RESUMO
Introduction: Osteopenia has been associated to several inflammatory conditions, including mycobacterial infections. How mycobacteria cause bone loss remains elusive, but direct bone infection may not be required. Methods: Genetically engineered mice and morphometric, transcriptomic, and functional analyses were used. Additionally, inflammatory mediators and bone turnover markers were measured in the serum of healthy controls, individuals with latent tuberculosis and patients with active tuberculosis. Results and discussion: We found that infection with Mycobacterium avium impacts bone turnover by decreasing bone formation and increasing bone resorption, in an IFNγ- and TNFα-dependent manner. IFNγ produced during infection enhanced macrophage TNFα secretion, which in turn increased the production of serum amyloid A (SAA) 3. Saa3 expression was upregulated in the bone of both M. avium- and M. tuberculosis-infected mice and SAA1 and 2 proteins (that share a high homology with murine SAA3 protein) were increased in the serum of patients with active tuberculosis. Furthermore, the increased SAA levels seen in active tuberculosis patients correlated with altered serum bone turnover markers. Additionally, human SAA proteins impaired bone matrix deposition and increased osteoclastogenesis in vitro. Overall, we report a novel crosstalk between the cytokine-SAA network operating in macrophages and bone homeostasis. These findings contribute to a better understanding of the mechanisms of bone loss during infection and open the way to pharmacological intervention. Additionally, our data and disclose SAA proteins as potential biomarkers of bone loss during infection by mycobacteria.
Assuntos
Mycobacterium tuberculosis , Proteína Amiloide A Sérica , Humanos , Camundongos , Animais , Proteína Amiloide A Sérica/genética , Proteína Amiloide A Sérica/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Osso e Ossos/metabolismo , Macrófagos/metabolismo , Citocinas/metabolismo , Mycobacterium tuberculosis/metabolismoRESUMO
Mycobacterium tuberculosis, the ancient master of causing tuberculosis, is one of the most successful pathogens capable of persistently colonizing host lungs. The EsxB (CFP-10) of ESX-1 system and PPE68 of the PPE family contribute to the virulence of M. tuberculosis. However, the virulence potential and pathogenetic characteristics of these two proteins during M. tuberculosis infection remain unclear. In this study, two prokaryotic expression plasmids for EsxB or PPE68 of M. tuberculosis were constructed and the recombinant proteins His-EsxB or His-PPE68 were purified. The proteome and transcriptome of MH-S cells treated with His-EsxB or His-PPE68 were explored, followed by validating the expression of the identified differentially expressed genes (DEGs) using quantitative PCR. A total of 159/439 specific proteins or 633/1117 DEGs were obtained between control and His-EsxB or His-PPE68 treated groups in the MH-S proteomes and transcriptomes. Additionally, 37/60 signal pathways were predicted in the His-EsxB or His-PPE68 treated groups and "Cytokine-cytokine receptor interaction" was the most represented pathway. Furthermore, the expression of the DEGs (IL-1ß, IL-6, and TNF-α) was significantly upregulated, suggesting that these DEGs contributed to the host response during EsxB or PPE68 treatment. These findings provide detailed information on developing an effective intervention strategy to control M. tuberculosis infection.
Assuntos
Mycobacterium tuberculosis , Tuberculose , Humanos , Mycobacterium tuberculosis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Fatores de Virulência/genética , MultiômicaRESUMO
Introduction: Tuberculosis (TB) is a major health problem characterized by an immuno-endocrine imbalance: elevated plasma levels of cortisol and pro- and anti-inflammatory mediators, as well as reduced levels of dehydroepiandrosterone. The etiological agent, Mycobacterium tuberculosis (Mtb), is captured by pulmonary macrophages (Mf), whose activation is necessary to cope with the control of Mtb, however, excessive activation of the inflammatory response also leads to tissue damage. Glucocorticoids (GC) are critical elements to counteract the immunoinflammatory reaction, and peroxisome proliferator-activated receptors (PPARs) are also involved in this regard. The primary forms of these receptors are PPARÏ, PPARα, and PPARß/δ, the former being the most involved in anti-inflammatory responses. In this work, we seek to gain some insight into the contribution of PPARÏ in immuno-endocrine-metabolic interactions by focusing on clinical studies in pulmonary TB patients and in vitro experiments on a Mf cell line. Methods and results: We found that TB patients, at the time of diagnosis, showed increased expression of the PPARÏ transcript in their peripheral blood mononuclear cells, positively associated with circulating cortisol and related to disease severity. Given this background, we investigated the expression of PPARÏ (RT-qPCR) in radiation-killed Mtb-stimulated human Mf. The Mtb stimulation of Mf derived from the human line THP1 significantly increased the expression of PPARÏ, while the activation of this receptor by a specific agonist decreased the expression of pro- and anti-inflammatory cytokines (IL-1ß and IL-10). As expected, the addition of GC to stimulated cultures reduced IL-1ß production, while cortisol treatment together with the PPARÏ agonist lowered the levels of this proinflammatory cytokine in stimulated cultures. The addition of RU486, a glucocorticoid receptor antagonist, only reversed the inhibition produced by the addition of GC. Conclusion: The current results provide a stimulating background for further analysis of the interconnection between PPARs and steroid hormones in the context of Mtb infection.
Assuntos
Mycobacterium tuberculosis , Tuberculose , Humanos , PPAR gama/metabolismo , PPAR gama/farmacologia , Hidrocortisona/farmacologia , Hidrocortisona/metabolismo , Leucócitos Mononucleares/metabolismo , Tuberculose/metabolismo , Mycobacterium tuberculosis/metabolismo , Citocinas/metabolismoRESUMO
In the course of evolution, Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis, has developed sophisticated strategies to evade host immune response, including the synthesis of small non-coding RNAs (sRNAs), which regulate post-transcriptional pathways involved in the stress adaptation of mycobacteria. sRNA MTS1338 is upregulated in Mtb during its infection of cultured macrophages and in the model of chronic tuberculosis, suggesting involvement in host-pathogen interactions. Here, we analyzed the role of MTS1338 in the Mtb response to macrophage-like stresses in vitro. The Mtb strain overexpressing MTS1338 demonstrated enhanced survival ability under low pH, nitrosative, and oxidative stress conditions simulating the antimicrobial environment inside macrophages. Transcriptomic analysis revealed that in MTS1338-overexpressing Mtb, the stress factors led to the activation of a number of transcriptional regulators, toxin-antitoxin modules, and stress chaperones, about half of which coincided with the genes induced in Mtb phagocytosed by macrophages. We determined the MTS1338 "core regulon", consisting of 11 genes that were activated in all conditions under MTS1338 overexpression. Our findings indicate that MTS1338 is a stress-induced sRNA that promotes Mtb survival in macrophages by triggering adaptive transcriptional mechanisms in response to host antimicrobial defense reactions.
Assuntos
Anti-Infecciosos , Mycobacterium tuberculosis , Pequeno RNA não Traduzido , Tuberculose , Humanos , Mycobacterium tuberculosis/metabolismo , Regulação Bacteriana da Expressão Gênica , Tuberculose/microbiologia , Anti-Infecciosos/metabolismo , Pequeno RNA não Traduzido/genética , Pequeno RNA não Traduzido/metabolismo , Interações Hospedeiro-PatógenoRESUMO
Emerging evidence suggests that long non-coding RNAs (LncRNAs) are involved in Mtb-induced programmed necrosis. Among these LncRNAs, LncRNA NR_003508 is associated with LPS-induced acute respiratory distress syndrome. However, whether LncRNA NR_003508 contributes to Mtb-induced programmed necrosis remains undocumented. Firstly, the expression of LncRNA NR_003508 was determined using RT-qPCR and FISH. The protein expression of RIPK1, p-RIPK1, RIPK3, p-RIPK3, MLKL, and p-MLKL was measured by Western blot in RAW264.7 and mouse lung tissues. Furthermore, luciferase reporter assays and bioinformatics were used to predict specific miRNA (miR-346-3p) and mRNA (RIPK1) regulated by LncRNA NR_003508. In addition, RT-qPCR was used to detect the RIPK1 expression in TB patients and healthy peripheral blood. The flow cytometry assay was performed to detect cell necrosis rates. Here we show that BCG infection-induced cell necrosis and increased LncRNA NR_003508 expression. si-NR_003508 inhibited BCG/H37Rv-induced programmed necrosis in vitro or in vivo. Functionally, LncRNA NR_003508 has been verified as a ceRNA for absorbing miR-346-3p, which targets RIPK1. Moreover, RIPK1 expression was elevated in the peripheral blood of TB patients compared with healthy people. Knockdown of LncRNA NR_003508 or miR-346-3p overexpression suppresses cell necrosis rate and ROS accumulation in RAW264.7 cells. In conclusion, LncRNA NR_003508 functions as a positive regulator of Mtb-induced programmed necrosis via sponging miR-346-3p to regulate RIPK1. Our findings may provide a promising therapeutic target for tuberculosis.
Assuntos
MicroRNAs , Mycobacterium tuberculosis , RNA Longo não Codificante , Camundongos , Animais , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Mycobacterium tuberculosis/metabolismo , Vacina BCG , Necrose/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Proliferação de Células/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismoRESUMO
Mycobacterium tuberculosis (Mtb), perhaps more than any other organism, is intrinsically appealing to chemical biologists. Not only does the cell envelope feature one of the most complex heteropolymers found in nature1 but many of the interactions between Mtb and its primary host (we humans) rely on lipid and not protein mediators.2,3 Many of the complex lipids, glycolipids, and carbohydrates biosynthesized by the bacterium still have unknown functions, and the complexity of the pathological processes by which tuberculosis (TB) disease progress offers many opportunities for these molecules to influence the human response. Because of the importance of TB in global public health, chemical biologists have applied a wide-ranging array of techniques to better understand the disease and improve interventions.
Assuntos
Mycobacterium tuberculosis , Tuberculose , Humanos , Tuberculose/tratamento farmacológico , Mycobacterium tuberculosis/metabolismo , Glicolipídeos , Membrana Celular/metabolismo , BiologiaRESUMO
The bacterial pathogen Mycobacterium tuberculosis binds to the C-type lectin DC-SIGN (dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin) on dendritic cells to evade the immune system. While DC-SIGN glycoconjugate ligands are ubiquitous among mycobacterial species, the receptor selectively binds pathogenic species from the M. tuberculosis complex (MTBC). Here, we unravel the molecular mechanism behind this intriguing selective recognition by means of a multidisciplinary approach combining single-molecule atomic force microscopy with Förster resonance energy transfer and bioassays. Molecular recognition imaging of mycobacteria demonstrates that the distribution of DC-SIGN ligands markedly differs between Mycobacterium bovis Bacille Calmette-Guérin (BCG) (model MTBC species) and Mycobacterium smegmatis (non-MTBC species), the ligands being concentrated into dense nanodomains on M. bovis BCG. Upon bacteria-host cell adhesion, ligand nanodomains induce the recruitment and clustering of DC-SIGN. Our study highlights the key role of clustering of both ligands on MTBC species and DC-SIGN host receptors in pathogen recognition, a mechanism that might be widespread in host-pathogen interactions.
Assuntos
Mycobacterium tuberculosis , Receptores de Superfície Celular , Ligantes , Receptores de Superfície Celular/metabolismo , Lectinas Tipo C/metabolismo , Mycobacterium tuberculosis/metabolismoRESUMO
The dormant phenotype of Mycobacterium tuberculosis that develops during infection poses a major challenge in disease treatment, since these bacilli show tolerance to front-line drugs. An in vitro hypoxia dormancy model was established, which produced phenotypically dormant Mycobacterium smegmatis after prolonged incubation under conditions of low oxygen, low pH, and nutrient limitation. Bacilli in this model displayed the classical dormancy characters, including loss of acid fastness, altered morphology, and, most importantly, tolerance to front-line drugs. The dormant form of M. smegmatis was treated with drugs and phytomolecules. Three phytomolecules exhibited activity against dormant bacilli, as shown by lack of regrowth in solid and liquid media. Further investigation of dormancy-active hits was carried out using in silico approaches to understand the druggable targets of these phytomolecules in dormant bacilli. For this study, molecular docking, molecular dynamics simulations (MDS), and molecular mechanics-generalized born solvent accessibility (MM-GBSA)-based binding energy (ΔGbind) calculations were performed. Five different targets, namely, isocitrate lyase (ICL), GMP synthase, LuxR, DosR, and serine/threonine protein kinase (STPK), from M. smegmatis and M. tuberculosis were studied in details. DosR and STPK were found to be the common targets in both the species that were more prone to the phytomolecules. The standard DosR inhibitor, HC104A, showed a lower dock score and binding energy of -4.27 and -34.50 kcal/mol, respectively, compared to the natural products under study. The phytomolecule, icariin, showed better docking score (dock score = -5.92 kcal/mol with and binding energy ΔGbind= -52.96 kcal/mol) with DosR compared to known DosR inhibitor, HC104A (dock score = -4.27 kcal/mol and binding energy ΔGbind = -34.50 kcal/mol). Similarly, the known STPK inhibitor MRCT67127 showed a lower dock score and binding energy of -4.25 and -29.43 kcal/mol, respectively, compared to the phytomolecule, icariin (dock score = -5.74 kcal/mol and ΔGbind= -43.41 kcal/mol). These compounds might ultimately lead to new therapeutics or may be useful as adjuvants to the first-line drugs to reduce the lengthy anti-TB therapy in the future.
Assuntos
Produtos Biológicos , Mycobacterium tuberculosis , Produtos Biológicos/farmacologia , Produtos Biológicos/metabolismo , Simulação de Acoplamento Molecular , Mycobacterium tuberculosis/metabolismo , Fenótipo , Simulação de Dinâmica MolecularRESUMO
Bacterial cell growth and division require the coordinated action of enzymes that synthesize and degrade cell wall polymers. Here, we identify enzymes that cleave the D-arabinan core of arabinogalactan, an unusual component of the cell wall of Mycobacterium tuberculosis and other mycobacteria. We screened 14 human gut-derived Bacteroidetes for arabinogalactan-degrading activities and identified four families of glycoside hydrolases with activity against the D-arabinan or D-galactan components of arabinogalactan. Using one of these isolates with exo-D-galactofuranosidase activity, we generated enriched D-arabinan and used it to identify a strain of Dysgonomonas gadei as a D-arabinan degrader. This enabled the discovery of endo- and exo-acting enzymes that cleave D-arabinan, including members of the DUF2961 family (GH172) and a family of glycoside hydrolases (DUF4185/GH183) that display endo-D-arabinofuranase activity and are conserved in mycobacteria and other microbes. Mycobacterial genomes encode two conserved endo-D-arabinanases with different preferences for the D-arabinan-containing cell wall components arabinogalactan and lipoarabinomannan, suggesting they are important for cell wall modification and/or degradation. The discovery of these enzymes will support future studies into the structure and function of the mycobacterial cell wall.
Assuntos
Mycobacterium tuberculosis , Polissacarídeos , Humanos , Polissacarídeos/metabolismo , Mycobacterium tuberculosis/metabolismo , Glicosídeo Hidrolases/metabolismo , Parede Celular/metabolismoRESUMO
Ribonucleases (RNases) are responsible for RNA metabolism. RNase J, the core enzyme of the RNA degradosome, plays an essential role in global mRNA decay. Emerging evidence showed that the RNase J of Mycobacterium tuberculosis (Mtb-RNase J) could be an excellent target for treating Mtb infection. Here, crystal structures of Mtb-RNase J in apo-state and complex with the single-strand RNA reveal the conformational change upon RNA binding and hydrolysis. Mtb-RNase J forms an active homodimer through the interactions between the ß-CASP and the ß-lactamase domain. Knockout of RNase J slows the growth rate and changes the colony morphologies and cell length in Mycobacterium smegmatis, which is restored by RNase J complementation. Finally, RNA-seq analysis shows that the knockout strain significantly changes the expression levels of 49 genes in metabolic pathways. Thus, our current study explores the structural basis of Mtb-RNase J and might provide a promising candidate in pharmacological treatment for tuberculosis.
Assuntos
Mycobacterium tuberculosis , Ribonucleases , Ribonucleases/metabolismo , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , RNA/metabolismo , Ribonuclease Pancreático/metabolismo , HidróliseRESUMO
Transcriptional pauses mediate regulation of RNA biogenesis. DNA-encoded pause signals trigger pausing by stabilizing RNA polymerase (RNAP) swiveling and inhibiting DNA translocation. The N-terminal domain (NGN) of the only universal transcription factor, NusG/Spt5, modulates pausing through contacts to RNAP and DNA. Pro-pausing NusGs enhance pauses, whereas anti-pausing NusGs suppress pauses. Little is known about pausing and NusG in the human pathogen Mycobacterium tuberculosis (Mtb). We report that MtbNusG is pro-pausing. MtbNusG captures paused, swiveled RNAP by contacts to the RNAP protrusion and nontemplate-DNA wedged between the NGN and RNAP gate loop. In contrast, anti-pausing Escherichia coli (Eco) NGN contacts the MtbRNAP gate loop, inhibiting swiveling and pausing. Using CRISPR-mediated genetics, we show that pro-pausing NGN is required for mycobacterial fitness. Our results define an essential function of mycobacterial NusG and the structural basis of pro- versus anti-pausing NusG activity, with broad implications for the function of all NusG orthologs.
Assuntos
Proteínas de Escherichia coli , Mycobacterium tuberculosis , Humanos , Fatores de Transcrição/genética , Fatores de Transcrição/química , Transcrição Gênica , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Proteínas de Escherichia coli/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , DNA , Fatores de Alongamento de Peptídeos/metabolismoRESUMO
The mycobacterial cell envelope consists of a typical plasma membrane, surrounded by a complex cell wall and a lipid-rich outer membrane. The biogenesis of this multilayer structure is a tightly regulated process requiring the coordinated synthesis and assembly of all its constituents. Mycobacteria grow by polar extension and recent studies showed that cell envelope incorporation of mycolic acids, the major constituent of the cell wall and outer membrane, is coordinated with peptidoglycan biosynthesis at the cell poles. However, there is no information regarding the dynamics of incorporation of other families of outer membrane lipids during cell elongation and division. Here, we establish that the translocation of non-essential trehalose polyphleates (TPP) occurs at different subcellular locations than that of the essential mycolic acids. Using fluorescence microscopy approaches, we investigated the subcellular localization of MmpL3 and MmpL10, respectively involved in the export of mycolic acids and TPP, in growing cells and their colocalization with Wag31, a protein playing a critical role in regulating peptidoglycan biosynthesis in mycobacteria. We found that MmpL3, like Wag31, displays polar localization and preferential accumulation at the old pole whereas MmpL10 is more homogenously distributed in the plasma membrane and slightly accumulates at the new pole. These results led us to propose a model in which insertion of TPP and mycolic acids into the mycomembrane is spatially uncoupled.
Assuntos
Mycobacterium tuberculosis , Mycobacterium , Trealose/metabolismo , Ácidos Micólicos/metabolismo , Peptidoglicano/metabolismo , Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Parede Celular/metabolismo , Mycobacterium/metabolismo , Mycobacterium tuberculosis/metabolismoRESUMO
Tuberculosis is an important chronic and often fatal infectious disease mainly caused by the bacterium Mycobacterium tuberculosis (Mtb). Mtb is one of the most successful pathogens that harbors several potential virulence factors not found in nonpathogenic mycobacteria. As the Mtb cell envelope is closely associated with its virulence and resistance, it is very important to understand the cell envelope for better treatment of causative pathogen. There is increasing evidence that Pro-Glu (PE) and Pro-Pro-Glu (PPE) proteins are the major effectors of virulence and persistence encoded in the Mtb H37Rv genome. However, the function of PE8 has not been explored to date. In this study, we heterologously expressed PE8 in nonpathogenic, fast-growing M. smegmatis to investigate the interaction between PE8 and the host to determine its possible biological functions. We found that recombinant M. smegmatis cells expressing PE8 were less susceptible to sodium dodecyl sulfate-induced surface stress compared with those expressing the empty vector, suggesting that PE8 may be involved in stress responses. In addition, macrophages infected with PE8-expressing M. smegmatis produced obviously lower levels of the proinflammatory factor IL-1ß, IL-6, and TNF-α and higher levels of the inhibitory factor IL-10. We further found that PE8 promoted M. smegmatis survival within macrophages by inhibiting late apoptosis of macrophages. Collectively, selective targeting of the PE/PPE protein family offers an untapped opportunity to the development of more effective and safer drugs against Mtb infection.
Assuntos
Citocinas , Mycobacterium tuberculosis , Citocinas/genética , Citocinas/metabolismo , Mycobacterium tuberculosis/metabolismo , Proteínas de Bactérias/genética , Interações Hospedeiro-Patógeno , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/metabolismo , ApoptoseRESUMO
Under suboptimal growth conditions, bacteria can transit to the dormant forms characterized by a significantly reduced metabolic activity, resistance to various stress factors, and absence of cell proliferation. Traditionally, the dormant state is associated with the formation of highly differentiated cysts and spores. However, non-spore-forming bacteria can transfer to the dormant-like hypobiotic state with the generation of less differentiated cyst-like forms (which are different from spores). This review focuses on morphological and biochemical changes occurred during formation of dormant forms of mycobacteria in particular pathogenic M. tuberculosis (Mtb) caused latent forms of tuberculosis. These forms are characterized by the low metabolic activity, the absence of cell division, resistance to some antibiotics, marked morphological changes, and loss of ability to grow on standard solid media ("non-culturable" state). Being produced in vitro, dormant Mtb retained ability to maintain latent infection in mice. After a long period of dormancy, mycobacteria retain a number of stable proteins with a potential enzymatic activity which could participate in maintaining of low-level metabolic activity in period of dormancy. Indeed, the metabolomic analysis showed significant levels of metabolites in the dormant cells even after a long period of dormancy, which may be indicative of residual metabolism in dormant mycobacteria. Special role may play intracellularly accumulated trehalose in dormant mycobacteria. Trehalose appears to stabilize dormant cells, as evidenced by the direct correlation between the trehalose content and cell viability during the long-term dormancy. In addition, trehalose can be considered as a reserve energy substrate consumed during reactivation of dormant mycobacteria due to the ATP-dependent conversion of trehalase from the latent to the active state. Another feature of dormant mycobacteria is a high representation of proteins participating in the enzymatic defense against stress factors and of low-molecular-weight compounds protecting cells in the absence of replication. Dormant mycobacteria contain a large number of hydrolyzing enzymes, which, on the one hand, ensure inactivation of biomolecules damaged by stress. On the other hand, the products of these enzymatic reactions can be used for the maintenance of energy state and vital activity of bacterial cells during their long-term survival in the dormant state, i.e., for creating a situation that we propose to refer to as the "catabolic survival". In general, dormant non-replicating mycobacterial cells can be described as morphologically altered forms that contain principal macromolecules and are stabilized and protected from the damaging factors by an arsenal of proteins and low-molecular-weight compounds. Because of the presumable occurrence of metabolic reactions in such cells, this form of survival should be referred to as hypobiosis.
Assuntos
Mycobacterium tuberculosis , Tuberculose , Animais , Camundongos , Trealose , Mycobacterium tuberculosis/metabolismo , Antibacterianos/metabolismoRESUMO
Acyl-CoA dehydrogenase (ChsE) is involved in the steroid side-chain degradation process. However, their function in vivo remains unclear. In this study, three ChsE, ChsE1-ChsE2, ChsE3, and ChsE4-ChsE5, were identified in Mycolicibacterium neoaurum, and their functions in vivo are studied and compared with those from Mycobacterium tuberculosis in vitro. By gene knockout, complementation, and the bioconversion of phytosterols, the function of ChsE was elucidated that ChsE4-ChsE5 could utilize C27, C24, and C22 steroids in vivo. ChsE3 could utilize C27 and C24 steroids in vivo. ChsE1-ChsE2 could utilize C27, C24, and C22 steroids in vivo. What is more, the production strain of a C22 steroid, 3-oxo-4,17-pregadiene-20-carboxylic acid methyl ester (PDCE), is constructed with ChsE overexpression. This study improved the understanding of the steroid bioconversion pathway and proposed a method of the production of a new C22 steroid. KEY POINTS: ⢠Three ChsE paralogs from M. neoaurum are identified and studied. ⢠The function of ChsE is overlapped in vivo. ⢠A C22 steroid (PDCE) producer was constructed with ChsE overexpression.
Assuntos
Mycobacterium tuberculosis , Fitosteróis , Esteroides/metabolismo , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Acil-CoA DesidrogenaseRESUMO
BACKGROUND: Mycobacterium tuberculosis (Mtb) forms physiologically relevant biofilms harboring drug-tolerant bacteria. This observation has brought the study of mycobacterial biofilms to the forefront of tuberculosis research. We established earlier that dithiothreitol (DTT) mediated reductive stress induces cellulose-rich biofilm formation in Mtb cultures. The molecular events associated with the DTT-induced biofilm formation are not known. Furthermore, there are only limited tools for monitoring the presence of cellulose in biofilms. RESULTS: To decipher the molecular events associated with DTT-induced biofilm formation, we used Mtb and non-pathogenic, fast-growing Mycobacterium smegmatis (Msm). We observed that DTT induces biofilm formation in Msm cultures. We explored whether media components facilitate biofilm formation in mycobacteria upon exposure to DTT. We observed that media component bovine serum albumin promotes mycobacterial biofilm formation in response to DTT. Furthermore, we analyzed the composition of extracellular polymeric substances of Msm biofilms. We found that, like Mtb biofilms, Msm biofilms are also rich in polysaccharides and proteins. We also developed a novel protein-based molecular probe for imaging cellulose by utilizing the cellulose-binding domain of cellulase CenA from Cellulomonas fimi and fusing it to fluorescent reporter mCherry. Characterization of this new probe revealed that it has a high affinity for cellulose and could be used for visualizing cellulose biosynthesis during the development of Agrobacterium biofilms. Furthermore, we have demonstrated that biological macromolecule cellulose is present in the extracellular polymeric substances of Msm biofilms using this novel probe. CONCLUSIONS: This study indicates that DTT-mediated reduction of media component BSA leads to the formation of nucleating foci. These nucleating foci are critical for subsequent attachment of bacterial cells and induction of EPS production. Furthermore, this new tool, IMT-CBD-mC, could be used for monitoring cellulose incorporation in plant cells, understanding cellulose biosynthesis dynamics during biofilm formation, etc.
Assuntos
Mycobacterium tuberculosis , Soroalbumina Bovina , Soroalbumina Bovina/farmacologia , Biofilmes , Mycobacterium tuberculosis/metabolismo , Mycobacterium smegmatis/metabolismo , Celulose/metabolismoRESUMO
CD8+ T cell recognition of Mycobacterium tuberculosis (Mtb)-specific peptides presented on major histocompatibility complex class I (MHC-I) contributes to immunity to tuberculosis (TB), but the principles that govern presentation of Mtb antigens on MHC-I are incompletely understood. In this study, mass spectrometry (MS) analysis of the MHC-I repertoire of Mtb-infected primary human macrophages reveals that substrates of Mtb's type VII secretion systems (T7SS) are overrepresented among Mtb-derived peptides presented on MHC-I. Quantitative, targeted MS shows that ESX-1 activity is required for presentation of Mtb peptides derived from both ESX-1 substrates and ESX-5 substrates on MHC-I, consistent with a model in which proteins secreted by multiple T7SSs access a cytosolic antigen processing pathway via ESX-1-mediated phagosome permeabilization. Chemical inhibition of proteasome activity, lysosomal acidification, or cysteine cathepsin activity did not block presentation of Mtb antigens on MHC-I, suggesting involvement of other proteolytic pathways or redundancy among multiple pathways. Our study identifies Mtb antigens presented on MHC-I that could serve as targets for TB vaccines, and reveals how the activity of multiple T7SSs interacts to contribute to presentation of Mtb antigens on MHC-I.
Assuntos
Apresentação de Antígeno , Mycobacterium tuberculosis , Humanos , Mycobacterium tuberculosis/metabolismo , Antígenos de Bactérias , Antígenos de Histocompatibilidade Classe I/metabolismo , Peptídeos/metabolismoRESUMO
ATP synthase is a key protein in the oxidative phosphorylation process, as it aids in the effective production of ATP (Adenosine triphosphate) in all life's of kingdoms. ATP synthases have distinctive properties that contribute to efficient ATP synthesis. The ATP synthase of mycobacterium is of special relevance since it has been identified as a target for potential anti-TB molecules, especially Bedaquiline (BDQ). Better knowledge of how mycobacterial ATP synthase functions and its peculiar characteristics will aid in our understanding of bacterial energy metabolism adaptations. Furthermore, identifying and understanding the important distinctions between human ATP synthase and bacterial ATP synthase may provide insight into the design and development of inhibitors that target specific ATP synthase. In recent years, many potential candidates targeting the ATP synthase of mycobacterium have been developed. In this review, we discuss the druggable targets of the Electron transport chain (ETC) and recently identified potent inhibitors (including clinical molecules) from 2015 to 2022 of diverse classes that target ATP synthase of M. tuberculosis.
Assuntos
Mycobacterium tuberculosis , Tuberculose , Humanos , Antituberculosos/farmacologia , Antituberculosos/metabolismo , Mycobacterium tuberculosis/metabolismo , Trifosfato de Adenosina/metabolismo , Tuberculose/tratamento farmacológico , Desenvolvimento de MedicamentosRESUMO
Ferritin, a key regulator of iron homeostasis in macrophages, has been reported to confer host defenses against Mycobacterium tuberculosis (Mtb) infection. Nuclear receptor coactivator 4 (NCOA4) was recently identified as a cargo receptor in ferritin degradation. Here, we show that Mtb infection enhanced NCOA4-mediated ferritin degradation in macrophages, which in turn increased the bioavailability of iron to intracellular Mtb and therefore promoted bacterial growth. Of clinical relevance, the upregulation of FTH1 in macrophages was associated with tuberculosis (TB) disease progression in humans. Mechanistically, Mtb infection enhanced NCOA4-mediated ferritin degradation through p38/AKT1- and TRIM21-mediated proteasomal degradation of HERC2, an E3 ligase of NCOA4. Finally, we confirmed that NCOA4 deficiency in myeloid cells expedites the clearance of Mtb infection in a murine model. Together, our findings revealed a strategy by which Mtb hijacks host ferritin metabolism for its own intracellular survival. Therefore, this represents a potential target for host-directed therapy against tuberculosis.
Assuntos
Mycobacterium tuberculosis , Tuberculose , Humanos , Animais , Camundongos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Coativadores de Receptor Nuclear/genética , Coativadores de Receptor Nuclear/metabolismo , Ferro/metabolismo , Ferritinas/genética , Ferritinas/metabolismo , Fatores de Transcrição/metabolismo , Tuberculose/genética , AutofagiaRESUMO
OBJECTIVES: The only approved vaccine, Bacillus Calmette Guérin (BCG) used in global tuberculosis (TB) immunization programmes has been very effective in childhood TB but not in adult pulmonary and latent TB. Moreover, the emergence of multi-drug resistance-TB cases demands either to increase efficiency of BCG or replace it with the one with improved efficacy. RESULTS: A novel combination of two most effective secreted protein antigens specific for Mycobacterium tuberculosis (Mtb), ESAT-6 and MPT-64 (but not present in BCG strains) fused with a cholera toxin B subunit (CTB) and tagged with 6xHis was expressed for the first time in Escherichia coli as well as in transgenic cucumber plants developed using Agrobacterium tumefaciens-mediated transformation. The recombinant fusion protein (His6x.CTB-ESAT6-MPT64) expressed in E. coli was purified by a single-step affinity chromatography and used to produce polyclonal antibodies in rabbit. The transgenic cucumber lines were confirmed by polymerase chain reaction (PCR), Southern blot hybridization, reverse transcriptase PCR (RT-PCR), real-time PCR (qRT-PCR) and expression of recombinant fusion protein by western blot analysis and its quantification by enzyme-linked immunosorbent assay (ELISA). A maximum value of the fusion protein, 478 ng.g-1 (0.030% of the total soluble protein) was obtained in a transgenic cucumber line. Rabbit immunized orally showed a significant increase in serum IgG levels against the fusion protein as compared to the non-immunized rabbit. CONCLUSIONS: Stable expression of Mtb antigens with CTB in edible cucumber plants (whose fruits are eaten raw) in sufficient amount possibly would facilitate development of a safe, affordable and orally delivered self-adjuvanted, novel dual antigen based subunit vaccine against TB.
