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1.
Folia Biol (Praha) ; 66(3): 91-103, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33069188

RESUMO

The most recent genome-editing system called CRISPR-Cas9 (clustered regularly interspaced short palindromic repeat system with associated protein 9-nuclease) was employed to delete four non-essential genes (i.e., Caeco1, Caidh1, Carom2, and Cataf10) individually to establish their gene functionality annotations in pathogen Candida albicans. The biological roles of these genes were investigated with respect to the cell wall integrity and biogenesis, calcium/calcineurin pathways, susceptibility of mutants towards temperature, drugs and salts. All the mutants showed increased vulnerability compared to the wild-type background strain towards the cell wall-perturbing agents, (antifungal) drugs and salts. All the mutants also exhibited repressed and defective hyphal growth and smaller colony size than control CA14. The cell cycle of all the mutants decreased enormously except for those with Carom2 deletion. The budding index and budding size also increased for all mutants with altered bud shape. The disposition of the mutants towards cell wall-perturbing enzymes disclosed lower survival and more rapid cell wall lysis events than in wild types. The pathogenicity and virulence of the mutants was checked by adhesion assay, and strains lacking rom2 and eco1 were found to possess the least adhesion capacity, which is synonymous to their decreased pathogenicity and virulence.


Assuntos
Candida albicans/fisiologia , Proteínas Fúngicas/fisiologia , Genes Fúngicos , Acetiltransferases/deficiência , Acetiltransferases/genética , Acetiltransferases/fisiologia , Antifúngicos/farmacologia , Sistemas CRISPR-Cas , Cálcio/fisiologia , Candida albicans/efeitos dos fármacos , Candida albicans/genética , Candida albicans/patogenicidade , Cátions/farmacologia , Adesão Celular , Ciclo Celular , Parede Celular/efeitos dos fármacos , Quitinases/farmacologia , Dano ao DNA , Proteínas Fúngicas/genética , Deleção de Genes , Glucana Endo-1,3-beta-D-Glucosidase/farmacologia , Hifas/crescimento & desenvolvimento , Isocitrato Desidrogenase/deficiência , Isocitrato Desidrogenase/genética , Isocitrato Desidrogenase/fisiologia , Fases de Leitura Aberta , Reprodução Assexuada , Fatores Associados à Proteína de Ligação a TATA/deficiência , Fatores Associados à Proteína de Ligação a TATA/genética , Fatores Associados à Proteína de Ligação a TATA/fisiologia , Virulência/genética
2.
J Transl Med ; 18(1): 329, 2020 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-32867854

RESUMO

BACKGROUND: The new Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), which was first detected in Wuhan (China) in December of 2019 is responsible for the current global pandemic. Phylogenetic analysis revealed that it is similar to other betacoronaviruses, such as SARS-CoV and Middle-Eastern Respiratory Syndrome, MERS-CoV. Its genome is ∼ 30 kb in length and contains two large overlapping polyproteins, ORF1a and ORF1ab that encode for several structural and non-structural proteins. The non-structural protein 1 (nsp1) is arguably the most important pathogenic determinant, and previous studies on SARS-CoV indicate that it is both involved in viral replication and hampering the innate immune system response. Detailed experiments of site-specific mutagenesis and in vitro reconstitution studies determined that the mechanisms of action are mediated by (a) the presence of specific amino acid residues of nsp1 and (b) the interaction between the protein and the host's small ribosomal unit. In fact, substitution of certain amino acids resulted in reduction of its negative effects. METHODS: A total of 17,928 genome sequences were obtained from the GISAID database (December 2019 to July 2020) from patients infected by SARS-CoV-2 from different areas around the world. Genomes alignment was performed using MAFFT (REFF) and the nsp1 genomic regions were identified using BioEdit and verified using BLAST. Nsp1 protein of SARS-CoV-2 with and without deletion have been subsequently modelled using I-TASSER. RESULTS: We identified SARS-CoV-2 genome sequences, from several Countries, carrying a previously unknown deletion of 9 nucleotides in position 686-694, corresponding to the AA position 241-243 (KSF). This deletion was found in different geographical areas. Structural prediction modelling suggests an effect on the C-terminal tail structure. CONCLUSIONS: Modelling analysis of a newly identified deletion of 3 amino acids (KSF) of SARS-CoV-2 nsp1 suggests that this deletion could affect the structure of the C-terminal region of the protein, important for regulation of viral replication and negative effect on host's gene expression. In addition, substitution of the two amino acids (KS) from nsp1 of SARS-CoV was previously reported to revert loss of interferon-alpha expression. The deletion that we describe indicates that SARS-CoV-2 is undergoing profound genomic changes. It is important to: (i) confirm the spreading of this particular viral strain, and potentially of strains with other deletions in the nsp1 protein, both in the population of asymptomatic and pauci-symptomatic subjects, and (ii) correlate these changes in nsp1 with potential decreased viral pathogenicity.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/virologia , Pneumonia Viral/virologia , Deleção de Sequência , Proteínas não Estruturais Virais/genética , Sequência de Aminoácidos , Sequência de Bases , Betacoronavirus/patogenicidade , Doenças Transmissíveis Emergentes/virologia , Infecções por Coronavirus/epidemiologia , Frequência do Gene , Genoma Viral , Geografia , Humanos , Lisina/genética , Modelos Moleculares , Pandemias/estatística & dados numéricos , Fenilalanina/genética , Pneumonia Viral/epidemiologia , Domínios Proteicos/genética , Serina/genética , Proteínas não Estruturais Virais/química , Virulência/genética , Replicação Viral/genética
3.
J Transl Med ; 18(1): 329, 2020 08 31.
Artigo em Inglês | MEDLINE | ID: covidwho-736398

RESUMO

BACKGROUND: The new Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), which was first detected in Wuhan (China) in December of 2019 is responsible for the current global pandemic. Phylogenetic analysis revealed that it is similar to other betacoronaviruses, such as SARS-CoV and Middle-Eastern Respiratory Syndrome, MERS-CoV. Its genome is ∼ 30 kb in length and contains two large overlapping polyproteins, ORF1a and ORF1ab that encode for several structural and non-structural proteins. The non-structural protein 1 (nsp1) is arguably the most important pathogenic determinant, and previous studies on SARS-CoV indicate that it is both involved in viral replication and hampering the innate immune system response. Detailed experiments of site-specific mutagenesis and in vitro reconstitution studies determined that the mechanisms of action are mediated by (a) the presence of specific amino acid residues of nsp1 and (b) the interaction between the protein and the host's small ribosomal unit. In fact, substitution of certain amino acids resulted in reduction of its negative effects. METHODS: A total of 17,928 genome sequences were obtained from the GISAID database (December 2019 to July 2020) from patients infected by SARS-CoV-2 from different areas around the world. Genomes alignment was performed using MAFFT (REFF) and the nsp1 genomic regions were identified using BioEdit and verified using BLAST. Nsp1 protein of SARS-CoV-2 with and without deletion have been subsequently modelled using I-TASSER. RESULTS: We identified SARS-CoV-2 genome sequences, from several Countries, carrying a previously unknown deletion of 9 nucleotides in position 686-694, corresponding to the AA position 241-243 (KSF). This deletion was found in different geographical areas. Structural prediction modelling suggests an effect on the C-terminal tail structure. CONCLUSIONS: Modelling analysis of a newly identified deletion of 3 amino acids (KSF) of SARS-CoV-2 nsp1 suggests that this deletion could affect the structure of the C-terminal region of the protein, important for regulation of viral replication and negative effect on host's gene expression. In addition, substitution of the two amino acids (KS) from nsp1 of SARS-CoV was previously reported to revert loss of interferon-alpha expression. The deletion that we describe indicates that SARS-CoV-2 is undergoing profound genomic changes. It is important to: (i) confirm the spreading of this particular viral strain, and potentially of strains with other deletions in the nsp1 protein, both in the population of asymptomatic and pauci-symptomatic subjects, and (ii) correlate these changes in nsp1 with potential decreased viral pathogenicity.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/virologia , Pneumonia Viral/virologia , Deleção de Sequência , Proteínas não Estruturais Virais/genética , Sequência de Aminoácidos , Sequência de Bases , Betacoronavirus/patogenicidade , Doenças Transmissíveis Emergentes/virologia , Infecções por Coronavirus/epidemiologia , Frequência do Gene , Genoma Viral , Geografia , Humanos , Lisina/genética , Modelos Moleculares , Pandemias/estatística & dados numéricos , Fenilalanina/genética , Pneumonia Viral/epidemiologia , Domínios Proteicos/genética , Serina/genética , Proteínas não Estruturais Virais/química , Virulência/genética , Replicação Viral/genética
4.
BMC Infect Dis ; 20(1): 585, 2020 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-32762666

RESUMO

BACKGROUND: The polymerase chain reaction (PCR) is commonly used to detect viral pathogens because of its high sensitivity and specificity. However, conventional PCR methods cannot determine virus infectivity. Virus infectivity is conventionally examined with methods such as the plaque assay, even though such assays require several days. Long-range reverse-transcription quantitative PCR (RT-qPCR) has previously been suggested for the rapid assessment of RNA virus infectivity where the loss of infectivity is attributable to genomic fragmentation. METHODS: IAV was irradiated with 253.7 nm ultraviolet (UV) rays to induce genomic strand breaks that were confirmed by a full-length RT-PCR assay. The IAV was then subjected to plaque assay, conventional RT-qPCR and long-range RT-qPCR to examine the relationship between infectious titer and copy number. A simple linear regression analysis was performed to examine the correlation between the results of these assays. RESULTS: A long-range RT-qPCR assay was developed and validated for influenza A virus (IAV). Although only a few minutes of UV irradiation was required to completely inactivate IAV, genomic RNA remained detectable by the conventional RT-qPCR and the full-length RT-PCR for NS of viral genome following inactivation. A long-range RT-qPCR assay was then designed using RT-priming at the 3' termini of each genomic segment and subsequent qPCR of the 5' regions. UV-mediated IAV inactivation was successfully analyzed by the long-range RT-qPCR assay especially when targeting PA of the viral genome. This was also supported by the regression analysis that the long-range RT-qPCR is highly correlated with plaque assay (Adjusted R2 = 0.931, P = 0.000066). CONCLUSIONS: This study suggests that IAV infectivity can be predicted without the infectivity assays. The rapid detection of pathogenic IAV has, therefore, been achieved with this sensing technology.


Assuntos
Vírus da Influenza A/genética , Vírus da Influenza A/patogenicidade , Infecções por Orthomyxoviridae/diagnóstico , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Virulência/genética , Animais , Cães , Genoma Viral/genética , Genoma Viral/efeitos da radiação , Vírus da Influenza A/isolamento & purificação , Vírus da Influenza A/efeitos da radiação , Células Madin Darby de Rim Canino , Infecções por Orthomyxoviridae/virologia , Estabilidade de RNA/efeitos da radiação , RNA Viral/genética , RNA Viral/efeitos da radiação , Raios Ultravioleta , Inativação de Vírus/efeitos da radiação
6.
Sci Rep ; 10(1): 14031, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32820179

RESUMO

The COVID-19 pandemic, caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), was declared on March 11, 2020 by the World Health Organization. As of the 31st of May, 2020, there have been more than 6 million COVID-19 cases diagnosed worldwide and over 370,000 deaths, according to Johns Hopkins. Thousands of SARS-CoV-2 strains have been sequenced to date, providing a valuable opportunity to investigate the evolution of the virus on a global scale. We performed a phylogenetic analysis of over 1,225 SARS-CoV-2 genomes spanning from late December 2019 to mid-March 2020. We identified a missense mutation, D614G, in the spike protein of SARS-CoV-2, which has emerged as a predominant clade in Europe (954 of 1,449 (66%) sequences) and is spreading worldwide (1,237 of 2,795 (44%) sequences). Molecular dating analysis estimated the emergence of this clade around mid-to-late January (10-25 January) 2020. We also applied structural bioinformatics to assess the potential impact of D614G on the virulence and epidemiology of SARS-CoV-2. In silico analyses on the spike protein structure suggests that the mutation is most likely neutral to protein function as it relates to its interaction with the human ACE2 receptor. The lack of clinical metadata available prevented our investigation of association between viral clade and disease severity phenotype. Future work that can leverage clinical outcome data with both viral and human genomic diversity is needed to monitor the pandemic.


Assuntos
Betacoronavirus/química , Infecções por Coronavirus/epidemiologia , Evolução Molecular , Pneumonia Viral/epidemiologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Sequência de Bases , Betacoronavirus/patogenicidade , Criança , Pré-Escolar , Simulação por Computador , Infecções por Coronavirus/virologia , Feminino , Genoma Viral/genética , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Pandemias , Peptidil Dipeptidase A/metabolismo , Filogenia , Pneumonia Viral/virologia , Conformação Proteica , Glicoproteína da Espícula de Coronavírus/metabolismo , Virulência/genética , Adulto Jovem
7.
Nat Commun ; 11(1): 4212, 2020 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-32839469

RESUMO

Phosphatases, together with kinases and transcription factors, are key components in cellular signalling networks. Here, we present a systematic functional analysis of the phosphatases in Cryptococcus neoformans, a fungal pathogen that causes life-threatening fungal meningoencephalitis. We analyse 230 signature-tagged mutant strains for 114 putative phosphatases under 30 distinct in vitro growth conditions, revealing at least one function for 60 of these proteins. Large-scale virulence and infectivity assays using insect and mouse models indicate roles in pathogenicity for 31 phosphatases involved in various processes such as thermotolerance, melanin and capsule production, stress responses, O-mannosylation, or retromer function. Notably, phosphatases Xpp1, Ssu72, Siw14, and Sit4 promote blood-brain barrier adhesion and crossing by C. neoformans. Together with our previous systematic studies of transcription factors and kinases, our results provide comprehensive insight into the pathobiological signalling circuitry of C. neoformans.


Assuntos
Cryptococcus neoformans/genética , Proteínas Fúngicas/genética , Perfilação da Expressão Gênica/métodos , Genoma Fúngico/genética , Estudo de Associação Genômica Ampla/métodos , Monoéster Fosfórico Hidrolases/genética , Animais , Análise por Conglomerados , Criptococose/microbiologia , Cryptococcus neoformans/patogenicidade , Feminino , Proteínas Fúngicas/classificação , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Camundongos Endogâmicos , Monoéster Fosfórico Hidrolases/classificação , Monoéster Fosfórico Hidrolases/metabolismo , Fosfotransferases/classificação , Fosfotransferases/genética , Fosfotransferases/metabolismo , Transdução de Sinais/genética , Termotolerância/genética , Fatores de Transcrição/classificação , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Virulência/genética
8.
BMC Infect Dis ; 20(1): 602, 2020 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-32799799

RESUMO

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


Assuntos
Nariz/microbiologia , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/veterinária , Staphylococcus aureus/efeitos dos fármacos , Animais , Animais Domésticos/microbiologia , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Portador Sadio , Farmacorresistência Bacteriana/efeitos dos fármacos , Farmacorresistência Bacteriana/genética , Humanos , Leucocidinas/genética , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Testes de Sensibilidade Microbiana , Nuclease do Micrococo/genética , Marrocos/epidemiologia , Proteínas de Ligação às Penicilinas/genética , Infecções Estafilocócicas/epidemiologia , Staphylococcus aureus/genética , Staphylococcus aureus/isolamento & purificação , Staphylococcus aureus/patogenicidade , Tetraciclina/farmacologia , Virulência/genética
9.
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
10.
PLoS One ; 15(8): e0237135, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32822422

RESUMO

DNA-binding Transcription Factors (TFs) play a central role in regulation of gene expression in prokaryotic organisms, and similarities at the sequence level have been reported. These proteins are predicted with different abundances as a consequence of genome size, where small organisms contain a low proportion of TFs and large genomes contain a high proportion of TFs. In this work, we analyzed a collection of 668 experimentally validated TFs across 30 different species from diverse taxonomical classes, including Escherichia coli K-12, Bacillus subtilis 168, Corynebacterium glutamicum, and Streptomyces coelicolor, among others. This collection of TFs, together with 111 hidden Markov model profiles associated with DNA-binding TFs collected from diverse databases such as PFAM and DBD, was used to identify the repertoire of proteins putatively devoted to gene regulation in 1321 representative genomes of Archaea and Bacteria. The predicted regulatory proteins were posteriorly analyzed in terms of their genomic context, allowing the prediction of functions for TFs and their neighbor genes, such as genes involved in virulence, enzymatic functions, phosphorylation mechanisms, and antibiotic resistance. The functional analysis associated with PFAM groups showed diverse functional categories were significantly enriched in the collection of TFs and the proteins encoded by the neighbor genes, in particular, small-molecule binding and amino acid transmembrane transporter activities associated with the LysR family and proteins devoted to cellular aromatic compound metabolic processes or responses to drugs, stress, or abiotic stimuli in the MarR family. We consider that with the increasing data derived from new technologies, novel TFs can be identified and help improve the predictions for this class of proteins in complete genomes. The complete collection of experimentally characterized and predicted TFs is available at http://web.pcyt.unam.mx/EntrafDB/.


Assuntos
Archaea/genética , Proteínas Arqueais/genética , Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/genética , Escherichia coli K12/genética , Fatores de Transcrição/genética , Archaea/patogenicidade , Proteínas Arqueais/metabolismo , Proteínas de Bactérias/metabolismo , Sítios de Ligação , DNA Arqueal/metabolismo , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/metabolismo , Escherichia coli K12/patogenicidade , Regulação da Expressão Gênica em Archaea , Regulação Bacteriana da Expressão Gênica , Genoma Arqueal , Genoma Bacteriano , Ligação Proteica , Fatores de Transcrição/metabolismo , Virulência/genética
11.
Sci Rep ; 10(1): 14031, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: covidwho-724696

RESUMO

The COVID-19 pandemic, caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), was declared on March 11, 2020 by the World Health Organization. As of the 31st of May, 2020, there have been more than 6 million COVID-19 cases diagnosed worldwide and over 370,000 deaths, according to Johns Hopkins. Thousands of SARS-CoV-2 strains have been sequenced to date, providing a valuable opportunity to investigate the evolution of the virus on a global scale. We performed a phylogenetic analysis of over 1,225 SARS-CoV-2 genomes spanning from late December 2019 to mid-March 2020. We identified a missense mutation, D614G, in the spike protein of SARS-CoV-2, which has emerged as a predominant clade in Europe (954 of 1,449 (66%) sequences) and is spreading worldwide (1,237 of 2,795 (44%) sequences). Molecular dating analysis estimated the emergence of this clade around mid-to-late January (10-25 January) 2020. We also applied structural bioinformatics to assess the potential impact of D614G on the virulence and epidemiology of SARS-CoV-2. In silico analyses on the spike protein structure suggests that the mutation is most likely neutral to protein function as it relates to its interaction with the human ACE2 receptor. The lack of clinical metadata available prevented our investigation of association between viral clade and disease severity phenotype. Future work that can leverage clinical outcome data with both viral and human genomic diversity is needed to monitor the pandemic.


Assuntos
Betacoronavirus/química , Infecções por Coronavirus/epidemiologia , Evolução Molecular , Pneumonia Viral/epidemiologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Sequência de Bases , Betacoronavirus/patogenicidade , Criança , Pré-Escolar , Simulação por Computador , Infecções por Coronavirus/virologia , Feminino , Genoma Viral/genética , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Pandemias , Peptidil Dipeptidase A/metabolismo , Filogenia , Pneumonia Viral/virologia , Conformação Proteica , Glicoproteína da Espícula de Coronavírus/metabolismo , Virulência/genética , Adulto Jovem
12.
PLoS One ; 15(8): e0238151, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32833990

RESUMO

Bacteria often possess relatively flexible genome structures and adaptive genetic variants that allow survival in unfavorable growth conditions. Bacterial survival tactics in disadvantageous microenvironments include mutations that are beneficial against threats in their niche. Here, we report that the aerobic rice bacterial pathogen Burkholderia glumae BGR1 changes a specific gene for improved survival in static culture conditions. Static culture triggered formation of colony variants with deletions or point mutations in the gene bspP (BGLU_RS28885), which putatively encodes a protein that contains PDC2, PAS-9, SpoIIE, and HATPase domains. The null mutant of bspP survived longer in static culture conditions and produced a higher level of bis-(3'-5')-cyclic dimeric guanosine monophosphate than the wild type. Expression of the bacterial cellulose synthase regulator (bcsB) gene was upregulated in the mutant, consistent with the observation that the mutant formed pellicles faster than the wild type. Mature pellicle formation was observed in the bspP mutant before pellicle formation in wild-type BGR1. However, the population density of the bspP null mutant decreased substantially when grown in Luria-Bertani medium with vigorous agitation due to failure of oxalate-mediated detoxification of the alkaline environment. The bspP null mutant was less virulent and exhibited less effective colonization of rice plants than the wild type. All phenotypes caused by mutations in bspP were recovered to those of the wild type by genetic complementation. Thus, although wild-type B. glumae BGR1 prolonged viability by spontaneous mutation under static culture conditions, such genetic changes negatively affected colonization in rice plants. These results suggest that adaptive gene sacrifice of B. glumae to survive unfavorable growth conditions is not always desirable as it can adversely affect adaptability in the host.


Assuntos
Adaptação Biológica/genética , Burkholderia/genética , Burkholderia/metabolismo , Burkholderia/patogenicidade , Regulação Bacteriana da Expressão Gênica/genética , Genoma Bacteriano/genética , Genômica/métodos , Mutação , Oryza/microbiologia , Doenças das Plantas/microbiologia , Percepção de Quorum/genética , Virulência/genética
13.
PLoS One ; 15(8): e0232305, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32785271

RESUMO

Shiga toxin-producing Escherichia coli (STEC) that cause severe disease predominantly carry the toxin gene variant stx2a. However, the role of Shiga toxin in the ruminant reservoirs of this zoonotic pathogen is poorly understood and strains that cause severe disease in humans (HUSEC) likely constitute a small and atypical subset of the overall STEC flora. The aim of this study was to investigate the presence of stx2a in samples from cattle and to isolate and characterize stx2a-positive E. coli. In nationwide surveys in Sweden and Norway samples were collected from individual cattle or from cattle herds, respectively. Samples were tested for Shiga toxin genes by real-time PCR and amplicon sequencing and stx2a-positive isolates were whole genome sequenced. Among faecal samples from Sweden, stx1 was detected in 37%, stx2 in 53% and stx2a in 5% and in skin (ear) samples in 64%, 79% and 2% respectively. In Norway, 79% of the herds were positive for stx1, 93% for stx2 and 17% for stx2a. Based on amplicon sequencing the most common stx2 types in samples from Swedish cattle were stx2a and stx2d. Multilocus sequence typing (MLST) of 39 stx2a-positive isolates collected from both countries revealed substantial diversity with 19 different sequence types. Only a few classical LEE-positive strains similar to HUSEC were found among the stx2a-positive isolates, notably a single O121:H19 and an O26:H11. Lineages known to include LEE-negative HUSEC were also recovered including, such as O113:H21 (sequence type ST-223), O130:H11 (ST-297), and O101:H33 (ST-330). We conclude that E. coli encoding stx2a in cattle are ranging from strains similar to HUSEC to unknown STEC variants. Comparison of isolates from human HUS cases to related STEC from the ruminant reservoirs can help identify combinations of virulence attributes necessary to cause HUS, as well as provide a better understanding of the routes of infection for rare and emerging pathogenic STEC.


Assuntos
Bovinos/microbiologia , Toxina Shiga II/genética , Escherichia coli Shiga Toxigênica/genética , Animais , Reservatórios de Doenças/microbiologia , Infecções por Escherichia coli/epidemiologia , Infecções por Escherichia coli/microbiologia , Variação Genética , Genoma Bacteriano , Síndrome Hemolítico-Urêmica/epidemiologia , Síndrome Hemolítico-Urêmica/microbiologia , Humanos , Tipagem de Sequências Multilocus , Noruega/epidemiologia , Prevalência , Reação em Cadeia da Polimerase em Tempo Real , Escherichia coli Shiga Toxigênica/citologia , Escherichia coli Shiga Toxigênica/isolamento & purificação , Suécia/epidemiologia , Virulência/genética , Zoonoses/epidemiologia , Zoonoses/microbiologia
14.
Appl Microbiol Biotechnol ; 104(19): 8427-8437, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32813067

RESUMO

Infectious bronchitis virus (IBV) is a member of genus gamma-coronavirus in the family Coronaviridae, causing serious economic losses to the poultry industry. Reverse genetics is a common technique to study the biological characteristics of viruses. So far, there is no BAC reverse genetic system available for rescue of IBV infectious clone. In the present study, a new strategy for the construction of IBV infectious cDNA clone was established. The full-length genomic cDNA of IBV vaccine strain H120 was constructed in pBAC vector from four IBV fragment subcloning vectors by homologous recombination, which contained the CMV promoter at the 5' end and the hepatitis D virus ribozyme (HDVR) sequence and bovine growth hormone polyadenylation (BGH) sequence after the polyA tail at the 3' end of the full-length cDNA. Subsequently, using the same technique, another plasmid pBAC-H120/SCS1 was also constructed, in which S1 gene from IBV H120 strain was replaced with that of a virulent SC021202 strain. Recombinant virus rH120 and rH120/SCS1 were rescued by transfecting the plasmids into BHK cells and passaged in embryonated chicken eggs. Finally, the pathogenicity of both the recombinant virus strains rH120 and rH120/SCS1 was evaluated in SPF chickens. The results showed that the chimeric rH120/SCS1 strain was not pathogenic compared with the wild-type IBV SC021202 strain and the chickens inoculated with rH120/SCS1 could resist challenge infection by IBV SC021202. Taken together, our results indicate that BAC reverse genetic system could be used to rescue IBV in vitro and IBV S1 protein alone might not be the key factor for IBV pathogenicity. KEY POINTS: • BAC vector was used to construct IBV full-length cDNA by homologous recombination. • Based on four subcloning vectors, a recombinant chimeric IBV H120/SCS1 was constructed and rescued. • Pathogenicity of H120/SCS1 was similar to that of H120, but different to that of SC021202.


Assuntos
Vírus da Bronquite Infecciosa/genética , Vírus da Bronquite Infecciosa/patogenicidade , Proteínas Virais/genética , Animais , Embrião de Galinha , Galinhas , Infecções por Coronavirus/veterinária , DNA Complementar , Recombinação Homóloga , Doenças das Aves Domésticas/virologia , Proteínas Recombinantes/genética , Vacinas Virais/genética , Vacinas Virais/imunologia , Virulência/genética
15.
PLoS Genet ; 16(7): e1008611, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32658892

RESUMO

Epimutations in fungal pathogens are emerging as novel phenomena that could explain the fast-developing resistance to antifungal drugs and other stresses. These epimutations are generated by RNA interference (RNAi) mechanisms that transiently silence specific genes to overcome stressful stimuli. The early-diverging fungus Mucor circinelloides exercises a fine control over two interacting RNAi pathways to produce epimutants: the canonical RNAi pathway and a new RNAi degradative pathway. The latter is considered a non-canonical RNAi pathway (NCRIP) because it relies on RNA-dependent RNA polymerases (RdRPs) and a novel ribonuclease III-like named R3B2 to degrade target transcripts. Here in this work, we uncovered the role of NCRIP in regulating virulence processes and transposon movements through key components of the pathway, RdRP1 and R3B2. Mutants in these genes are unable to launch a proper virulence response to macrophage phagocytosis, resulting in a decreased virulence potential. The transcriptomic profile of rdrp1Δ and r3b2Δ mutants revealed a pre-exposure adaptation to the stressful phagosomal environment even when the strains are not confronted by macrophages. These results suggest that NCRIP represses key targets during regular growth and releases its control when a stressful environment challenges the fungus. NCRIP interacts with the RNAi canonical core to protect genome stability by controlling the expression of centromeric retrotransposable elements. In the absence of NCRIP, these retrotransposons are robustly repressed by the canonical RNAi machinery; thus, supporting the antagonistic role of NCRIP in containing the epimutational pathway. Both interacting RNAi pathways might be essential to govern host-pathogen interactions through transient adaptations, contributing to the unique traits of the emerging infection mucormycosis.


Assuntos
Mucorales/genética , Mucormicose/genética , Interferência de RNA , Ribonuclease III/genética , Antifúngicos/farmacologia , Farmacorresistência Fúngica/genética , Epigênese Genética/genética , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica/genética , Instabilidade Genômica/efeitos dos fármacos , Interações Hospedeiro-Patógeno/genética , Mucorales/patogenicidade , Mucormicose/microbiologia , Mutação/genética , RNA Mensageiro/genética , Transdução de Sinais/efeitos dos fármacos , Virulência/genética
16.
PLoS One ; 15(7): e0233945, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32701964

RESUMO

The survival of Listeria (L.) monocytogenes in foods and food production environments (FPE) is dependent on several genes that increase tolerance to stressors; this includes competing with intrinsic bacteria. We aimed to uncover genes that are differentially expressed (DE) in L. monocytogenes sequence type (ST) 121 strain 6179 when co-cultured with cheese rind bacteria. L. monocytogenes was cultivated in broth or on plates with either a Psychrobacter or Brevibacterium isolate from cheese rinds. RNA was extracted from co-cultures in broth after two or 12 hours and from plates after 24 and 72 hours. Broth co-cultivations with Brevibacterium or Psychrobacter yielded up to 392 and 601 DE genes, while plate co-cultivations significantly affected the expression of up to 190 and 485 L. monocytogenes genes, respectively. Notably, the transcription of virulence genes encoding the Listeria adhesion protein and Listeriolysin O were induced during plate and broth co-cultivations. The expression of several systems under the control of the global stress gene regulator, σB, increased during co-cultivation. A cobalamin-dependent gene cluster, responsible for the catabolism of ethanolamine and 1,2-propanediol, was upregulated in both broth and plate co-cultures conditions. Finally, a small non-coding (nc)RNA, Rli47, was induced after 72 hours of co-cultivation on plates and accounted for 50-90% of the total reads mapped to L. monocytogenes. A recent study has shown that Rli47 may contribute to L. monocytogenes stress survival by slowing growth during stress conditions through the suppression of branch-chained amino acid biosynthesis. We hypothesize that Rli47 may have an impactful role in the response of L. monocytogenes to co-cultivation by regulating a complex network of metabolic and virulence mechanisms.


Assuntos
Brevibacterium/metabolismo , Queijo/microbiologia , Etanolamina/metabolismo , Microbiologia de Alimentos , Regulação Bacteriana da Expressão Gênica , Listeria monocytogenes/genética , Propilenoglicol/metabolismo , Psychrobacter/metabolismo , Transcriptoma , Aclimatação , Ágar , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Técnicas de Cocultura , Meios de Cultura , Transporte de Elétrons/genética , Fermentação/genética , Listeria monocytogenes/metabolismo , Listeria monocytogenes/patogenicidade , Plasmídeos , RNA Bacteriano/biossíntese , RNA Bacteriano/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Pequeno RNA não Traduzido/biossíntese , Pequeno RNA não Traduzido/genética , Virulência/genética
17.
BMC Infect Dis ; 20(1): 453, 2020 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-32600258

RESUMO

INTRODUCTION: Uropathogenic E. coli is the leading cause of Urinary tract infections (UTIs), contributing to 80-90% of all community-acquired and 30-50% of all hospital-acquired UTIs. Biofilm forming Uropathogenic E. coli are associated with persistent and chronic inflammation leading to complicated and or recurrent UTIs. Biofilms provide an environment for poor antibiotic penetration and horizontal transfer of virulence genes which favors the development of Multidrug-resistant organisms (MDRO). Understanding biofilm formation and antimicrobial resistance determinants of Uropathogenic E. coli strains will provide insight into the development of treatment options for biofilm-associated UTIs. The aim of this study was to determine the biofilm forming capability, presence of virulence genes and antimicrobial susceptibility pattern of Uropathogenic E. coli isolates in Uganda. METHODS: This was a cross-sectional study carried in the Clinical Microbiology and Molecular biology laboratories at the Department of Medical Microbiology, Makerere University College of Health Sciences. We randomly selected 200 Uropathogenic E. coli clinical isolates among the stored isolates collected between January 2018 and December 2018 that had significant bacteriuria (> 105 CFU). All isolates were subjected to biofilm detection using the Congo Red Agar method and Antimicrobial susceptibility testing was performed using the Kirby disk diffusion method. The isolates were later subjected PCR for the detection of Urovirulence genes namely; Pap, Fim, Sfa, Afa, Hly and Cnf, using commercially designed primers. RESULTS: In this study, 62.5% (125/200) were positive biofilm formers and 78% (156/200) of these were multi-drug resistant (MDR). The isolates were most resistant to Trimethoprim sulphamethoxazole and Amoxicillin (93%) followed by gentamycin (87%) and the least was imipenem (0.5%). Fim was the most prevalent Urovirulence gene (53.5%) followed by Pap (21%), Sfa (13%), Afa (8%), Cnf (5.5%) and Hyl (0%). CONCLUSIONS: We demonstrate a high prevalence of biofilm-forming Uropathogenic E. coli strains that are highly associated with the MDR phenotype. We recommend routine surveillance of antimicrobial resistance and biofilm formation to understand the antibiotics suitable in the management of biofilm-associated UTIs.


Assuntos
Antibacterianos/uso terapêutico , Biofilmes/crescimento & desenvolvimento , Infecções por Escherichia coli/epidemiologia , Infecções Urinárias/epidemiologia , Escherichia coli Uropatogênica/genética , Escherichia coli Uropatogênica/patogenicidade , Estudos Transversais , Farmacorresistência Bacteriana Múltipla/genética , Infecções por Escherichia coli/tratamento farmacológico , Infecções por Escherichia coli/microbiologia , Humanos , Testes de Sensibilidade Microbiana , Fenótipo , Reação em Cadeia da Polimerase , Prevalência , Uganda/epidemiologia , Infecções Urinárias/tratamento farmacológico , Infecções Urinárias/microbiologia , Escherichia coli Uropatogênica/efeitos dos fármacos , Escherichia coli Uropatogênica/isolamento & purificação , Virulência/genética , Fatores de Virulência/genética
18.
BMC Infect Dis ; 20(1): 472, 2020 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-32616018

RESUMO

BACKGROUND: Carbapenem-resistant hypervirulent Klebsiella pneumoniae strains have recently come into existence worldwide; however, researchers in northeast China are not aware of their clinical features and molecular characteristics. METHODS: Here, the molecular and virulent characteristics of 44 carbapenem-resistant K. pneumoniae (CRKP) isolates collected from January 2015 to December 2017 were studied. Multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) were carried out to define the clonal relatedness among the isolates. PCR and capsular serotyping of the virulence-associated genes, as well as biofilm formation and serum complement-mediated killing assays, were employed to determine the virulent potential. The genomic features and associated mobile genetic elements of JmsCRE57 were detected by whole genome sequencing. RESULTS: The only positive isolate was JmsCRE57, which belonged to the ST375 serotype K2 that expressed uge, mrkD, fimH, kpn, aerobactin and rmpA virulence-associated genes and showed strong biofilm formation and serum sensitivity. Sequencing results showed that the JmsCRE57 genome mainly consisted of a circular chromosome, three antimicrobial resistant plasmids and a virulent plasmid. The antimicrobial resistant plasmid expressing blaKPC-2, blaCTX-M-15, aph(3″)-Ib, aph(6)-Id, qnrB1, aac(3)-IIa, aac(6')-Ib-cr, blaOXA-1, blaTEM-1B, catB4, sul2, dfrA14 and blaSHV-99. The virulent plasmid belonged to the IncHI1B group, which is mainly composed of mucoid phenotype genes and siderophore-associated genes. The remaining CRKP strains that expressed uge, fimH, mrkD and kpn virulence-associated genes were not successfully typed. CONCLUSION: Our results provide new insights on the epidemiology of carbapenem-resistant K2 hypervirulent K. pneumoniae ST375 and CRKP ST76 strains in northeast China, which may help control their future outbreaks.


Assuntos
Enterobacteriáceas Resistentes a Carbapenêmicos/genética , Enterobacteriáceas Resistentes a Carbapenêmicos/patogenicidade , Surtos de Doenças , Infecções por Klebsiella/epidemiologia , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/patogenicidade , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Antibacterianos/uso terapêutico , Enterobacteriáceas Resistentes a Carbapenêmicos/isolamento & purificação , Carbapenêmicos/uso terapêutico , China/epidemiologia , Eletroforese em Gel de Campo Pulsado , Feminino , Humanos , Infecções por Klebsiella/sangue , Infecções por Klebsiella/tratamento farmacológico , Infecções por Klebsiella/microbiologia , Klebsiella pneumoniae/imunologia , Klebsiella pneumoniae/isolamento & purificação , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Tipagem de Sequências Multilocus , Plasmídeos/genética , Sorogrupo , Sorotipagem , Virulência/genética , Sequenciamento Completo do Genoma , Adulto Jovem
19.
PLoS One ; 15(7): e0236016, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32649692

RESUMO

Cholera remains a major global public health threat and continuous emergence of new Vibrio cholerae strains is of major concern. We conducted a molecular epidemiological study to detect virulence markers and antimicrobial resistance patterns of V. cholerae isolates obtained from the 2012-2015 cholera outbreaks in Ghana. Archived clinical isolates obtained from the 2012, 2014 and 2015 cholera outbreaks in Ghana were revived by culture and subjected to microscopy, biochemical identification, serotyping, antibiotic susceptibility testing, molecular detection of distinct virulence factors and Multi-Locus Variable-Number of Tandem-Repeat Analysis (MLVA). Of 277 isolates analysed, 168 (60.6%) were confirmed to be V. cholerae and 109 (39.4%) isolates constituted other bacteria (Escherichia coli, Aeromonas sobria, Pseudomonas aeruginosa, Enterobacter cloacae and Enterococci faecalis). Serotyping the V. cholerae isolates identified 151 (89.9%) as Ogawa, 3 (1.8%) as Inaba and 14 (8.3%) as non-O1/O139 serogroup. The O1 serogroup isolates (154/168, 91.7%) carried the cholera toxin ctxB gene as detected by PCR. Additional virulence genes detected include zot, tcpA, ace, rtxC, toxR, rtxA, tcpP, hlyA and tagA. The most common and rare virulence factors detected among the isolates were rtxC (165 isolates) and tcpP (50 isolates) respectively. All isolates from 2014 and 2015 were multidrug resistant against the selected antibiotics. MLVA differentiated the isolates into 2 large unique clones A and B, with each predominating in a particular year. Spatial analysis showed clustering of most isolates at Ablekuma sub-district. Identification of several virulence genes among the two different genotypes of V. cholerae isolates and resistance to first- and second-line antibiotics, calls for scaleup of preventive strategies to reduce transmission, and strengthening of public health laboratories for rapid antimicrobial susceptibility testing to guide accurate treatment. Our findings support the current WHO licensed cholera vaccines which include both O1 Inaba and Ogawa serotypes.


Assuntos
Cólera/epidemiologia , Vibrio cholerae/metabolismo , Antibacterianos/farmacologia , Cólera/diagnóstico , Cólera/microbiologia , Toxina da Cólera/genética , Toxina da Cólera/metabolismo , Surtos de Doenças , Farmacorresistência Bacteriana Múltipla/genética , Gana/epidemiologia , Humanos , Testes de Sensibilidade Microbiana , Filogenia , Sorogrupo , Sequências de Repetição em Tandem/genética , Vibrio cholerae/classificação , Vibrio cholerae/isolamento & purificação , Vibrio cholerae/patogenicidade , Virulência/genética
20.
J Infect Public Health ; 13(10): 1544-1550, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: covidwho-633894

RESUMO

BACKGROUND: Middle East respiratory syndrome coronavirus (MERS-CoV) has continued to cause sporadic outbreaks of severe respiratory tract infection over the last 8 years. METHODS: Complete genome sequencing using next-generation sequencing was performed for MERS-CoV isolates from cases that occurred in Riyadh between 2015 and 2019. Phylogenetic analysis and molecular mutational analysis were carried out to investigate disease severity. RESULTS: A total of eight MERS-CoV isolates were subjected to complete genome sequencing. Phylogenetic analysis resulted in the assembly of 7/8 sequences within lineage 3 and one sequence within lineage 4 showing complex genomic recombination. The isolates contained a variety of unique amino acid substitutions in ORF1ab (41), the N protein (10), the S protein (9) and ORF4b (5). CONCLUSION: Our study shows that MERS-CoV is evolving. The emergence of new variants carries the potential for increased virulence and could impose a challenge to the global health system. We recommend the sequencing every new MERS-CoV isolate to observe the changes in the virus and relate them to clinical outcomes.


Assuntos
Infecções por Coronavirus/virologia , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Mutação , RNA Viral/análise , Adulto , Idoso , Aminoácidos/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas do Nucleocapsídeo/genética , Filogenia , Arábia Saudita , Virulência/genética , Sequenciamento Completo do Genoma
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