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1.
Mol Syst Biol ; 17(5): e10280, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33943004

RESUMO

The co-catabolism of multiple host-derived carbon substrates is required by Mycobacterium tuberculosis (Mtb) to successfully sustain a tuberculosis infection. However, the metabolic plasticity of this pathogen and the complexity of the metabolic networks present a major obstacle in identifying those nodes most amenable to therapeutic interventions. It is therefore critical that we define the metabolic phenotypes of Mtb in different conditions. We applied metabolic flux analysis using stable isotopes and lipid fingerprinting to investigate the metabolic network of Mtb growing slowly in our steady-state chemostat system. We demonstrate that Mtb efficiently co-metabolises either cholesterol or glycerol, in combination with two-carbon generating substrates without any compartmentalisation of metabolism. We discovered that partitioning of flux between the TCA cycle and the glyoxylate shunt combined with a reversible methyl citrate cycle is the critical metabolic nodes which underlie the nutritional flexibility of Mtb. These findings provide novel insights into the metabolic architecture that affords adaptability of bacteria to divergent carbon substrates and expand our fundamental knowledge about the methyl citrate cycle and the glyoxylate shunt.

2.
ACS Infect Dis ; 7(1): 174-188, 2021 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-33356117

RESUMO

Tuberculosis (TB) is the most lethal bacterial infectious disease worldwide. It is notoriously difficult to treat, requiring a cocktail of antibiotics administered over many months. The dense, waxy outer membrane of the TB-causing agent, Mycobacterium tuberculosis (Mtb), acts as a formidable barrier against uptake of antibiotics. Subsequently, enzymes involved in maintaining the integrity of the Mtb cell wall are promising drug targets. Recently, we demonstrated that Mtb lacking malic enzyme (MEZ) has altered cell wall lipid composition and attenuated uptake by macrophages. These results suggest that MEZ contributes to lipid biosynthesis by providing reductants in the form of NAD(P)H. Here, we present the X-ray crystal structure of MEZ to 3.6 Å. We use biochemical assays to demonstrate MEZ is dimeric in solution and to evaluate the effects of pH and allosteric regulators on its kinetics and thermal stability. To assess the interactions between MEZ and its substrate malate and cofactors, Mn2+ and NAD(P)+, we ran a series of molecular dynamics (MD) simulations. First, the MD analysis corroborates our empirical observations that MEZ is unusually flexible, which persists even with the addition of substrate and cofactors. Second, the MD simulations reveal that dimeric MEZ subunits alternate between open and closed states, and that MEZ can stably bind its NAD(P)+ cofactor in multiple conformations, including an inactive, compact NAD+ form. Together the structure of MEZ and insights from its dynamics can be harnessed to inform the design of MEZ inhibitors that target Mtb and not human malic enzyme homologues.


Assuntos
Mycobacterium tuberculosis , Preparações Farmacêuticas , Tuberculose , Antituberculosos , Humanos , Simulação de Dinâmica Molecular
3.
Nat Methods ; 17(3): 311-318, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32015544

RESUMO

Tissues and organs are composed of diverse cell types, which poses a major challenge for cell-type-specific profiling of gene expression. Current metabolic labeling methods rely on exogenous pyrimidine analogs that are only incorporated into RNA in cells expressing an exogenous enzyme. This approach assumes that off-target cells cannot incorporate these analogs. We disprove this assumption and identify and characterize the enzymatic pathways responsible for high background incorporation. We demonstrate that mammalian cells can incorporate uracil analogs and characterize the enzymatic pathways responsible for high background incorporation. To overcome these limitations, we developed a new small molecule-enzyme pair consisting of uridine/cytidine kinase 2 and 2'-azidouridine. We demonstrate that 2'-azidouridine is only incorporated in cells expressing uridine/cytidine kinase 2 and characterize selectivity mechanisms using molecular dynamics and X-ray crystallography. Furthermore, this pair can be used to purify and track RNA from specific cellular populations, making it ideal for high-resolution cell-specific RNA labeling. Overall, these results reveal new aspects of mammalian salvage pathways and serve as a new benchmark for designing, characterizing and evaluating methodologies for cell-specific labeling of biomolecules.


Assuntos
RNA/química , Uracila/química , Animais , Azidas/química , Biotinilação , Domínio Catalítico , Técnicas de Cocultura , Desoxiuridina/análogos & derivados , Desoxiuridina/química , Células HEK293 , Células HeLa , Humanos , Cinética , Camundongos , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Células NIH 3T3 , Núcleosídeo-Fosfato Quinase/metabolismo , Domínios Proteicos , RNA Interferente Pequeno/genética , Uridina/química , Uridina Quinase/metabolismo
5.
Biochemistry ; 58(46): 4610-4620, 2019 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-31638374

RESUMO

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, requires iron for survival. In Mtb, MhuD is the cytosolic protein that degrades imported heme. MhuD is distinct, in both sequence and structure, from canonical heme oxygenases (HOs) but homologous with IsdG-type proteins. Canonical HO is found mainly in eukaryotes, while IsdG-type proteins are predominantly found in prokaryotes, including pathogens. While there are several published structures of MhuD and other IsdG-type proteins in complex with the heme substrate, no structures of IsdG-type proteins in complex with a product have been reported, unlike the case for HOs. We recently showed that the Mtb variant MhuD-R26S produces biliverdin IXα (αBV) rather than the wild-type mycobilin isomers. Given that mycobilin and other IsdG-type protein products like staphylobilin are difficult to isolate in quantities sufficient for structure determination, here we use the MhuD-R26S variant and its product αBV as a proxy to study the IsdG-type protein-product complex. First, we show that αBV has a nanomolar affinity for MhuD and the R26S variant. Second, we determined the MhuD-R26S-αBV complex structure to 2.5 Å, which reveals two notable features: (1) two αBV molecules bound per active site and (2) a novel α-helix (α3) that was not observed in previous MhuD-heme structures. Finally, through molecular dynamics simulations, we show that α3 is stable with the proximal αBV alone. MhuD's high affinity for the product and the observed structural and electrostatic changes that accompany substrate turnover suggest that there may be an unidentified class of proteins that are responsible for the extraction of products from MhuD and other IsdG-type proteins.


Assuntos
Proteínas de Bactérias/química , Biliverdina/metabolismo , Heme/metabolismo , Oxigenases de Função Mista/química , Mycobacterium tuberculosis/metabolismo , Proteínas de Bactérias/metabolismo , Biliverdina/química , Cristalografia por Raios X , Heme/química , Humanos , Oxigenases de Função Mista/metabolismo , Modelos Moleculares , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/genética , Mutação Puntual , Conformação Proteica , Especificidade por Substrato , Tuberculose/microbiologia
6.
Structure ; 27(11): 1660-1674.e5, 2019 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-31515004

RESUMO

Contact-dependent growth inhibition (CDI) is a form of interbacterial competition mediated by CdiB-CdiA two-partner secretion systems. CdiA effector proteins carry polymorphic C-terminal toxin domains (CdiA-CT), which are neutralized by specific CdiI immunity proteins to prevent self-inhibition. Here, we present the crystal structures of CdiA-CT⋅CdiI complexes from Klebsiella pneumoniae 342 and Escherichia coli 3006. The toxins adopt related folds that resemble the ribonuclease domain of colicin D, and both are isoacceptor-specific tRNases that cleave the acceptor stem of deacylated tRNAGAUIle. Although the toxins are similar in structure and substrate specificity, CdiA-CTKp342 activity requires translation factors EF-Tu and EF-Ts, whereas CdiA-CTEC3006 is intrinsically active. Furthermore, the corresponding immunity proteins are unrelated in sequence and structure. CdiIKp342 forms a dimeric ß sandwich, whereas CdiIEC3006 is an α-solenoid monomer. Given that toxin-immunity genes co-evolve as linked pairs, these observations suggest that the similarities in toxin structure and activity reflect functional convergence.


Assuntos
Proteínas de Bactérias/química , Toxinas Bacterianas/química , Colicinas/química , Proteínas de Escherichia coli/química , Evolução Molecular , Proteínas de Membrana/química , Ribonucleases/química , Sistemas Toxina-Antitoxina , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Sítios de Ligação , Colicinas/genética , Colicinas/metabolismo , Escherichia coli , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Klebsiella pneumoniae/enzimologia , Klebsiella pneumoniae/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Ligação Proteica , RNA de Transferência/química , RNA de Transferência/metabolismo , Ribonucleases/genética , Ribonucleases/metabolismo
8.
Microbiol Spectr ; 7(3)2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31172908

RESUMO

Mycobacterium tuberculosis is an ancient master of the art of causing human disease. One important weapon within its fully loaded arsenal is the type VII secretion system. M. tuberculosis has five of them: ESAT-6 secretion systems (ESX) 1 to 5. ESX-1 has long been recognized as a major cause of attenuation of the FDA-licensed vaccine Mycobacterium bovis BCG, but its importance in disease progression and transmission has recently been elucidated in more detail. This review summarizes the recent advances in (i) the understanding of the ESX-1 structure and components, (ii) our knowledge of ESX-1's role in hijacking macrophage function to set a path for infection and dissemination, and (iii) the development of interventions that utilize ESX-1 for diagnosis, drug interventions, host-directed therapies, and vaccines.


Assuntos
Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Mycobacterium tuberculosis/imunologia , Mycobacterium tuberculosis/metabolismo , Tuberculose/imunologia , Sistemas de Secreção Tipo VII/imunologia , Sistemas de Secreção Tipo VII/metabolismo , Vacina BCG/imunologia , Sistemas de Secreção Bacterianos/metabolismo , Quimiocinas , Interações Hospedeiro-Patógeno , Humanos , Macrófagos/imunologia , Mycobacterium tuberculosis/patogenicidade , Necrose , Fagossomos , Tuberculose/diagnóstico , Tuberculose/tratamento farmacológico , Tuberculose/prevenção & controle , Vacinas , Virulência
9.
Biochemistry ; 58(6): 489-492, 2019 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-30605595

RESUMO

Mycobacterium tuberculosis heme-degrading protein MhuD degrades heme to mycobilin isomers and iron, while its closest homologues from Staphylococcus aureus, IsdG and IsdI, degrade heme to staphylobilin isomers, formaldehyde, and iron. Superposition of the structures of the heme-bound complexes reveals that the heme molecule in the MhuD active site is rotated ∼90° about the tetrapyrrole plane with respect to IsdG and IsdI active site heme molecules. Therefore, the variation in IsdG/IsdI and MhuD chromophore products may be attributed to the different heme orientations. In MhuD, two arginines, Arg22 and Arg26, stabilize the heme propionates and may account for the heme orientation. Herein, we demonstrate that the MhuD-R26S variant alters the resulting chromophore product from mycobilin to biliverdin IXα (α-BV), whereas the R22S variant does not. Surprisingly, unlike canonical heme oxygenase (HO) that also degrades heme to α-BV, the MhuD-R26S variant produces the C1 product formaldehyde rather than carbon monoxide as observed for HO. The MhuD-R26S variant is an important tool for further probing the mechanism of action of MhuD and for studying the fate of the MhuD product in mycobacterium.


Assuntos
Proteínas de Bactérias/metabolismo , Heme Oxigenase (Desciclizante)/metabolismo , Heme/metabolismo , Mutação , Mycobacterium tuberculosis/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Biliverdina/metabolismo , Monóxido de Carbono/metabolismo , Formaldeído/metabolismo , Heme/química , Heme Oxigenase (Desciclizante)/química , Heme Oxigenase (Desciclizante)/genética , Modelos Moleculares , Conformação Proteica
10.
Chem Rev ; 119(2): 1193-1220, 2019 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-30474981

RESUMO

The highly contagious disease tuberculosis (TB) is caused by the bacterium Mycobacterium tuberculosis (Mtb), which has been evolving drug resistance at an alarming rate. Like all human pathogens, Mtb requires iron for growth and virulence. Consequently, Mtb iron transport is an emerging drug target. However, the development of anti-TB drugs aimed at these metabolic pathways has been restricted by the dearth of information on Mtb iron acquisition. In this Review, we describe the multiple strategies utilized by Mtb to acquire ferric iron and heme iron. Mtb iron uptake is a complex process, requiring biosynthesis and subsequent export of Mtb siderophores, followed by ferric iron scavenging and ferric-siderophore import into Mtb. Additionally, Mtb possesses two possible heme uptake pathways and an Mtb-specific mechanism of heme degradation that yields iron and novel heme-degradation products. We conclude with perspectives for potential therapeutics that could directly target Mtb heme and iron uptake machineries. We also highlight how hijacking Mtb heme and iron acquisition pathways for drug import may facilitate drug transport through the notoriously impregnable Mtb cell wall.


Assuntos
Ferro/metabolismo , Mycobacterium tuberculosis/metabolismo , Tuberculose/microbiologia , Proteínas de Bactérias/metabolismo , Transporte Biológico , Heme/metabolismo , Heme Oxigenase (Desciclizante)/metabolismo , Humanos , Ferro/química , Mycobacterium tuberculosis/patogenicidade , Sideróforos/química , Sideróforos/metabolismo , Tuberculose/tratamento farmacológico , Virulência
11.
Metallomics ; 10(11): 1560-1563, 2018 11 14.
Artigo em Inglês | MEDLINE | ID: mdl-30239544

RESUMO

MhuD is a protein found in mycobacteria that can bind up to two heme molecules per protein monomer and catalyze the degradation of heme to mycobilin in vitro. Here the Kd1 for heme dissociation from heme-bound MhuD was determined to be 7.6 ± 0.8 nM and the Kd2 for heme dissocation from diheme-bound MhuD was determined to be 3.3 ± 1.1 µM. These data strongly suggest that MhuD is a competent heme oxygenase in vivo.


Assuntos
Proteínas de Bactérias/metabolismo , Heme Oxigenase (Desciclizante)/metabolismo , Heme/metabolismo , Mycobacterium tuberculosis/enzimologia
12.
Mol Microbiol ; 109(4): 509-527, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29923643

RESUMO

Bacteria use several different secretion systems to deliver toxic EndoU ribonucleases into neighboring cells. Here, we present the first structure of a prokaryotic EndoU toxin in complex with its cognate immunity protein. The contact-dependent growth inhibition toxin CdiA-CTSTECO31 from Escherichia coli STEC_O31 adopts the eukaryotic EndoU fold and shares greatest structural homology with the nuclease domain of coronavirus Nsp15. The toxin contains a canonical His-His-Lys catalytic triad in the same arrangement as eukaryotic EndoU domains, but lacks the uridylate-specific ribonuclease activity that characterizes the superfamily. Comparative sequence analysis indicates that bacterial EndoU domains segregate into at least three major clades based on structural variations in the N-terminal subdomain. Representative EndoU nucleases from clades I and II degrade tRNA molecules with little specificity. In contrast, CdiA-CTSTECO31 and other clade III toxins are specific anticodon nucleases that cleave tRNAGlu between nucleotides C37 and m2 A38. These findings suggest that the EndoU fold is a versatile scaffold for the evolution of novel substrate specificities. Such functional plasticity may account for the widespread use of EndoU effectors by diverse inter-bacterial toxin delivery systems.


Assuntos
Antibacterianos/metabolismo , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Endorribonucleases/genética , Endorribonucleases/metabolismo , Escherichia coli/metabolismo , Sequência de Aminoácidos , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , RNA de Transferência/metabolismo , Análise de Sequência de Proteína
14.
RNA Biol ; 15(1): 9-12, 2018 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-29099294

RESUMO

Bovine pancreatic ribonuclease (RNase A) is the founding member of the RNase A superfamily. Members of this superfamily of ribonucleases have high sequence diversity, but possess a similar structural fold, together with a conserved His-Lys-His catalytic triad and structural disulfide bonds. Until recently, RNase A proteins had exclusively been identified in eukaryotes within vertebrae. Here, we discuss the discovery by Batot et al. of a bacterial RNase A superfamily member, CdiA-CTYkris: a toxin that belongs to an inter-bacterial competition system from Yersinia kristensenii. CdiA-CTYkris exhibits the same structural fold as conventional RNase A family members and displays in vitro and in vivo ribonuclease activity. However, CdiA-CTYkris shares little to no sequence similarity with RNase A, and lacks the conserved disulfide bonds and catalytic triad of RNase A. Interestingly, the CdiA-CTYkris active site more closely resembles the active site composition of various eukaryotic endonucleases. Despite lacking sequence similarity to eukaryotic RNase A family members, CdiA-CTYkris does share high sequence similarity with numerous Gram-negative and Gram-positive bacterial proteins/domains. Nearly all of these bacterial homologs are toxins associated with virulence and bacterial competition, suggesting that the RNase A superfamily has a distinct bacterial subfamily branch, which likely arose by way of convergent evolution. Finally, RNase A interacts directly with the immunity protein of CdiA-CTYkris, thus the cognate immunity protein for the bacterial RNase A member could be engineered as a new eukaryotic RNase A inhibitor.


Assuntos
Toxinas Bacterianas/química , Endonucleases/química , Ribonuclease Pancreático/química , Sequência de Aminoácidos , Animais , Toxinas Bacterianas/genética , Domínio Catalítico , Bovinos , Cristalografia por Raios X , Endonucleases/antagonistas & inibidores , Endonucleases/genética , Família Multigênica , Domínios Proteicos , Dobramento de Proteína , Ribonuclease Pancreático/genética , Yersinia/enzimologia
15.
Nucleic Acids Res ; 45(17): 10306-10320, 2017 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-28973472

RESUMO

Contact-dependent growth inhibition (CDI) is a mechanism of inter-cellular competition in which Gram-negative bacteria exchange polymorphic toxins using type V secretion systems. Here, we present structures of the CDI toxin from Escherichia coli NC101 in ternary complex with its cognate immunity protein and elongation factor Tu (EF-Tu). The toxin binds exclusively to domain 2 of EF-Tu, partially overlapping the site that interacts with the 3'-end of aminoacyl-tRNA (aa-tRNA). The toxin exerts a unique ribonuclease activity that cleaves the single-stranded 3'-end from tRNAs that contain guanine discriminator nucleotides. EF-Tu is required to support this tRNase activity in vitro, suggesting the toxin specifically cleaves substrate in the context of GTP·EF-Tu·aa-tRNA complexes. However, superimposition of the toxin domain onto previously solved GTP·EF-Tu·aa-tRNA structures reveals potential steric clashes with both aa-tRNA and the switch I region of EF-Tu. Further, the toxin induces conformational changes in EF-Tu, displacing a ß-hairpin loop that forms a critical salt-bridge contact with the 3'-terminal adenylate of aa-tRNA. Together, these observations suggest that the toxin remodels GTP·EF-Tu·aa-tRNA complexes to free the 3'-end of aa-tRNA for entry into the nuclease active site.


Assuntos
Toxinas Bacterianas/química , Proteínas de Escherichia coli/metabolismo , Fator Tu de Elongação de Peptídeos/metabolismo , RNA Bacteriano/metabolismo , RNA de Transferência/metabolismo , Toxinas Bacterianas/metabolismo , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Guanina/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , Conformação Proteica , Domínios Proteicos , Proteínas Recombinantes de Fusão/metabolismo , Relação Estrutura-Atividade , Especificidade por Substrato
16.
Biochem J ; 474(18): 3089-3092, 2017 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-28860337

RESUMO

Trypanosomatids are parasitic eukaryotic organisms that cause human disease. These organisms have complex lifestyles; cycling between vertebrate and insect hosts and alternating between two morphologies; a replicating form and an infective, nonreplicating one. Because trypanosomatids are one of the few organisms that do not synthesize the essential cofactor, heme, these parasites sequester the most common form, heme B, from their hosts. Once acquired, the parasites derivatize heme B to heme A by two sequential enzyme reactions. Although heme C is found in many cytochrome c and c1 proteins, heme A is the cofactor of only one known protein, cytochrome c oxidase (CcO). In a recent issue of the Biochemical Journal, Merli et al. [Biochem. J. (2017) 474, 2315-2332] demonstrate that the final step in the synthesis of heme A by heme A synthase (TcCox15) and the subsequent activity of CcO are essential for infectivity and replication of Trypanosoma cruzi.


Assuntos
Heme/química , Parasitos , Animais , Citocromos c , Complexo IV da Cadeia de Transporte de Elétrons , Humanos , Trypanosoma cruzi
17.
18.
Nucleic Acids Res ; 45(9): 5013-5025, 2017 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-28398546

RESUMO

Contact-dependent growth inhibition (CDI) is an important mechanism of inter-bacterial competition found in many Gram-negative pathogens. CDI+ cells express cell-surface CdiA proteins that bind neighboring bacteria and deliver C-terminal toxin domains (CdiA-CT) to inhibit target-cell growth. CDI+ bacteria also produce CdiI immunity proteins, which specifically neutralize cognate CdiA-CT toxins to prevent self-inhibition. Here, we present the crystal structure of the CdiA-CT/CdiIYkris complex from Yersinia kristensenii ATCC 33638. CdiA-CTYkris adopts the same fold as angiogenin and other RNase A paralogs, but the toxin does not share sequence similarity with these nucleases and lacks the characteristic disulfide bonds of the superfamily. Consistent with the structural homology, CdiA-CTYkris has potent RNase activity in vitro and in vivo. Structure-guided mutagenesis reveals that His175, Arg186, Thr276 and Tyr278 contribute to CdiA-CTYkris activity, suggesting that these residues participate in substrate binding and/or catalysis. CdiIYkris binds directly over the putative active site and likely neutralizes toxicity by blocking access to RNA substrates. Significantly, CdiA-CTYkris is the first non-vertebrate protein found to possess the RNase A superfamily fold, and homologs of this toxin are associated with secretion systems in many Gram-negative and Gram-positive bacteria. These observations suggest that RNase A-like toxins are commonly deployed in inter-bacterial competition.


Assuntos
Toxinas Bacterianas/química , Endorribonucleases/química , Ribonuclease Pancreático/química , Yersinia/enzimologia , Toxinas Bacterianas/metabolismo , Cristalografia por Raios X , Modelos Moleculares , Conformação Proteica , RNA/metabolismo , Ribonuclease Pancreático/metabolismo
19.
mBio ; 7(5)2016 10 25.
Artigo em Inglês | MEDLINE | ID: mdl-27795400

RESUMO

Heme oxygenase-1 (HO-1) is a stress response antioxidant enzyme which catalyzes the degradation of heme released during inflammation. HO-1 expression is upregulated in both experimental and human Mycobacterium tuberculosis infection, and in patients it is a biomarker of active disease. Whether the enzyme plays a protective versus pathogenic role in tuberculosis has been the subject of debate. To address this controversy, we administered tin protoporphyrin IX (SnPPIX), a well-characterized HO-1 enzymatic inhibitor, to mice during acute M. tuberculosis infection. These SnPPIX-treated animals displayed a substantial reduction in pulmonary bacterial loads comparable to that achieved following conventional antibiotic therapy. Moreover, when administered adjunctively with antimycobacterial drugs, the HO-1 inhibitor markedly enhanced and accelerated pathogen clearance. Interestingly, both the pulmonary induction of HO-1 expression and the efficacy of SnPPIX treatment in reducing bacterial burden were dependent on the presence of host T lymphocytes. Although M. tuberculosis expresses its own heme-degrading enzyme, SnPPIX failed to inhibit its enzymatic activity or significantly restrict bacterial growth in liquid culture. Together, the above findings reveal mammalian HO-1 as a potential target for host-directed monotherapy and adjunctive therapy of tuberculosis and identify the immune response as a critical regulator of this function. IMPORTANCE: There is no reliable vaccine against tuberculosis (TB), and conventional antibiotic therapy is administered over at least 6 months. This prolonged treatment period can lead to noncompliance resulting in relapsed infection as well as the emergence of multidrug resistance. Thus, there is an urgent need for improved therapeutic regimens that can more rapidly and efficiently control M. tuberculosis in infected patients. Here, we describe a potential strategy for treating TB based on pharmacological inhibition of the host heme-degrading enzyme HO-1. This approach results in significantly reduced bacterial burdens in mice, and when administered in conjunction with conventional antibiotic therapy, leads to faster, more effective pathogen clearance without detectable direct effects on the mycobacteria themselves. Interestingly, the effects of HO-1 inhibition on M. tuberculosis infection in vivo are dependent on the presence of an intact host immune system. These observations establish mammalian HO-1 as a potential target for host-directed therapy of TB.


Assuntos
Inibidores Enzimáticos/administração & dosagem , Heme Oxigenase-1/antagonistas & inibidores , Fatores Imunológicos/administração & dosagem , Metaloporfirinas/administração & dosagem , Mycobacterium tuberculosis/imunologia , Protoporfirinas/administração & dosagem , Linfócitos T/imunologia , Tuberculose/tratamento farmacológico , Animais , Carga Bacteriana , Modelos Animais de Doenças , Pulmão/microbiologia , Pulmão/patologia , Camundongos , Resultado do Tratamento , Tuberculose/imunologia
20.
Infect Immun ; 84(12): 3408-3422, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27647868

RESUMO

Bacillus anthracis is a sporulating Gram-positive bacterium that is the causative agent of anthrax and a potential weapon of bioterrorism. The U.S.-licensed anthrax vaccine is made from an incompletely characterized culture supernatant of a nonencapsulated, toxigenic strain (anthrax vaccine absorbed [AVA]) whose primary protective component is thought to be protective antigen (PA). AVA is effective in protecting animals and elicits toxin-neutralizing antibodies in humans, but enthusiasm is dampened by its undefined composition, multishot regimen, recommended boosters, and potential for adverse reactions. Improving next-generation anthrax vaccines is important to safeguard citizens and the military. Here, we report that vaccination with recombinant forms of a conserved domain (near-iron transporter [NEAT]), common in Gram-positive pathogens, elicits protection in a murine model of B. anthracis infection. Protection was observed with both Freund's and alum adjuvants, given subcutaneously and intramuscularly, respectively, with a mixed composite of NEATs. Protection correlated with an antibody response against the NEAT domains and a decrease in the numbers of bacteria in major organs. Anti-NEAT antibodies promote opsonophagocytosis of bacilli by alveolar macrophages. To guide the development of inactive and safe NEAT antigens, we also report the crystal structure of one of the NEAT domains (Hal) and identify critical residues mediating its heme-binding and acquisition activity. These results indicate that we should consider NEAT proteins in the development of an improved antianthrax vaccine.


Assuntos
Vacinas contra Antraz/imunologia , Antraz/prevenção & controle , Proteínas de Bactérias/imunologia , Animais , Vacinas contra Antraz/administração & dosagem , Anticorpos Antibacterianos/sangue , Bacillus anthracis , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Clonagem Molecular , Injeções Intramusculares , Camundongos , Modelos Moleculares , Fagócitos , Conformação Proteica
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