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1.
Biochem Cell Biol ; 102(3): 226-237, 2024 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-38377487

RESUMO

We here describe the structure-based design of small molecule inhibitors of the type IV secretion system of Helicobacter pylori. The secretion system is encoded by the cag pathogenicity island, and we chose Cagα, a hexameric ATPase and member of the family of VirB11-like proteins, as target for inhibitor design. We first solved the crystal structure of Cagα in a complex with the previously identified small molecule inhibitor 1G2. The molecule binds at the interface between two Cagα subunits and mutagenesis of the binding site identified Cagα residues F39 and R73 as critical for 1G2 binding. Based on the inhibitor binding site we synthesized 98 small molecule derivates of 1G2 to improve binding of the inhibitor. We used the production of interleukin-8 of gastric cancer cells during H. pylori infection to screen the potency of inhibitors and we identified five molecules (1G2_1313, 1G2_1338, 1G2_2886, 1G2_2889, and 1G2_2902) that have similar or higher potency than 1G2. Differential scanning fluorimetry suggested that these five molecules bind Cagα, and enzyme assays demonstrated that some are more potent ATPase inhibitors than 1G2. Finally, scanning electron microscopy revealed that 1G2 and its derivatives inhibit the assembly of T4SS-determined extracellular pili suggesting a mechanism for their anti-virulence effect.


Assuntos
Adenosina Trifosfatases , Proteínas de Bactérias , Helicobacter pylori , Helicobacter pylori/enzimologia , Humanos , Adenosina Trifosfatases/antagonistas & inibidores , Adenosina Trifosfatases/metabolismo , Adenosina Trifosfatases/química , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/química , Sistemas de Secreção Tipo IV/metabolismo , Sistemas de Secreção Tipo IV/química , Sistemas de Secreção Tipo IV/antagonistas & inibidores , Desenho de Fármacos , Infecções por Helicobacter/tratamento farmacológico , Infecções por Helicobacter/microbiologia , Cristalografia por Raios X , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/química , Modelos Moleculares , Sítios de Ligação , Relação Estrutura-Atividade , Linhagem Celular Tumoral , Interleucina-8/metabolismo
2.
Can J Microbiol ; 70(4): 119-127, 2024 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-38176008

RESUMO

Helicobacter pylori resistance to antibiotics is a growing problem and it increasingly leads to treatment failure. While the bacterium is present worldwide, the severity of clinical outcomes is highly dependent on the geographical origin and genetic characteristics of the strains. One of the major virulence factors identified in H. pylori is the cag pathogenicity island (cagPAI), which encodes a type IV secretion system (T4SS) used to translocate effectors into human cells. Here, we investigated the genetic variability of the cagPAI among 13 antibiotic-resistant H. pylori strains that were isolated from patient biopsies in Québec. Seven of the clinical strains carried the cagPAI, but only four could be readily cultivated under laboratory conditions. We observed variability of the sequences of CagA and CagL proteins that are encoded by the cagPAI. All clinical isolates induce interleukin-8 secretion and morphological changes upon co-incubation with gastric cancer cells and two of them produce extracellular T4SS pili. Finally, we demonstrate that molecule 1G2, a small molecule inhibitor of the Cagα protein from the model strain H. pylori 26695, reduces interleukin-8 secretion in one of the clinical isolates. Co-incubation with 1G2 also inhibits the assembly of T4SS pili, suggesting a mechanism for its action on T4SS function.


Assuntos
Infecções por Helicobacter , Helicobacter pylori , Humanos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Antígenos de Bactérias/genética , Sistemas de Secreção Tipo IV/genética , Sistemas de Secreção Tipo IV/metabolismo , Helicobacter pylori/genética , Helicobacter pylori/metabolismo , Interleucina-8/metabolismo , Infecções por Helicobacter/microbiologia
3.
Proc Natl Acad Sci U S A ; 115(23): 5950-5955, 2018 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-29784815

RESUMO

Type IV secretion systems (T4SSs) are multiprotein assemblies that translocate macromolecules across the cell envelope of bacteria. X-ray crystallographic and electron microscopy (EM) analyses have increasingly provided structural information on individual T4SS components and on the entire complex. As of now, relatively little information has been available on the exact localization of the inner membrane-bound T4SS components, notably the mostly periplasmic VirB8 protein and the very hydrophobic VirB6 protein. We show here that the membrane-bound, full-length version of the VirB8 homolog TraE from the plasmid pKM101 secretion system forms a high-molecular-mass complex that is distinct from the previously characterized periplasmic portion of the protein that forms dimers. Full-length TraE was extracted from the membranes with detergents, and analysis by size-exclusion chromatography, cross-linking, and size exclusion chromatography (SEC) multiangle light scattering (MALS) shows that it forms a high-molecular-mass complex. EM and small-angle X-ray scattering (SAXS) analysis demonstrate that full-length TraE forms a hexameric complex with a central pore. We also overproduced and purified the VirB6 homolog TraD and show by cross-linking, SEC, and EM that it binds to TraE. Our results suggest that TraE and TraD interact at the substrate translocation pore of the secretion system.


Assuntos
Proteínas da Membrana Bacteriana Externa/química , Proteínas de Escherichia coli/química , Proteínas de Membrana/química , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas da Membrana Bacteriana Externa/ultraestrutura , Conjugação Genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/ultraestrutura , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana/ultraestrutura , Plasmídeos/genética , Multimerização Proteica , Sistemas de Secreção Tipo IV
4.
Glob Chang Biol ; 26(10): 5942-5964, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32628332

RESUMO

Smallholder farmers in sub-Saharan Africa (SSA) currently grow rainfed maize with limited inputs including fertilizer. Climate change may exacerbate current production constraints. Crop models can help quantify the potential impact of climate change on maize yields, but a comprehensive multimodel assessment of simulation accuracy and uncertainty in these low-input systems is currently lacking. We evaluated the impact of varying [CO2 ], temperature and rainfall conditions on maize yield, for different nitrogen (N) inputs (0, 80, 160 kg N/ha) for five environments in SSA, including cool subhumid Ethiopia, cool semi-arid Rwanda, hot subhumid Ghana and hot semi-arid Mali and Benin using an ensemble of 25 maize models. Models were calibrated with measured grain yield, plant biomass, plant N, leaf area index, harvest index and in-season soil water content from 2-year experiments in each country to assess their ability to simulate observed yield. Simulated responses to climate change factors were explored and compared between models. Calibrated models reproduced measured grain yield variations well with average relative root mean square error of 26%, although uncertainty in model prediction was substantial (CV = 28%). Model ensembles gave greater accuracy than any model taken at random. Nitrogen fertilization controlled the response to variations in [CO2 ], temperature and rainfall. Without N fertilizer input, maize (a) benefited less from an increase in atmospheric [CO2 ]; (b) was less affected by higher temperature or decreasing rainfall; and (c) was more affected by increased rainfall because N leaching was more critical. The model intercomparison revealed that simulation of daily soil N supply and N leaching plays a crucial role in simulating climate change impacts for low-input systems. Climate change and N input interactions have strong implications for the design of robust adaptation approaches across SSA, because the impact of climate change in low input systems will be modified if farmers intensify maize production with balanced nutrient management.


Assuntos
Mudança Climática , Zea mays , Fertilizantes , Mali , Nitrogênio
5.
J Biol Chem ; 293(35): 13415-13426, 2018 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-29976757

RESUMO

Many bacterial pathogens employ multicomponent protein complexes such as type IV secretion systems (T4SSs) to transfer virulence factors into host cells. Here we studied the interaction between two essential T4SS components: the very hydrophobic inner membrane protein VirB6, which may be a component of the translocation channel, and VirB10, which links the inner and outer bacterial membranes. To map the interaction site between these two T4SS components, we conducted alanine scanning and deleted six-amino acid stretches from the N-terminal periplasmic domain of VirB6 from Brucella suis Using the bacterial two-hybrid system to analyze the effects of these alterations on the VirB6-VirB10 interaction, we identified the amino acid regions 16-21 and 28-33 and Leu-18 in VirB6 as being required for this interaction. SDS-PAGE coupled with Western blotting of cell lysates and native PAGE of detergent-extracted membrane proteins revealed that the corresponding VirB6 residues in Agrobacterium tumefaciens (Phe-20 and amino acids 18-23 and 30-35) modulate the stability of both VirB6 and VirB5. However, the results from immuno-EM and super-resolution microscopy suggested that these regions and residues are not required for membrane association or for polar localization of VirB6. The six-amino acid deletions in the N terminus of VirB6 abolished pilus formation and virulence of A. tumefaciens, and the corresponding deletions in the VirB6 homolog TraD from the plasmid pKM101-T4SS abrogated plasmid transfer. Our results indicate that specific residues of the VirB6 N-terminal domain are required for VirB6 stabilization, its interaction with VirB10, and the incorporation of VirB2 and VirB5 into T-pili.


Assuntos
Agrobacterium tumefaciens/metabolismo , Proteínas de Bactérias/metabolismo , Doenças das Plantas/microbiologia , Mapas de Interação de Proteínas , Sistemas de Secreção Tipo IV/metabolismo , Agrobacterium tumefaciens/química , Sequência de Aminoácidos , Proteínas de Bactérias/química , Brucella suis/química , Brucella suis/metabolismo , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Alinhamento de Sequência , Sistemas de Secreção Tipo IV/química
6.
Biochem Cell Biol ; 97(3): 215-223, 2019 06.
Artigo em Francês | MEDLINE | ID: mdl-30142282

RESUMO

Agrobacterium tumefaciens is a well studied phytopathogen given its various applications for deciphering host-pathogen interactions, bacterial communication, and capacity to transfer DNA fragments into host cells via a membrane protein system, the type IV secretion system (T4SS). T4SS mechanism is similar to the one responsible for antibiotic resistance gene transmission, and new knowledge gained could be applied to other organisms using such a mechanism. As well, A. tumefaciens is of economic importance in biotechnology due to its capacity to generate genetically modified plants. Agrobacterium tumefaciens harbours a plasmid known as Ti plasmid encoding T4SS function genes used for transferring genetic information and plant colonization. In this review, the authors describe the molecular basis of infection, from detection of host signals, to the description of different regions of Ti plasmid key to infection, ending with substrate transfer through bacterial wall. [Journal translation].


Agrobacterium tumefaciens est un phytopathogène étudié en raison de ses nombreuses applications pour des études d'interactions hôte­pathogène, la communication bactérienne et la capacité à transférer un fragment d'ADN dans la cellule hôte avec un système de protéines membranaires, le système de sécrétion de type IV (T4SS). Le mécanisme du T4SS est similaire à celui de la transmission des gènes de résistances aux antibiotiques et les connaissances obtenues pourront ensuite être appliquées à d'autres organismes utilisant ce genre de mécanisme. Également A. tumefaciens a une importance économique pour la biotechnologie grâce à sa capacité à générer des plantes génétiquement modifiées. Agrobacterium tumefaciens contient un plasmide, le plasmide Ti, qui code les fonctions du T4SS pour le transfert de l'information génétique et la colonisation des plantes. Dans cette revue nous décrivons les bases moléculaires de l'infection, allant de la détection des signaux provenant de l'hôte, en passant par la description des différentes régions du plasmide Ti importantes pour l'infection et en finissant avec le transfert du substrat à travers la paroi bactérienne.


Assuntos
Agrobacterium tumefaciens/metabolismo , Doenças das Plantas , Sistemas de Secreção Tipo IV/metabolismo , Interações Hospedeiro-Parasita , Doenças das Plantas/genética , Sistemas de Secreção Tipo IV/genética
7.
Eur J Oral Sci ; 127(4): 313-322, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31230388

RESUMO

The junctional epithelium (JE) is a specialized portion of the gingiva that seals off the tooth-supporting tissues from the oral environment. This relationship is achieved via a unique adhesive extracellular matrix that is, in fact, a specialized basal lamina (sBL). Three unique proteins - amelotin (AMTN), odontogenic ameloblast-associated (ODAM), and secretory calcium-binding phosphoprotein proline-glutamine rich 1 (SCPPPQ1) - together with laminin-332 structure the supramolecular organization of this sBL and determine its adhesive capacity. Despite the constant challenge of the JE by the oral microbiome, little is known of the susceptibility of the sBL to bacterial degradation. Assays with trypsin-like proteases, as well as incubation with Porphyromonas gingivalis, Prevotella intermedia, and Treponema denticola, revealed that all constituents, except SCPPPQ1, were rapidly degraded. Porphyromonas gingivalis was also shown to alter the supramolecular network of reconstituted and native sBLs. These results provide evidence that proteolytic enzymes and selected gram-negative periodontopathogenic bacteria can attack this adhesive extracellular matrix, intimating that its degradation could contribute to progression of periodontal diseases.


Assuntos
Membrana Basal/microbiologia , Inserção Epitelial/microbiologia , Matriz Extracelular/patologia , Gengiva , Dente , Amiloide , Proteínas de Ligação ao Cálcio , Proteínas do Esmalte Dentário , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Neoplasias , Fosfoproteínas , Porphyromonas gingivalis , Prevotella intermedia , Treponema denticola
8.
Curr Top Microbiol Immunol ; 413: 169-186, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29536359

RESUMO

The Agrobacterium tumefaciens VirB/D4 type IV secretion system (T4SS) comprises 12 membrane-bound proteins, and it assembles a surface-exposed T-pilus. It is considered to be the archetypical system that is generally used to orient the nomenclature of other T4SS. Whereas the sequence similarities between T4SSs from different organisms are often limited, the general mechanism of action appears to be conserved, and the evolutionary relationship to bacterial conjugation systems and to T4SSs from animal pathogens is well established. Agrobacterium is a natural genetic engineer that is extensively used for the generation of transgenic plants for research and for agro-biotechnological applications. It also served as an early model for the understanding of pathogen-host interactions and for the transfer of macromolecular virulence factors into host cells. The knowledge on the mechanism of its T4SS inspired the search for small molecules that inhibit the virulence of bacterial pathogens and of bacterial conjugation. Inhibitors of bacterial virulence and of conjugation have interesting potential as alternatives to antibiotics and as inhibitors of antimicrobial resistance gene transfer. Mechanistic work on the Agrobacterium T4SS will continue to inspire the search for inhibitor target sites and drug design.


Assuntos
Agrobacterium tumefaciens , Animais , Proteínas de Bactérias , Fatores de Virulência
9.
Biochemistry ; 56(17): 2261-2270, 2017 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-28414460

RESUMO

In mammalian cells, the incorporation of the 21st amino acid, selenocysteine, into proteins is guided by the Sec machinery. The function of this protein complex requires several protein-protein and protein-RNA interactions, leading to the incorporation of selenocysteine at UGA codons. It is guided by stem-loop structures localized in the 3' untranslated regions of the selenoprotein-encoding genes. Here, we conducted a global analysis of interactions between the Sec biosynthesis and incorporation components using a bioluminescence resonance energy transfer assay in mammalian cells that showed that selenocysteine synthase (SEPSECS), SECp43, and selenophosphate synthetases SEPHS1 and SEPHS2 form oligomers in eukaryotic cells. We also showed that SEPHS2 interacts with SEPSECS and SEPHS1; these interactions were confirmed by co-immunoprecipitation. To further analyze the interactions of SECp43, the protein was expressed in Escherichia coli, and small-angle X-ray scattering analysis revealed that it is a globular protein comprising two RNA-binding domains. Using phage display, we identified potential interaction sites and highlighted two residues (K166 and P167) required for its dimerization. The SECp43 structural model presented here constitutes the basis of future exploration of the protein-protein interactions among early components of the selenocysteine biosynthesis and incorporation pathway.


Assuntos
Aminoacil-tRNA Sintetases/metabolismo , Modelos Moleculares , Fosfotransferases/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transferases/metabolismo , Substituição de Aminoácidos , Aminoacil-tRNA Sintetases/química , Aminoacil-tRNA Sintetases/genética , Técnicas de Transferência de Energia por Ressonância de Bioluminescência , Técnicas de Visualização da Superfície Celular , Reagentes de Ligações Cruzadas/farmacologia , Dimerização , Células HEK293 , Humanos , Imunoprecipitação , Mutação , Proteínas Nucleares , Fosfotransferases/química , Fosfotransferases/genética , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Espalhamento a Baixo Ângulo , Succinimidas/farmacologia , Transferases/química , Transferases/genética , Difração de Raios X
10.
J Biol Chem ; 291(45): 23817-23829, 2016 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-27634044

RESUMO

Gram-negative bacteria use type IV secretion systems (T4SSs) for a variety of macromolecular transport processes that include the exchange of genetic material. The pKM101 plasmid encodes a T4SS similar to the well-studied model systems from Agrobacterium tumefaciens and Brucella suis Here, we studied the structure and function of TraE, a homolog of VirB8 that is an essential component of all T4SSs. Analysis by X-ray crystallography revealed a structure that is similar to other VirB8 homologs but displayed an altered dimerization interface. The dimerization interface observed in the X-ray structure was corroborated using the bacterial two-hybrid assay, biochemical characterization of the purified protein, and in vivo complementation, demonstrating that there are different modes of dimerization among VirB8 homologs. Analysis of interactions using the bacterial two-hybrid and cross-linking assays showed that TraE and its homologs from Agrobacterium, Brucella, and Helicobacter pylori form heterodimers. They also interact with heterologous VirB10 proteins, indicating a significant degree of plasticity in the protein-protein interactions of VirB8-like proteins. To further assess common features of VirB8-like proteins, we tested a series of small molecules derived from inhibitors of Brucella VirB8 dimerization. These molecules bound to TraE in vitro, docking predicted that they bind to a structurally conserved surface groove of the protein, and some of them inhibited pKM101 plasmid transfer. VirB8-like proteins thus share functionally important sites, and these can be exploited for the design of specific inhibitors of T4SS function.


Assuntos
Proteínas de Bactérias/química , Bactérias Gram-Negativas/química , Plasmídeos/química , Sistemas de Secreção Tipo IV/química , Agrobacterium tumefaciens/química , Agrobacterium tumefaciens/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/metabolismo , Brucella suis/química , Brucella suis/metabolismo , Cristalografia por Raios X , Bactérias Gram-Negativas/metabolismo , Helicobacter pylori/química , Helicobacter pylori/metabolismo , Modelos Moleculares , Plasmídeos/antagonistas & inibidores , Plasmídeos/metabolismo , Conformação Proteica , Mapas de Interação de Proteínas , Multimerização Proteica , Bibliotecas de Moléculas Pequenas/farmacologia , Sistemas de Secreção Tipo IV/antagonistas & inibidores , Sistemas de Secreção Tipo IV/metabolismo
11.
Glob Chang Biol ; 20(7): 2301-20, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24395589

RESUMO

Potential consequences of climate change on crop production can be studied using mechanistic crop simulation models. While a broad variety of maize simulation models exist, it is not known whether different models diverge on grain yield responses to changes in climatic factors, or whether they agree in their general trends related to phenology, growth, and yield. With the goal of analyzing the sensitivity of simulated yields to changes in temperature and atmospheric carbon dioxide concentrations [CO2 ], we present the largest maize crop model intercomparison to date, including 23 different models. These models were evaluated for four locations representing a wide range of maize production conditions in the world: Lusignan (France), Ames (USA), Rio Verde (Brazil) and Morogoro (Tanzania). While individual models differed considerably in absolute yield simulation at the four sites, an ensemble of a minimum number of models was able to simulate absolute yields accurately at the four sites even with low data for calibration, thus suggesting that using an ensemble of models has merit. Temperature increase had strong negative influence on modeled yield response of roughly -0.5 Mg ha(-1) per °C. Doubling [CO2 ] from 360 to 720 µmol mol(-1) increased grain yield by 7.5% on average across models and the sites. That would therefore make temperature the main factor altering maize yields at the end of this century. Furthermore, there was a large uncertainty in the yield response to [CO2 ] among models. Model responses to temperature and [CO2 ] did not differ whether models were simulated with low calibration information or, simulated with high level of calibration information.


Assuntos
Mudança Climática , Água/metabolismo , Zea mays/crescimento & desenvolvimento , Zea mays/metabolismo , Dióxido de Carbono/metabolismo , Produtos Agrícolas/crescimento & desenvolvimento , Produtos Agrícolas/metabolismo , Geografia , Modelos Biológicos , Temperatura
12.
Structure ; 31(4): 375-384.e4, 2023 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-36513067

RESUMO

Agrobacterium tumefaciens is a natural genetic engineer that transfers DNA into plants, which is the most applied process for generation of genetically modified plants. DNA transfer is mediated by a type IV secretion system in the cell envelope and extracellular T-pili. We here report the cryo-electron microscopic structures of the T-pilus at 3.2-Å resolution and of the plasmid pKM101-determined N-pilus at 3-Å resolution. Both pili contain a main pilus protein (VirB2 in A. tumefaciens, TraM in pKM101) and phospholipids arranged in a five-start helical assembly. They contain positively charged amino acids in the lumen, and the lipids are positively charged in the T-pilus (phosphatidylcholine) conferring overall positive charge. Mutagenesis of the lumen-exposed Arg91 in VirB2 results in protein destabilization and loss of pilus formation. Our results reveal that different phospholipids can be incorporated into type IV secretion pili and that the charge of the lumen may be of functional importance.


Assuntos
Agrobacterium tumefaciens , Proteínas de Bactérias , Agrobacterium tumefaciens/genética , Agrobacterium tumefaciens/química , Agrobacterium tumefaciens/metabolismo , Proteínas de Bactérias/química , Microscopia Crioeletrônica , Fímbrias Bacterianas/metabolismo , Membrana Celular/metabolismo
13.
Philos Trans R Soc Lond B Biol Sci ; 378(1871): 20220040, 2023 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-36633286

RESUMO

We present a potential mechanism for emergence of catalytic activity that is essential for survival, from a non-catalytic protein fold. The type B dihydrofolate reductase (DfrB) family of enzymes were first identified in pathogenic bacteria because their dihydrofolate reductase activity is sufficient to provide trimethoprim (TMP) resistance. DfrB enzymes are described as poorly evolved as a result of their unusual structural and kinetic features. No characterized protein shares sequence homology with DfrB enzymes; how they evolved to emerge in the modern resistome is unknown. In this work, we identify DfrB homologues from a database of putative and uncharacterized proteins. These proteins include an SH3-like fold homologous to the DfrB enzymes, embedded in a variety of additional structural domains. By means of functional, structural and biophysical characterization, we demonstrate that these distant homologues and their extracted SH3-like fold can display dihydrofolate reductase activity and confer TMP resistance. We provide evidence of tetrameric assembly and catalytic mechanism analogous to that of DfrB enzymes. These results contribute, to our knowledge, the first insights into a potential evolutionary path taken by this SH3-like fold to emerge in the modern resistome following introduction of TMP. This article is part of the theme issue 'Reactivity and mechanism in chemical and synthetic biology'.


Assuntos
Oxirredutases , Tetra-Hidrofolato Desidrogenase , Tetra-Hidrofolato Desidrogenase/genética , Tetra-Hidrofolato Desidrogenase/química , Tetra-Hidrofolato Desidrogenase/metabolismo , Antibacterianos , Farmacorresistência Bacteriana
14.
Nat Commun ; 14(1): 2006, 2023 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-37037821

RESUMO

The acidification of water in mining areas is a global environmental issue primarily catalyzed by sulfur-oxidizing bacteria (SOB). Little is known about microbial sulfur cycling in circumneutral pH mine tailing impoundment waters. Here we investigate biological sulfur oxidation over four years in a mine tailings impoundment water cap, integrating aqueous sulfur geochemistry, genome-resolved metagenomics and metatranscriptomics. The microbial community is consistently dominated by neutrophilic, chemolithoautotrophic SOB (relative abundances of ~76% in 2015, ~55% in 2016/2017 and ~60% in 2018). Results reveal two SOB strategies alternately dominate across the four years, influencing acid generation and sulfur speciation. Under oxic conditions, novel Halothiobacillus drive lower pH conditions (as low as 4.3) and lower [S2O32-] via the complete Sox pathway coupled to O2. Under anoxic conditions, Thiobacillus spp. dominate in activity, via the incomplete Sox and rDSR pathways coupled to NO3-, resulting in higher [S2O32-] and no net significant acidity generation. This study provides genomic evidence explaining acidity generation and thiosulfate accumulation patterns in a circumneutral mine tailing impoundment and has significant environmental applications in preventing the discharge of sulfur compounds that can impact downstream environments. These insights illuminate opportunities for in situ biotreatment of reduced sulfur compounds and prediction of acidification events using gene-based monitoring and in situ RNA detection.


Assuntos
Bactérias , Tiossulfatos , Tiossulfatos/metabolismo , Oxirredução , Bactérias/genética , Bactérias/metabolismo , Enxofre/metabolismo , Compostos de Enxofre/metabolismo , Água/metabolismo
15.
Biochemistry ; 51(18): 3881-90, 2012 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-22515661

RESUMO

Type IV secretion systems are macromolecular assemblies in the cell envelopes of bacteria that function in macromolecular translocation. Structural biology approaches have provided insights into the interaction of core complex components, but information about proteins that undergo transient interactions with membrane components has not been forthcoming. We have pursued an unbiased approach using peptide arrays and phage display to identify interaction partners and interaction domains of type IV secretion system assembly factor VirB8. These approaches identified the globular domain from the VirB5 protein to interact with VirB8. This interaction was confirmed in cross-linking, pull-down, and fluorescence resonance energy transfer (FRET)-based interaction assays. In addition, using phage display analysis, we identified different regions of VirB6 as potential interaction partners of VirB8. Using a FRET-based interaction assay, we provide the first direct experimental evidence of the interaction of a VirB6 periplasmic domain with VirB8. These results will allow us to conduct directed structural biological work and structure-function analyses aimed at defining the molecular details and biological significance of these interactions with VirB8 in the future.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Brucella/metabolismo , Transferência Ressonante de Energia de Fluorescência , Proteínas de Membrana Transportadoras/genética , Biblioteca de Peptídeos , Periplasma/metabolismo , Multimerização Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Transporte Proteico , Fatores de Virulência/metabolismo
16.
bioRxiv ; 2022 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-35233570

RESUMO

SARS-CoV-2 infection of host cells starts by binding of the Spike glycoprotein (S) to the ACE2 receptor. The S-ACE2 interaction is a potential target for therapies against COVID-19 as demonstrated by the development of immunotherapies blocking this interaction. Here, we present the commercially available VE607, comprised of three stereoisomers, that was originally described as an inhibitor of SARS-CoV-1. We show that VE607 specifically inhibits infection of SARS-CoV-1 and SARS-CoV-2 S-expressing pseudoviral particles as well as authentic SARS-CoV-2. VE607 stabilizes the receptor binding domain (RBD) in its "up" conformation. In silico docking and mutational analysis map the VE607 binding site at the RBD-ACE2 interface. The IC 50 values are in the low micromolar range for pseudoparticles derived from SARS-CoV-2 Wuhan/D614G as well as from variants of concern (Alpha, Beta, Gamma, Delta and Omicron), suggesting that VE607 has potential for the development of drugs against SARS-CoV-2 infections.

17.
iScience ; 25(7): 104528, 2022 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-35677392

RESUMO

SARS-CoV-2 infection of host cells starts by binding the Spike glycoprotein (S) to the ACE2 receptor. The S-ACE2 interaction is a potential target for therapies against COVID-19 as demonstrated by the development of immunotherapies blocking this interaction. VE607 - a commercially available compound composed of three stereoisomers - was described as an inhibitor of SARS-CoV-1. Here, we show that VE607 broadly inhibits pseudoviral particles bearing the Spike from major VOCs (D614G, Alpha, Beta, Gamma, Delta, Omicron - BA.1, and BA.2) as well as authentic SARS-CoV-2 at low micromolar concentrations. In silico docking, mutational analysis, and smFRET revealed that VE607 binds to the receptor binding domain (RBD)-ACE2 interface and stabilizes RBD in its "up" conformation. Prophylactic treatment with VE607 did not prevent SARS-CoV-2-induced mortality in K18-hACE2 mice, but it did reduce viral replication in the lungs by 37-fold. Thus, VE607 is an interesting lead for drug development for the treatment of SARS-CoV-2 infection.

18.
J Bacteriol ; 193(9): 2097-106, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21398549

RESUMO

Type IV secretion systems are virulence factors used by many gram-negative bacteria to translocate macromolecules across the cell envelope. VirB8 is an essential inner membrane component of type IV secretion systems, and it is believed to form a homodimer. In the absence of VirB8, the levels of several other VirB proteins were reduced (VirB1, VirB3, VirB4, VirB5, VirB6, VirB7, and VirB11) in Agrobacterium tumefaciens, underlining its importance for complex stability. To assess the importance of dimerization, we changed residues at the predicted dimer interface (V97, A100, Q93, and E94) in order to strengthen or to abolish dimerization. We verified the impact of the changes on dimerization in vitro with purified V97 variants, followed by analysis of the in vivo consequences in a complemented virB8 deletion strain. Dimer formation was observed in vivo after the introduction of a cysteine residue at the predicted interface (V97C), and this variant supported DNA transfer, but the formation of elongated T pili was not detected by the standard pilus isolation technique. Variants with changes at V97 and A100 that weaken dimerization did not support type IV secretion system functions. The T-pilus component VirB2 cofractionated with high-molecular-mass core protein complexes extracted from the membranes, and the presence of VirB8 as well as its dimer interface were important for this association. We conclude that the VirB8 dimer interface is required for T4SS function, for the stabilization of many VirB proteins, and for targeting of VirB2 to the T-pilus assembly site.


Assuntos
Agrobacterium tumefaciens/metabolismo , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Agrobacterium tumefaciens/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Teste de Complementação Genética , Variação Genética , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Fatores de Virulência
19.
J Biol Chem ; 285(26): 19757-66, 2010 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-20427270

RESUMO

Agrobacterium tumefaciens is a plant pathogen that utilizes a type IV secretion system (T4SS) to transfer DNA and effector proteins into host cells. In this study we discovered that an alpha-crystallin type small heat-shock protein (alpha-Hsp), HspL, is a molecular chaperone for VirB8, a T4SS assembly factor. HspL is a typical alpha-Hsp capable of protecting the heat-labile model substrate citrate synthase from thermal aggregation. It forms oligomers in a concentration-dependent manner in vitro. Biochemical fractionation revealed that HspL is mainly localized in the inner membrane and formed large complexes with certain VirB protein subassemblies. Protein-protein interaction studies indicated that HspL interacts with VirB8, a bitopic integral inner membrane protein that is essential for T4SS assembly. Most importantly, HspL is able to prevent the aggregation of VirB8 fused with glutathione S-transferase in vitro, suggesting that it plays a role as VirB8 chaperone. The chaperone activity of two HspL variants with amino acid substitutions (F98A and G118A) for both citrate synthase and glutathione S-transferase-VirB8 was reduced and correlated with HspL functions in T4SS-mediated DNA transfer and virulence. This study directly links in vitro and in vivo functions of an alpha-Hsp and reveals a novel alpha-Hsp function in T4SS stability and bacterial virulence.


Assuntos
Agrobacterium tumefaciens/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Choque Térmico Pequenas/metabolismo , Chaperonas Moleculares/metabolismo , Agrobacterium tumefaciens/genética , Agrobacterium tumefaciens/patogenicidade , Proteínas de Bactérias/genética , Western Blotting , Membrana Celular/metabolismo , Citrato (si)-Sintase/metabolismo , Proteínas de Choque Térmico Pequenas/química , Proteínas de Choque Térmico Pequenas/genética , Chaperonas Moleculares/genética , Mutação , Multimerização Proteica , Estabilidade Proteica , Especificidade por Substrato , Temperatura , Transformação Genética , Virulência
20.
Infect Immun ; 79(3): 1033-43, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21173315

RESUMO

As bacterial pathogens develop resistance against most currently used antibiotics, novel alternatives for treatment of microbial infectious diseases are urgently needed. Targeting bacterial virulence functions in order to disarm pathogens represents a promising alternative to classical antibiotic therapy. Type IV secretion systems, which are multiprotein complexes in the cell envelope that translocate effectors into host cells, are critical bacterial virulence factors in many pathogens and excellent targets for such "antivirulence" drugs. The VirB8 protein from the mammalian pathogen Brucella was chosen as a specific target, since it is an essential type IV secretion system component, it participates in multiple protein-protein interactions, and it is essential for the assembly of this translocation machinery. The bacterial two-hybrid system was adapted to assay VirB8 interactions, and a high-throughput screen identified specific small-molecule inhibitors. VirB8 interaction inhibitors also reduced the levels of VirB8 and of other VirB proteins, and many of them inhibited virB gene transcription in Brucella abortus 2308, suggesting that targeting of the secretion system has complex regulatory effects in vivo. One compound strongly inhibited the intracellular proliferation of B. abortus 2308 in a J774 macrophage infection model. The results presented here show that in vivo screens with the bacterial two-hybrid assay are suited to the identification of inhibitors of Brucella type IV secretion system function.


Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Sistemas de Secreção Bacterianos/efeitos dos fármacos , Brucella abortus/efeitos dos fármacos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Animais , Brucella abortus/crescimento & desenvolvimento , Brucella abortus/metabolismo , Linhagem Celular , Macrófagos/efeitos dos fármacos , Macrófagos/microbiologia , Camundongos , Viabilidade Microbiana , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Técnicas do Sistema de Duplo-Híbrido
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