Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 30.050
Filtrar
1.
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
2.
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
3.
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
4.
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
5.
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
6.
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
7.
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
8.
Proc Natl Acad Sci U S A ; 117(33): 20235-20243, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32753384

RESUMO

All cells require Mg2+ to replicate and proliferate. The macrophage protein Slc11a1 is proposed to protect mice from invading microbes by causing Mg2+ starvation in host tissues. However, the Mg2+ transporter MgtB enables the facultative intracellular pathogen Salmonella enterica serovar Typhimurium to cause disease in mice harboring a functional Slc11a1 protein. Here, we report that, unexpectedly, the Salmonella small protein MgtR promotes MgtB degradation by the protease FtsH, which raises the question: How does Salmonella preserve MgtB to promote survival inside macrophages? We establish that the Salmonella small protein MgtU prevents MgtB proteolysis, even when MgtR is absent. Like MgtB, MgtU is necessary for survival in Slc11a1 +/+ macrophages, resistance to oxidative stress, and growth under Mg2+ limitation conditions. The Salmonella Mg2+ transporter MgtA is not protected by MgtU despite sharing 50% amino acid identity with MgtB and being degraded in an MgtR- and FtsH-dependent manner. Surprisingly, the mgtB, mgtR, and mgtU genes are part of the same transcript, providing a singular example of transcript-specifying proteins that promote and hinder degradation of the same target. Our findings demonstrate that small proteins can confer pathogen survival inside macrophages by altering the abundance of related transporters, thereby furthering homeostasis.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Macrófagos/microbiologia , Magnésio/metabolismo , Salmonella typhimurium/fisiologia , Animais , Proteínas de Bactérias/genética , Proteínas de Transporte de Cátions/genética , Linhagem Celular , Macrófagos/fisiologia , Camundongos , Plasmídeos/genética , Salmonella typhimurium/genética , Virulência
9.
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
10.
PLoS One ; 15(8): e0238218, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32845909

RESUMO

One of the most studied mechanisms involved in bacterial evolution and diversification is conjugative transfer (CT) of plasmids. Plasmids able to transfer by CT often encode beneficial traits for bacterial survival under specific environmental conditions. Rhizobium etli CFN42 is a Gram-negative bacterium of agricultural relevance due to its symbiotic association with Phaseolus vulgaris through the formation of Nitrogen-fixing nodules. The genome of R. etli CFN42 consists of one chromosome and six large plasmids. Among these, pRet42a has been identified as a conjugative plasmid. The expression of the transfer genes is regulated by a quorum sensing (QS) system that includes a traI gene, which encodes an acyl-homoserine lactone (AHL) synthase and two transcriptional regulators (TraR and CinR). Recently, we have shown that pRet42a can perform CT on the root surface and inside nodules. The aim of this work was to determine the role of plant-related compounds in the CT of pRet42a. We found that bean root exudates or root and nodule extracts induce the CT of pRet42a in the plant rhizosphere. One possibility is that these compounds are used as nutrients, allowing the bacteria to increase their growth rate and reach the population density leading to the activation of the QS system in a shorter time. We tested if P. vulgaris compounds could substitute the bacterial AHL synthesized by TraI, to activate the conjugation machinery. The results showed that the transfer of pRet42a in the presence of the plant is dependent on the bacterial QS system, which cannot be substituted by plant compounds. Additionally, individual compounds of the plant exudates were evaluated; among these, some increased and others decreased the CT. With these results, we suggest that the plant could participate at different levels to modulate the CT, and that some compounds could be activating genes in the conjugation machinery.


Assuntos
Conjugação Genética/genética , Compostos Fitoquímicos/farmacologia , Plasmídeos/genética , Rhizobium etli/genética , DNA Helicases/genética , DNA Helicases/metabolismo , Phaseolus/química , Phaseolus/microbiologia , Percepção de Quorum/fisiologia , Rizosfera , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
11.
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
12.
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
13.
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
14.
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
15.
PLoS One ; 15(8): e0237886, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32810191

RESUMO

Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) causes gastroenteritis in many countries. However, in Brazil there are few studies that have conducted a virulence characterization of this serovar. The aim of this study was to evaluate the virulence potential of S. Typhimurium strains isolated in Brazil. Forty S. Typhimurium strains isolated from humans (n = 20) and food (n = 20) from Brazil were studied regarding their invasion and survival in human epithelial cells (Caco-2) and macrophages (U937). Their virulence potential was determined using the Galleria mellonella larvae model combined with the analysis of virulence genes by whole genome sequencing (WGS). A total of 67.5% of the S. Typhimurium studied (32.5% isolated from humans and 35% isolated from food) invaded Caco-2 epithelial cells at levels similar to or greater than the S. Typhimurium SL1344 prototype strain. In addition, 37.5% of the studied strains (25% isolated from humans and 12.5% isolated from food) survived in U937 human macrophages at levels similar to or greater than SL1344. S. Typhimurium strains isolated from humans (40%) and food (25%) showed high or intermediate virulence in G. mellonella larvae after seven days exposure. Approximately, 153 virulence genes of chromosomal and plasmidial origin were detected in the strains studied. In conclusion, the ability of the S. Typhimurium to invade Caco-2 epithelial cells was strain dependent and was not related to the source or the year of isolation. However, S. Typhimurium strains isolated from humans showed greater survival rates in U937 human macrophages, and presented higher proportion of isolates with a virulent profile in G. mellonella in comparison to strains isolated from food suggesting that this difference may be related to the higher frequency of human isolates which contained plasmid genes, such as spvABCDR operon, pefABCD operon, rck and mig-5.


Assuntos
Microbiologia de Alimentos , Salmonella typhimurium/genética , Salmonella typhimurium/isolamento & purificação , Animais , Brasil , Células CACO-2 , Sobrevivência Celular , Células Epiteliais/microbiologia , Genes Bacterianos , Genótipo , Humanos , Macrófagos/microbiologia , Mariposas/microbiologia , Fenótipo , Plasmídeos/genética , Salmonella typhimurium/patogenicidade , Células U937 , Virulência/genética
16.
BMC Infect Dis ; 20(1): 569, 2020 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-32753067

RESUMO

BACKGROUND: HIV-1 produces defective mutants in the process of reproduction. The significance of the mutants has not been well investigated. METHODS: The plasmids of wild type (HIV-1NL4-3) and Env-defective (HIV-1SG3ΔEnv) HIV-1 were co-transfected into HEK293T cells. The progeny virus was collected to infect MT4 cells. The env gene and near-full-length genome (NFLG) of HIV-1 were amplified and sequenced. The phylogenetic diversity, recombinant patterns and hotspots, and the functionality of HIV-1 Env were determined. RESULTS: A total of 42 env genes and 8 NFLGs were successfully amplified and sequenced. Five types of recombinant patterns of env were identified and the same recombinant sites were detected in different patterns. The recombination hotspots were found distributing mainly in conservative regions of env. The recombination between genes of HIV-1NL4-3 and HIV-1SG3Δenv increased the variety of viral quasispecies and resulted in progeny viruses with relative lower infectious ability than that of HIVNL4-3. The defective env genes as well as NFLG could be detected after 20 passages. CONCLUSION: The existence of the defective HIV-1 promotes the phylogenetic evolution of the virus, thus increasing the diversity of virus population. The role of defective genes may be converted from junk genes to useful materials and cannot be neglected in the study of HIV-1 reservoir.


Assuntos
Evolução Molecular , Infecções por HIV/patologia , HIV-1/fisiologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/genética , Células HEK293 , Infecções por HIV/virologia , HIV-1/classificação , HIV-1/genética , Humanos , Filogenia , Plasmídeos/genética , Plasmídeos/metabolismo , Recombinação Genética , Produtos do Gene env do Vírus da Imunodeficiência Humana/química , Produtos do Gene env do Vírus da Imunodeficiência Humana/metabolismo
17.
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
18.
G3 (Bethesda) ; 10(9): 3399-3402, 2020 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-32763951

RESUMO

The world is facing a global pandemic of COVID-19 caused by the SARS-CoV-2 coronavirus. Here we describe a collection of codon-optimized coding sequences for SARS-CoV-2 cloned into Gateway-compatible entry vectors, which enable rapid transfer into a variety of expression and tagging vectors. The collection is freely available. We hope that widespread availability of this SARS-CoV-2 resource will enable many subsequent molecular studies to better understand the viral life cycle and how to block it.


Assuntos
Betacoronavirus/genética , Fases de Leitura Aberta/genética , Betacoronavirus/isolamento & purificação , Clonagem Molecular , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Escherichia coli/metabolismo , Humanos , Pandemias , Plasmídeos/genética , Plasmídeos/metabolismo , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Potyvirus/genética
19.
PLoS One ; 15(7): e0235641, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32614888

RESUMO

We sequenced 25 isolates of phenotypically multidrug-resistant Salmonella Indiana (n = 11), Typhimurium (n = 8), and Enteritidis (n = 6) using both MinION long-read [SQK-LSK109 and flow cell (R9.4.1)] and MiSeq short-read (Nextera XT and MiSeq Reagent Kit v2) sequencing technologies to determine the advantages of each approach in terms of the characteristics of genome structure, antimicrobial resistance (AMR), virulence potential, whole-genome phylogeny, and pan-genome. The MinION reads were base-called in real-time using MinKnow 3.4.8 integrated with Guppy 3.0.7. The long-read-only assembly, Illumina-only assembly, and hybrid assembly pipelines of Unicycler 0.4.8 were used to generate the MinION, MiSeq, and hybrid assemblies, respectively. The MinION assemblies were highly contiguous compared to the MiSeq assemblies but lacked accuracy, a deficiency that was mitigated by adding the MiSeq short reads through the Unicycler hybrid assembly which corrected erroneous single nucleotide polymorphisms (SNPs). The MinION assemblies provided similar predictions of AMR and virulence potential compared to the MiSeq and hybrid assemblies, although they produced more total false negatives of AMR genotypes, primarily due to failure in identifying tetracycline resistance genes in 11 of the 19 MinION assemblies of tetracycline-resistant isolates. The MinION assemblies displayed a large genetic distance from their corresponding MiSeq and hybrid assemblies on the whole-genome phylogenetic tree, indicating that the lower read accuracy of MinION sequencing caused incorrect clustering. The pan-genome of the MinION assemblies contained significantly more accessory genes and less core genes compared to the MiSeq and hybrid assemblies, suggesting that although these assemblies were more contiguous, their sequencing errors reduced accurate genome annotations. Our research demonstrates that MinION sequencing by itself provides an efficient assessment of the genome structure, antimicrobial resistance, and virulence potential of Salmonella; however, it is not sufficient for whole-genome phylogenetic and pan-genome analyses. MinION in combination with MiSeq facilitated the most accurate genomic analyses.


Assuntos
Farmacorresistência Bacteriana Múltipla/genética , Genoma Bacteriano , Salmonella enterica/genética , Sequenciamento Completo do Genoma/métodos , Antibacterianos/farmacologia , Genótipo , Testes de Sensibilidade Microbiana , Fenótipo , Filogenia , Plasmídeos/genética , Plasmídeos/metabolismo , Mutação Puntual , Salmonella enterica/classificação , Salmonella enterica/efeitos dos fármacos , Salmonella enterica/patogenicidade , Salmonella enteritidis/classificação , Salmonella enteritidis/efeitos dos fármacos , Salmonella enteritidis/genética , Salmonella enteritidis/patogenicidade , Salmonella typhimurium/classificação , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/genética , Salmonella typhimurium/patogenicidade , Virulência
20.
PLoS One ; 15(7): e0235633, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32628709

RESUMO

The antibacterial efficacy of the tetracycline antibiotics has been greatly reduced by the development of resistance, hence a decline in their clinical use. The hok/sok locus is a type I toxin/antitoxin plasmid stability element, often associated with multi-drug resistance plasmids, especially ESBL-encoding plasmids. It enhances host cell survivability and pathogenicity in stressful growth conditions, and increases bacterial tolerance to ß-lactam antibiotics. The hok/sok locus forms dsRNA by RNA:RNA interactions between the toxin encoding mRNA and antitoxin non-coding RNA, and doxycycline has been reported to bind dsRNA structures and inhibit their cleavage/processing by the dsRNase, RNase III. This study investigated the antibacterial activities of doxycycline in hok/sok host bacteria cells, the effects on hok/sok-induced changes in growth and the mechanism(s) involved. Diverse strains of E. coli were transformed with hok/sok plasmids and assessed for doxycycline susceptibility and growth changes. The results show that the hok/sok locus increases bacterial susceptibility to doxycycline, which is more apparent in strains with more pronounced hok/sok-induced growth effects. The increased doxycycline susceptibility occurs despite ß-lactam resistance imparted by hok/sok. Doxycycline was found to induce bacterial death in a manner phenotypically characteristic of Hok toxin expression, suggesting that it inhibits the toxin/antitoxin dsRNA degradation, leading to Hok toxin expression and cell death. In this way, doxycycline could counteract the multi-drug resistance plasmid maintenance/propagation, persistence and pathogenicity mechanisms associated with the hok/sok locus, which could potentially help in efforts to mitigate the rise of antimicrobial resistance.


Assuntos
Antibacterianos/farmacologia , Toxinas Bacterianas/genética , Doxiciclina/farmacologia , Proteínas de Escherichia coli/genética , Escherichia coli/efeitos dos fármacos , RNA Bacteriano/genética , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Plasmídeos/genética , Plasmídeos/metabolismo , RNA Bacteriano/metabolismo , RNA de Cadeia Dupla/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA