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1.
GM Crops Food ; 12(1): 47-56, 2021 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-32862762

RESUMO

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.


Assuntos
Saccharum/genética , Agrobacterium , Animais , Proteínas de Bactérias/genética , Endotoxinas , Proteínas Hemolisinas/genética , Plantas Geneticamente Modificadas
2.
GM Crops Food ; 12(1): 1-17, 2021 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-32762312

RESUMO

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.


Assuntos
Proteínas Hemolisinas/genética , Mariposas , Animais , Proteínas de Bactérias/genética , Endotoxinas , Gossypium , Resistência a Inseticidas , Larva , Paquistão , Plantas Geneticamente Modificadas
3.
Nat Commun ; 11(1): 5065, 2020 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-33033237

RESUMO

The type VI protein secretion system (T6SS) is a powerful needle-like machinery found in Gram-negative bacteria that can penetrate the cytosol of receiving cells in milliseconds by physical force. Anchored by its membrane-spanning complex (MC) and a baseplate (BP), the T6SS sheath-tube is assembled in a stepwise process primed by TssA and terminated by TagA. However, the molecular details of its assembly remain elusive. Here, we systematically examined the initiation and termination of contractile and non-contractile T6SS sheaths in MC-BP, tssA and tagA mutants by fluorescence microscopy. We observe long pole-to-pole sheath-tube structures in the non-contractile MC-BP defective mutants but not in the Hcp tube or VgrG spike mutants. Combining overexpression and genetic mutation data, we demonstrate complex effects of TssM, TssA and TagA interactions on T6SS sheath-tube dynamics. We also report promiscuous interactions of TagA with multiple T6SS components, similar to TssA. Our results demonstrate that priming of the T6SS sheath-tube assembly is not dependent on TssA, nor is the assembly termination dependent on the distal end TssA-TagA interaction, and highlight the tripartite control of TssA-TssM-TagA on sheath-tube initiation and termination.


Assuntos
Proteínas de Bactérias/metabolismo , Sistemas de Secreção Tipo VI/metabolismo , Vibrio cholerae/metabolismo , Proteínas de Bactérias/química , Membrana Celular/metabolismo , Escherichia coli/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Membrana/metabolismo , Viabilidade Microbiana , Modelos Biológicos , Mutação/genética , Ligação Proteica , Domínios Proteicos
4.
Nat Commun ; 11(1): 4906, 2020 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-32999292

RESUMO

The CRISPR-Cas12a RNA-guided complexes have tremendous potential for nucleic acid detection but are limited to the picomolar detection limit without an amplification step. Here, we develop a platform with engineered crRNAs and optimized conditions that enabled us to detect various clinically relevant nucleic acid targets with higher sensitivity, achieving a limit of detection in the femtomolar range without any target pre-amplification step. By extending the 3'- or 5'-ends of the crRNA with different lengths of ssDNA, ssRNA, and phosphorothioate ssDNA, we discover a self-catalytic behavior and an augmented rate of LbCas12a-mediated collateral cleavage activity as high as 3.5-fold compared to the wild-type crRNA and with significant improvement in specificity for target recognition. Particularly, the 7-mer DNA extension to crRNA is determined to be universal and spacer-independent for enhancing the sensitivity and specificity of LbCas12a-mediated nucleic acid detection. We perform a detailed characterization of our engineered ENHANCE system with various crRNA modifications, target types, reporters, and divalent cations. With isothermal amplification of SARS-CoV-2 RNA using RT-LAMP, the modified crRNAs are incorporated in a paper-based lateral flow assay that can detect the target with up to 23-fold higher sensitivity within 40-60 min.


Assuntos
Proteínas de Bactérias/metabolismo , Betacoronavirus/genética , Proteínas Associadas a CRISPR/metabolismo , Endodesoxirribonucleases/metabolismo , Técnicas de Amplificação de Ácido Nucleico/métodos , RNA Viral/isolamento & purificação , Transativadores/metabolismo , Betacoronavirus/isolamento & purificação , Sistemas CRISPR-Cas , Técnicas de Laboratório Clínico , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/virologia , DNA de Cadeia Simples , Pandemias , Pneumonia Viral , RNA Guia/genética , RNA Viral/genética
5.
Nat Commun ; 11(1): 4947, 2020 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-33009392

RESUMO

Pseudomonas syringae is a Gram-negative and model pathogenic bacterium that causes plant diseases worldwide. Here, we set out to identify binding motifs for all 301 annotated transcription factors (TFs) of P. syringae using HT-SELEX. We successfully identify binding motifs for 100 TFs. We map functional interactions between the TFs and their targets in virulence-associated pathways, and validate many of these interactions and functions using additional methods such as ChIP-seq, electrophoretic mobility shift assay (EMSA), RT-qPCR, and reporter assays. Our work identifies 25 virulence-associated master regulators, 14 of which had not been characterized as TFs before.


Assuntos
Proteínas de Bactérias/metabolismo , DNA Bacteriano/metabolismo , Pseudomonas syringae/metabolismo , Fatores de Transcrição/metabolismo , Sistemas de Secreção Bacterianos , Sítios de Ligação , Matrizes de Pontuação de Posição Específica , Ligação Proteica , Multimerização Proteica , Pseudomonas syringae/patogenicidade , Reprodutibilidade dos Testes , Técnica de Seleção de Aptâmeros , Virulência
6.
PLoS Pathog ; 16(8): e1008822, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32866204

RESUMO

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.


Assuntos
Disenteria Bacilar , Shigella flexneri , Transdução de Sinais , Vacúolos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Disenteria Bacilar/genética , Disenteria Bacilar/metabolismo , Células HeLa , Humanos , Shigella flexneri/genética , Shigella flexneri/metabolismo , Shigella flexneri/patogenicidade , Vacúolos/genética , Vacúolos/metabolismo , Vacúolos/microbiologia , Fatores de Virulência/genética , Fatores de Virulência/metabolismo , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo
7.
S Afr Med J ; 110(8): 783-790, 2020 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-32880307

RESUMO

BACKGROUND: Rates of healthcare-associated infections (HAIs) among babies born in developing countries are higher than among those born in resource-rich countries, as a result of suboptimal infection prevention and control (IPC) practices. Following two reported deaths of neonates with carbapenem-resistant Klebsiella pneumoniae bloodstream infections (BSIs), we conducted an outbreak investigation in a neonatal unit of a regional hospital in Gauteng Province, South Africa. OBJECTIVES: To confirm an outbreak of K. pneumoniae BSIs and assess the IPC programme in the neonatal unit. METHODS: We calculated total and organism-specific BSI incidence risks for culture-confirmed cases in the neonatal unit for baseline and outbreak periods. We conducted a clinical record review for a subset of cases with K. pneumoniae BSI that had been reported to the investigating team by the neonatal unit. An IPC audit was performed in different areas of the neonatal unit. We confirmed species identification and antimicrobial susceptibility, and used polymerase chain reaction for confirmation of carbapenemase genes and pulsed-field gel electrophoresis (PFGE) for typing of submitted clinical isolates. RESULTS: From January 2017 to August 2018, 5 262 blood cultures were submitted, of which 11% (560/5 262) were positive. Of 560 positive blood cultures, 52% (n=292) were positive for pathogenic organisms associated with healthcare-associated BSIs. K. pneumoniae comprised the largest proportion of these cases (32%; 93/292). The total incidence risk of healthcare-associated BSI for the baseline period (January 2017 - March 2018) was 6.8 cases per 100 admissions, and that for the outbreak period (April - September 2018) was 10.1 cases per 100 admissions. The incidence risk of K. pneumoniae BSI for the baseline period was 1.6 cases per 100 admissions, compared with 5.0 cases per 100 admissions during the outbreak period. Average bed occupancy for the entire period was 118% (range 101 - 133%), that for the baseline period was 117%, and that for the outbreak period was 121%. In a subset of 12 neonates with K. pneumoniae bacteraemia, the median (interquartile range (IQR)) gestational age at birth was 27 (26 - 29) weeks, and the median (IQR) birth weight was 1 100 (880 - 1 425) g. Twelve bloodstream and 31 colonising K. pneumoniae isolates were OXA-48-positive. All isolates were genetically related by PFGE analysis (89% similarity). Inadequate IPC practices were noted, including suboptimal adherence to aseptic technique and hand hygiene (57% overall score in the neonatal intensive care unit), with poor monitoring and reporting of antimicrobial use (pharmacy score 55%). CONCLUSIONS: Overcrowding and inadequate IPC and antimicrobial stewardship contributed to a large outbreak of BSIs caused by genetically related carbapenemase-producing K. pneumoniae isolates in the neonatal unit.


Assuntos
Bacteriemia/microbiologia , Infecção Hospitalar/microbiologia , Surtos de Doenças , Unidades Hospitalares , Infecções por Klebsiella/epidemiologia , Gestão de Antimicrobianos , Bacteriemia/epidemiologia , Proteínas de Bactérias/metabolismo , Auditoria Clínica , Infecção Hospitalar/epidemiologia , Aglomeração , Humanos , Incidência , Recém-Nascido , Controle de Infecções , Klebsiella pneumoniae/enzimologia , Klebsiella pneumoniae/isolamento & purificação , Programas Médicos Regionais , África do Sul/epidemiologia , beta-Lactamases/metabolismo
8.
PLoS Pathog ; 16(9): e1008867, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32925969

RESUMO

Surface attachment, an early step in the colonization of multiple host environments, activates the virulence of the human pathogen P. aeruginosa. However, the downstream toxins that mediate surface-dependent P. aeruginosa virulence remain unclear, as do the signaling pathways that lead to their activation. Here, we demonstrate that alkyl-quinolone (AQ) secondary metabolites are rapidly induced upon surface association and act directly on host cells to cause cytotoxicity. Surface-induced AQ cytotoxicity is independent of other AQ functions like quorum sensing or PQS-specific activities like iron sequestration. We further show that packaging of AQs in outer-membrane vesicles (OMVs) increases their cytotoxicity to host cells but not their ability to stimulate downstream quorum sensing pathways in bacteria. OMVs lacking AQs are significantly less cytotoxic, suggesting these molecules play a role in OMV cytotoxicity, in addition to their previously characterized role in OMV biogenesis. AQ reporters also enabled us to dissect the signal transduction pathways downstream of the two known regulators of surface-dependent virulence, the quorum sensing receptor, LasR, and the putative mechanosensor, PilY1. Specifically, we show that PilY1 regulates surface-induced AQ production by repressing the AlgR-AlgZ two-component system. AlgR then induces RhlR, which can induce the AQ biosynthesis operon under specific conditions. These findings collectively suggest that the induction of AQs upon surface association is both necessary and sufficient to explain surface-induced P. aeruginosa virulence.


Assuntos
Proteínas de Bactérias/metabolismo , Infecções por Pseudomonas/metabolismo , Pseudomonas aeruginosa , Quinolonas/farmacologia , Percepção de Quorum/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Fatores de Virulência/metabolismo , Células A549 , Animais , Humanos , Camundongos , Infecções por Pseudomonas/patologia , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/patogenicidade
9.
PLoS Pathog ; 16(9): e1008871, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32936831

RESUMO

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.


Assuntos
Proteínas de Bactérias/imunologia , Vacinas Bacterianas/imunologia , Imunização , Lipoproteínas/imunologia , Sífilis/imunologia , Treponema pallidum/imunologia , Animais , Proteínas de Bactérias/genética , Vacinas Bacterianas/genética , Humanos , Lipoproteínas/genética , Domínios Proteicos , Dobramento de Proteína , Coelhos , Sífilis/genética , Sífilis/patologia , Sífilis/prevenção & controle , Treponema pallidum/genética , Treponema pallidum/patogenicidade
10.
PLoS Pathog ; 16(9): e1008878, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32946535

RESUMO

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.


Assuntos
Proteínas de Bactérias , Membrana Celular/metabolismo , Chlamydia trachomatis , Proteína Neuronal da Síndrome de Wiskott-Aldrich/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Membrana Celular/genética , Membrana Celular/patologia , Chlamydia trachomatis/genética , Chlamydia trachomatis/metabolismo , Chlamydia trachomatis/patogenicidade , Células HeLa , Humanos , Mutação , Domínios Proteicos , Pseudópodes/genética , Pseudópodes/metabolismo , Proteína Neuronal da Síndrome de Wiskott-Aldrich/genética
11.
PLoS Pathog ; 16(9): e1008852, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32960931

RESUMO

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.


Assuntos
Proteínas de Bactérias , Toxinas Bacterianas , Clostridium difficile , Enterocolite Pseudomembranosa , Glucosiltransferases , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Clostridium difficile/enzimologia , Clostridium difficile/genética , Clostridium difficile/patogenicidade , Cricetinae , Modelos Animais de Doenças , Enterocolite Pseudomembranosa/enzimologia , Enterocolite Pseudomembranosa/genética , Enterocolite Pseudomembranosa/patologia , Feminino , Deleção de Genes , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Masculino , Camundongos
12.
Nat Commun ; 11(1): 4817, 2020 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-32968056

RESUMO

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.


Assuntos
Bdellovibrio bacteriovorus/enzimologia , Bdellovibrio bacteriovorus/metabolismo , Muramidase/química , Muramidase/metabolismo , Periplasma/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bdellovibrio bacteriovorus/genética , Parede Celular , Escherichia coli , Regulação Bacteriana da Expressão Gênica , Modelos Moleculares , Muramidase/genética , Mutação , Peptidoglicano/metabolismo , Fenótipo , Conformação Proteica , Especificidade por Substrato
13.
PLoS One ; 15(8): e0237883, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32866169

RESUMO

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.


Assuntos
Aminoácidos/genética , Reagentes para Ligações Cruzadas/metabolismo , Luz , Neisseria meningitidis/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Encéfalo/irrigação sanguínea , Endocitose , Células Endoteliais/microbiologia , Fímbrias Bacterianas/metabolismo , Humanos , Microvasos/patologia , Mutação/genética , Plasmídeos/genética
14.
Nat Commun ; 11(1): 4554, 2020 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-32917865

RESUMO

Non-ribosomal peptide synthetase (NRPS) enzymes form modular assembly-lines, wherein each module governs the incorporation of a specific monomer into a short peptide product. Modules are comprised of one or more key domains, including adenylation (A) domains, which recognise and activate the monomer substrate; condensation (C) domains, which catalyse amide bond formation; and thiolation (T) domains, which shuttle reaction intermediates between catalytic domains. This arrangement offers prospects for rational peptide modification via substitution of substrate-specifying domains. For over 20 years, it has been considered that C domains play key roles in proof-reading the substrate; a presumption that has greatly complicated rational NRPS redesign. Here we present evidence from both directed and natural evolution studies that any substrate-specifying role for C domains is likely to be the exception rather than the rule, and that novel non-ribosomal peptides can be generated by substitution of A domains alone. We identify permissive A domain recombination boundaries and show that these allow us to efficiently generate modified pyoverdine peptides at high yields. We further demonstrate the transferability of our approach in the PheATE-ProCAT model system originally used to infer C domain substrate specificity, generating modified dipeptide products at yields that are inconsistent with the prevailing dogma.


Assuntos
Monofosfato de Adenosina/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Domínios Proteicos , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Embaralhamento de DNA , Modelos Moleculares , Família Multigênica , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Peptídeo Sintases/genética , Peptídeo Sintases/metabolismo , Conformação Proteica , Pseudomonas , Especificidade por Substrato
15.
Adv Exp Med Biol ; 1267: 45-58, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32894476

RESUMO

In this chapter, we will focus on ParABS: an apparently simple, three-component system, required for the segregation of bacterial chromosomes and plasmids. We will specifically describe how biophysical measurements combined with physical modeling advanced our understanding of the mechanism of ParABS-mediated complex assembly, segregation and positioning.


Assuntos
Proteínas de Bactérias/metabolismo , Segregação de Cromossomos , Cromossomos Bacterianos/metabolismo , Posicionamento Cromossômico , DNA Bacteriano/metabolismo , Plasmídeos/metabolismo
16.
Nat Commun ; 11(1): 4648, 2020 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-32938927

RESUMO

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).


Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Farmacorresistência Bacteriana/genética , Infecções por Flavobacteriaceae/epidemiologia , Flavobacteriaceae/genética , Tigeciclina/farmacologia , China/epidemiologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Evolução Molecular , Flavobacteriaceae/efeitos dos fármacos , Flavobacteriaceae/isolamento & purificação , Infecções por Flavobacteriaceae/microbiologia , Humanos , Filogenia
17.
Yonsei Med J ; 61(9): 789-796, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32882763

RESUMO

PURPOSE: The prevalence of Mycobacterium tuberculosis (M. tb) and the status of M. bovis BCG vaccination may affect host immune responses to M. tb antigens. Understanding of the predominant local M. tb strain and immune signatures induced by its strain-specific antigens may contribute to an improved diagnosis of tuberculosis (TB). The aim of this study was to determine immune responses to M. tb antigen which was identified from the hyper-virulent Beijing/K strain in South Korea. MATERIALS AND METHODS: Pulmonary TB patients (n=52) and healthy subjects (n=92) including individuals with latent TB infection (n=31) were recruited, and QuantiFERON-TB Gold In-Tube tests were performed. The Beijing/K-antigen specific immune signatures were examined by diluted whole blood assays and multiplex bead arrays in a setting where nationwide BCG vaccination is employed. RESULTS: Statistical analyses demonstrated that three [C-X-C motif chemokine (CXCL10), interleukin (IL)-6, interferon (IFN)-α] of 17 cytokines/chemokines distinguished active cases from healthy controls following stimulation with the Beijing/K-specific antigen. IFN-α also differentiated between active diseases and latent TB infection (p<0.01), and the detection rate of TB was dramatically increased in combination with IL-6 and CXCL10 at the highest levels of specificity (95-100%). CONCLUSION: Our data indicate that immune signatures to the M. tb Beijing/K-specific antigen can provide useful information for improved TB diagnostics. The antigen may be developed as a diagnostic marker or a vaccine candidate, particularly in regions where the M. tb Beijing/K strain is endemic.


Assuntos
Tuberculose Latente/diagnóstico , Mycobacterium tuberculosis/isolamento & purificação , Tuberculose Pulmonar/diagnóstico , Adolescente , Adulto , Antígenos de Bactérias/sangue , Antígenos de Bactérias/genética , Antígenos de Superfície/sangue , Antígenos de Superfície/genética , Proteínas de Bactérias , Pequim , Estudos de Casos e Controles , Citocinas/sangue , Feminino , Humanos , Mycobacterium tuberculosis/imunologia , Mycobacterium tuberculosis/metabolismo , República da Coreia , Sensibilidade e Especificidade
18.
Nat Commun ; 11(1): 4501, 2020 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-32908132

RESUMO

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.


Assuntos
Proteínas de Bactérias/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Streptomyces/metabolismo , Estreptovaricina/análogos & derivados , Acetilação , Proteínas de Bactérias/genética , Proteínas de Bactérias/ultraestrutura , Biocatálise , Cristalografia por Raios X , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/ultraestrutura , Ensaios Enzimáticos , Metilação , Mutagênese Sítio-Dirigida , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Estreptovaricina/biossíntese , Estreptovaricina/química , Estreptovaricina/metabolismo
19.
Nat Commun ; 11(1): 4522, 2020 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-32908144

RESUMO

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.


Assuntos
Infecções por Acinetobacter/tratamento farmacológico , Acinetobacter baumannii/genética , Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Infecção Hospitalar/tratamento farmacológico , Farmacorresistência Bacteriana Múltipla/genética , Infecções por Acinetobacter/microbiologia , Acinetobacter baumannii/efeitos dos fármacos , Antibacterianos/uso terapêutico , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Parede Celular/efeitos dos fármacos , Parede Celular/genética , Parede Celular/metabolismo , Infecção Hospitalar/microbiologia , Análise Mutacional de DNA , Elementos de DNA Transponíveis/genética , DNA Bacteriano/genética , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Sinergismo Farmacológico , Humanos , Testes de Sensibilidade Microbiana , Mutação
20.
Wei Sheng Yan Jiu ; 49(4): 569-573, 2020 Jul.
Artigo em Chinês | MEDLINE | ID: mdl-32928347

RESUMO

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.


Assuntos
Alimentos Geneticamente Modificados , Zea mays/genética , Animais , Proteínas de Bactérias/genética , Endotoxinas , Proteínas Hemolisinas/genética , Plantas Geneticamente Modificadas , Ratos , Ratos Sprague-Dawley
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