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1.
Vet Parasitol ; 273: 45-51, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31442892

RESUMO

Fasciola gigantica and hybrid Fasciola are distributed throughout Asia. Herein, we investigated the species of the Fasciola fluke distributed in three hotspots of fascioliasis in Cambodia. A total of 92 flukes collected from 21 slaughtered cattle from Kandal (44), Battambang (41), and Kratie (7) Provinces were identified as F. gigantica using multiplex PCR for a nuclear phosphoenolpyruvate carboxykinase (PEPCK) gene. The overall prevalence of F. gigantica infestation was 7.14% (21/294). Phylogenetic as well as population genetics analyses were performed using the mitochondrial NADH dehydrogenase subunit 1 (ND1). The 19 ND1 haplotypes were identified from Cambodian F. gigantica (haplotype diversity, 0.83). All of the haplotypes were classified into F. gigantica haplogroup C, which includes ND1 haplotypes detected from Thailand, Vietnam, Indonesia, Myanmar, and China. Among haplogroup C, novel and unique haplotypes of Cambodia were found in the Battambang and Kandal Provinces, and the nucleotide diversity of the Cambodian population (0.00532) was the highest. Pairwise fixation indices among the F. gigantica populations from these countries indicated that the Cambodian and Thailand populations were related to each other. The highest genetic diversity in the Cambodian population suggests that F. gigantica in Cambodia may be the ancestor of the populations in Southeast Asian countries. Most likely, livestock movement, including Zebu cattle, played an important role in the transmission of F. gigantica. In this study, the hybrid Fasciola flukes that are commonly found in neighboring countries, were not found in Cambodia. Further comprehensive investigations of Fasciola prevalence should be conducted by analyzing a wider range of hosts throughout Cambodia to reach a more solid conclusion about the absence of hybrid flukes.


Assuntos
Distribuição Animal , Doenças dos Bovinos/parasitologia , Fasciola/classificação , Fasciola/genética , Fasciolíase/veterinária , Variação Genética , Animais , Ásia Sudeste/epidemiologia , Camboja , Bovinos , Doenças dos Bovinos/epidemiologia , Fasciolíase/epidemiologia , Fasciolíase/parasitologia , Haplótipos , NADH Desidrogenase/genética , Fosfotransferases/genética , Prevalência
2.
Nature ; 572(7768): 270-274, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31291642

RESUMO

Receptor kinases of the Catharanthus roseus RLK1-like (CrRLK1L) family have emerged as important regulators of plant reproduction, growth and responses to the environment1. Endogenous RAPID ALKALINIZATION FACTOR (RALF) peptides2 have previously been proposed as ligands for several members of the CrRLK1L family1. However, the mechanistic basis of this perception is unknown. Here we report that RALF23 induces a complex between the CrRLK1L FERONIA (FER) and LORELEI (LRE)-LIKE GLYCOSYLPHOSPHATIDYLINOSITOL (GPI)-ANCHORED PROTEIN 1 (LLG1) to regulate immune signalling. Structural and biochemical data indicate that LLG1 (which is genetically important for RALF23 responses) and the related LLG2 directly bind RALF23 to nucleate the assembly of RALF23-LLG1-FER and RALF23-LLG2-FER heterocomplexes, respectively. A conserved N-terminal region of RALF23 is sufficient for the biochemical recognition of RALF23 by LLG1, LLG2 or LLG3, and binding assays suggest that other RALF peptides that share this conserved N-terminal region may be perceived by LLG proteins in a similar manner. Structural data also show that RALF23 recognition is governed by the conformationally flexible C-terminal sides of LLG1, LLG2 and LLG3. Our work reveals a mechanism of peptide perception in plants by GPI-anchored proteins that act together with a phylogenetically unrelated receptor kinase. This provides a molecular framework for understanding how diverse RALF peptides may regulate multiple processes, through perception by distinct heterocomplexes of CrRLK1L receptor kinases and GPI-anchored proteins of the LRE and LLG family.


Assuntos
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/imunologia , Arabidopsis/metabolismo , Proteínas Ligadas por GPI/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/química , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Fragmentos de Peptídeos/metabolismo , Fosfotransferases/metabolismo , Proteínas de Arabidopsis/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Modelos Moleculares , Mutagênese , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fosfotransferases/genética , Maleabilidade , Ligação Proteica/genética , Conformação Proteica , Multimerização Proteica
3.
Oncogene ; 38(38): 6479-6490, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31324890

RESUMO

Diffuse intrinsic pontine glioma (or DIPG) are pediatric high-grade gliomas associated with a dismal prognosis. They harbor specific substitution in histone H3 at position K27 that induces major epigenetic dysregulations. Most clinical trials failed so far to increase survival, and radiotherapy remains the most efficient treatment, despite only transient tumor control. We conducted the first lentiviral shRNA dropout screen in newly diagnosed DIPG to generate a cancer-lethal signature as a basis for the development of specific treatments with increased efficacy and reduced side effects compared to existing anticancer therapies. The analysis uncovered 41 DIPG essential genes among the 672 genes of human kinases tested, for which several distinct interfering RNAs impaired cell expansion of three different DIPG stem-cell cultures without deleterious effect on two control neural stem cells. Among them, PLK1, AURKB, CHEK1, EGFR, and GSK3A were previously identified by similar approach in adult GBM indicating common dependencies of these cancer cells and pediatric gliomas. As expected, we observed an enrichment of genes involved in proliferation and cell death processes with a significant number of candidates belonging to PTEN/PI3K/AKT and EGFR pathways already under scrutiny in clinical trials in this disease. We highlighted VRK3, a gene involved especially in cell cycle regulation, DNA repair, and neuronal differentiation, as a non-oncogenic addiction in DIPG. Its repression totally blocked DIPG cell growth in the four cellular models evaluated, and induced cell death in H3.3-K27M cells specifically but not in H3.1-K27M cells, supporting VRK3 as an interesting and promising target in DIPG.


Assuntos
Neoplasias do Tronco Encefálico/genética , Fosfotransferases/genética , Proteínas Serina-Treonina Quinases/fisiologia , RNA Interferente Pequeno/fisiologia , Análise de Sequência de RNA/métodos , Neoplasias do Tronco Encefálico/diagnóstico , Neoplasias do Tronco Encefálico/patologia , Sobrevivência Celular/genética , Células Cultivadas , /patologia , Genes Essenciais , Células HEK293 , Humanos , Fosfatidilinositol 3-Quinases/análise , Fosfatidilinositol 3-Quinases/genética , Fosfotransferases/análise , Prognóstico , Proteínas Serina-Treonina Quinases/análise , Proteínas Serina-Treonina Quinases/genética , RNA Interferente Pequeno/análise
4.
Clin Lab ; 65(7)2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31307186

RESUMO

BACKGROUND: Acinetobacter baumannii (A. baumannii) is an opportunistic pathogen associated with serious hospital acquired infections. It is resistant to multiple antibiotics. The therapy for infections due to this bacterium is a combination of aminoglycosides with carbapenem antibiotics. There are recent reports of the prevalence of ami-noglycoside resistance among A. baumannii. The aim of the present study was to determine the prevalence of phosphotransferases APH (3')-Via (aphA6), acetyltransferases AAC (3)-Ia (aacC1), nucleotidyl transferases ANT (2'')-Ia (aadB), and ANT (3") -Ia (aadA1) genes among clinical isolates of A. baumannii and its relation to resis-tance to amikacin and gentamicin. METHODS: The study included all clinical samples from intensive care units (ICUs) patients with suspected hospital acquired infections. Positive cultures were identified by Gram stain and complete biochemical identifications. An-tibiotic susceptibility was carried out by disc diffusion method. Minimum inhibitory concentration determination (MIC) to amikacin and gentamicin was performed by microdilution method. Polymerase chain reaction (PCR) was performed for detection of aadB, aadA1, aphA6, aadC1 genes. RESULTS: The study included 1,200 bacterial isolates from patients admitted to ICUs during the period of the study. A. baumannii represented 100 isolates from Gram negative bacilli. The study of MIC of A. baumannii to amikacin and gentamicin revealed that 50 (50%) of the isolates had resistance by MIC study with 36 (72%) of those having high level aminoglycoside resistance (HLAR) and 14 (28%) had non-high-level aminoglycoside resistance. The most common prevalent resistant genes among A. baumannii resistance to aminoglycosides was aadB (42%), fol-lowed by aphA6 (26%). Less prevalent genes were aadA1 (18%) and aacC1 (12%). There were 24 isolates with negative PCR for the studied genes. In a comparison between the prevalence of resistant genes among A. baumannii with HLAR and non HLAR, there was a non-significant increase of aphA6 (30.6%) and aadB (44.4%) in HLAR isolates compared to non HLAR (14.3%, 35.7%, respectively; p = 0.2), with a significant increase in aacC1 28.6% in HLAR compared to 5.6% in non HLAR (p = 0.02). CONCLUSIONS: Half of the clinical isolates of A. baumannii have resistance to the aminoglycosides amikacin and gen-tamicin. Most isolates have a high resistance level to aminoglycosides. The isolates have different types of amino-glycoside modifying genes. Further studies are required to detect other genes associated with resistance to amino-glycosides.


Assuntos
Acinetobacter baumannii/efeitos dos fármacos , Aminoglicosídeos/farmacologia , Farmacorresistência Bacteriana Múltipla/genética , Genes Bacterianos/genética , Acetiltransferases/genética , Infecções por Acinetobacter/tratamento farmacológico , Infecções por Acinetobacter/microbiologia , Acinetobacter baumannii/genética , Acinetobacter baumannii/fisiologia , Amicacina/farmacologia , Antibacterianos/farmacologia , Infecção Hospitalar/tratamento farmacológico , Infecção Hospitalar/microbiologia , Estudos Transversais , Gentamicinas/farmacologia , Humanos , Testes de Sensibilidade Microbiana , Nucleotidiltransferases/genética , Fosfotransferases/genética
5.
Nat Commun ; 10(1): 3354, 2019 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-31350417

RESUMO

How microbes dynamically coordinate uptake and simultaneous utilization of nutrients in complex nutritional ecosystems is still an open question. Here, we develop a constraint-based modeling approach that exploits non-targeted exo-metabolomics data to unravel adaptive decision-making processes in dynamic nutritional environments. We thereby investigate metabolic adaptation of Escherichia coli to continuously changing conditions during batch growth in complex medium. Unexpectedly, model-based analysis of time resolved exo-metabolome data revealed that fastest growth coincides with preferred catabolism of amino acids, which, in turn, reduces glucose uptake and increases acetate overflow. We show that high intracellular levels of the amino acid degradation metabolites pyruvate and oxaloacetate can directly inhibit the phosphotransferase system (PTS), and reveal their functional role in mediating regulatory decisions for uptake and catabolism of alternative carbon sources. Overall, the proposed methodology expands the spectrum of possible applications of flux balance analysis to decipher metabolic adaptation mechanisms in naturally occurring habitats and diverse organisms.


Assuntos
Aminoácidos/metabolismo , Escherichia coli/metabolismo , Glucose/metabolismo , Acetatos/metabolismo , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Ácido Oxaloacético/metabolismo , Fosfotransferases/genética , Fosfotransferases/metabolismo , Ácido Pirúvico/metabolismo
6.
PLoS Pathog ; 15(7): e1007900, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31269090

RESUMO

The Pseudomonas syringae acetyltransferase HopZ1a is delivered into host cells by the type III secretion system to promote bacterial growth. However, in the model plant host Arabidopsis thaliana, HopZ1a activity results in an effector-triggered immune response (ETI) that limits bacterial proliferation. HopZ1a-triggered immunity requires the nucleotide-binding, leucine-rich repeat domain (NLR) protein, ZAR1, and the pseudokinase, ZED1. Here we demonstrate that HopZ1a can acetylate members of a family of 'receptor-like cytoplasmic kinases' (RLCK family VII; also known as PBS1-like kinases, or PBLs) and promote their interaction with ZED1 and ZAR1 to form a ZAR1-ZED1-PBL ternary complex. Interactions between ZED1 and PBL kinases are determined by the pseudokinase features of ZED1, and mutants designed to restore ZED1 kinase motifs can (1) bind to PBLs, (2) recruit ZAR1, and (3) trigger ZAR1-dependent immunity in planta, all independently of HopZ1a. A ZED1 mutant that mimics acetylation by HopZ1a also triggers immunity in planta, providing evidence that effector-induced perturbations of ZED1 also activate ZAR1. Overall, our results suggest that interactions between these two RLCK families are promoted by perturbations of structural features that distinguish active from inactive kinase domain conformations. We propose that effector-induced interactions between ZED1/ZRK pseudokinases (RLCK family XII) and PBL kinases (RLCK family VII) provide a sensitive mechanism for detecting perturbations of either kinase family to activate ZAR1-mediated ETI.


Assuntos
Proteínas de Arabidopsis/imunologia , Proteínas de Arabidopsis/metabolismo , Arabidopsis/imunologia , Arabidopsis/metabolismo , Fosfotransferases/imunologia , Fosfotransferases/metabolismo , Imunidade Vegetal , Acetilação , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/imunologia , Proteínas de Transporte/metabolismo , Interações entre Hospedeiro e Microrganismos/genética , Interações entre Hospedeiro e Microrganismos/imunologia , Modelos Imunológicos , Mutação , Fosfotransferases/genética , Domínios e Motivos de Interação entre Proteínas , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Pseudomonas syringae/imunologia , Pseudomonas syringae/metabolismo , Pseudomonas syringae/patogenicidade
7.
mSphere ; 4(3)2019 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-31189560

RESUMO

The azithromycin resistance conferred by phosphotransferase is encoded in the gene mph(A). This gene has been discovered in and reported for many bacterial species. We examined the prevalence of azithromycin resistance in Vibrio fluvialis (AR-VF) isolated during 2014 to 2015 from the hospitalized acute diarrheal patients in Kolkata, India. Most of the V. fluvialis isolates are identified as the sole pathogen (54%). The prevalence of AR-VF was higher in 2015 (19 [68%]) than in 2014 (9 [32%]). Among AR-VF isolates, the azithromycin MICs ranged from 4 to >256 mg/liter. Twenty-eight of the 48 (58%) V. fluvialis isolates harbored the gene mph(A) and phenotypically resistant to azithromycin. All the AR-VF isolates remained susceptible to doxycycline. In addition to azithromycin, other antimicrobial resistance-encoding genes of AR-VF were also characterized. All the AR-VF isolates were positive for class 1 integron, and most of them (17/28) carried the dfrA1 gene cassettes. Only one isolate was positive for the ereA gene, which encodes resistance to erythomycin. The majority of the isolates were resistant to ß-lactam antibiotics (bla OXA-1 [96%], bla OXA-7 [93%], and bla TEM-9 [68%]) and aminoglycoside actetyltransferase, conferring resistance to ciprofloxacin-modifying enzyme [aac(6')Ib-cr] (96%). Analyses by pulsed-field gel electrophoresis (PFGE) showed that the AR-VF isolates belonged to different genetic lineages. This is the first study to report azithromycin resistance and the presence of the mph(A) gene in V. fluvialis isolates. Circulation of AR-VF isolates with high azithromycin MICs is worrisome, since it may limit the treatment options for diarrheal infections.IMPORTANCE The progressive rise in antibiotic resistance among enteric pathogens in developing countries is becoming a big concern. India is one of the largest consumers of antibiotics, and their use is not well regulated. V. fluvialis is increasingly recognized as an emerging diarrheal pathogen of public health importance. Here we report the emergence of azithromycin resistance in V. fluvialis isolates from diarrheal patients in Kolkata, India. Azithromycin has been widely used in the treatment of various infections, both in children and in adults. Resistance to azithromycin is encoded in the gene mph(A). Emerging azithromycin resistance in V. fluvialis is a major public health challenge, and future studies should be focused on identifying ways to prevent the dissemination of this antibiotic resistance gene.


Assuntos
Antibacterianos/farmacologia , Azitromicina/farmacologia , Diarreia/microbiologia , Farmacorresistência Bacteriana Múltipla , Fosfotransferases/genética , Vibrio/efeitos dos fármacos , Vibrio/enzimologia , Criança , Pré-Escolar , Feminino , Humanos , Índia , Masculino , Testes de Sensibilidade Microbiana , Vibrio/genética
8.
PLoS One ; 14(5): e0217828, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31150492

RESUMO

The discovery of 20 unconventional kinetochore proteins in Trypanosoma brucei has opened a new and interesting area of evolutionary research to study a biological process previously thought to be highly conserved in all eukaryotes. In addition, the discovery of novel proteins involved in a critical cellular process provides an opportunity to exploit differences between kinetoplastid and human kinetochore proteins to develop therapeutics for diseases caused by kinetoplastid parasites. Consequently, we identified two of the unconventional kinetochore proteins as key targets (the highly related kinases KKT10 and KKT19). Recombinant T. brucei KKT19 (TbKKT19) protein was produced, a peptide substrate phosphorylated by TbKKT19 identified (KKLRRTLSVA), Michaelis constants for KKLRRTLSVA and ATP were determined (179 µM and 102 µM respectively) and a robust high-throughput compatible biochemical assay developed. This biochemical assay was validated pharmacologically with inhibition by staurosporine and hypothemycin (IC50 values of 288 nM and 65 nM respectively). Surprisingly, a subsequent high-throughput screen of a kinase-relevant compound library (6,624 compounds) yielded few hits (8 hits; final hit rate 0.12%). The low hit rate observed was unusual for a kinase target, particularly when screened against a compound library enriched with kinase hinge binding scaffolds. In an attempt to understand the low hit rate a TbKKT19 homology model, based on human cdc2-like kinase 1 (CLK1), was generated. Analysis of the TbKKT19 sequence and structure revealed no obvious features that could explain the low hit rates. Further work will therefore be necessary to explore this unique kinetochore kinase as well as to assess whether the few hits identified can be developed into tool molecules or new drugs.


Assuntos
Peptídeos/antagonistas & inibidores , Fosfotransferases/antagonistas & inibidores , Trypanosoma brucei brucei/efeitos dos fármacos , Tripanossomíase Africana/dietoterapia , Animais , Descoberta de Drogas , Ensaios de Triagem em Larga Escala , Humanos , Cinetocoros/efeitos dos fármacos , Cinetocoros/enzimologia , Peptídeos/química , Fosfotransferases/química , Fosfotransferases/genética , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Estaurosporina/farmacologia , Trypanosoma brucei brucei/enzimologia , Tripanossomíase Africana/parasitologia , Zearalenona/análogos & derivados , Zearalenona/farmacologia
9.
Mol Cell ; 74(5): 1086-1102.e5, 2019 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-31101498

RESUMO

Kinase and phosphatase overexpression drives tumorigenesis and drug resistance. We previously developed a mass-cytometry-based single-cell proteomics approach that enables quantitative assessment of overexpression effects on cell signaling. Here, we applied this approach in a human kinome- and phosphatome-wide study to assess how 649 individually overexpressed proteins modulated cancer-related signaling in HEK293T cells in an abundance-dependent manner. Based on these data, we expanded the functional classification of human kinases and phosphatases and showed that the overexpression effects include non-catalytic roles. We detected 208 previously unreported signaling relationships. The signaling dynamics analysis indicated that the overexpression of ERK-specific phosphatases sustains proliferative signaling. This suggests a phosphatase-driven mechanism of cancer progression. Moreover, our analysis revealed a drug-resistant mechanism through which overexpression of tyrosine kinases, including SRC, FES, YES1, and BLK, induced MEK-independent ERK activation in melanoma A375 cells. These proteins could predict drug sensitivity to BRAF-MEK concurrent inhibition in cells carrying BRAF mutations.


Assuntos
Carcinogênese/genética , Melanoma/genética , Monoéster Fosfórico Hidrolases/genética , Fosfotransferases/genética , Proteínas Proto-Oncogênicas B-raf/genética , Proliferação de Células/genética , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Melanoma/enzimologia , Melanoma/patologia , Mutação , Fosforilação/genética , Inibidores de Proteínas Quinases/farmacologia , Proteômica , Transdução de Sinais/efeitos dos fármacos
10.
Infect Immun ; 87(7)2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31036600

RESUMO

Enterococcus faecalis strains are resident intestinal bacteria associated with invasive infections, inflammatory bowel diseases, and colon cancer. Although factors promoting E. faecalis colonization of intestines are not fully known, one implicated pathway is a phosphotransferase system (PTS) in E. faecalis strain OG1RF that phosphorylates gluconate and contains the genes OG1RF_12399 to OG1RF_12402 (OG1RF_12399-12402). We hypothesize that this PTS permits growth in gluconate, facilitates E. faecalis intestinal colonization, and exacerbates colitis. We generated E. faecalis strains containing deletions/point mutations in this PTS and measured bacterial growth and PTS gene expression in minimal medium supplemented with selected carbohydrates. We show that E. faecalis upregulates OG1RF_12399 transcription specifically in the presence of gluconate and that E. faecalis strains lacking, or harboring a single point mutation in, OG1RF_12399-12402 are unable to grow in minimal medium containing gluconate. We colonized germfree wild-type and colitis-prone interleukin-10-deficient mice with defined bacterial consortia containing the E. faecalis strains and measured inflammation and bacterial abundance in the colon. We infected macrophage and intestinal epithelial cell lines with the E. faecalis strains and measured intracellular bacterial survival and proinflammatory cytokine secretion. The presence of OG1RF_12399-12402 is not required for E. faecalis colonization of the mouse intestine but is associated with an accelerated onset of experimental colitis in interleukin-10-deficient mice, altered bacterial composition in the colon, enhanced E. faecalis survival within macrophages, and increased proinflammatory cytokine secretion by colon tissue and macrophages. Further studies of bacterial carbohydrate metabolism in general, and E. faecalis PTS-gluconate in particular, during inflammation may identify new mechanisms of disease pathogenesis.


Assuntos
Proteínas de Bactérias/metabolismo , Colite/microbiologia , Enterococcus faecalis/enzimologia , Macrófagos/imunologia , Fosfotransferases/metabolismo , Animais , Proteínas de Bactérias/genética , Colite/genética , Colite/imunologia , Enterococcus faecalis/genética , Enterococcus faecalis/crescimento & desenvolvimento , Feminino , Gluconatos/metabolismo , Humanos , Interleucina-10/genética , Interleucina-10/imunologia , Intestinos/imunologia , Intestinos/microbiologia , Macrófagos/microbiologia , Masculino , Camundongos , Óperon , Fosfotransferases/genética
11.
mBio ; 10(3)2019 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-31088917

RESUMO

Actinobacteria have long been the main source of antibiotics, secondary metabolites with tightly controlled biosynthesis by environmental and physiological factors. Phosphorylation of exogenous glucosamine has been suggested as a mechanism for incorporation of this extracellular material into secondary metabolite biosynthesis, but experimental evidence of specific glucosamine kinases in Actinobacteria is lacking. Here, we present the molecular fingerprints for the identification of a unique family of actinobacterial glucosamine kinases. Structural and biochemical studies on a distinctive kinase from the soil bacterium Streptacidiphilus jiangxiensis unveiled its preference for glucosamine and provided structural evidence of a phosphoryl transfer to this substrate. Conservation of glucosamine-contacting residues across a large number of uncharacterized actinobacterial proteins unveiled a specific glucosamine binding sequence motif. This family of kinases and their genetic context may represent the missing link for the incorporation of environmental glucosamine into the antibiotic biosynthesis pathways in Actinobacteria and can be explored to enhance antibiotic production.IMPORTANCE The discovery of novel enzymes involved in antibiotic biosynthesis pathways is currently a topic of utmost importance. The high levels of antibiotic resistance detected worldwide threaten our ability to combat infections and other 20th-century medical achievements, namely, organ transplantation or cancer chemotherapy. We have identified and characterized a unique family of enzymes capable of phosphorylating glucosamine to glucosamine-6-phosphate, a crucial molecule directly involved in the activation of antibiotic production pathways in Actinobacteria, nature's main source of antimicrobials. The consensus sequence identified for these glucosamine kinases will help establish a molecular fingerprint to reveal yet-uncharacterized sequences in antibiotic producers, which should have an important impact in biotechnological and biomedical applications, including the enhancement and optimization of antibiotic production.


Assuntos
Actinobacteria/enzimologia , Actinobacteria/genética , Glucosamina/análogos & derivados , Glucose-6-Fosfato/análogos & derivados , Fosfotransferases/genética , Fosfotransferases/metabolismo , Antibacterianos/biossíntese , Impressões Digitais de DNA , Glucosamina/metabolismo , Glucose-6-Fosfato/metabolismo , Fosforilação , Ligação Proteica , RNA Ribossômico 16S/genética , Microbiologia do Solo
12.
Nat Commun ; 10(1): 1919, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-31015472

RESUMO

Bacteria of the genera Pseudomonas and Bacillus can promote plant growth and protect plants from pathogens. However, the interactions between these plant-beneficial bacteria are understudied. Here, we explore the interaction between Bacillus subtilis 3610 and Pseudomonas chlororaphis PCL1606. We show that the extracellular matrix protects B. subtilis colonies from infiltration by P. chlororaphis. The absence of extracellular matrix results in increased fluidity and loss of structure of the B. subtilis colony. The P. chlororaphis type VI secretion system (T6SS) is activated upon contact with B. subtilis cells, and stimulates B. subtilis sporulation. Furthermore, we find that B. subtilis sporulation observed prior to direct contact with P. chlororaphis is mediated by histidine kinases KinA and KinB. Finally, we demonstrate the importance of the extracellular matrix and the T6SS in modulating the coexistence of the two species on melon plant leaves and seeds.


Assuntos
Bacillus subtilis/genética , Cucurbitaceae/microbiologia , Matriz Extracelular/metabolismo , Regulação Bacteriana da Expressão Gênica , Interações Microbianas/genética , Pseudomonas chlororaphis/genética , Bacillus subtilis/crescimento & desenvolvimento , Bacillus subtilis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Contagem de Colônia Microbiana , Fosfotransferases/genética , Fosfotransferases/metabolismo , Folhas de Planta/microbiologia , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Pseudomonas chlororaphis/crescimento & desenvolvimento , Pseudomonas chlororaphis/metabolismo , Sementes/microbiologia , Esporos Bacterianos/genética , Esporos Bacterianos/crescimento & desenvolvimento , Esporos Bacterianos/metabolismo , Simbiose/fisiologia , Sistemas de Secreção Tipo VI/genética , Sistemas de Secreção Tipo VI/metabolismo
13.
Plant Sci ; 280: 348-354, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30824014

RESUMO

This work reports the molecular cloning and heterologous expression of the genes coding for α and ß subunits of pyrophosphate-dependent phosphofructokinase (PPi-PFK) from orange. When expressed individually, both recombinant subunits were produced as highly purified monomeric proteins able to phosphorylate fructose-6-phosphate at the expenses of PPi (specific activity of 0.075 and 0.017 units. mg-1 for α and ß subunits, respectively). On the other hand, co-expression rendered a α3ß3 hexamer with specific activity three orders of magnitude higher than the single subunits. All the conformations of the enzyme were characterized with respect to its kinetic properties and sensitivity to the regulator fructose-2,6-bisphosphate. A thorough review of current knowledge on the matter indicates that this is the first report of the recombinant production of active plant PPi-PFK and the characterization of its different conformations. This is a main contribution for future studies focused to better understand the enzyme properties and how it accomplishes its relevant role in plant metabolism.


Assuntos
Citrus sinensis/enzimologia , Fosfofrutoquinases/metabolismo , Fosfotransferases/metabolismo , Citrus sinensis/genética , Clonagem Molecular , Difosfatos/metabolismo , Frutosedifosfatos/metabolismo , Frutosefosfatos/metabolismo , Expressão Gênica , Cinética , Complexos Multiproteicos , Fosfofrutoquinases/genética , Fosforilação , Fosfotransferases/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas Recombinantes
14.
Food Funct ; 10(3): 1736-1746, 2019 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-30855043

RESUMO

This study evaluated the possible prebiotic effects of dietary fucosylated chondroitin sulfate from Acaudina molpadioides (Am-CHS) on the modulation of the gut microbiota and the improvement in the risk factors for chronic inflammation in high fat diet-fed mice. The results showed that the Am-CHS treatment greatly modified the gut microbiota, including the decrease in Bacteroidetes, increase in Firmicutes, elevation in Lactobacillus (intestinal barrier protector) and short chain fatty acid (SCFA)-producing bacteria (Lactobacillus, Bifidobacterium, and Lachnospiraceae NK4A136 group), and reduction in the lipopolysaccharide (LPS) producer (Escherichia coli). This modulation inhibited inflammatory response, manifesting the decreases in circulating proinflammatory cytokines and their mRNA expression, and the increases in interleukin-10. Dietary Am-CHS caused reductions in serum and fecal LPS concentrations and inhibition of transcription of toll-like receptor 4 (TLR4) and its downstream proteins. In addition, there were increases in the portal levels of fecal SCFAs, which probably contributed to an increase in the adenosine monophosphate-activated protein kinase (AMPK) protein in Am-CHS-treated mice. These results suggest that modulation of gut microbiota by Am-CHS can improve chronic inflammation by reducing LPS levels and TLR4 signaling. Modulation also appears to increase the levels of fecal SCFAs, which activates AMPK and finally leads to inflammation resistance.


Assuntos
Sulfatos de Condroitina/farmacologia , Microbioma Gastrointestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Intestinos/efeitos dos fármacos , Obesidade/induzido quimicamente , Pepinos-do-Mar/química , Animais , Sulfatos de Condroitina/química , Citocinas/genética , Citocinas/metabolismo , DNA Bacteriano/química , DNA Bacteriano/genética , Proteínas de Escherichia coli/classificação , Proteínas de Escherichia coli/genética , Ácidos Graxos Voláteis/química , Ácidos Graxos Voláteis/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Inflamação/tratamento farmacológico , Lipopolissacarídeos/química , Lipopolissacarídeos/metabolismo , Masculino , Proteínas de Membrana/classificação , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/tratamento farmacológico , Fosfotransferases/classificação , Fosfotransferases/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
15.
mBio ; 10(1)2019 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-30782654

RESUMO

The widely conserved LytR-CpsA-Psr (LCP) family of enzymes in Gram-positive bacteria is known to attach glycopolymers, including wall teichoic acid, to the cell envelope. However, it is undetermined if these enzymes are capable of catalyzing glycan attachment to surface proteins. In the actinobacterium Actinomyces oris, an LCP homolog here named LcpA is genetically linked to GspA, a glycoprotein that is covalently attached to the bacterial peptidoglycan by the housekeeping sortase SrtA. Here we show by X-ray crystallography that LcpA adopts an α-ß-α structural fold, akin to the conserved LCP domain, which harbors characteristic catalytic arginine residues. Consistently, alanine substitution for these residues, R149 and R266, abrogates GspA glycosylation, leading to accumulation of an intermediate form termed GspALMM, which is also observed in the lcpA mutant. Unlike other LCP proteins characterized to date, LcpA contains a stabilizing disulfide bond, mutations of which severely affect LcpA stability. In line with the established role of disulfide bond formation in oxidative protein folding in A. oris, deletion of vkor, coding for the thiol-disulfide oxidoreductase VKOR, also significantly reduces LcpA stability. Biochemical studies demonstrated that the recombinant LcpA enzyme possesses pyrophosphatase activity, enabling hydrolysis of diphosphate bonds. Furthermore, this recombinant enzyme, which weakly interacts with GspA in solution, catalyzes phosphotransfer to GspALMM Altogether, the findings support that A. oris LcpA is an archetypal LCP enzyme that glycosylates a cell wall-anchored protein, a process that may be conserved in Actinobacteria, given the conservation of LcpA and GspA in these high-GC-content organisms.IMPORTANCE In Gram-positive bacteria, the conserved LCP family enzymes studied to date are known to attach glycopolymers, including wall teichoic acid, to the cell envelope. It is unknown if these enzymes catalyze glycosylation of surface proteins. We show here in the actinobacterium Actinomyces oris by X-ray crystallography and biochemical analyses that A. oris LcpA is an LCP homolog, possessing pyrophosphatase and phosphotransferase activities known to belong to LCP enzymes that require conserved catalytic Arg residues, while harboring a unique disulfide bond critical for protein stability. Importantly, LcpA mediates glycosylation of the surface protein GspA via phosphotransferase activity. Our studies provide the first experimental evidence of an archetypal LCP enzyme that promotes glycosylation of a cell wall-anchored protein in Gram-positive bacteria.


Assuntos
Actinomyces/enzimologia , Proteínas de Bactérias/metabolismo , Proteínas de Choque Térmico/metabolismo , Fosfotransferases/química , Fosfotransferases/metabolismo , Substituição de Aminoácidos , Domínio Catalítico , Cristalografia por Raios X , Análise Mutacional de DNA , Glicosilação , Modelos Moleculares , Fosfotransferases/genética , Conformação Proteica
16.
Mol Microbiol ; 111(5): 1367-1381, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30767351

RESUMO

Heme is an essential cofactor and alternative iron source for almost all bacterial species but may cause severe toxicity upon elevated levels and consequently, regulatory mechanisms coordinating heme homeostasis represent an important fitness trait. A remarkable scenario is found in several corynebacterial species, e.g. Corynebacterium glutamicum and Corynebacterium diphtheriae, which dedicate two paralogous, heme-responsive two-component systems, HrrSA and ChrSA, to cope with the Janus nature of heme. Here, we combined experimental reporter profiling with a quantitative mathematical model to understand how this particular regulatory network architecture shapes the dynamic response to heme. Our data revealed an instantaneous activation of the detoxification response (hrtBA) upon stimulus perception and we found that kinase activity of both kinases contribute to this fast onset. Furthermore, instant deactivation of the PhrtBA promoter is achieved by a strong ChrS phosphatase activity upon stimulus decline. While the activation of detoxification response is uncoupled from further factors, heme utilization is additionally governed by the global iron regulator DtxR integrating information on iron availability into the regulatory network. Altogether, our data provide comprehensive insights how TCS cross-regulation and network hierarchy shape the temporal dynamics of detoxification (hrtBA) and utilization (hmuO) as part of a global homeostatic response to heme.


Assuntos
Proteínas de Bactérias/metabolismo , Corynebacterium glutamicum/enzimologia , Regulação Bacteriana da Expressão Gênica , Heme/metabolismo , Fosfotransferases/metabolismo , Transdução de Sinais , Proteínas de Bactérias/genética , Corynebacterium glutamicum/genética , Homeostase , Ferro/metabolismo , Modelos Teóricos , Fosforilação , Fosfotransferases/genética , Regiões Promotoras Genéticas
17.
J Microbiol Biotechnol ; 29(3): 367-372, 2019 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-30661323

RESUMO

Deactivation of aminoglycosides by their modifying enzymes, including a number of aminoglycoside O-phosphotransferases, is the most ubiquitous resistance mechanism in aminoglycoside-resistant pathogens. Nonetheless, in a couple of biosynthetic pathways for gentamicins, fortimicins, and istamycins, phosphorylation of aminoglycosides seems to be a unique and initial step for the creation of a natural defensive structural feature such as a 3',4'- dideoxy scaffold. Our aim was to elucidate the biochemical details on the beginning of these C3',4'-dideoxygenation biosynthetic steps for aminoglycosides. The biosynthesis of istamycins must surely involve these 3',4'-didehydroxylation steps, but much less has been reported in terms of characterization of istamycin biosynthetic genes, especially about the phosphotransferase-encoding gene. In the disruption and complementation experiments pointing to a putative gene, istP, in the genome of wild-type Streptomyces tenjimariensis, the function of the istP gene was proved here to be a phosphotransferase. Next, an in-frame deletion of a known phosphotransferase-encoding gene forP from the genome of wild-type Micromonospora olivasterospora resulted in the appearance of a hitherto unidentified fortimicin shunt product, namely 3-O-methyl-FOR-KK1, whereas complementation of forP restored the natural fortimicin metabolite profiles. The bilateral complementation of an istP gene (or forP) in the ΔforP mutant ( or ΔistP mutant strain) successfully restored the biosynthesis of 3',4'- dideoxy fortimicins and istamycins , thus clearly indicating that they are interchangeable launchers of the biosynthesis of 3',4'-dideoxy types of 1,4-diaminocyclitol antibiotics.


Assuntos
Aminoglicosídeos/biossíntese , Antibacterianos/biossíntese , Vias Biossintéticas/genética , Vias Biossintéticas/fisiologia , Genes Bacterianos/genética , Fosfotransferases/genética , Sequência de Aminoácidos , Aminoglicosídeos/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Nucleotídeos de Desoxiguanina/biossíntese , Nucleotídeos de Desoxiguanina/genética , Didesoxinucleotídeos/biossíntese , Didesoxinucleotídeos/genética , Gentamicinas/biossíntese , Micromonospora/genética , Micromonospora/metabolismo , Alinhamento de Sequência , Streptomyces/genética , Streptomyces/metabolismo
18.
Fungal Biol ; 123(1): 87-93, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30654961

RESUMO

Mosquitoes are the most important medical species by transmitting some of deadly infectious diseases to human. In recent years extensive studies of vector control have been focused on biological control agents due to the grave issue raised by continuous application of chemical compounds. Pythium guiyangense X.Q. Su was first isolated from infected larvae of Aedes albopictus in 2006 in China and it has been proven to be a promising mosquito control agent. However, the molecular mechanisms of this oomycete pathogenic to mosquitoes are still not clear. In this study, we identified a new gene from the genome of P. guiyangense, PgAGC1 that belongs to the AGC kinase group and we found that the transcriptional expression levels of this gene were significantly up-regulated during infection of mosquito Culex pipiens pallens. Disruption of the PgAGC1gene via genetic transformation methods affects colony growth and stress responses and results in reduced mortality and infection rates. All the evidence revealed that, besides its role in growth and stress resistance, PgAGC1 is putative determinants of P. guiyangense virulence. The results of this study become of particular importance in understanding the mechanisms of oomycete-mosquito interactions.


Assuntos
Culex/microbiologia , Fosfotransferases/metabolismo , Pythium/enzimologia , Pythium/crescimento & desenvolvimento , Fatores de Virulência/metabolismo , Animais , Perfilação da Expressão Gênica , Técnicas de Inativação de Genes , Fosforilação , Fosfotransferases/genética , Processamento de Proteína Pós-Traducional , Pythium/patogenicidade , Transcrição Genética
19.
IEEE Trans Cybern ; 49(5): 1932-1943, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-29993676

RESUMO

Class labels are required for supervised learning but may be corrupted or missing in various applications. In binary classification, for example, when only a subset of positive instances is labeled whereas the remaining are unlabeled, positive-unlabeled (PU) learning is required to model from both positive and unlabeled data. Similarly, when class labels are corrupted by mislabeled instances, methods are needed for learning in the presence of class label noise (LN). Here we propose adaptive sampling (AdaSampling), a framework for both PU learning and learning with class LN. By iteratively estimating the class mislabeling probability with an adaptive sampling procedure, the proposed method progressively reduces the risk of selecting mislabeled instances for model training and subsequently constructs highly generalizable models even when a large proportion of mislabeled instances is present in the data. We demonstrate the utilities of proposed methods using simulation and benchmark data, and compare them to alternative approaches that are commonly used for PU learning and/or learning with LN. We then introduce two novel bioinformatics applications where AdaSampling is used to: 1) identify kinase-substrates from mass spectrometry-based phosphoproteomics data and 2) predict transcription factor target genes by integrating various next-generation sequencing data.


Assuntos
Biologia Computacional/métodos , Aprendizado de Máquina , Proteínas , Algoritmos , Modelos Estatísticos , Fosfoproteínas/química , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosfotransferases/química , Fosfotransferases/genética , Fosfotransferases/metabolismo , Proteínas/química , Proteínas/genética , Proteínas/metabolismo , Fatores de Transcrição
20.
J Cell Physiol ; 234(3): 2984-2996, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30058720

RESUMO

The extracellular matrix (ECM) physically supports cells and influences stem cell behaviour, modulating kinase-mediated signalling cascades. Cell-derived ECMs have emerged in bone regeneration as they reproduce physiological tissue-architecture and ameliorate mesenchymal stromal cell (MSC) properties. Titanium scaffolds show good mechanical properties, facilitate cell adhesion, and have been routinely used for bone tissue engineering (BTE). We analyzed the kinomic signature of human MSCs in adhesion to an osteopromotive osteoblast-derived ECM, and compared it to MSCs on titanium. PamChip kinase-array analysis revealed 63 phosphorylated peptides on ECM and 59 on titanium, with MSCs on ECM exhibiting significantly higher kinase activity than on titanium. MSCs on the two substrates showed overlapping kinome profiles, with activation of similar signalling pathways (FAK, ERK, and PI3K signalling). Inhibition of PI3K signalling in cells significantly reduced adhesion to ECM and increased the number of nonadherent cells on both substrates. In summary, this study comprehensively characterized the kinase activity in MSCs on cell-derived ECM and titanium, highlighting the role of PI3K signalling in kinomic changes regulating osteoblast viability and adhesion. Kinome profile analysis represents a powerful tool to select pathways to better understand cell behaviour. Osteoblast-derived ECM could be further investigated as titanium scaffold-coating to improve BTE.


Assuntos
Regeneração Óssea/genética , Matriz Extracelular/genética , Osteogênese/genética , Fosfotransferases/genética , Osso e Ossos/efeitos dos fármacos , Osso e Ossos/metabolismo , Adesão Celular/genética , Diferenciação Celular/genética , Proliferação de Células/genética , Humanos , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/metabolismo , Engenharia Tecidual , Titânio/farmacologia
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