Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 106.982
Filtrar
1.
Annu Int Conf IEEE Eng Med Biol Soc ; 2020: 2372-2375, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-33018483

RESUMO

To advance synthetic biology approaches that utilize S. oneidensis as host for biotechnology applications, we have investigated the variation in plasmid copy number of a modular vector set resulting from distinct origins of replication under different conditions. The replicons yielded a ≈9X-fold range for plasmid copy number variation in S. oneidensis (while the same origins yielded a ≈3X-fold range in Escherichia coli). This provides a sizeable range to control gene expression levels in S. oneidensis for synthetic biology applications. In addition, plasmid harboring the pBBR1 origin resulted in stable copy numbers in S. oneidensis under different conditions (mid-logarithmic, stationary, multi-plasmid). This may enable the realization of synthetic circuits in S. oneidensis where predictable, quantitative behavior is desired (in either single- or double-plasmid contexts).


Assuntos
Variações do Número de Cópias de DNA , Shewanella , Escherichia coli/genética , Plasmídeos/genética , Shewanella/genética
2.
Nat Commun ; 11(1): 4963, 2020 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-33009406

RESUMO

Bacterial nanotubes are membranous structures that have been reported to function as conduits between cells to exchange DNA, proteins, and nutrients. Here, we investigate the morphology and formation of bacterial nanotubes using Bacillus subtilis. We show that nanotube formation is associated with stress conditions, and is highly sensitive to the cells' genetic background, growth phase, and sample preparation methods. Remarkably, nanotubes appear to be extruded exclusively from dying cells, likely as a result of biophysical forces. Their emergence is extremely fast, occurring within seconds by cannibalizing the cell membrane. Subsequent experiments reveal that cell-to-cell transfer of non-conjugative plasmids depends strictly on the competence system of the cell, and not on nanotube formation. Our study thus supports the notion that bacterial nanotubes are a post mortem phenomenon involved in cell disintegration, and are unlikely to be involved in cytoplasmic content exchange between live cells.


Assuntos
Bacillus subtilis/citologia , Bacillus subtilis/metabolismo , Viabilidade Microbiana , Nanotubos/química , Bacillus subtilis/genética , Bacillus subtilis/ultraestrutura , Conjugação Genética , DNA Bacteriano/genética , Plasmídeos/genética
3.
Nan Fang Yi Ke Da Xue Xue Bao ; 40(6): 806-813, 2020 Jun 30.
Artigo em Chinês | MEDLINE | ID: mdl-32895213

RESUMO

OBJECTIVE: To prepare the recombinant peptide MVF-HER3 I composed of the 183-227aa peptide segment of human epidermal growth factor receptor 3 (HER3 I) and the measles virus protein 288-302 peptide segment (MVF), and prepare polyclonal antibodies (PcAb) against this recombinant peptide. METHODS: The MVF-HER3 I gene was synthesized chemically and subcloned into pET21b or pET32a plasmid containing Thioredoxin (Trx) tag gene. The recombinant plasmids were identified by endonuclease digestion. MVF-HER3 I was expressed in E.coli BL21(DE3) cells under an optimal bacterial expression condition. The fusion protein Trx-MVF-HER3 I was purified using nickel ion affinity chromatography, and the purified protein was digested by enterokinase to remove Trx tag. The digested mixture underwent further nickel ion affinity chromatography to obtain purified MVF-HER3 I. The purified MVF-HER3 I was used to immunize SD rats subcutaneously for preparing anti-MVF-HER3 I PcAb. The titer of PcAb was determined using ELISA. The bindings of anti-MVF-HER3 I PcAb to MVF-HER3 I, native HER3 and MCF7 cells were analyzed using immunoblotting, immunoprecipitation and laser confocal microscopy. The growth inhibition effect of the antibodies on MCF7 cells cultured in the absence or presence of NRG was assessed using sulforhodamine B. RESULTS: The recombinant peptide gene could not be expressed alone, but could be efficiently expressed after fusion with Trx gene under optimized conditions. The fusion peptide MVF-HER3 I was successfully prepared from Trx-MVF-HER3 I. The anti-MVF-HER3 I PcAb, with a titer reaching 1: 512 000, specifically bound to MVF-HER3 I, recognized native HER3 and bound to the membrane of MCF7 cells. The obtained PcAb could dose-dependently inhibit the growth of MCF7 cells irrespective of the presence or absence of NRG. CONCLUSIONS: We successfully obtained the recombinant peptide MVF-HER3 I and prepared its PcAb, which can facilitate further functional analysis of HER3 signaling pathway.


Assuntos
Receptor ErbB-3/imunologia , Animais , Anticorpos , Ensaio de Imunoadsorção Enzimática , Escherichia coli , Humanos , Plasmídeos , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes de Fusão
4.
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
5.
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
6.
Zhongguo Xue Xi Chong Bing Fang Zhi Za Zhi ; 32(4): 355-360, 2020 Aug 24.
Artigo em Chinês | MEDLINE | ID: mdl-32935508

RESUMO

OBJECTIVE: To investigate the biological properties of Schistosoma japonicum SjGrpE protein, and to express and purify the recombinant SjGrpE protein and test its immunogenicity. METHODS: The amino acid composition, molecular weight, hydrophilicity and hydrophobicity, transmembrane region, signal peptide, localization, phosphorylation site, ubiquitination site, glycosylation site, secondary and tertiary structures and B cell epitopes of the SjGrpE protein were predicted using bioinformatics analyses. The SjGrpE gene was amplified using PCR assay using S. japonicum cDNA as a template, double enzyme-digested and linked to the pET28a vector to yield the recombinant plasmid pET28a-SjGrpE. The recombinant plasmid pET28a-SjGrpE was transformed into Escherichia coli BL21, and then IPTG was employed to induce the expression of the target protein, which was purified by nickel ion affinity chromatography. After mice were immunized with the recombinant SjGrpE protein, mouse sera were collected, and the polyclonal antibody against the SjGrpE protein was characterized. RESULTS: SjGrpE protein, which was identified as a hydrophilic protein, was predicted to have a molecular weight of approximately 24.3 kDa without transmembrane regions or signal peptides, and locate in the mitochondrion. SjGrpE protein contained 18 phosphorylation sites and 2 ubiquitination sites, but had no glycosylation sites. In addition, SjGrpE protein contained 5 B-cell epitopes. The full length of SjGrpE gene was approximately 660 bp. The recombinant pET28a-SjGrpE plasmid was successfully generated, and the recombinant SjGrpE protein was obtained following the affinity chromatography, which stimulated mice to secrete high-titer antibodies. CONCLUSIONS: The recombinant SjGrpE protein has been successfully prepared and this recombinant protein has a high immunogenicity, which provides a basis for evaluating its value as a vaccine candidate for S. japonicum infections.


Assuntos
Proteínas de Helminto , Proteínas Recombinantes , Schistosoma japonicum , Animais , Anticorpos Anti-Helmínticos/isolamento & purificação , Clonagem Molecular , Escherichia coli/genética , Proteínas de Helminto/química , Proteínas de Helminto/genética , Proteínas de Helminto/imunologia , Proteínas de Helminto/isolamento & purificação , Camundongos , Plasmídeos/genética , Reação em Cadeia da Polimerase , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Schistosoma japonicum/genética , Schistosoma japonicum/metabolismo
7.
Sheng Wu Gong Cheng Xue Bao ; 36(8): 1659-1671, 2020 Aug 25.
Artigo em Chinês | MEDLINE | ID: mdl-32924364

RESUMO

To construct TeI3c/4c-based and temperature-inducible gene inactivation system (Thermotargetron) and to apply it to gene inactivation of mesophilic bacteria. The subunit of flagellum (fliC) and C4 dicarboxylate orotate:H⁺ symporter (dctA) genes were chosen as targets in the genome of Escherichia coli HMS174 (DE3) strain. According to recognition roles of TeI3c/4c intron, the fliC489a, fliC828s, fliC1038s and dctA2a sites were chosen as target sites. Gene-targeting plasmids were constructed based on pHK-TT1A by using overlap PCR method and transformed into HMS174 cells. An aliquot mid-log phase cultures of the transformants were shocked at 48 °C and plated on LB plate (containing chloramphenicol). Afterwards, gene mutants were screened by using colony PCR and DNA sequencing. After the mutants were obtained, the phenotypes of ΔfliC and ΔdctA gene mutants were characterized by using agar puncture and carbon metabolism experiments. Colony PCR and sequencing results show that TeI3c/4c intron was inserted in the designed sites of fliC and dctA genes. The gene-targeting efficiency of Thermotargetron system was 100%. Phenotype verification experiments of the mutants demonstrated that the cell motility of all ΔfliC mutants was damaged and the malate assimilation ability of ΔdctA mutant was deprived comparing to wild-type HMS174 strain. In our study, a temperature-inducible and high-efficiency gene inactivation system was established for mesophilic bacteria. This system could achieve high efficiency and precise gene inactivation by modulation of the incubation duration of the transformants at 48 °C.


Assuntos
Escherichia coli , Inativação Gênica , Marcação de Genes , Técnicas Genéticas , Temperatura , Escherichia coli/genética , Flagelos , Marcação de Genes/métodos , Mutação , Plasmídeos
9.
BMC Infect Dis ; 20(1): 703, 2020 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-32977759

RESUMO

BACKGROUND: Treatment of gonorrhea is complicated by the development of antimicrobial resistance in Neisseria gonorrhoeae (GC) to the antibiotics recommended for treatment. Knowledge on types of plasmids and the antibiotic resistance genes they harbor is useful in monitoring the emergence and spread of bacterial antibiotic resistance. In Kenya, studies on gonococcal antimicrobial resistance are few and data on plasmid mediated drug resistance is limited. The present study characterizes plasmid mediated resistance in N. gonorrhoeae isolates recovered from Kenya between 2013 and 2018. METHODS: DNA was extracted from 36 sub-cultured GC isolates exhibiting varying drug resistance profiles. Whole genome sequencing was done on Illumina MiSeq platform and reads assembled de-novo using CLC Genomics Workbench. Genome annotation was performed using Rapid Annotation Subsystem Technology. Comparisons in identified antimicrobial resistance determinants were done using Bioedit sequence alignment editor. RESULTS: Twenty-four (66.7%) isolates had both ß-lactamase (TEM) and TetM encoding plasmids. 8.3% of the isolates lacked both TEM and TetM plasmids and had intermediate to susceptible penicillin and tetracycline MICs. Twenty-six (72%) isolates harbored TEM encoding plasmids. 25 of the TEM plasmids were of African type while one was an Asian type. Of the 36 isolates, 31 (86.1%) had TetM encoding plasmids, 30 of which harbored American TetM, whereas 1 carried a Dutch TetM. All analyzed isolates had non-mosaic penA alleles. All the isolates expressing TetM were tetracycline resistant (MIC> 1 mg/L) and had increased doxycycline MICs (up to 96 mg/L). All the isolates had S10 ribosomal protein V57M amino acid substitution associated with tetracycline resistance. No relation was observed between PenB and MtrR alterations and penicillin and tetracycline MICs. CONCLUSION: High-level gonococcal penicillin and tetracycline resistance in the sampled Kenyan regions was found to be mediated by plasmid borne blaTEM and tetM genes. While the African TEM plasmid, TEM1 and American TetM are the dominant genotypes, Asian TEM plasmid, a new TEM239 and Dutch TetM have emerged in the regions.


Assuntos
Antibacterianos/uso terapêutico , Farmacorresistência Bacteriana Múltipla/genética , Gonorreia/tratamento farmacológico , Gonorreia/epidemiologia , Neisseria gonorrhoeae/genética , Penicilinas/uso terapêutico , Plasmídeos/genética , Resistência a Tetraciclina/genética , Tetraciclina/uso terapêutico , DNA Bacteriano/genética , Feminino , Genótipo , Gonorreia/microbiologia , Humanos , Quênia/epidemiologia , Masculino , Testes de Sensibilidade Microbiana , Neisseria gonorrhoeae/isolamento & purificação , Sequenciamento Completo do Genoma , beta-Lactamases/genética
10.
Ecotoxicol Environ Saf ; 205: 111300, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-32961492

RESUMO

Bacterial resistance caused by the abuse of antibiotics has attracted worldwide attention. However, there are few studies exploring bacterial resistance under the environmental exposure condition of antibiotics that is featured by low-dose and mixture. In this study, sulfonamides (SAs), sulfonamide potentiators (SAPs) and tetracyclines (TCs) were used to determine the effects of antibiotics on plasmid RP4 conjugative transfer of Escherichia coli (E. coli) under single or combined exposure, and the relationship between the effects of antibiotics on conjugative transfer and growth was investigated. The results show that the effects of single or binary antibiotics on plasmid RP4 conjugative transfer all exhibit a hormetic phenomenon. The linear regression reveals that the concentrations of the three antibiotics promoting conjugative transfer are correlated with the concentrations promoting growth and the physicochemical properties of the compounds. The combined effects of SAs-SAPs and SAs-TCs on plasmid conjugative transfer are mainly synergistic and antagonistic. While SAPs provide more effective concentrations for the promotion of conjugative transfer in SAs-SAPs mixtures, SAs play a more important role in promoting conjugative transfer in SAs-TCs mixtures. Mechanism explanation shows that SAs, SAPs and TCs inhibit bacterial growth by acting on their target proteins DHPS, DHFR and 30S ribosomal subunit, respectively. This study indicates that toxic stress stimulates the occurrence of conjugative transfer and promotes the development of bacterial resistance, which will provide a reference for resistance risk assessment of antibiotic exposure.


Assuntos
Antibacterianos/toxicidade , Conjugação Genética/efeitos dos fármacos , Poluentes Ambientais/toxicidade , Escherichia coli/efeitos dos fármacos , Hormese , Plasmídeos , Antagonismo de Drogas , Sinergismo Farmacológico , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Plasmídeos/efeitos dos fármacos , Plasmídeos/genética , Sulfonamidas/toxicidade , Tetraciclinas/toxicidade
11.
Viruses ; 12(9)2020 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-32927639

RESUMO

The recent outbreak of a novel Coronavirus (SARS-CoV-2) and its rapid spread across the continents has generated an urgent need for assays to detect the neutralising activity of human sera or human monoclonal antibodies against SARS-CoV-2 spike protein and to evaluate the serological immunity in humans. Since the accessibility of live virus microneutralisation (MN) assays with SARS-CoV-2 is limited and requires enhanced bio-containment, the approach based on "pseudotyping" can be considered a useful complement to other serological assays. After fully characterising lentiviral pseudotypes bearing the SARS-CoV-2 spike protein, we employed them in pseudotype-based neutralisation assays in order to profile the neutralising activity of human serum samples from an Italian sero-epidemiological study. The results obtained with pseudotype-based neutralisation assays mirrored those obtained when the same panel of sera was tested against the wild type virus, showing an evident convergence of the pseudotype-based neutralisation and MN results. The overall results lead to the conclusion that the pseudotype-based neutralisation assay is a valid alternative to using the wild-type strain, and although this system needs to be optimised and standardised, it can not only complement the classical serological methods, but also allows serological assessments to be made when other methods cannot be employed, especially in a human pandemic context.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/virologia , Lentivirus/genética , Testes de Neutralização/métodos , Pandemias , Pneumonia Viral/virologia , Animais , Anticorpos Neutralizantes , Anticorpos Antivirais/imunologia , Betacoronavirus/imunologia , Linhagem Celular , Infecções por Coronavirus/epidemiologia , Humanos , Soros Imunes/imunologia , Itália/epidemiologia , Plasmídeos/genética , Pneumonia Viral/epidemiologia , Estudos Soroepidemiológicos , Glicoproteína da Espícula de Coronavírus/biossíntese , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/fisiologia , Transfecção , Vesiculovirus/genética , Carga Viral
12.
Ecotoxicol Environ Saf ; 204: 111119, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32798757

RESUMO

The rapid spread of antibiotic resistance genes (ARGs) is a great challenge to the ecological safety and human health. The intestine of humans and animals is an important site for the increase and spread of ARGs due to the great diversity and abundance of microorganisms in the intestinal microecology. ARGs, including the intracellular (iARGs) and the extracellular (eARGs) ARGs, are usually introduced into the intestinal tract through the diet, and the iARGs are colonized and spread in the intestinal microbiota with the help of the host bacteria. However, whether the eARGs can enter the intestinal microorganisms in the absence of host bacteria is not known. Here, we show the transformation and the diffusion of the ampramycin resistance gene (Ap) carried by the free plasmid RK2 in the intestinal microbiota of mice. After two days of consecutive gavage with free RK2, the intracellular Ap gene increases from days 0-8 in the feces of mice, and has remained constant. Bacterial transformation happens in the small intestine, including proximal and distal jejuna and proximal and distal ilea, at the early stage (first two days), and the intracellular RK2 is diffused into the intestinal microbiota of mice by conjugation on days 2-8 day, which is based on the distribution of eARG and iARG and the mRNA expression levels of trbBp, trfAp, korA, korB, and trbA. The characteristics of ARGs susceptible microbiota for transformation are analyzed using 16s rRNA gene sequencing, transmission electron microscopy, and flow cytometric. The ingestion of RK2 affects the composition of intestinal microbiota especially for Proteobacteria, and the antibiotic residue promotes the increase in Escherichia coli. These findings are important to assess the risk of ARGs, especially the eARGs in the intestinal microecology.


Assuntos
Resistência Microbiana a Medicamentos/genética , Microbioma Gastrointestinal/fisiologia , Genes Bacterianos , Camundongos/microbiologia , Animais , Antibacterianos , Bactérias , Escherichia coli/efeitos dos fármacos , Fezes , Humanos , Intestinos , Microbiota , Plasmídeos , RNA Ribossômico 16S/genética
13.
PLoS One ; 15(8): e0235942, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32804931

RESUMO

Genome editing is now widely used in plant science for both basic research and molecular crop breeding. The clustered regularly interspaced short palindromic repeats (CRISPR) technology, through its precision, high efficiency and versatility, allows for editing of many sites in plant genomes. This system has been highly successful to produce knock-out mutants through the introduction of frameshift mutations due to error-prone repair pathways. Nevertheless, recent new CRISPR-based technologies such as base editing and prime editing can generate precise and on demand nucleotide conversion, allowing for fine-tuning of protein function and generating gain-of-function mutants. However, genome editing through CRISPR systems still have some drawbacks and limitations, such as the PAM restriction and the need for more diversity in CRISPR tools to mediate different simultaneous catalytic activities. In this study, we successfully used the CRISPR-Cas9 system from Staphylococcus aureus (SaCas9) for the introduction of frameshift mutations in the tetraploid genome of the cultivated potato (Solanum tuberosum). We also developed a S. aureus-cytosine base editor that mediate nucleotide conversions, allowing for precise modification of specific residues or regulatory elements in potato. Our proof-of-concept in potato expand the plant dicot CRISPR toolbox for biotechnology and precision breeding applications.


Assuntos
Proteína 9 Associada à CRISPR/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Mutação INDEL , Solanum tuberosum/genética , Staphylococcus aureus/enzimologia , Sistemas CRISPR-Cas , Mutação da Fase de Leitura , Edição de Genes/métodos , Genoma de Planta , Plasmídeos/genética , Staphylococcus aureus/genética
14.
PLoS One ; 15(8): e0233325, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32756562

RESUMO

Antibiotics discovery was a significant breakthrough in the field of therapeutic medicines, but the over (mis)use of such antibiotics (in parallel) caused the increasing number of resistant bacterial species at an ever-higher rate. This study was thus devised to assess the multi-drug resistant bacteria present in sanitation-related facilities in human workplaces. In this regard, samples were collected from different gender, location, and source-based facilities, and subsequent antibiotic sensitivity testing was performed on isolated bacterial strains. Four classes of the most commonly used antibiotics i.e., ß-lactam, Aminoglycosides, Macrolides, and Sulphonamides, were evaluated against the isolated bacteria. The antibiotic resistance profile of different (70) bacterial strains showed that the antibiotic resistance-based clusters also followed the grouping based on their isolation sources, mainly the gender. Twenty-three bacterial strains were further selected for their 16s rRNA gene based molecular identification and for phylogenetic analysis to evaluate the taxonomic evolution of antibiotic resistant bacteria (ARB). Moreover, the bacterial resistance to Sulphonamides and beta lactam was observed to be the most and to Aminoglycosides and macrolides as the least. Plasmid curing was also performed for multidrug resistant (MDR) bacterial strains, which significantly abolished the resistance potential of bacterial strains for different antibiotics. These curing results suggested that the antibiotic resistance determinants in these purified bacterial strains are present on respective plasmids. Altogether, the data suggested that the human workplaces are the hotspot for the prevalence of MDR bacteria and thus may serve as the source of horizontal gene transfer and further transmission to other environments.


Assuntos
Bactérias/efeitos dos fármacos , Bactérias/isolamento & purificação , Farmacorresistência Bacteriana Múltipla , Toaletes , Local de Trabalho , Bactérias/genética , Farmacorresistência Bacteriana Múltipla/genética , Microbiologia Ambiental , Feminino , Humanos , Higiene , Masculino , Exposição Ocupacional , Paquistão , Filogenia , Plasmídeos , Prevalência , Fatores R , RNA Ribossômico 16S/genética , Saneamento
15.
PLoS One ; 15(8): e0232806, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32785265

RESUMO

There is an increasing consumer demand for minimally processed, preservative free and microbiologically safe food. These factors, combined with risks of antibiotic resistance, have led to interest in bacteriocins produced by lactic acid bacteria (LAB) as natural food preservatives and as potential protein therapeutics. We previously reported the discovery of plantacyclin B21AG, a circular bacteriocin produced by Lactobacillus plantarum B21. Here, we describe the cloning and functional expression of the bacteriocin gene cluster in the probiotic Lactobacillus plantarum WCFS1. Genome sequencing demonstrated that the bacteriocin is encoded on a 20 kb native plasmid, designated as pB21AG01. Seven open reading frames (ORFs) putatively involved in bacteriocin production, secretion and immunity were cloned into an E. coli/Lactobacillus shuttle vector, pTRKH2. The resulting plasmid, pCycB21, was transformed into L. plantarum WCFS1. The cell free supernatants (CFS) of both B21 and WCFS1 (pCycB21) showed an antimicrobial activity of 800 AU/mL when tested against WCFS1 (pTRKH2) as the indicator strain, showing that functional expression of plantacyclin B21AG had been achieved. Real-time PCR analysis revealed that the relative copy number of pB21AG01 was 7.60 ± 0.79 in L. plantarum B21 whilst pCycB21 and pTRKH2 was 0.51 ± 0.05 and 25.19 ± 2.68 copies respectively in WCFS1. This indicates that the bacteriocin gene cluster is located on a highly stable low copy number plasmid pB21AG01 in L. plantarum B21. Inclusion of the native promoter for the bacteriocin operon from pB21AG01 results in similar killing activity being observed in both the wild type and recombinant hosts despite the lower copy number of pCycB21.


Assuntos
Bacteriocinas/genética , Microbiologia de Alimentos , Lactobacillus plantarum/genética , Probióticos , Mapeamento Cromossômico , Clonagem Molecular , Conservantes de Alimentos , Dosagem de Genes , Genes Bacterianos , Humanos , Família Multigênica , Plasmídeos/genética
16.
PLoS One ; 15(8): e0237474, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32857767

RESUMO

The effective treatment of carbapenemase-producing Klebsiella pneumoniae infection has been limited and required novel potential agents. Due to the novel drug development crisis, using old antimicrobial agents and combination therapy have been highlighted. This study focused on fosfomycin which inhibits cell wall synthesis and has potential activity on Enterobacteriaceae. We evaluated fosfomycin activity against carbapenemase-producing K. pneumoniae and characterized fosfomycin resistance mechanisms. Fosfomycin revealed effective activity against only 31.8% of carbapenemase-producing K. pneumoniae isolates. The major resistance mechanism was FosA3 production. The co-occurrence of FosA3 overexpression with the mutation of glpT (or loss of glpT) and/or uhpT was mediated high-level resistance (MIC>256 mg/L) to fosfomycin. Moreover, fosA3 silenced in sixteen fosfomycin-susceptible isolates and the plasmid carrying fosA3 of these isolates increased 32- to 64-fold of fosfomycin MICs in Escherichia coli DH5α transformants. The in vitro activity of fosfomycin combination with amikacin by checkerboard assay showed synergism and no interaction in six (16.2%) and sixteen isolates (43.3%), respectively. No antagonism of fosfomycin and amikacin was observed. Notably, the silence of aac (6)'-Ib and aphA6 was observed in amikacin-susceptible isolates. Our study suggests that the combination of fosfomycin and amikacin may be insufficient for the treatment of carbapenemase-producing K. pneumoniae isolates.


Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Farmacorresistência Bacteriana/genética , Fosfomicina/farmacologia , Klebsiella pneumoniae/efeitos dos fármacos , beta-Lactamases/metabolismo , Amicacina/farmacologia , Substituição de Aminoácidos , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/genética , Escherichia coli/metabolismo , Humanos , Infecções por Klebsiella/microbiologia , Infecções por Klebsiella/patologia , Klebsiella pneumoniae/enzimologia , Klebsiella pneumoniae/isolamento & purificação , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Testes de Sensibilidade Microbiana , Plasmídeos/genética , Plasmídeos/metabolismo , RNA Mensageiro/metabolismo , beta-Lactamases/genética
17.
PLoS Negl Trop Dis ; 14(8): e0008542, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32810151

RESUMO

Presently, the principal tools to combat malaria are restricted to killing the parasite in infected people and killing the mosquito vector to thwart transmission. While successful, these approaches are losing effectiveness in view of parasite resistance to drugs and mosquito resistance to insecticides. Clearly, new approaches to fight this deadly disease need to be developed. Recently, one such approach-engineering mosquito resident bacteria to secrete anti-parasite compounds-has proven in the laboratory to be highly effective. However, implementation of this strategy requires approval from regulators as it involves introduction of recombinant bacteria into the field. A frequent argument by regulators is that if something unexpectedly goes wrong after release, there must be a recall mechanism. This report addresses this concern. Previously we have shown that a Serratia bacterium isolated from a mosquito ovary is able to spread through mosquito populations and is amenable to be engineered to secrete anti-plasmodial compounds. We have introduced a plasmid into this bacterium that carries a fluorescent protein gene and show that when cultured in the laboratory, the plasmid is completely lost in about 130 bacterial generations. Importantly, when these bacteria were introduced into mosquitoes, the bacteria were transmitted from one generation to the next, but the plasmid was lost after three mosquito generations, rendering the bacteria non-recombinant (wild type). Furthermore, no evidence was obtained for horizontal transfer of the plasmid to other bacteria either in culture or in the mosquito. Prior to release, it is imperative to demonstrate that the genes that thwart parasite development in the mosquito are safe to the environment. This report describes a methodology to safely achieve this goal, utilizing transient expression from a plasmid that is gradually lost, returning the bacterium to wild type status.


Assuntos
Anopheles/microbiologia , Agentes de Controle Biológico/farmacologia , Mosquitos Vetores/microbiologia , Serratia/genética , Serratia/metabolismo , Animais , Bactérias/genética , Bactérias/metabolismo , Transmissão de Doença Infecciosa , Feminino , Malária , Masculino , Ovário/microbiologia , Plasmídeos/genética
18.
PLoS Genet ; 16(8): e1008965, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32760058

RESUMO

The mobilizable resistance island Salmonella genomic island 1 (SGI1) is specifically mobilized by IncA and IncC conjugative plasmids. SGI1, its variants and IncC plasmids propagate multidrug resistance in pathogenic enterobacteria such as Salmonella enterica serovars and Proteus mirabilis. SGI1 modifies and uses the conjugation apparatus encoded by the helper IncC plasmid, thus enhancing its own propagation. Remarkably, although SGI1 needs a coresident IncC plasmid to excise from the chromosome and transfer to a new host, these elements have been reported to be incompatible. Here, the stability of SGI1 and its helper IncC plasmid, each expressing a different fluorescent reporter protein, was monitored using fluorescence-activated cell sorting (FACS). Without selective pressure, 95% of the cells segregated into two subpopulations containing either SGI1 or the helper plasmid. Furthermore, FACS analysis revealed a high level of SGI1-specific fluorescence in IncC+ cells, suggesting that SGI1 undergoes active replication in the presence of the helper plasmid. SGI1 replication was confirmed by quantitative PCR assays, and extraction and restriction of its plasmid form. Deletion of genes involved in SGI1 excision from the chromosome allowed a stable coexistence of SGI1 with its helper plasmid without selective pressure. In addition, deletion of S003 (rep) or of a downstream putative iteron-based origin of replication, while allowing SGI1 excision, abolished its replication, alleviated the incompatibility with the helper plasmid and enabled its cotransfer to a new host. Like SGI1 excision functions, rep expression was found to be controlled by AcaCD, the master activator of IncC plasmid transfer. Transient SGI1 replication seems to be a key feature of the life cycle of this family of genomic islands. Sequence database analysis revealed that SGI1 variants encode either a replication initiator protein with a RepA_C domain, or an alternative replication protein with N-terminal replicase and primase C terminal 1 domains.


Assuntos
Proteínas de Bactérias/genética , Conjugação Genética/genética , Ilhas Genômicas/genética , Fosfoproteínas/genética , Plasmídeos/genética , Antibacterianos/farmacologia , Cromossomos/efeitos dos fármacos , Cromossomos/genética , DNA Helicases/genética , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Farmacorresistência Bacteriana Múltipla/genética , Plasmídeos/efeitos dos fármacos , Proteus mirabilis/genética , Salmonella enterica/genética , Transativadores/genética
19.
Nat Commun ; 11(1): 4070, 2020 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-32792502

RESUMO

Human astroviruses are small non-enveloped viruses with positive-sense single-stranded RNA genomes. Astroviruses cause acute gastroenteritis in children worldwide and have been associated with encephalitis and meningitis in immunocompromised individuals. It is still unknown how astrovirus particles exit infected cells following replication. Through comparative genomic analysis and ribosome profiling we here identify and confirm the expression of a conserved alternative-frame ORF, encoding the protein XP. XP-knockout astroviruses are attenuated and pseudo-revert on passaging. Further investigation into the function of XP revealed plasma and trans Golgi network membrane-associated roles in virus assembly and/or release through a viroporin-like activity. XP-knockout replicons have only a minor replication defect, demonstrating the role of XP at late stages of infection. The discovery of XP advances our knowledge of these important human viruses and opens an additional direction of research into their life cycle and pathogenesis.


Assuntos
Canais Iônicos/metabolismo , Mamastrovirus/metabolismo , Proteínas não Estruturais Virais/metabolismo , Animais , Linhagem Celular , Cricetinae , Eletroforese em Gel de Poliacrilamida , Genômica/métodos , Células HeLa , Humanos , Immunoblotting , Imunoprecipitação , Canais Iônicos/genética , Mamastrovirus/genética , Microscopia de Fluorescência , Plasmídeos/genética , Ribossomos , Proteínas não Estruturais Virais/genética , Replicação Viral/genética , Replicação Viral/fisiologia
20.
PLoS Biol ; 18(8): e3000814, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32797039

RESUMO

Plasmid-mediated horizontal gene transfer of antibiotic resistance and virulence in pathogenic bacteria underlies a major public health issue. Understanding how, in the absence of antibiotic-mediated selection, plasmid-bearing cells avoid being outnumbered by plasmid-free cells is key to developing counterstrategies. Here, we quantified the induction of the plasmidial sex pheromone pathway of Enterococcus faecalis to show that the integration of the stimulatory (mate-sensing) and inhibitory (self-sensing) signaling modules from the pCF10 conjugative plasmid provides a precise measure of the recipient-to-donor ratio, agnostic to variations in population size. Such ratiometric control of conjugation favors vertical plasmid transfer under low mating likelihood and allows activation of conjugation functions only under high mating likelihood. We further show that this strategy constitutes a cost-effective investment into mating effort because overstimulation produces unproductive self-aggregation and growth rate reduction. A mathematical model suggests that ratiometric control of conjugation increases plasmid fitness and predicts a robust long-term, stable coexistence of donors and recipients. Our results demonstrate how population-level parameters can control transfer of antibiotic resistance in bacteria, opening the door for biotic control strategies.


Assuntos
Proteínas de Bactérias/genética , Resistência Microbiana a Medicamentos/genética , Enterococcus faecalis/genética , Transferência Genética Horizontal , Feromônios/genética , Percepção de Quorum/efeitos dos fármacos , Antibacterianos/farmacologia , Carga Bacteriana , Proteínas de Bactérias/metabolismo , Conjugação Genética , Enterococcus faecalis/efeitos dos fármacos , Enterococcus faecalis/crescimento & desenvolvimento , Enterococcus faecalis/metabolismo , Expressão Gênica , Aptidão Genética , Modelos Estatísticos , Feromônios/biossíntese , Plasmídeos/química , Plasmídeos/metabolismo , Percepção de Quorum/genética , Virulência
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA