RESUMEN
Mass spectrometry-based single-cell proteomic analysis has recently gained momentum and is now an emerging area with established protocols and promising results. Traditional proteomic studies, especially involving bacteria, have been limited to suspension cultures with large protein yields. Such studies, however, remain population centered with the uniqueness of individual responses to environmental challenges becoming diluted. To enable bacterial single-colony proteomics, we describe a quantitative mass spectrometry-based protocol to isolate and analyze the proteome of a single mycobacterial colony from 7H10 media, with growth supplements for optimal growth. Following protein purification and digestion, tryptic peptides are analyzed by UHPLC coupled to a hybrid Q Exactive mass spectrometer. Raw data were analyzed using the MaxQuant Suite, and downstream statistical analysis was performed using Perseus software. A total of 7805 unique peptides and 1387 proteins were identified. Data are available via ProteomeXchange with identifier PXD018168. In this chapter, we identify steps most prone to sample loss and describe measures of alleviation that allows the preservation of protein yield and boosts quantitative power while increasing reproducibility, of "very limiting samples."
Asunto(s)
Proteínas Bacterianas/análisis , Mycobacterium/química , Proteómica/métodos , Cromatografía Liquida/métodos , Humanos , Mycobacterium/citología , Infecciones por Mycobacterium/microbiología , Mycobacterium smegmatis/química , Mycobacterium smegmatis/citología , Proteoma/análisis , Espectrometría de Masas en Tándem/métodosRESUMEN
Mycobacteria grow by inserting new cell wall material in discrete zones at the cell poles. This pattern implies that polar growth zones must be assembled de novo at each division, but the mechanisms that control the initiation of new pole growth are unknown. Here, we combine time-lapse optical and atomic force microscopy to measure single-cell pole growth in mycobacteria with nanometer-scale precision. We show that single-cell growth is biphasic due to a lag phase of variable duration before the new pole transitions from slow to fast growth. This transition and cell division are independent events. The difference between the lag and interdivision times determines the degree of single-cell growth asymmetry, which is high in fast-growing species and low in slow-growing species. We propose a biphasic growth model that is distinct from previous unipolar and bipolar models and resembles "new end take off" (NETO) dynamics of polar growth in fission yeast.
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Modelos Biológicos , Mycobacterium/citología , Mycobacterium/crecimiento & desarrollo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , División Celular , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Microscopía de Fuerza Atómica , Mycobacterium/genética , Análisis Espacio-Temporal , Imagen de Lapso de TiempoRESUMEN
BACKGROUND: Direct sputum smear is still the first-choice tool for screening of tuberculosis worldwide. Variants of this technique, to improve the sensitivity are desired. METHODS: Two microbiological variants of the standard sputum smear ("pellet" and "diluted-pellet") for both Ziehl-Neelsen (ZN) and auramine fluorescence (AF) staining were evaluated. In addition, two methods for concentration of mycobacteria in sputum, using positive and negative pressure filtration, were tested and compared. The evaluation of the microbiological variants was performed on 98 culture positive sputum samples from different TB patients. The diagnostics sensitivity and the level of detritus in the processed sputum were determined. Bacilli load in the smear variants was determined by microscopic observation and by manual inspection of microscopic digital images. The comparison of the mycobacteria filtration methods was performed on 76 smear positive sputum samples. Filters retaining the concentrated mycobacteria were stained with AF and compared with the direct smear. Bacilli load, detritus level, filtered volume, filtration time and background noise level, were determined. RESULTS: The sensitivity of microscopy with the microbiological variants was 7.1% and 2% higher in ZN and AF respectively, compared to direct smear. The sensitivity of AF in diluted pellet was significantly higher than all ZN variants (P < 0.05). Detritus level observed in slides was significantly lower in the diluted pellet than the pellet and direct smear in ZN and AF (P < 0.001). A significant increase in the bacilli load in microscopic observation and digital images analysis was observed in pellet and diluted pellet than the direct method (P <0.0001). The concentration of mycobacteria using positive-pressure filtration showed a trend to produce a higher bacilli load compared to the negative-pressure filtration and direct smear, although it was not significant. Detritus levels were significantly higher in both variants of filtration (P < 0.0001). Filtered volumes were higher in positive-pressure compared to negative-pressure filtration. Filtration times were significantly higher in negative-pressure compared to positive-pressure filtration (P < 0.0001). CONCLUSION: The proposed variants improved the performance of the standard sputum smear, making it an important test for settings with high rates of smear-negative TB cases.
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Pruebas Diagnósticas de Rutina/métodos , Microscopía/métodos , Mycobacterium/citología , Manejo de Especímenes/métodos , Esputo/microbiología , Tuberculosis/diagnóstico , Humanos , Tamizaje Masivo/métodos , Sensibilidad y Especificidad , Coloración y Etiquetado/métodosRESUMEN
Energy metabolism has recently gained interest as a target space for antibiotic drug development in mycobacteria. Of particular importance is bedaquiline (Sirturo), which kills mycobacteria by inhibiting the F1F0 ATP synthase. Other components of the electron transport chain such as the NADH dehydrogenases (NDH-2 and NdhA) and the terminal respiratory oxidase bc1:aa3 are also susceptible to chemical inhibition. Because antituberculosis drugs are prescribed as part of combination therapies, the interaction between novel drugs targeting energy metabolism and classical first and second line antibiotics must be considered to maximize treatment efficiency. Here, we show that subinhibitory concentration of drugs targeting the F1F0 ATP synthase and the cytochrome bc1:aa3, as well as energy uncouplers, interfere with the bactericidal potency of isoniazid and moxifloxacin. Isoniazid- and moxifloxacin-induced mycobacterial death correlated with a transient increase in intracellular ATP that was dissipated by co-incubation with energy metabolism inhibitors. Although oxidative phosphorylation is a promising target space for drug development, a better understanding of the link between energy metabolism and antibiotic-induced mycobacterial death is essential to develop potent drug combinations for the treatment of tuberculosis.
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Antibacterianos/farmacología , Metabolismo Energético/efectos de los fármacos , Mycobacterium/efectos de los fármacos , Adenosina Trifosfato/metabolismo , Antituberculosos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Diseño de Fármacos , Proteínas del Complejo de Cadena de Transporte de Electrón/antagonistas & inhibidores , Isoniazida/farmacología , Moxifloxacino/farmacología , Mycobacterium/citología , Fosforilación Oxidativa/efectos de los fármacos , ATPasas de Translocación de Protón/antagonistas & inhibidoresRESUMEN
Mycobacterium tuberculosis (MTB) infections rely on continued growth and division. Despite the substantial global burden of tuberculosis, the underlying mechanism governing growth is incompletely understood. Bifunctional penicillin-binding protein (PBP1), encoded by Rv0050 (ponA1) of MTB, is a key peptidoglycan synthase and plays a central role in mycobacterial growth and division by its interaction with Rpf-interacting protein A (RipA, peptidoglycan endopeptidase). Our previous work suggested that the hyper-variable proline repeats are located at the N end of PBP1. In this study, we prove that altered secondary structure resulting from polymorphic proline repeats modulates the interaction between PBP1 and RipA. Without proper coordination of peptidoglycan synthase and hydrolase, cell elongation and division is also altered resulting in phenotypic changes in the population as indicated by altered dispersion, slowed growth, or shortened cell length. Together, our data reveal that polymorphisms in Rv0050 induce mycobacterial growth and morphologic changes, and hence are responsible for giving bacteria their shape.
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Mycobacterium smegmatis/genética , Mycobacterium/crecimiento & desarrollo , Proteínas de Unión a las Penicilinas/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Mycobacterium/citología , Mycobacterium/genética , Mycobacterium smegmatis/citología , Mycobacterium smegmatis/crecimiento & desarrollo , Proteínas de Unión a las Penicilinas/química , Dominios Proteicos Ricos en ProlinaRESUMEN
In order to obtain high 4-androstene-3,17-dione (AD) yield, nucleus degradation needs to be avoided during phytosterol bioconversion process with Mycobacterium neoaurum NwIB-R10hsd4A. 3-Ketosteroid-Δ1-dehydrogenase (KstD) catalyzes 1,2-desaturation of steroids and is a key enzyme involved in steroid nucleus oxidation. Heterogeneous expression and characterization of two KstDs (KstD2, KstD3) from M. neoaurum NwIB-R10hsd4A showed that their activities were inhibited by shifting temperature from 30 to 37 °C. However, the total activities of KstD2 and KstD3 were replenished when M. neoaurum NwIB-R10hsd4A was cultured at 37 °C because the transcription levels of kstD2 and kstD3 were upregulated 1.61- and 1.43-fold respectively compared with the cultivation at 30 °C. As the optimal temperature for cell growth was 30 °C, we developed a two-step bioprocess, cell culture at 30 °C and bioconversion with resting cells at 37 °C avoiding higher transcriptional level of kstD2 and kstD3. This process repressed the activities of KstDs, resulted in the decrease of 1,2-desaturation products, and reduced the nucleus degradation (17.6%). AD production increased to 24.7 g l-1 at higher substrate concentration (50 g l-1). These results indicated that the two-step bioprocess was potential in phytosterol biotransformation industrially.
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Núcleo Celular/metabolismo , Mycobacterium/metabolismo , Fitosteroles/metabolismo , Biotransformación , División Celular , Calor , Mycobacterium/citología , Oxidorreductasas/metabolismo , Transcripción GenéticaRESUMEN
Mycobacterium tuberculosis (Mtb) infects millions of people each year. These bacilli can survive inside macrophages. To favor their survival, pathogen alters various signal transduction pathways in host cells. Phospholipase C (PLC) signaling regulates various processes in mammalian cells but has never been investigated for their roles in regulating phagocytosis and killing of mycobacteria by macrophages. Here, we report that infection with Mtb but not Mycobacterium smegmatis (MS) induces phosphorylation of PLC-γ2 at tyrosine 1217 in J774A.1 cells. Small interfering RNA-mediated knockdown of PLC-γ2 expression leads to the enhanced killing of both MS and Mtb by these cells suggesting that Mtb activates PLC-γ2 to promote its intracellular survival within macrophages. Knockdown of PLC-γ2 also lead to increased uptake of Mtb but not MS by J774.A.1 cells. Further, we have observed that PLC-γ2 was required for Mtb-induced inhibition of expression of proinflammatory cytokine tumor necrosis factor-α, inducible nitric oxide synthase, and chemokine (C-C motif) ligand 5 (RANTES). Altogether, our results for the first time demonstrate that Mtb induces activation of macrophages PLC-γ2 to inhibit their mycobactericidal response.
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Espacio Intracelular/microbiología , Macrófagos/enzimología , Macrófagos/microbiología , Viabilidad Microbiana , Mycobacterium/citología , Fosfolipasa C gamma/metabolismo , Animales , Línea Celular , Citocinas/metabolismo , Mediadores de Inflamación/metabolismo , Ratones , Infecciones por Mycobacterium/microbiología , Infecciones por Mycobacterium/patología , FosforilaciónRESUMEN
Diadenosine polyphosphates, Ap(2-7)A, which contain two adenosines in a 5',5' linkage through phosphodiester bonds involving 2-7 phosphates, regulate diverse cellular functions in all organisms, from bacteria to humans, under normal and stress conditions. We had earlier reported consistent occurrence of asymmetric constriction during division (ACD) in 20-30% of dividing mycobacterial cells in culture, irrespective of different growth media, implying exogenous action of some factor of mycobacterial origin. Consistent with this premise, concentrated culture supernatant (CCS), but not the equivalent volume-wise concentrated unused medium, dramatically enhanced the ACD proportion to 70-90%. Mass spectrometry and biochemical analyses of the bioactive fraction from CCS revealed the ACD-effecting factor to be Ap6A. Synthetic Ap6A showed a mass spectrometry profile, biochemical characteristics, and bioactivity identical to native Ap6A in the CCS. Thus, the present work reveals a novel role for Ap6A in generating cell-length asymmetry during mycobacterial cell-division and thereby cell-length heterogeneity in the population.
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División Celular/efectos de los fármacos , Fosfatos de Dinucleósidos/metabolismo , Mycobacterium/citología , Mycobacterium/metabolismo , Fosfatos de Dinucleósidos/aislamiento & purificación , Mycobacterium smegmatis/citología , Mycobacterium smegmatis/metabolismo , Mycobacterium tuberculosis/citología , Mycobacterium tuberculosis/metabolismoRESUMEN
Canine Leproid Granuloma Syndrome (CLGS), also known as canine leprosy, is a cutaneous nodular infectious disease caused by Mycobacterium sp.. Despite being reported worldwide, it is still quite unknown and underdiagnosed. Diagnosis may be achieved by cytopathology or histopathology of skin lesions, but identification of the infectious agent is complex, since bacterial in vitro growth is not possible, relying upon molecular techniques such as PCR to confirm Mycobacterium DNA in the sample. We report a CLGS case in Niteroi, Rio de Janeiro state, Brazil, diagnosed by cytopathology and submitted to molecular identification of the agent. PCR amplification of hsp65 gene was performed and revealed 100% genetic homology to M. murphy strain. This is the first CLGS report with molecular identification in Rio de Janeiro state, and this finding should raise awareness about CLGS as a differential diagnosis among granulomatous skin diseases in this region.(AU)
A síndrome de granuloma leproide canino (SGLC), também conhecida como lepra canina, é uma doença infecciosa cutânea nodular causada por Mycobacterium sp. Apesar de ser relatada mundialmente, ainda é bastante desconhecida e subdiagnosticada. O diagnóstico pode ser conseguido por citopatologia ou histopatologia de lesões cutâneas, mas a identificação do agente infeccioso é complexa, uma vez que o crescimento in vitro bacteriano não é possível, dependendo de técnicas moleculares como a PCR para confirmar o DNA de Mycobacterium na amostra. Relatou-se um caso da SGLC em Niterói, estado do Rio de Janeiro, Brasil, diagnosticado por citopatologia e submetido à identificação molecular do agente. Foi realizada amplificação por PCR do gene hsp65, que revelou 100% de homologia genética com a cepa M. murphy. Este é o primeiro relato da SGLC com identificação molecular no estado do Rio de Janeiro, o que mostra a importância de se acrescentar a SGLC ao diagnóstico diferencial das doenças granulomatosas de pele nessa região.(AU)
Asunto(s)
Animales , Perros , Reacción en Cadena de la Polimerasa/estadística & datos numéricos , Mycobacterium/citología , Mycobacterium/patogenicidad , Infecciones por Mycobacterium , PerrosRESUMEN
BACKGROUND: The complex architecture of the mycobacterial cell wall is responsible for many physiological and infection-associated properties of these pathogens. Long chain mycolic acids with chiral functional groups constitute a major lipid envelope component, and therefore play a crucial role in determining the permeability and fluidity of the cell wall. Varying proportions of the different mycolate types are present in various mycobacteria. This serves to designate a specific mycolic acid profile, allowing the differentiation of species and subspecies. OBJECTIVE: In this review, we explore the diversity in mycolic acid chemical structure, its influence on cell wall permeability and consequences for virulence, and present how they might be exploited as research targets in diagnosis and chemotherapy. METHODS: The search of bibliographic databases for adequate, informative and valuable peerreviewed literature was undertaken. The proper papers were identified by the hand and electronic searching through the relevant to the topic-leading journals, key words, named authors, reference scanning, etc. RESULTS: The review is based on the ninety two carefully selected articles that allow this review to provide up-to-date information. Twenty nine papers were published within last 5 years, including 10 showed up in 2016-2017. CONCLUSION: The revision of the relevant literature showed that mycolic acids played a crucial role in host-pathogen interactions and thus might serve as a promising tool for differentiation of mycobacterial species and discovering novel tuberculocidal therapeutic strategies.
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Interacciones Huésped-Patógeno , Mycobacterium/química , Ácidos Micólicos/química , Permeabilidad de la Membrana Celular , Mycobacterium/citología , Mycobacterium/metabolismo , Ácidos Micólicos/metabolismoRESUMEN
Mycobacteria are endowed with a highly impermeable mycomembrane that confers intrinsic resistance to many antibiotics. Several unique mycomembrane glycolipids have been isolated and structurally characterized, but the underlying organization and dynamics of glycolipids within the cell envelope remain poorly understood. We report here a study of mycomembrane dynamics that was enabled by trehalose-fluorophore conjugates capable of labeling trehalose glycolipids in live actinomycetes. We identified fluorescein-trehalose analogues that are metabolically incorporated into the trehalose mycolates of representative Mycobacterium, Corynebacterium, Nocardia, and Rhodococcus species. Using these probes, we studied the mobilities of labeled glycolipids by time-lapse microscopy and fluorescence recovery after photobleaching experiments and found that mycomembrane fluidity varies widely across species and correlates with mycolic acid structure. Finally, we discovered that treatment of mycobacteria with ethambutol, a front-line tuberculosis (TB) drug, significantly increases mycomembrane fluidity. These findings enhance our understanding of mycobacterial cell envelope structure and dynamics and have implications for development of TB drug cocktails.
Asunto(s)
Membrana Celular/metabolismo , Mycobacterium/citología , Termodinámica , Membrana Celular/efectos de los fármacos , Supervivencia Celular , Etambutol/farmacología , Fluidez de la Membrana/efectos de los fármacos , Mycobacterium/efectos de los fármacosRESUMEN
In steroid biotransformation, soybean oil can improve the productivity of steroids by increasing substrate solubility and strengthen the cell membrane permeability. However, little is known of its role as oxygen carrier and its mechanism of promoting the steroid biotransformation. In this work, soybean oil used as oxygen vector for the enhancement of androst-4-ene-3,17-dione (AD) production by Mycobacterium neoaurum TCCC 11979 (MNR) was investigated. Upon the addition of 16% (v/v) soybean oil, the volumetric oxygen transfer coefficient (K L a) value increased by 44%, and the peak molar yield of AD (55.76%) was achieved. Analysis of intracellular cofactor levels showed high NAD+, ATP level, and a low NADH/NAD+ ratio. Meanwhile, the two key enzymes of the tricarboxylic acid (TCA) cycle, namely, isocitrate dehydrogenase and α-ketoglutarate dehydrogenase, were upregulated after incubation with soybean oil. These enhancements induced by the increasing of oxygen supply showed positive effects on phytosterol (PS) bioconversion. Results could contribute to the understanding of effects of soybean oil as oxygen vector on steroid biotransformation and provided a convenient method for enhancing the efficiency of aerobic steroid biocatalysis.
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Androstadienos/metabolismo , Androstenodiona/metabolismo , Mycobacterium/metabolismo , Oxígeno/metabolismo , Biotransformación/efectos de los fármacos , Ciclo del Ácido Cítrico/efectos de los fármacos , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/metabolismo , Mycobacterium/citología , Mycobacterium/efectos de los fármacos , Aceite de Soja/farmacologíaRESUMEN
Macrophages play an essential role in the immune system by ingesting and degrading invading pathogens, initiating an inflammatory response and instructing adaptive immune cells, and resolving inflammation to restore homeostasis. More interesting is the fact that some bacteria have evolved to use macrophages as a natural habitat and tools of spread in the host, e.g., Mycobacterium tuberculosis (Mtb) and some non-tuberculous mycobacteria (NTM). Mtb is considered one of humanity's most successful pathogens and is the causal agent of tuberculosis, while NTMs cause opportunistic infections all of which are of significant public health concern. Here, we describe mechanisms by which intracellular pathogens, with an emphasis on mycobacteria, manipulate macrophage functions to circumvent killing and live inside these cells even under considerable immunological pressure. Such macrophage functions include the selective evasion or engagement of pattern recognition receptors, production of cytokines, reactive oxygen and nitrogen species, phagosome maturation, as well as other killing mechanisms like autophagy and cell death. A clear understanding of host responses elicited by a specific pathogen and strategies employed by the microbe to evade or exploit these is of significant importance for the development of effective vaccines and targeted immunotherapy against persistent intracellular infections like tuberculosis.
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Macrófagos/microbiología , Viabilidad Microbiana , Mycobacterium/citología , Animales , Interacciones Huésped-Patógeno , Humanos , Fagocitosis , FagosomasRESUMEN
A closed droplet based lab-on-a-chip (LOC) device has been developed for the differentiation of six species of mycobacteria, i.e., both Mycobacterium tuberculosis complex (MTC) and nontuberculous mycobacteria (NTM), using surface-enhanced Raman spectroscopy (SERS). The combination of a fast and simple bead-beating module for the disruption of the bacterial cell with the LOC-SERS device enables the application of an easy and reliable system for bacteria discrimination. Without extraction or further treatment of the sample, the obtained SERS spectra are dominated by the cell-wall component mycolic acid. For the differentiation, a robust data set was recorded using a droplet based LOC-SERS device. Thus, more than 2100 individual SERS spectra of the bacteria suspension were obtained in 1 h. The differentiation of bacteria using LOC-SERS provides helpful information for physicians to define the conditions for the treatment of individual patients.
Asunto(s)
Dispositivos Laboratorio en un Chip , Mycobacterium/aislamiento & purificación , Mycobacterium/citología , Especificidad de la Especie , Espectrometría Raman/instrumentación , Propiedades de SuperficieRESUMEN
CONTEXT: Clinical presentation of Mycobacterium tuberculosis complex (MTBC) and non-tuberculous mycobacteria (NTM) infections may or may not be the same, but the treatment is always different. Hence accurate differentiation between MTBC and NTM is of utmost importance. AIMS: To assess in parallel, the utility of MPT64 antigen immunochromatography assay (MPT64 ICT) and bacillary morphology on liquid culture smear, for rapid differentiation between MTBC and NTM in clinical isolates. SETTINGS AND DESIGNS: Private sector reference laboratory, prospective. SUBJECTS AND METHODS: Thousand and ninety-three mycobacterial isolates, recovered using Mycobacteria Growth Indicator Tube 960 liquid culture system (BD, USA), were subjected to MPT64 ICT (Standard Diagnostics Inc., Korea), para amino nitrobenzoicacid (PNB), niacin, and nitrate reduction tests. Smears prepared from culture vials were subjected to Ziehl-Neelsen staining and observed microscopically for typical patterns (chords, single cells, etc.,). PNB, nitrate and niacin tests served as the reference method for MTBC identification. RESULTS: Thousand and fourteen and 79 isolates were identified as MTBC and NTM, respectively. MPT64 ICT correctly identified 955/1014 MTBC and all NTM isolates, yielding sensitivity and specificity of 94.2% and 100%, respectively. 936/1014 (92.3%) MTBC isolates revealed characteristic serpentine chording on culture smear including 56/59 MPT64 ICT negative isolates. Sensitivity and specificity of liquid culture smear were 98.1% and 82.3%, respectively. CONCLUSION: Correlation of MPT64 ICT results with liquid culture smear was useful, especially in MPT64 ICT negative isolates, where the latter could help to determine need and/or type of additional confirmatory testing. Liquid culture smear, however, lacked specificity and cannot be used as a stand alone test.
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Antígenos Bacterianos/análisis , Proteínas Bacterianas/análisis , Cromatografía de Afinidad/métodos , Microscopía/métodos , Infecciones por Mycobacterium no Tuberculosas/diagnóstico , Mycobacterium/clasificación , Mycobacterium/aislamiento & purificación , Tuberculosis/diagnóstico , Técnicas de Tipificación Bacteriana/métodos , Humanos , Mycobacterium/química , Mycobacterium/citología , Infecciones por Mycobacterium no Tuberculosas/microbiología , Sensibilidad y Especificidad , Tuberculosis/microbiologíaRESUMEN
The global pathogen Mycobacterium tuberculosis and other species in the suborder Corynebacterineae possess a distinctive outer membrane called the mycomembrane (MM). The MM is composed of mycolic acids, which are either covalently linked to an underlying arabinogalactan layer or incorporated into trehalose glycolipids that associate with the MM non-covalently. These structures are generated through a process called mycolylation, which is central to mycobacterial physiology and pathogenesis and is an important target for tuberculosis drug development. Current approaches to investigating mycolylation rely on arduous analytical methods that occur outside the context of a whole cell. Herein, we describe mycobacteria-specific chemical reporters that can selectively probe either covalent arabinogalactan mycolates or non-covalent trehalose mycolates in live mycobacteria. These probes, in conjunction with bioorthogonal chemistry, enable selective inâ situ detection of the major MM components.
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Membrana Celular/química , Sondas Moleculares/química , Mycobacterium/química , Mycobacterium/citología , Ácidos Micólicos/análisis , Ácidos Micólicos/química , Estructura MolecularRESUMEN
Effects of different carbon sources on growth, membrane permeability, ß-sitosterol consumption, androstadienedione and androstenedione (AD(D)) production by Mycobacterium neoaurum were investigated. The results indicated that glucose was advantageous to the growth and resulted in the adverse effects on the phytosterols consumption and AD(D) production compared to the results of propanol and isopropanol as sole carbon source. The cell wall widths of 9.76 by propanol and 8.00 nm by isopropanol were 38.3 and 49.4 % thinner than that of 15.82 nm by glucose, respectively. The partition coefficient of the cell grown in propanol and isopropanol was 18.1 and 22.2, which were 7.23- and 9.09-fold higher than that of the cell grown in glucose.
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Androstadienos/metabolismo , Androstenodiona/biosíntesis , Carbono/farmacología , Permeabilidad de la Membrana Celular/efectos de los fármacos , Mycobacterium/crecimiento & desarrollo , Mycobacterium/metabolismo , Sitoesteroles/metabolismo , Pared Celular/efectos de los fármacos , Pared Celular/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Lípidos/análisis , Mycobacterium/citología , Mycobacterium/efectos de los fármacosRESUMEN
The mycobacterial tlyA gene product, Rv1694 (MtbTlyA), has been annotated as "hemolysin" which was re-annotated as 2'-O rRNA methyl transferase. In order to function as a hemolysin, it must reach the extracellular milieu with the help of signal sequence(s) and/or transmembrane segment(s). However, the MtbTlyA neither has classical signals sequences that signify general/Sec/Tat pathways nor transmembrane segments. Interestingly, the tlyA gene appears to be restricted to pathogenic strains such as H37Rv, M. marinum, M. leprae, than M. smegmatis, M. vaccae, M. kansasii etc., which highlights the need for a detailed investigation to understand its functions. In this study, we have provided several evidences which highlight the presence of TlyA on the surface of M. marinum (native host) and upon expression in M. smegmatis (surrogate host) and E. coli (heterologous host). The TlyA was visualized at the bacterial-surface by confocal microscopy and accessible to Proteinase K. In addition, sub-cellular fractionation has revealed the presence of TlyA in the membrane fractions and this sequestration is not dependent on TatA, TatC or SecA2 pathways. As a consequence of expression, the recombinant bacteria exhibit distinct hemolysis. Interestingly, the MtbTlyA was also detected in both membrane vesicles secreted by M. smegmatis and outer membrane vesicles secreted by E. coli. Our experimental evidences unambiguously confirm that the mycobacterial TlyA can reach the extra cellular milieu without any signal sequence. Hence, the localization of TlyA class of proteins at the bacterial surface may highlight the existence of non-classical bacterial secretion mechanisms.
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Proteínas Bacterianas/análisis , Proteínas Bacterianas/genética , Pared Celular/química , Mycobacterium/química , Señales de Clasificación de Proteína , Animales , Endopeptidasa K/metabolismo , Escherichia coli/química , Escherichia coli/genética , Ratones , Microscopía Confocal , Mycobacterium/citología , Mycobacterium/genética , Proteolisis , Conejos , Vesículas Secretoras/químicaRESUMEN
Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a major pathogen responsible for 1.5 million deaths annually. This bacterium is characterized by a highly unusual and impermeable cell envelope, which plays a key role in mycobacterial survival and virulence. Although many studies have focused on the composition and functioning of the mycobacterial cell envelope, the capsular α-glucan has received relatively minor attention. Here we show that a murine monoclonal antibody (Mab) directed against glycogen cross-reacts with mycobacterial α-glucans, polymers of α(1-4)-linked glucose residues with α(1-6)-branch points. We identified the Mab epitope specificity by saturation transfer difference NMR and show that the α(1-4)-linked glucose residues are important in glucan-Mab interaction. The minimal epitope is formed by (linear) maltotriose. Notably, a Mycobacterium mutant lacking the branching enzyme GlgB does not react with the Mab; this suggests that the α(1-6)-branches form part of the epitope. These seemingly conflicting data can be explained by the fact that in the mutant the linear form of the α-glucan (amylose) is insoluble. This Mab was subsequently used to develop several techniques helpful in capsular α-glucan research. By using a capsular glucan-screening methodology based on this Mab we were able to identify several unknown genes involved in capsular α-glucan biogenesis. Additionally, we developed two methods for the detection of capsular α-glucan levels. This study therefore opens new ways to study capsular α-glucan and to identify possible targets for further research.