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1.
GM Crops Food ; 12(1): 47-56, 2021 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-32862762

RESUMEN

The sugarcane (Saccharum X officinarum) is one of the most important crops used to produce sugar and raw material for biofuel in the world. One of the main causes for sucrose content and yield losses is the attack by insect. In this investigation, cry1Ac gene was introduced into sugarcane variety GT54-9(C9) using the Agrobacterium tumefaciens transformation method for transgenic sugarcane production presenting insect-resistance. The A. tumefaciens strain GV1303 including pARTcry1Ac vector was used for the production of transformed sugarcane. The Bacillus thuringiensis cry gene were successfully used to produce transgenic plants used for the improvement of both agronomic efficiency and product quality by acquiring insect resistance. PCR and Southern hybridization techniques were used to confirm the cry1Ac gene incorporation into sugarcane genome. Transformation percentage was 22.2% using PCR analysis with specific primers for cry1Ac and npt-II (Neomycin phosphotransferase) genes. The expression of cry1Ac gene was determined using reverse transcriptase polymerase chain reaction (RT-PCR), QuickStix test, and insect bioassays. Bioassays for transformed sugarcane plants showed high level of toxicity to Sesamia cretica giving 100% mortality of the larvae. Sugarcane insect resistance was improved significantly by using cry1Ac gene transformation.


Asunto(s)
Saccharum/genética , Agrobacterium , Animales , Proteínas Bacterianas/genética , Endotoxinas , Proteínas Hemolisinas/genética , Plantas Modificadas Genéticamente
2.
GM Crops Food ; 12(1): 1-17, 2021 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-32762312

RESUMEN

A biophysical survey was conducted in 15 cotton-growing districts of Pakistan. Four hundred cotton growers were approached and inquired about the production technology of Bt cotton. Further, 25 strip tests using combo strips (Cry1Ac, Cry2Ab, Vip3Aa and Cp4, EPSPS gene) were performed at each farmer's field. Out of 10,000 total-tested samples, farmers claimed 9682 samples as Bt and 318 samples as non-Bt. After performing a strip test, 1009 and 87 samples were found false negative and false positive, respectively. Only 53 samples were found positive for Cry2Ab, 214 for EPSPS and none for Vip3Aa gene. Quantification of Cry endotoxin and bioassay studies were performed by taking leaves from upper, middle, and lower canopies, and fruiting parts at approximately 80 days after sowing from 89 varieties. Expression was highly variable among different canopies and fruiting parts. Moreover, Cry endotoxin expression and insect mortality varied significantly among varieties from 0.26 µg g-1 to 3.54 µg g-1 with mortality ranging from 28 to 97%, respectively. Highest Cry1Ac expression (3.54 µg g-1) and insect mortality (97%) were observed for variety FH-142 from DG Khan. Cry endotoxin expression varied significantly across various plant parts, i.e., IUB-13 variety from upper canopy documented 0.34 µg g-1 expression with 37% insect mortality in Layyah to 3.42 µg g-1 expression and 96% insect mortality from DG Khan. Lethal dose, LD95 (2.20 µg g-1) of Cry1Ac endotoxin was optimized for effective control of H. armigera. Our results provided evidence of practical resistance in H. armigera and way forward.


Asunto(s)
Proteínas Hemolisinas/genética , Mariposas Nocturnas , Animales , Proteínas Bacterianas/genética , Endotoxinas , Gossypium , Resistencia a los Insecticidas , Larva , Pakistán , Plantas Modificadas Genéticamente
3.
Wei Sheng Yan Jiu ; 49(4): 569-573, 2020 Jul.
Artículo en Chino | MEDLINE | ID: mdl-32928347

RESUMEN

OBJECTIVE: To evaluate the effects of genetically modified maize with Cry1Ab and epsps genes on immune function in F3 rats. METHODS: A total of 180 weaning SD rats for F0 generation were randomly divided into three groups, which were treated with AIN-93 G feed control diet, parental maize diet and genetically modified maize diet respectively. After three generations of breeding, antibody producing cells determination, concanavalin A(ConA)-induced lymphocyte transformation test, natural killer(NK)cells activities assay, whole blood lymphocyte subtype detection, delayed type hypersensitivity test and immunity organ index were performed. RESULTS: There were no significant differences between parental maize diet and genetically modified maize diet in terms of the number of antibody-producing cells, ConA-induced spleen lymphocyte proliferation, NK cell activity, whole blood lymphocyte subsets, delayed type hypersensitivity and thymus index(P>0. 05). CONCLUSION: Under the conditions of this experiment, no significant effects were found on immune function of F3 SD rats through the three generation development study of genetically modified maize with CrylAb and epsps genes.


Asunto(s)
Alimentos Modificados Genéticamente , Zea mays/genética , Animales , Proteínas Bacterianas/genética , Endotoxinas , Proteínas Hemolisinas/genética , Plantas Modificadas Genéticamente , Ratas , Ratas Sprague-Dawley
4.
PLoS Pathog ; 16(8): e1008822, 2020 08.
Artículo en Inglés | MEDLINE | ID: mdl-32866204

RESUMEN

Shigella flexneri invades host cells by entering within a bacteria-containing vacuole (BCV). In order to establish its niche in the host cytosol, the bacterium ruptures its BCV. Contacts between S. flexneri BCV and infection-associated macropinosomes (IAMs) formed in situ have been reported to enhance BCV disintegration. The mechanism underlying S. flexneri vacuolar escape remains however obscure. To decipher the molecular mechanism priming the communication between the IAMs and S. flexneri BCV, we performed mass spectrometry-based analysis of the magnetically purified IAMs from S. flexneri-infected cells. While proteins involved in host recycling and exocytic pathways were significantly enriched at the IAMs, we demonstrate more precisely that the S. flexneri type III effector protein IpgD mediates the recruitment of the exocyst to the IAMs through the Rab8/Rab11 pathway. This recruitment results in IAM clustering around S. flexneri BCV. More importantly, we reveal that IAM clustering subsequently facilitates an IAM-mediated unwrapping of the ruptured vacuole membranes from S. flexneri, enabling the naked bacterium to be ready for intercellular spread via actin-based motility. Taken together, our work untangles the molecular cascade of S. flexneri-driven host trafficking subversion at IAMs to develop its cytosolic lifestyle, a crucial step en route for infection progression at cellular and tissue level.


Asunto(s)
Disentería Bacilar , Shigella flexneri , Transducción de Señal , Vacuolas , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Disentería Bacilar/genética , Disentería Bacilar/metabolismo , Células HeLa , Humanos , Shigella flexneri/genética , Shigella flexneri/metabolismo , Shigella flexneri/patogenicidad , Vacuolas/genética , Vacuolas/metabolismo , Vacuolas/microbiología , Factores de Virulencia/genética , Factores de Virulencia/metabolismo , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/metabolismo
5.
Nat Commun ; 11(1): 4648, 2020 09 16.
Artículo en Inglés | MEDLINE | ID: mdl-32938927

RESUMEN

Emergence of tigecycline-resistance tet(X) gene orthologues rendered tigecycline ineffective as last-resort antibiotic. To understand the potential origin and transmission mechanisms of these genes, we survey the prevalence of tet(X) and its orthologues in 2997 clinical E. coli and K. pneumoniae isolates collected nationwide in China with results showing very low prevalence on these two types of strains, 0.32% and 0%, respectively. Further surveillance of tet(X) orthologues in 3692 different clinical Gram-negative bacterial strains collected during 1994-2019 in hospitals in Zhejiang province, China reveals 106 (2.7%) tet(X)-bearing strains with Flavobacteriaceae being the dominant (97/376, 25.8%) bacteria. In addition, tet(X)s are found to be predominantly located on the chromosomes of Flavobacteriaceae and share similar GC-content as Flavobacteriaceae. It also further evolves into different orthologues and transmits among different species. Data from this work suggest that Flavobacteriaceae could be the potential ancestral source of the tigecycline resistance gene tet(X).


Asunto(s)
Antibacterianos/farmacología , Proteínas Bacterianas/genética , Farmacorresistencia Bacteriana/genética , Infecciones por Flavobacteriaceae/epidemiología , Flavobacteriaceae/genética , Tigeciclina/farmacología , China/epidemiología , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Evolución Molecular , Flavobacteriaceae/efectos de los fármacos , Flavobacteriaceae/aislamiento & purificación , Infecciones por Flavobacteriaceae/microbiología , Humanos , Filogenia
6.
PLoS Pathog ; 16(9): e1008871, 2020 09.
Artículo en Inglés | MEDLINE | ID: mdl-32936831

RESUMEN

Deconvolution of syphilis pathogenesis and selection of candidate syphilis vaccinogens requires detailed knowledge of the molecular architecture of the Treponema pallidum outer membrane (OM). The T. pallidum OM contains a low density of integral OM proteins, while the spirochete's many lipoprotein immunogens are periplasmic. TP0751, a lipoprotein with a lipocalin fold, is reportedly a surface-exposed protease/adhesin and protective antigen. The rapid expansion of calycin/lipocalin structures in the RCSB PDB database prompted a comprehensive reassessment of TP0751. Small angle X-ray scattering analysis of full-length protein revealed a bipartite topology consisting of an N-terminal, intrinsically disordered region (IDR) and the previously characterized C-terminal lipocalin domain. A DALI server query using the lipocalin domain yielded 97 hits, 52 belonging to the calycin superfamily, including 15 bacterial lipocalins, but no Gram-negative surface proteins. Surprisingly, Tpp17 (TP0435) was identified as a structural ortholog of TP0751. In silico docking predicted that TP0751 can bind diverse ligands along the rim of its eight-stranded ß-barrel; high affinity binding of one predicted ligand, heme, to the lipocalin domain was demonstrated. qRT-PCR and immunoblotting revealed very low expression of TP0751 compared to other T. pallidum lipoproteins. Immunoblot analysis of immune rabbit serum failed to detect TP0751 antibodies, while only one of five patients with secondary syphilis mounted a discernible TP0751-specific antibody response. In opsonophagocytosis assays, neither TP0751 nor Tpp17 antibodies promoted uptake of T. pallidum by rabbit peritoneal macrophages. Rabbits immunized with intact, full-length TP0751 showed no protection against local or disseminated infection following intradermal challenge with T. pallidum. Our data argue that, like other lipoprotein lipocalins in dual-membrane bacteria, TP0751 is periplasmic and binds small molecules, and we propose that its IDR facilitates ligand binding by and offloading from the lipocalin domain. The inability of TP0751 to elicit opsonic or protective antibodies is consistent with a subsurface location.


Asunto(s)
Proteínas Bacterianas/inmunología , Vacunas Bacterianas/inmunología , Inmunización , Lipoproteínas/inmunología , Sífilis/inmunología , Treponema pallidum/inmunología , Animales , Proteínas Bacterianas/genética , Vacunas Bacterianas/genética , Humanos , Lipoproteínas/genética , Dominios Proteicos , Pliegue de Proteína , Conejos , Sífilis/genética , Sífilis/patología , Sífilis/prevención & control , Treponema pallidum/genética , Treponema pallidum/patogenicidad
7.
PLoS Pathog ; 16(9): e1008878, 2020 09.
Artículo en Inglés | MEDLINE | ID: mdl-32946535

RESUMEN

As an obligate intracellular pathogen, host cell invasion is paramount to Chlamydia trachomatis proliferation. While the mechanistic underpinnings of this essential process remain ill-defined, it is predicted to involve delivery of prepackaged effector proteins into the host cell that trigger plasma membrane remodeling and cytoskeletal reorganization. The secreted effector proteins TmeA and TarP, have risen to prominence as putative key regulators of cellular invasion and bacterial pathogenesis. Although several studies have begun to unravel molecular details underlying the putative function of TarP, the physiological function of TmeA during host cell invasion is unknown. Here, we show that TmeA employs molecular mimicry to bind to the GTPase binding domain of N-WASP, which results in recruitment of the actin branching ARP2/3 complex to the site of chlamydial entry. Electron microscopy revealed that TmeA mutants are deficient in filopodia capture, suggesting that TmeA/N-WASP interactions ultimately modulate host cell plasma membrane remodeling events necessary for chlamydial entry. Importantly, while both TmeA and TarP are necessary for effective host cell invasion, we show that these effectors target distinct pathways that ultimately converge on activation of the ARP2/3 complex. In line with this observation, we show that a double mutant suffers from a severe entry defect nearly identical to that observed when ARP3 is chemically inhibited or knocked down. Collectively, our study highlights both TmeA and TarP as essential regulators of chlamydial invasion that modulate the ARP2/3 complex through distinct signaling platforms, resulting in plasma membrane remodeling events that are essential for pathogen uptake.


Asunto(s)
Proteínas Bacterianas , Membrana Celular/metabolismo , Chlamydia trachomatis , Proteína Neuronal del Síndrome de Wiskott-Aldrich/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/genética , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Membrana Celular/genética , Membrana Celular/patología , Chlamydia trachomatis/genética , Chlamydia trachomatis/metabolismo , Chlamydia trachomatis/patogenicidad , Células HeLa , Humanos , Mutación , Dominios Proteicos , Seudópodos/genética , Seudópodos/metabolismo , Proteína Neuronal del Síndrome de Wiskott-Aldrich/genética
8.
PLoS Pathog ; 16(9): e1008852, 2020 09.
Artículo en Inglés | MEDLINE | ID: mdl-32960931

RESUMEN

Enzymatic inactivation of Rho-family GTPases by the glucosyltransferase domain of Clostridioides difficile Toxin B (TcdB) gives rise to various pathogenic effects in cells that are classically thought to be responsible for the disease symptoms associated with C. difficile infection (CDI). Recent in vitro studies have shown that TcdB can, under certain circumstances, induce cellular toxicities that are independent of glucosyltransferase (GT) activity, calling into question the precise role of GT activity. Here, to establish the importance of GT activity in CDI disease pathogenesis, we generated the first described mutant strain of C. difficile producing glucosyltransferase-defective (GT-defective) toxin. Using allelic exchange (AE) technology, we first deleted tcdA in C. difficile 630Δerm and subsequently introduced a deactivating D270N substitution in the GT domain of TcdB. To examine the role of GT activity in vivo, we tested each strain in two different animal models of CDI pathogenesis. In the non-lethal murine model of infection, the GT-defective mutant induced minimal pathology in host tissues as compared to the profound caecal inflammation seen in the wild-type and 630ΔermΔtcdA (ΔtcdA) strains. In the more sensitive hamster model of CDI, whereas hamsters in the wild-type or ΔtcdA groups succumbed to fulminant infection within 4 days, all hamsters infected with the GT-defective mutant survived the 10-day infection period without primary symptoms of CDI or evidence of caecal inflammation. These data demonstrate that GT activity is indispensable for disease pathogenesis and reaffirm its central role in disease and its importance as a therapeutic target for small-molecule inhibition.


Asunto(s)
Proteínas Bacterianas , Toxinas Bacterianas , Clostridium difficile , Enterocolitis Seudomembranosa , Glucosiltransferasas , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Clostridium difficile/enzimología , Clostridium difficile/genética , Clostridium difficile/patogenicidad , Cricetinae , Modelos Animales de Enfermedad , Enterocolitis Seudomembranosa/enzimología , Enterocolitis Seudomembranosa/genética , Enterocolitis Seudomembranosa/patología , Femenino , Eliminación de Gen , Glucosiltransferasas/genética , Glucosiltransferasas/metabolismo , Masculino , Ratones
9.
Nat Commun ; 11(1): 4817, 2020 09 23.
Artículo en Inglés | MEDLINE | ID: mdl-32968056

RESUMEN

Lysozymes are among the best-characterized enzymes, acting upon the cell wall substrate peptidoglycan. Here, examining the invasive bacterial periplasmic predator Bdellovibrio bacteriovorus, we report a diversified lysozyme, DslA, which acts, unusually, upon (GlcNAc-) deacetylated peptidoglycan. B. bacteriovorus are known to deacetylate the peptidoglycan of the prey bacterium, generating an important chemical difference between prey and self walls and implying usage of a putative deacetyl-specific "exit enzyme". DslA performs this role, and ΔDslA strains exhibit a delay in leaving from prey. The structure of DslA reveals a modified lysozyme superfamily fold, with several adaptations. Biochemical assays confirm DslA specificity for deacetylated cell wall, and usage of two glutamate residues for catalysis. Exogenous DslA, added ex vivo, is able to prematurely liberate B. bacteriovorus from prey, part-way through the predatory lifecycle. We define a mechanism for specificity that invokes steric selection, and use the resultant motif to identify wider DslA homologues.


Asunto(s)
Bdellovibrio bacteriovorus/enzimología , Bdellovibrio bacteriovorus/metabolismo , Muramidasa/química , Muramidasa/metabolismo , Periplasma/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Bdellovibrio bacteriovorus/genética , Pared Celular , Escherichia coli , Regulación Bacteriana de la Expresión Génica , Modelos Moleculares , Muramidasa/genética , Mutación , Peptidoglicano/metabolismo , Fenotipo , Conformación Proteica , Especificidad por Sustrato
10.
PLoS Pathog ; 16(9): e1008552, 2020 09.
Artículo en Inglés | MEDLINE | ID: mdl-32966346

RESUMEN

Type VI secretion systems (T6SSs) are complex macromolecular injection machines which are widespread in Gram-negative bacteria. They are involved in host-cell interactions and pathogenesis, required to eliminate competing bacteria, or are important for the adaptation to environmental stress conditions. Here we identified regulatory elements controlling the T6SS4 of Yersinia pseudotuberculosis and found a novel type of hexameric transcription factor, RovC. RovC directly interacts with the T6SS4 promoter region and activates T6SS4 transcription alone or in cooperation with the LysR-type regulator RovM. A higher complexity of regulation was achieved by the nutrient-responsive global regulator CsrA, which controls rovC expression on the transcriptional and post-transcriptional level. In summary, our work unveils a central mechanism in which RovC, a novel key activator, orchestrates the expression of the T6SS weapons together with a global regulator to deploy the system in response to the availability of nutrients in the species' native environment.


Asunto(s)
Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Regiones Promotoras Genéticas , Sistemas de Secreción Tipo VI/metabolismo , Yersinia pseudotuberculosis/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Conformación Proteica , Estrés Fisiológico , Sistemas de Secreción Tipo VI/química , Sistemas de Secreción Tipo VI/genética , Yersinia pseudotuberculosis/genética
11.
Mol Genet Genomics ; 295(6): 1529-1535, 2020 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-32894358

RESUMEN

Lanthipeptides are a subgroup of ribosomally encoded and post-translationally modified peptides (RiPPs) which frequently possess potent biological activity. Here we provide the first comprehensive bioinformatic analysis of the lanthipeptide-producing capability of the Salinispora genus, a marine actinomycete. One hundred twenty-two Salinispora arenicola, tropica, and pacifica genomic sequences were analyzed for lanthipeptide gene clusters, and the resulting 182 clusters were divided into seven groups based on sequence similarities. Group boundaries were defined based on LanB and LanM sequences with greater than 80% similarity within groups. Of the seven groups, six are predicted to encode class I lanthipeptides while only one group is predicted to encode class II lanthipeptides. Leader and core peptides were predicted for each cluster along with the number of possible lanthionine bridges. Notably, all of the predicted products of these clusters would represent novel lanthipeptide scaffolds. Of the 122 Salinispora genomes analyzed in this study, 92% contained at least one lanthipeptide gene cluster suggesting that Salinispora is a rich, yet untapped, source of lanthipeptides.


Asunto(s)
Alanina/análogos & derivados , Proteínas Bacterianas/metabolismo , Genoma Bacteriano , Micromonosporaceae/metabolismo , Fragmentos de Péptidos/metabolismo , Sulfuros/metabolismo , Alanina/aislamiento & purificación , Alanina/metabolismo , Proteínas Bacterianas/genética , Genómica , Micromonosporaceae/genética , Micromonosporaceae/crecimiento & desarrollo , Fragmentos de Péptidos/aislamiento & purificación , Sulfuros/aislamiento & purificación
12.
PLoS One ; 15(8): e0237883, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32866169

RESUMEN

Although whole-genome sequencing has provided novel insights into Neisseria meningitidis, many open reading frames have only been annotated as hypothetical proteins with unknown biological functions. Our previous genetic analyses revealed that the hypothetical protein, NMB1345, plays a crucial role in meningococcal infection in human brain microvascular endothelial cells; however, NMB1345 has no homology to any identified protein in databases and its physiological function could not be elucidated using pre-existing methods. Among the many biological technologies to examine transient protein-protein interaction in vivo, one of the developed methods is genetic code expansion with non-canonical amino acids (ncAAs) utilizing a pyrrolysyl-tRNA synthetase/tRNAPyl pair from Methanosarcina species: However, this method has never been applied to assign function-unknown proteins in pathogenic bacteria. In the present study, we developed a new method to genetically incorporate ncAAs-encoded photocrosslinking probes into N. meningitidis by utilizing a pyrrolysyl-tRNA synthetase/tRNAPyl pair and elucidated the biological function(s) of the NMB1345 protein. The results revealed that the NMB1345 protein directly interacts with PilE, a major component of meningococcal pili, and further physicochemical and genetic analyses showed that the interaction between the NMB1345 protein and PilE was important for both functional pilus formation and meningococcal infectious ability in N. meningitidis. The present study using this new methodology for N. meningitidis provides novel insights into meningococcal pathogenesis by assigning the function of a hypothetical protein.


Asunto(s)
Aminoácidos/genética , Reactivos de Enlaces Cruzados/metabolismo , Luz , Neisseria meningitidis/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Encéfalo/irrigación sanguínea , Endocitosis , Células Endoteliales/microbiología , Fimbrias Bacterianas/metabolismo , Humanos , Microvasos/patología , Mutación/genética , Plásmidos/genética
13.
Nat Commun ; 11(1): 4501, 2020 09 09.
Artículo en Inglés | MEDLINE | ID: mdl-32908132

RESUMEN

Streptovaricin C is a naphthalenic ansamycin antibiotic structurally similar to rifamycins with potential anti-MRSA bioactivities. However, the formation mechanism of the most fascinating and bioactivity-related methylenedioxy bridge (MDB) moiety in streptovaricins is unclear. Based on genetic and biochemical evidences, we herein clarify that the P450 enzyme StvP2 catalyzes the MDB formation in streptovaricins, with an atypical substrate inhibition kinetics. Furthermore, X-ray crystal structures in complex with substrate and structure-based mutagenesis reveal the intrinsic details of the enzymatic reaction. The mechanism of MDB formation is proposed to be an intramolecular nucleophilic substitution resulting from the hydroxylation by the heme core and the keto-enol tautomerization via a crucial catalytic triad (Asp89-His92-Arg72) in StvP2. In addition, in vitro reconstitution uncovers that C6-O-methylation and C4-O-acetylation of streptovaricins are necessary prerequisites for the MDB formation. This work provides insight for the MDB formation and adds evidence in support of the functional versatility of P450 enzymes.


Asunto(s)
Proteínas Bacterianas/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Streptomyces/metabolismo , Estreptovaricina/análogos & derivados , Acetilación , Proteínas Bacterianas/genética , Proteínas Bacterianas/ultraestructura , Biocatálisis , Cristalografía por Rayos X , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/ultraestructura , Pruebas de Enzimas , Metilación , Mutagénesis Sitio-Dirigida , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestructura , Estreptovaricina/biosíntesis , Estreptovaricina/química , Estreptovaricina/metabolismo
14.
Nat Commun ; 11(1): 4522, 2020 09 09.
Artículo en Inglés | MEDLINE | ID: mdl-32908144

RESUMEN

A unique, protective cell envelope contributes to the broad drug resistance of the nosocomial pathogen Acinetobacter baumannii. Here we use transposon insertion sequencing to identify A. baumannii mutants displaying altered susceptibility to a panel of diverse antibiotics. By examining mutants with antibiotic susceptibility profiles that parallel mutations in characterized genes, we infer the function of multiple uncharacterized envelope proteins, some of which have roles in cell division or cell elongation. Remarkably, mutations affecting a predicted cell wall hydrolase lead to alterations in lipooligosaccharide synthesis. In addition, the analysis of altered susceptibility signatures and antibiotic-induced morphology patterns allows us to predict drug synergies; for example, certain beta-lactams appear to work cooperatively due to their preferential targeting of specific cell wall assembly machineries. Our results indicate that the pathogen may be effectively inhibited by the combined targeting of multiple pathways critical for envelope growth.


Asunto(s)
Infecciones por Acinetobacter/tratamiento farmacológico , Acinetobacter baumannii/genética , Antibacterianos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Infección Hospitalaria/tratamiento farmacológico , Farmacorresistencia Bacteriana Múltiple/genética , Infecciones por Acinetobacter/microbiología , Acinetobacter baumannii/efectos de los fármacos , Antibacterianos/uso terapéutico , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Pared Celular/efectos de los fármacos , Pared Celular/genética , Pared Celular/metabolismo , Infección Hospitalaria/microbiología , Análisis Mutacional de ADN , Elementos Transponibles de ADN/genética , ADN Bacteriano/genética , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Sinergismo Farmacológico , Humanos , Pruebas de Sensibilidad Microbiana , Mutación
15.
Nat Commun ; 11(1): 4097, 2020 08 14.
Artículo en Inglés | MEDLINE | ID: mdl-32796861

RESUMEN

Staphylococcus aureus is generally thought to divide in three alternating orthogonal planes over three consecutive division cycles. Although this mode of division was proposed over four decades ago, the molecular mechanism that ensures this geometry of division has remained elusive. Here we show, for three different strains, that S. aureus cells do not regularly divide in three alternating perpendicular planes as previously thought. Imaging of the divisome shows that a plane of division is always perpendicular to the previous one, avoiding bisection of the nucleoid, which segregates along an axis parallel to the closing septum. However, one out of the multiple planes perpendicular to the septum which divide the cell in two identical halves can be used in daughter cells, irrespective of its orientation in relation to the penultimate division plane. Therefore, division in three orthogonal planes is not the rule in S. aureus.


Asunto(s)
Proteínas Bacterianas/metabolismo , Staphylococcus aureus/citología , Staphylococcus aureus/metabolismo , Proteínas Bacterianas/genética , Microbiología , Imagen de Lapso de Tiempo
16.
Nat Commun ; 11(1): 4126, 2020 08 17.
Artículo en Inglés | MEDLINE | ID: mdl-32807804

RESUMEN

Neisseria gonorrhoeae is an urgent public health threat due to rapidly increasing incidence and antibiotic resistance. In contrast with the trend of increasing resistance, clinical isolates that have reverted to susceptibility regularly appear, prompting questions about which pressures compete with antibiotics to shape gonococcal evolution. Here, we used genome-wide association to identify loss-of-function (LOF) mutations in the efflux pump mtrCDE operon as a mechanism of increased antibiotic susceptibility and demonstrate that these mutations are overrepresented in cervical relative to urethral isolates. This enrichment holds true for LOF mutations in another efflux pump, farAB, and in urogenitally-adapted versus typical N. meningitidis, providing evidence for a model in which expression of these pumps in the female urogenital tract incurs a fitness cost for pathogenic Neisseria. Overall, our findings highlight the impact of integrating microbial population genomics with host metadata and demonstrate how host environmental pressures can lead to increased antibiotic susceptibility.


Asunto(s)
Proteínas Bacterianas/metabolismo , Cuello del Útero/microbiología , Neisseria gonorrhoeae/efectos de los fármacos , Neisseria gonorrhoeae/genética , Animales , Proteínas Bacterianas/genética , Farmacorresistencia Microbiana/genética , Femenino , Regulación Bacteriana de la Expresión Génica , Estudio de Asociación del Genoma Completo , Humanos , Pruebas de Sensibilidad Microbiana , Mutación/genética , Neisseria gonorrhoeae/metabolismo , Operón/genética , Regiones Promotoras Genéticas/genética
17.
Rev Bras Parasitol Vet ; 29(3): e005820, 2020 Aug 03.
Artículo en Inglés | MEDLINE | ID: mdl-32756774

RESUMEN

Ehrlichia canis is the main etiological agent of canine monocytic ehrlichiosis (CME), a globally canine infectious disease. In Brazil, CME is considered to be endemic, and its prevalence can reach 65% in some states. The diagnosis of ehrlichiosis is important for treatment and epidemiological purposes. The E. canis TRP36 (Tandem Repeat Protein) protein elicits the earliest acute-phase antibody response observed during the course of the disease. This study aimed to generate the recombinant TRP36 protein from E. canis São Paulo strain and to evaluate its potential as a tool for the serologic diagnosis of CME. The E. canis São Paulo isolate was cultivated in DH82 lineage cells, and its genomic DNA was obtained. The bacterial DNA fragment encoding the entire ORF of TRP36 was cloned into the pBAD/Thio-TOPO vector and transformed into Escherichia coli DH10B competent cells with the trp36-bearing plasmid for protein expression. To evaluate the protein antigenicity, 16 canine serum samples were previously tested (by PCR and the commercial SNAP®4Dx® serological test). The results were in accordance with the SNAP®4Dx® test. Experiments using this recombinant protein as an antigen, targeting the development of a serologic test based on ELISA methodology, are the next step to produce a reliable, affordable and useful diagnostic tool for CME in Brazil.


Asunto(s)
Proteínas Bacterianas , Enfermedades de los Perros , Ehrlichia canis , Ehrlichiosis/veterinaria , Proteínas Recombinantes , Pruebas Serológicas/veterinaria , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Brasil , Línea Celular , Enfermedades de los Perros/diagnóstico , Perros , Ehrlichia canis/genética , Ehrlichiosis/diagnóstico , Escherichia coli/genética , Expresión Génica , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología
18.
BMC Infect Dis ; 20(1): 602, 2020 Aug 14.
Artículo en Inglés | MEDLINE | ID: mdl-32799799

RESUMEN

BACKGROUND: The objectives of this study were to determine for the first time, in Morocco, the nasal carriage rate, antimicrobial susceptibility profiles and virulence genes of Staphylococcus. aureus isolated from animals and breeders in close contact. METHODS: From 2015 to 2016, 421 nasal swab samples were collected from 26 different livestock areas in Tangier. Antimicrobial susceptibility phenotypes were determined by disk diffusion according to EUCAST 2015. The presence of nuc, mecA, mecC, lukS/F-PV, and tst genes were determined by Polymerase Chain Reaction (PCR) for all isolates. RESULTS: The overall S. aureus nasal carriage rate was low in animals (9.97%) and high in breeders (60%) with a statistically significant difference, (OR = 13.536; 95% CI = 7.070-25.912; p < 0.001). In general, S. aureus strains were susceptible to the majority of antibiotics and the highest resistance rates were found against tetracycline (16.7% in animals and 10% in breeders). No Methicillin-Resistant S. aureus (MRSA) was detected in animals and breeders. A high rate of tst and lukS/F-PV genes has been recovered only from animals (11.9 and 16.7%, respectively). CONCLUSION: Despite the lower rate of nasal carriage of S. aureus and the absence of MRSA strains in our study, S. aureus strains harbored a higher frequency of tst and lukS/F-PV virulence genes, which is associated to an increased risk of infection dissemination in humans. This highlights the need for further larger and multi-center studies to better define the transmission of the pathogenic S. aureus between livestock, environment, and humans.


Asunto(s)
Nariz/microbiología , Infecciones Estafilocócicas/microbiología , Infecciones Estafilocócicas/veterinaria , Staphylococcus aureus/efectos de los fármacos , Animales , Animales Domésticos/microbiología , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Portador Sano , Farmacorresistencia Bacteriana/efectos de los fármacos , Farmacorresistencia Bacteriana/genética , Humanos , Leucocidinas/genética , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Staphylococcus aureus Resistente a Meticilina/aislamiento & purificación , Pruebas de Sensibilidad Microbiana , Nucleasa Microcócica/genética , Marruecos/epidemiología , Proteínas de Unión a las Penicilinas/genética , Infecciones Estafilocócicas/epidemiología , Staphylococcus aureus/genética , Staphylococcus aureus/aislamiento & purificación , Staphylococcus aureus/patogenicidad , Tetraciclina/farmacología , Virulencia/genética
19.
PLoS Biol ; 18(8): e3000762, 2020 08.
Artículo en Inglés | MEDLINE | ID: mdl-32760088

RESUMEN

Centrosomes, the main microtubule organizing centers (MTOCs) of metazoan cells, contain an older "mother" and a younger "daughter" centriole. Stem cells either inherit the mother or daughter-centriole-containing centrosome, providing a possible mechanism for biased delivery of cell fate determinants. However, the mechanisms regulating centrosome asymmetry and biased centrosome segregation are unclear. Using 3D-structured illumination microscopy (3D-SIM) and live-cell imaging, we show in fly neural stem cells (neuroblasts) that the mitotic kinase Polo and its centriolar protein substrate Centrobin (Cnb) accumulate on the daughter centriole during mitosis, thereby generating molecularly distinct mother and daughter centrioles before interphase. Cnb's asymmetric localization, potentially involving a direct relocalization mechanism, is regulated by Polo-mediated phosphorylation, whereas Polo's daughter centriole enrichment requires both Wdr62 and Cnb. Based on optogenetic protein mislocalization experiments, we propose that the establishment of centriole asymmetry in mitosis primes biased interphase MTOC activity, necessary for correct spindle orientation.


Asunto(s)
Proteínas de Ciclo Celular/genética , Centriolos/metabolismo , Centrosoma/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Mitosis , Proteínas Serina-Treonina Quinasas/genética , Animales , Animales Modificados Genéticamente , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Centriolos/ultraestructura , Centrosoma/ultraestructura , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citología , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/metabolismo , Embrión no Mamífero , Regulación del Desarrollo de la Expresión Génica , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Interfase , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Optogenética/métodos , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal
20.
PLoS Biol ; 18(8): e3000790, 2020 08.
Artículo en Inglés | MEDLINE | ID: mdl-32776918

RESUMEN

Concentrative nucleoside transporters (CNTs), members of the solute carrier (SLC) 28 transporter family, facilitate the salvage of nucleosides and therapeutic nucleoside derivatives across the plasma membrane. Despite decades of investigation, the structures of human CNTs remain unknown. We determined the cryogenic electron microscopy (cryo-EM) structure of human CNT (hCNT) 3 at an overall resolution of 3.6 Å. As with its bacterial homologs, hCNT3 presents a trimeric architecture with additional N-terminal transmembrane helices to stabilize the conserved central domains. The conserved binding sites for the substrate and sodium ions unravel the selective nucleoside transport and distinct coupling mechanism. Structural comparison of hCNT3 with bacterial homologs indicates that hCNT3 is stabilized in an inward-facing conformation. This study provides the molecular determinants for the transport mechanism of hCNTs and potentially facilitates the design of nucleoside drugs.


Asunto(s)
Proteínas Bacterianas/química , Proteínas de Transporte de Membrana/química , Uridina/química , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Baculoviridae/genética , Baculoviridae/metabolismo , Sitios de Unión , Transporte Biológico , Clonación Molecular , Microscopía por Crioelectrón , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Humanos , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Modelos Moleculares , Unión Proteica , Conformación Proteica en Hélice alfa , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células Sf9 , Spodoptera , Homología Estructural de Proteína , Especificidad por Sustrato , Uridina/metabolismo
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