Gotowa bibliografia na temat „Mtb Rv1860”

Iron-sulfur (Fe-S) clusters are inorganic prosthetic groups in proteins composed exclusively of iron and inorganic sulfide. These cofactors are required in a wide range of critical cellular pathways. Iron-sulfur clusters do not form spontaneously in vivo; several proteins are required to mobilize sulfur and iron, assemble and traffic-nascent clusters. Bacteria have developed several Fe-S assembly systems, such as the ISC, NIF, and SUF systems. Interestingly, in Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), the SUF machinery is the primary Fe-S biogenesis system. This operon is essential for the viability of Mtb under normal growth conditions, and the genes it contains are known to be vulnerable, revealing the Mtb SUF system as an interesting target in the fight against tuberculosis. In the present study, two proteins of the Mtb SUF system were characterized for the first time: Rv1464(sufS) and Rv1465(sufU). The results presented reveal how these two proteins work together and thus provide insights into Fe-S biogenesis/metabolism by this pathogen. Combining biochemistry and structural approaches, we showed that Rv1464 is a type II cysteine-desulfurase enzyme and that Rv1465 is a zinc-dependent protein interacting with Rv1464. Endowed with a sulfurtransferase activity, Rv1465 significantly enhances the cysteine-desulfurase activity of Rv1464 by transferring the sulfur atom from persulfide on Rv1464 to its conserved Cys40 residue. The zinc ion is important for the sulfur transfer reaction between SufS and SufU, and His354 in SufS plays an essential role in this reaction. Finally, we showed that Mtb SufS-SufU is more resistant to oxidative stress than E. coli SufS-SufE and that the presence of zinc in SufU is likely responsible for this improved resistance. This study on Rv1464 and Rv1465 will help guide the design of future anti-tuberculosis agents.

The widespread use of antibiotics has placed bacterial pathogens under intense pressure to evolve new survival mechanisms. Genomic analysis of 51,229 Mycobacterium tuberculosis ( Mtb ) clinical isolates has identified an essential transcriptional regulator, Rv1830 , herein called resR for resilience regulator, as a frequent target of positive (adaptive) selection. resR mutants do not show canonical drug resistance or drug tolerance but instead shorten the post-antibiotic effect, meaning that they enable Mtb to resume growth after drug exposure substantially faster than wild-type strains. We refer to this phenotype as antibiotic resilience. ResR acts in a regulatory cascade with other transcription factors controlling cell growth and division, which are also under positive selection in clinical isolates of Mtb . Mutations of these genes are associated with treatment failure and the acquisition of canonical drug resistance.

The widely administered tuberculosis (TB) vaccine, Bacillus Calmette-Guerin (BCG), is the only licensed vaccine, but has highly variable efficiency against childhood and pulmonary TB. Therefore, the BCG prime-boost strategy is a rational solution for the development of new TB vaccines. Studies have shown that Mycobacterium tuberculosis (Mtb) culture filtrates contain proteins that have promising vaccine potential. In this study, Rv1876 bacterioferritin was identified from the culture filtrate fraction with strong immunoreactivity. Its immunobiological potential has not been reported previously. We found that recombinant Rv1876 protein induced dendritic cells’ (DCs) maturation by MAPK and NF-κB signaling activation, induced a T helper type 1 cell-immune response, and expanded the population of the effector/memory T cell. Boosting BCG with Rv1876 protein enhanced the BCG-primed Th1 immune response and reduced the bacterial load in the lung compared to those of BCG alone. Thus, Rv1876 is a good target for the prime-boost strategy.

Iron-sulfur (Fe-S) cluster proteins carry out essential cellular functions in diverse organisms, including the human pathogen Mycobacterium tuberculosis (Mtb). The mechanisms underlying Fe-S cluster biogenesis are poorly defined in Mtb. Here, we show that Mtb SufT (Rv1466), a DUF59 domain-containing essential protein, is required for the Fe-S cluster maturation. Mtb SufT homodimerizes and interacts with Fe-S cluster biogenesis proteins; SufS and SufU. SufT also interacts with the 4Fe-4S cluster containing proteins; aconitase and SufR. Importantly, a hyperactive cysteine in the DUF59 domain mediates interaction of SufT with SufS, SufU, aconitase, and SufR. We efficiently repressed the expression of SufT to generate a SufT knock-down strain in Mtb (SufT-KD) using CRISPR interference. Depleting SufT reduces aconitase’s enzymatic activity under standard growth conditions and in response to oxidative stress and iron limitation. The SufT-KD strain exhibited defective growth and an altered pool of tricarboxylic acid cycle intermediates, amino acids, and sulfur metabolites. Using Seahorse Extracellular Flux analyzer, we demonstrated that SufT depletion diminishes glycolytic rate and oxidative phosphorylation in Mtb. The SufT-KD strain showed defective survival upon exposure to oxidative stress and nitric oxide. Lastly, SufT depletion reduced the survival of Mtb in macrophages and attenuated the ability of Mtb to persist in mice. Altogether, SufT assists in Fe-S cluster maturation and couples this process to bioenergetics of Mtb for survival under low and high demand for Fe-S clusters.

The tryptophan-biosynthesis pathway is essential for Mycobacterium tuberculosis (Mtb) to cause disease, but not all of the enzymes that catalyse this pathway in this organism have been identified. The structure and function of the enzyme complex that catalyses the first committed step in the pathway, the anthranilate synthase (AS) complex, have been analysed. It is shown that the open reading frames Rv1609 (trpE) and Rv0013 (trpG) encode the chorismate-utilizing (AS-I) and glutamine amidotransferase (AS-II) subunits of the AS complex, respectively. Biochemical assays show that when these subunits are co-expressed a bifunctional AS complex is obtained. Crystallization trials on Mtb-AS unexpectedly gave crystals containing only AS-I, presumably owing to its selective crystallization from solutions containing a mixture of the AS complex and free AS-I. The three-dimensional structure reveals that Mtb-AS-I dimerizes via an interface that has not previously been seen in AS complexes. As is the case in other bacteria, it is demonstrated that Mtb-AS shows cooperative allosteric inhibition by tryptophan, which can be rationalized based on interactions at this interface. Comparative inhibition studies on Mtb-AS-I and related enzymes highlight the potential for single inhibitory compounds to target multiple chorismate-utilizing enzymes for TB drug discovery.

Mycobacterium tuberculosis is considered a successful pathogen with multiple strategies to undermine host immunity. The YrbE3A is encoded by Rv1964 within the RD15 region present in the genome of Mtb, but missing in M. bovis, M. bovis BCG (Pasteur) strain, and M. smegmatis (Ms). However, little is known about its function. In this study, the YrbE3A gene was cloned into pMV261 and expressed in Ms and BCG, while the strains with the vector served as the controls. The YrbE3A was expressed on the mycobacterial membrane, and the purified protein could stimulate RAW264.7 cells to produce IL-6. Furthermore, the effect of the recombinant strains on cytokine secretion by RAW264.7 was confirmed, which varied with the host strains. Ms_YrbE3A increased significantly higher levels of TNF-α and IL-6 than did Ms_vec, while BCG_YrbE3A enhanced higher TNF-α than BCG_vec. The pathways associated with NF-κB p65 and MAPK p38/JNK, other than Erk1/2, regulated this process. In addition, mice were infected with Ms_YrbE3A and Ms-vec and were kinetically examined. Compared to Ms-vec, Ms_YrbE3A induced more serious inflammatory damage, higher levels of TNF-α and IL-6, higher numbers of lymphocytes, neutrophils, and monocytes in a time-dependent way, but lower lung bacterial load in lung. These findings may contribute to a better understanding of Mtb pathogenesis.

Abstract Episodes of virus-induced exacerbations of asthma are accompanied by increased eosinophils (EOS) in respiratory secretions and evidence of EOS degranulation. Although rhinoviruses (RV) are the viruses most often implicated in exacerbations of asthma in both children and adults, little is known about the immune response to this group of viruses and, in particular, EOS-RV interactions. To define such interactions, we incubated human rhinovirus type 16 (RV16), a serotype using ICAM-1 as a receptor, with EOS purified from PBMC, and measured EOS-RV binding, EOS-mediated Ag presentation and T cell activation, and EOS cell surface marker expression and superoxide production. Significant RV16 binding occurred to EOS that were pretreated with granulocyte-macrophage CSF, and this binding was inhibited by anti-ICAM-1 mAb. EOS also presented viral Ags to RV16-specific T cells, causing T cell proliferation and secretion of IFN-γ. RV16 induced a significant shift from CD18dim to CD18bright, but did not affect EOS expression of CD54, CD69, or HLA-DR. Finally, RV16 did not induce superoxide production from peripheral blood EOS. These findings suggest that RV16 also binds to airway EOS, which resemble granulocyte-macrophage CSF-treated blood EOS in terms of high expression of ICAM-1. Furthermore, our findings suggest that EOS could participate in RV-induced immune responses through Ag presentation and T cell activation. By activating RV-specific T cells, EOS may play an important role in the initiation of antiviral T cell responses, and these effects could also contribute to enhanced airway inflammation and increased asthma symptoms in susceptible individuals.

ABSTRACT Tuberculous meningitis (TBM) is the most common form of chronic infection of the central nervous system. Despite the magnitude of the problem, the general diagnostic outlook is discouraging. Specifically, there is no generally accepted early confirmative diagnosis protocol available for TBM. Various Mycobacterium tuberculosis antigens are now recognized as potential markers for diagnosis of TBM. However, their presence remains questionable, and many of these antigens are reported in the blood but not in the cerebrospinal fluid (CSF). This study identifies a specific protein marker in CSF which will be useful in early diagnosis of TBM. We have demonstrated the presence of a 30-kDa protein band in CSF of 100% (n = 5) of confirmed and 90% (n = 138) of suspected TBM patients out of 153 TBM patients. The 30-kDa band was excised from the gel, destained extensively, and digested with trypsin. The resulting peptides were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Partially purified proteins from CSF samples of TBM were analyzed by two-dimensional polyacrylamide gel electrophoresis and Western blotting. Immunoblotting and enzyme-linked immunosorbent assay (ELISA) were performed to confirm the presence of proteins in the 30-kDa protein band. The antigen 85 (Ag 85) complex was detected in CSF of TBM patients by indirect ELISA using antibodies against Ag 85 complex. The results of this study showed the 30-kDa protein band contained MTB proteins Rv3804c (Ag85A) and Rv1886c (Ag 85B), both members of the Ag85 complex. This was also confirmed by using immunotechniques such as indirect ELISA and the dot immunobinding assay. Detection of Ag85 complex was observed in CSF of 89% (71 out of 80) of suspected TBM patients that were 30-kDa protein positive. The observed 30-kDa protein in the CSF is comprised of the MTB Ag85 complex. This protein was earlier reported to be present in the blood of patients with extra-central nervous system tuberculosis. Therefore, this finding suggests that this protein can be used as a molecular marker for any type of tuberculous infection. It also provides a more sensitive immunoassay option for the early and confirmatory diagnosis of TBM.

Abstract There is evidence that rhinovirus (RV) infections are frequent causes of increased asthmatic symptoms and can specifically enhance allergic inflammation in the airway. To further define effects of RV infection on cellular immunity, we have begun to develop in vitro models for study. When human PBMC were incubated with 35S-labeled RV16, specific binding via ICAM-1 on monocytes was observed. Incubation of PBMC with RV also led to a dose-related increase in the expression of the early activation marker CD69 on 30 to 70% of T cells. The RV16-induced increases in CD69 were blocked by anti-ICAM-1 mAb, and were not elicited by UV-inactivated (noninfectious) virus. The degree of CD69 enhancement correlated with the number of monocytes in mixtures of PBMC, did not occur in monocyte-depleted cultures, and was mediated by one or more soluble factor(s). RV also induced secretion of IFN-gamma from both peripheral blood T cells and NK cells, and IFN-gamma mRNA was greatest in T cells that were CD69+. Finally, supernatant from RV-activated CD3+CD69+ cells had biologic activity that promoted eosinophil survival in vitro; this RV16-associated activity was blocked when co-incubations were performed with IFN-gamma mAbs. These observations suggest that RV nonspecifically activates a large proportion of T cells through a monocyte-dependent mechanism. Such changes in vivo could enhance airway inflammation, and this may include effects on inflammatory cells in the airways of allergic individuals.

Abstract Potential interactions between rhinovirus (RV) and both the airway macrophage and its precursor cell, the blood monocyte, were investigated in terms of direct binding, intracellular replication, cell survival, and cytokine production. When HeLa cell suspensions are inoculated with RV as a positive control, virus titer increases by 100-fold in the first 24 h, confirming intracellular replication. In contrast, RV titer in monocyte and macrophage suspensions steadily decreased. Despite a lack of productive RV replication, cell-associated RV RNA was detectable using a biotin-labeled cDNA probe as early as 6 h after inoculation. Direct binding of RV16 to macrophages was confirmed using radiolabeled virus, although preincubation with anti-ICAM-1 mAb did not block this interaction. Synthesis of RV RNA, as indicated by [3H]uridine incorporation in actinomycin D-treated cells, was detected in HeLa cells but not macrophages, suggesting that the viral RNA detected inside macrophages was from input virus and was not newly synthesized. RV inoculation did not adversely affect monocyte or macrophage viability. Finally, RV caused macrophage activation, as indicated by the induction of TNF-alpha secretion. These in vitro findings suggest that macrophages interact with major group RV in vivo, and raise the possibility that there is a second cellular receptor for these viruses. Furthermore, macrophages do not serve as permissive host cells during in vivo RV infection, but instead may be active participants in anti-RV immunity and RV-induced airway inflammation.

Le gaz toxique CO peut agir à faibles quantités comme molécule de signalisation ; détectée par les protéines senseurs à base d’hème.La première partie de ce travail concerne le régulateur de transcription bactérien CO-dépendant RcoM-2, qui possède une affinité extrêmement élevée pour le CO, tout en étant insensible à l'O₂. RcoM-2 se lie à son ADN cible seulement lorsque du CO est lié à l’hème. Nous avons caractérisé l'interaction hème-CO dans la protéine entière RcoM-2 et l'avons comparée à l’hémo-domaine isolé RcoMH-2. RcoM-2 peut lier du CO avec une affinité effective plus faible que RcoMH-2. Le CO se dissocie avec un taux 20 fois plus élevé par rapport à RcoMH-2, où la liaison du CO est quasi irréversible. Une petite fraction du CO peut échapper de la protéine, ainsi permettant RcoM-2 à agir comme senseur du CO. La présence du domaine de liaison à l'ADN influence les propriétés de fixation du CO à l'hème. Les études sur l'origine moléculaire précise des propriétés dynamiques doivent attendre la structure 3D de RcoM-2.La détection du CO est cruciale pour Mycobacterium tuberculosis, l’agent infectieux de la tuberculose, qui doit répondre aux mécanismes de défense de l'hôte, dont le CO. Le gène cor (rv1829) pourrait y jouer un rôle. Nous avons démontré que Cor est un dimère très stable, capable de lier un hème de façon stoechiométrique, suggérant ainsi une fonction potentielle de senseur direct du CO. Un résidu histidine a été identifié comme ligand potentiel de l’hème. La dynamique interne du CO est très similaire de celle d’autres senseurs à CO bactériens. Cor présente une activité de liaison à l'ADN dépendante de l'hème et du CO, qui est aboli dans le mutant H70A. Nos études ont également montré que le régulateur de transcription mycobactérien Rv0081, induit en réponse aux changements gazeux, peut se lier sur la région régulatrice prédite de cor. La création d’une souche Δcor dans le modèle non-pathogène M. smegmatis se poursuit et constituera un premier pas vers des futures analyses transcriptomiques
The toxic gas CO can act in low quantities as signaling molecule; detected by heme-based sensor proteins.The CO-dependent transcription factor RcoM-2 has a very high affinity for CO, while being insensitive to O₂. RcoM-2 binds to DNA only when CO is bound to heme. We characterized the heme-CO interaction in full-length RcoM-2 and compared it with the isolated heme domain RcoMH-2. RcoM-2 can bind CO with lower effective affinity than RcoMH-2. CO dissociates with a 20-fold higher rate than in RcoMH-2, where CO binding is almost irreversible. A small fraction of CO can escape from the protein, thus allowing RcoM-2 to act as CO sensor. The presence of the DNA binding domain influences the binding properties of CO to heme. Identification of the precise molecular origin of the dynamic properties must await the 3D structure RcoM-2.CO detection is crucial for Mtb, the infectious agent of tuberculosis that must overcome the host's defense mechanisms, including CO. The gene cor (rv1829) has been implicated in these processes. We have shown that Cor is a highly stable dimer that is able to stoichiometrically bind a heme cofactor, suggesting a potential function as direct CO sensor. A histidine residue was identified as potential heme ligand. The internal CO dynamics is very similar to other bacterial CO sensors. Cor exhibits DNA binding activity that depends on the presence of heme and CO, which is abolished in the H70A mutant. Our studies also showed that the transcriptional regulator Rv0081, induced in response to gaz changes, can bind to the predicted regulatory region of cor. The creation of a Δcor strain in the non- pathogenic model M. smegmatis continues and will be a first step towards transcriptomic analyzes

Tuberculosis continues to prevail as the major cause of mortality from any single pathogen around the world, despite implementation of control programs and the availability of effective drugs and the vaccine BCG. Mycobacterium bovis Bacillus Calmette Guerin (BCG) is the only vaccine available for clinical use and protects children against disseminated disease; however, it has shown variable levels of efficacy against pulmonary TB in adults. The drawbacks of anti-TB chemotherapy such as the inability of currently used drugs to kill bacteria that are “dormant”, long term treatment required for the current drugs, and emergence of multidrug resistant M. tuberculosis (Mtb) pose an urgent need for a more effective vaccine against TB. Thus, one of the challenges is to develop effective TB vaccines which can confer long term protection against pulmonary TB and also better understanding of protective immune response against TB is essential to develop better vaccines. The general aim of the thesis was to understand role of Mtb Rv1860 in modulating the host macrophage immune response. Macrophages are the first line of defense against Mtb, but Mtb continues to reside in them for years. Macrophages also function as APC’s during the course of infection and they process Mtb antigens and present them to T cells by MHC molecules to trigger adaptive immunity. Glycoconjugate structures present on the surface of invading pathogens are of importance in the first contact with the host innate immune system. This interaction has a decisive role in the establishment of the initial immune response and in the outcome of the infection. M. tuberculosis is able to subvert the host innate immune response and survive within macrophages and modulate the immune response using its unique outer cell wall components like lipoarabinomannan (LAM), lipomannan (LM) and phosphatidyl-myo inositol mannosides (PIM’s). Previous studies form our laboratory showed human CD8+ T cells in healthy latently infected individuals correlated with protection from TB disease. Conversely, we also reported that the glycosylated form of Apa abrogated the activation of and T cell polarization by dendritic cells infected with BCG expressing Rv1860 from Mtb. In M. tuberculosis Rv1860 codes for an alanine-proline-rich antigen, also known as the 45/47-kDa protein complex. Its expression is restricted to members of Mtb complex; based on initial binding to concanavalin-A and later mass spectrometry analysis, Rv1860 was characterized as a mannosylated protein. In the present study, in chapter 1, we have investigated the effect of Rv1860 on the functions of mouse bone marrow derived primary macrophages (BMDM) which are innate immune cells and also identified the interacting partners of Rv1860 in BCG. Infection of BMDM with recombinant Mycobacterium bovis BCG expressing Mtb Rv1860 (BCG-Rv1860) increased the production of pro- inflammatory cytokines over the levels produced by infection with wild-type parent strain whereas expression of Rv1860 in M. smegmatis inhibited the production of pro- inflammatory cytokines by macrophages. BCG-Rv1860 enhances inflammation, evidenced by increase in cytokines like TNF-α, IL-6 and Il-12p40 which is transcriptionally mediated. In chapter 2, we have shown that BCG-Rv1860 induces enhanced phosphorylation of JNK and induce ROS production. BCG-Rv1860 induces early autophagy evidenced by conversion of LC3-I to LC3-II and enhanced by stability of p62. On early stage phagophore, p62 can contribute to the assembly of the necrosome complex on these membranes and induces necroptotic death. BCG-Rv1860 inhibits caspase activation, thereby inhibiting apoptosis. Death pathway induced by BCG-Rv1860 was unequivocally shown to be necroptosis confirmed by phosphorylation of MLKL, necrosome assembly, inhibition of cell death and inflammation by necrostatin-1.