO
The mycobacterial cell envelope is a complex multilayered structure that provides the strength to the rod-shaped cell and creates the permeability barrier against antibiotics and host immune attack. In this review, we will discuss the spatial coordination of cell envelope biosynthesis and how plasma membrane compartmentalization plays a role in this process. The spatial organization of cell envelope biosynthetic enzymes as well as other membrane-associated proteins is crucial for cellular processes such as polar growth and midcell septum formation. We will highlight metabolic enzymes involved in the localized biosynthesis of envelope components such as peptidoglycan, arabinogalactan and outer/inner membrane lipids. The known and potential roles of cytoskeletal and coiled coil proteins in driving subcellular protein localization will also be summarized. Finally, we provide a comprehensive overview of known lateral heterogeneities in mycobacterial plasma membrane, with a particular focus on the intracellular membrane domain, recently revealed by biochemical fractionation and fluorescence microscopy. We consider how this dynamic and multifunctional membrane microdomain contributes to the subcellular localization of membrane proteins and spatially restricted cell envelope biosynthesis in mycobacteria.
Assuntos
Proteínas de Bactérias/química , Membrana Celular/química , Parede Celular/química , Microdomínios da Membrana/química , Proteínas de Membrana/química , Mycobacterium tuberculosis/química , Proteínas de Bactérias/imunologia , Sequência de Carboidratos , Divisão Celular , Membrana Celular/imunologia , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Parede Celular/imunologia , Parede Celular/metabolismo , Parede Celular/ultraestrutura , Galactanos/química , Galactanos/imunologia , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunidade Inata , Microdomínios da Membrana/imunologia , Microdomínios da Membrana/metabolismo , Microdomínios da Membrana/ultraestrutura , Proteínas de Membrana/imunologia , Mycobacterium tuberculosis/imunologia , Mycobacterium tuberculosis/patogenicidade , Peptidoglicano/química , Peptidoglicano/imunologia , Tuberculose Pulmonar/imunologia , Tuberculose Pulmonar/microbiologiaRESUMO
In the last several decades, exposure to pesticides has become a concern to environmental and human health. Many pesticides are environmentally persistent and are characterized by varying degrees of toxicity and adverse effects, including DNA damage. The present study was undertaken to evaluate the genotoxic potential of organophosphate pesticide fenthion in Wistar rats, as assessed by the comet assay. Adult male Wistar rats were treated with a solution of fenthion at a concentration of 40 mg/kg/day, administered intraperitoneally for 18 consecutive days. Rats were killed 24 hours after the last pesticide administration, and the comet assay was performed in peripheral blood cells. The comet assay results revealed that the damage index (19.29 ± 3.59 vs. 7.80 ± 2.25) and the damage frequency (17.00 ± 3.46 vs. 7.5 ± 2.46) found in fenthion-treated rats were significantly higher than those found in the control group (p = 0.001 and p = 0.0006, respectively). The results show that fenthion affects the DNA integrity of rat cells and may induce DNA damage in exposed organisms.