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1.
Mol Cell ; 80(2): 175-177, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-33065017

RESUMO

Eisenbart et al. (2020) find an SSR-associated sRNA, NikS, that is subject to variable repeat-controlled expression. NikS regulates H. pylori virulence by post-transcriptionally repressing pathogenicity factors, including CagA and VacA, via base-pairing to their mRNAs.


Assuntos
Helicobacter pylori , Fatores de Virulência , DNA , Regulação Bacteriana da Expressão Gênica , Helicobacter pylori/genética , RNA Bacteriano/genética , Virulência/genética
2.
Nat Commun ; 11(1): 4531, 2020 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-32913225

RESUMO

RNAs begin to fold and function during transcription. Riboswitches undergo cotranscriptional switching in the context of transcription elongation, RNA folding, and ligand binding. To investigate how these processes jointly modulate the function of the folate stress-sensing Fusobacterium ulcerans ZTP riboswitch, we apply a single-molecule vectorial folding (VF) assay in which an engineered superhelicase Rep-X sequentially releases fluorescently labeled riboswitch RNA from a heteroduplex in a 5'-to-3' direction, at ~60 nt s-1 [comparable to the speed of bacterial RNA polymerase (RNAP)]. We demonstrate that the ZTP riboswitch is kinetically controlled and that its activation is favored by slower unwinding, strategic pausing between but not before key folding elements, or a weakened transcription terminator. Real-time single-molecule monitoring captures folding riboswitches in multiple states, including an intermediate responsible for delayed terminator formation. These results show how individual nascent RNAs occupy distinct channels within the folding landscape that controls the fate of the riboswitch.


Assuntos
Fusobacterium/genética , Regulação Bacteriana da Expressão Gênica , Dobramento de RNA/genética , RNA Bacteriano/genética , Riboswitch/genética , Aminoimidazol Carboxamida/metabolismo , Fusobacterium/metabolismo , Conformação de Ácido Nucleico , RNA Bacteriano/metabolismo , Ribonucleotídeos/metabolismo , Imagem Individual de Molécula , Transcrição Genética
3.
Proc Natl Acad Sci U S A ; 117(28): 16333-16338, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32601241

RESUMO

Bacterial transfer RNAs (tRNAs) contain evolutionarily conserved sequences and modifications that ensure uniform binding to the ribosome and optimal translational accuracy despite differences in their aminoacyl attachments and anticodon nucleotide sequences. In the tRNA anticodon stem-loop, the anticodon sequence is correlated with a base pair in the anticodon loop (nucleotides 32 and 38) to tune the binding of each tRNA to the decoding center in the ribosome. Disruption of this correlation renders the ribosome unable to distinguish correct from incorrect tRNAs. The molecular basis for how these two tRNA features combine to ensure accurate decoding is unclear. Here, we solved structures of the bacterial ribosome containing either wild-type [Formula: see text] or [Formula: see text] containing a reversed 32-38 pair on cognate and near-cognate codons. Structures of wild-type [Formula: see text] bound to the ribosome reveal 23S ribosomal RNA (rRNA) nucleotide A1913 positional changes that are dependent on whether the codon-anticodon interaction is cognate or near cognate. Further, the 32-38 pair is destabilized in the context of a near-cognate codon-anticodon pair. Reversal of the pairing in [Formula: see text] ablates A1913 movement regardless of whether the interaction is cognate or near cognate. These results demonstrate that disrupting 32-38 and anticodon sequences alters interactions with the ribosome that directly contribute to misreading.


Assuntos
Biossíntese de Proteínas/genética , RNA de Transferência/química , RNA de Transferência/genética , Anticódon/química , Anticódon/genética , Anticódon/metabolismo , Pareamento de Bases , Códon/genética , Códon/metabolismo , Cristalografia por Raios X , Modelos Moleculares , Mutação , Conformação de Ácido Nucleico , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Ribossômico 23S/química , RNA Ribossômico 23S/genética , RNA Ribossômico 23S/metabolismo , RNA de Transferência/metabolismo , Ribossomos/química , Ribossomos/metabolismo , Thermus thermophilus/genética , Thermus thermophilus/metabolismo
4.
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
5.
Nat Commun ; 11(1): 3557, 2020 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-32678091

RESUMO

Bacteria of the genus Bacteroides are common members of the human intestinal microbiota and important degraders of polysaccharides in the gut. Among them, the species Bacteroides thetaiotaomicron has emerged as the model organism for functional microbiota research. Here, we use differential RNA sequencing (dRNA-seq) to generate a single-nucleotide resolution transcriptome map of B. thetaiotaomicron grown under defined laboratory conditions. An online browser, called 'Theta-Base' ( www.helmholtz-hiri.de/en/datasets/bacteroides ), is launched to interrogate the obtained gene expression data and annotations of ~4500 transcription start sites, untranslated regions, operon structures, and 269 noncoding RNA elements. Among the latter is GibS, a conserved, 145 nt-long small RNA that is highly expressed in the presence of N-acetyl-D-glucosamine as sole carbon source. We use computational predictions and experimental data to determine the secondary structure of GibS and identify its target genes. Our results indicate that sensing of N-acetyl-D-glucosamine induces GibS expression, which in turn modifies the transcript levels of metabolic enzymes.


Assuntos
Bacteroides thetaiotaomicron/genética , Microbioma Gastrointestinal , Pequeno RNA não Traduzido/genética , Transcriptoma , Acetilglucosamina/metabolismo , Proteínas de Bactérias/genética , Bacteroides thetaiotaomicron/crescimento & desenvolvimento , Bacteroides thetaiotaomicron/metabolismo , Meios de Cultura/metabolismo , Regulação Bacteriana da Expressão Gênica , Humanos , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas , RNA Bacteriano/genética , Pequeno RNA não Traduzido/química , RNA não Traduzido/genética , Reprodutibilidade dos Testes , Navegador
6.
Mol Cell ; 79(3): 416-424.e5, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32645367

RESUMO

CRISPR-Cas12c/d proteins share limited homology with Cas12a and Cas9 bacterial CRISPR RNA (crRNA)-guided nucleases used widely for genome editing and DNA detection. However, Cas12c (C2c3)- and Cas12d (CasY)-catalyzed DNA cleavage and genome editing activities have not been directly observed. We show here that a short-complementarity untranslated RNA (scoutRNA), together with crRNA, is required for Cas12d-catalyzed DNA cutting. The scoutRNA differs in secondary structure from previously described tracrRNAs used by CRISPR-Cas9 and some Cas12 enzymes, and in Cas12d-containing systems, scoutRNA includes a conserved five-nucleotide sequence that is essential for activity. In addition to supporting crRNA-directed DNA recognition, biochemical and cell-based experiments establish scoutRNA as an essential cofactor for Cas12c-catalyzed pre-crRNA maturation. These results define scoutRNA as a third type of transcript encoded by a subset of CRISPR-Cas genomic loci and explain how Cas12c/d systems avoid requirements for host factors including ribonuclease III for bacterial RNA-mediated adaptive immunity.


Assuntos
Bactérias/genética , Proteínas de Bactérias/genética , Sistemas CRISPR-Cas , Endodesoxirribonucleases/genética , Genoma Bacteriano/imunologia , RNA Bacteriano/genética , Pequeno RNA não Traduzido/genética , Bactérias/classificação , Bactérias/imunologia , Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Sequência de Bases , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Endodesoxirribonucleases/metabolismo , Escherichia coli/genética , Escherichia coli/imunologia , Escherichia coli/metabolismo , Conformação de Ácido Nucleico , Filogenia , RNA Bacteriano/química , RNA Bacteriano/metabolismo , RNA Guia/genética , RNA Guia/metabolismo , Pequeno RNA não Traduzido/química , Pequeno RNA não Traduzido/metabolismo , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico
7.
Nat Commun ; 11(1): 3363, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32620750

RESUMO

Studying emerging or neglected pathogens is often challenging due to insufficient information and absence of genetic tools. Dual RNA-seq provides insights into host-pathogen interactions, and is particularly informative for intracellular organisms. Here we apply dual RNA-seq to Orientia tsutsugamushi (Ot), an obligate intracellular bacterium that causes the vector-borne human disease scrub typhus. Half the Ot genome is composed of repetitive DNA, and there is minimal collinearity in gene order between strains. Integrating RNA-seq, comparative genomics, proteomics, and machine learning to study the transcriptional architecture of Ot, we find evidence for wide-spread post-transcriptional antisense regulation. Comparing the host response to two clinical isolates, we identify distinct immune response networks for each strain, leading to predictions of relative virulence that are validated in a mouse infection model. Thus, dual RNA-seq can provide insight into the biology and host-pathogen interactions of a poorly characterized and genetically intractable organism such as Ot.


Assuntos
Regulação Bacteriana da Expressão Gênica/imunologia , Interações Hospedeiro-Patógeno/imunologia , Doenças Negligenciadas/imunologia , Orientia tsutsugamushi/genética , Tifo por Ácaros/imunologia , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/metabolismo , Linhagem Celular , Modelos Animais de Doenças , Estudos de Viabilidade , Feminino , Genoma Bacteriano , Células Endoteliais da Veia Umbilical Humana , Humanos , Interferon Tipo I/imunologia , Interferon Tipo I/metabolismo , Sequências Repetitivas Dispersas/genética , Camundongos , Doenças Negligenciadas/microbiologia , Orientia tsutsugamushi/imunologia , Orientia tsutsugamushi/patogenicidade , Proteômica , RNA Bacteriano/genética , RNA Bacteriano/isolamento & purificação , RNA Bacteriano/metabolismo , RNA não Traduzido/genética , RNA não Traduzido/metabolismo , RNA-Seq , Tifo por Ácaros/microbiologia , Transcrição Genética , Sequenciamento Completo do Exoma
8.
RNA ; 26(10): 1431-1447, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32611709

RESUMO

RNA structure influences numerous processes in all organisms. In bacteria, these processes include transcription termination and attenuation, small RNA and protein binding, translation initiation, and mRNA stability, and can be regulated via metabolite availability and other stresses. Here we use Structure-seq2 to probe the in vivo RNA structurome of Bacillus subtilis grown in the presence and absence of amino acids. Our results reveal that amino acid starvation results in lower overall dimethyl sulfate (DMS) reactivity of the transcriptome, indicating enhanced protection owing to protein binding or RNA structure. Starvation-induced changes in DMS reactivity correlated inversely with transcript abundance changes. This correlation was particularly pronounced in genes associated with the stringent response and CodY regulons, which are involved in adaptation to nutritional stress, suggesting that RNA structure contributes to transcript abundance change in regulons involved in amino acid metabolism. Structure-seq2 accurately reported on four known amino acid-responsive riboswitches: T-box, SAM, glycine, and lysine riboswitches. Additionally, we discovered a transcription attenuation mechanism that reduces yfmG expression when amino acids are added to the growth medium. We also found that translation of a leader peptide (YfmH) encoded just upstream of yfmG regulates yfmG expression. Our results are consistent with a model in which a slow rate of yfmH translation caused by limitation of the amino acids encoded in YfmH prevents transcription termination in the yfmG leader region by favoring formation of an overlapping antiterminator structure. This novel RNA switch offers a way to simultaneously monitor the levels of multiple amino acids.


Assuntos
Aminoácidos/genética , Bacillus subtilis/genética , Proteínas de Bactérias/genética , RNA Bacteriano/genética , Regulação Bacteriana da Expressão Gênica/genética , Conformação de Ácido Nucleico , Estabilidade de RNA/genética , Transcrição Genética/genética , Transcriptoma/genética
9.
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
10.
Nat Commun ; 11(1): 2837, 2020 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-32503992

RESUMO

Group II introns are ubiquitous self-splicing ribozymes and retrotransposable elements evolutionarily and chemically related to the eukaryotic spliceosome, with potential applications as gene-editing tools. Recent biochemical and structural data have captured the intron in multiple conformations at different stages of catalysis. Here, we employ enzymatic assays, X-ray crystallography, and molecular simulations to resolve the spatiotemporal location and function of conformational changes occurring between the first and the second step of splicing. We show that the first residue of the highly-conserved catalytic triad is protonated upon 5'-splice-site scission, promoting a reversible structural rearrangement of the active site (toggling). Protonation and active site dynamics induced by the first step of splicing facilitate the progression to the second step. Our insights into the mechanism of group II intron splicing parallels functional data on the spliceosome, thus reinforcing the notion that these evolutionarily-related molecular machines share the same enzymatic strategy.


Assuntos
Íntrons/genética , Precursores de RNA/metabolismo , Processamento de RNA , RNA Bacteriano/metabolismo , Spliceossomos/metabolismo , Bacillaceae/genética , Domínio Catalítico/genética , Cristalografia por Raios X , Simulação de Dinâmica Molecular , Mutagênese , Conformação de Ácido Nucleico , Precursores de RNA/genética , RNA Bacteriano/genética , Análise Espaço-Temporal
11.
Nucleic Acids Res ; 48(12): 6970-6979, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32479610

RESUMO

Recently, prokaryotic riboswitches have been identified that regulate transcription in response to change of the concentration of secondary messengers. The ZMP (5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR))-sensing riboswitch from Thermosinus carboxydivorans is a transcriptional ON-switch that is involved in purine and carbon-1 metabolic cycles. Its aptamer domain includes the pfl motif, which features a pseudoknot, impeding rho-independent terminator formation upon stabilization by ZMP interaction. We herein investigate the conformational landscape of transcriptional intermediates including the expression platform of this riboswitch and characterize the formation and unfolding of the important pseudoknot structure in the context of increasing length of RNA transcripts. NMR spectroscopic data show that even surprisingly short pre-terminator stems are able to disrupt ligand binding and thus metabolite sensing. We further show that the pseudoknot structure, a prerequisite for ligand binding, is preformed in transcription intermediates up to a certain length. Our results describe the conformational changes of 13 transcription intermediates of increasing length to delineate the change in structure as mRNA is elongated during transcription. We thus determine the length of the key transcription intermediate to which addition of a single nucleotide leads to a drastic drop in ZMP affinity.


Assuntos
Aptâmeros de Nucleotídeos/genética , Conformação de Ácido Nucleico , Ribonucleotídeos/genética , Riboswitch/genética , Aptâmeros de Nucleotídeos/química , Firmicutes/genética , Firmicutes/ultraestrutura , Ligantes , Purinas/metabolismo , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , Ribonucleotídeos/química
12.
BMC Infect Dis ; 20(1): 419, 2020 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-32546213

RESUMO

BACKGROUND: Four new variants of Chlamydia trachomatis (nvCTs), detected in several countries, cause false-negative or equivocal results using the Aptima Combo 2 assay (AC2; Hologic). We evaluated the clinical sensitivity and specificity, as well as the analytical inclusivity and exclusivity of the updated AC2 for the detection of CT and Neisseria gonorrhoeae (NG) on the automated Panther system (Hologic). METHODS: We examined 1004 clinical AC2 samples and 225 analytical samples spiked with phenotypically and/or genetically diverse NG and CT strains, and other potentially cross-reacting microbial species. The clinical AC2 samples included CT wild type (WT)-positive (n = 488), all four described AC2 diagnostic-escape nvCTs (n = 170), NG-positive (n = 214), and CT/NG-negative (n = 202) specimens. RESULTS: All nvCT-positive samples (100%) and 486 (99.6%) of the CT WT-positive samples were positive in the updated AC2. All NG-positive, CT/NG-negative, Trichomonas vaginalis (TV)-positive, bacterial vaginosis-positive, and Candida-positive AC2 specimens gave correct results. The clinical sensitivity and specificity of the updated AC2 for CT detection was 99.7 and 100%, respectively, and for NG detection was 100% for both. Examining spiked samples, the analytical inclusivity and exclusivity were 100%, i.e., in clinically relevant concentrations of spiked microbe. CONCLUSIONS: The updated AC2, including two CT targets and one NG target, showed a high sensitivity, specificity, inclusivity and exclusivity for the detection of CT WT, nvCTs, and NG. The updated AC2 on the fully automated Panther system offers a simple, rapid, high-throughput, sensitive, and specific diagnosis of CT and NG, which can easily be combined with detection of Mycoplasma genitalium and TV.


Assuntos
Infecções por Chlamydia/diagnóstico , Chlamydia trachomatis/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Análise de Sequência de RNA/métodos , Candida/genética , Candidíase/diagnóstico , Candidíase/microbiologia , Infecções por Chlamydia/microbiologia , Reações Cruzadas , Feminino , Gonorreia/diagnóstico , Gonorreia/microbiologia , Humanos , Neisseria gonorrhoeae/genética , RNA Bacteriano/genética , RNA Ribossômico 23S/genética , Sensibilidade e Especificidade , Tricomoníase/diagnóstico , Tricomoníase/parasitologia , Trichomonas vaginalis/genética
13.
PLoS Pathog ; 16(5): e1008423, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32365143

RESUMO

Post-transcriptional regulation via small regulatory RNAs (sRNAs) has been implicated in diverse regulatory processes in bacteria, including virulence. One class of sRNAs, termed trans-acting sRNAs, can affect the stability and/or the translational efficiency of regulated transcripts. In this study, we utilized a collaborative approach that employed data from infection with the Borrelia burgdorferi Tn library, coupled with Tn-seq, together with borrelial sRNA and total RNA transcriptomes, to identify an intergenic trans-acting sRNA, which we designate here as ittA for infectivity-associated and tissue-tropic sRNA locus A. The genetic inactivation of ittA resulted in a significant attenuation in infectivity, with decreased spirochetal load in ear, heart, skin and joint tissues. In addition, the ittA mutant did not disseminate to peripheral skin sites or heart tissue, suggesting a role for ittA in regulating a tissue-tropic response. RNA-Seq analysis determined that 19 transcripts were differentially expressed in the ittA mutant relative to its genetic parent, including vraA, bba66, ospD and oms28 (bba74). Subsequent proteomic analyses also showed a significant decrease of OspD and Oms28 (BBA74) proteins. To our knowledge this is the first documented intergenic sRNA that alters the infectivity potential of B. burgdorferi.


Assuntos
Borrelia burgdorferi/genética , Pequeno RNA não Traduzido/metabolismo , Tropismo/genética , Borrelia burgdorferi/metabolismo , Borrelia burgdorferi/patogenicidade , Regulação Bacteriana da Expressão Gênica/genética , Biblioteca Gênica , Genoma Bacteriano , Doença de Lyme/microbiologia , Proteômica , RNA Bacteriano/genética , Pequeno RNA não Traduzido/genética , Transcriptoma/genética , Virulência
14.
PLoS One ; 15(5): e0233170, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32433701

RESUMO

BACKGROUND: Recent evidence suggests a role for the gut microbiome in the development and progression of many diseases and many studies have been carried out to analyse the microbiome using a variety of methods. In this study, we compare MinION sequencing with meta-transcriptomics and amplicon-based sequencing for microbiome analysis of colorectal tumour tissue samples. METHODS: DNA and RNA were extracted from 11 colorectal tumour samples. 16S rRNA amplicon sequencing and MinION sequencing was carried out using genomic DNA, and RNA-Sequencing for meta-transcriptomic analysis. Non-human MinION and RNA-Sequencing reads, and 16S rRNA amplicon sequencing reads were taxonomically classified using a database built from available RefSeq bacterial and archaeal genomes and a k-mer based algorithm in Kraken2. Concordance between the three platforms at different taxonomic levels was tested on a per-sample basis using Spearman's rank correlation. RESULTS: The average number of reads per sample using RNA-Sequencing was greater than 129 times that generated using MinION sequencing. However, the average read length of MinION sequences was more than 13 times that of RNA or 16S rRNA amplicon sequencing. Taxonomic assignment using 16S sequencing was less reliable beyond the genus level, and both RNA-Sequencing and MinION sequencing could detect greater numbers of phyla and genera in the same samples, compared to 16S sequencing. Bacterial species associated with colorectal cancer, Fusobacterium nucleatum, Parvimonas micra, Bacteroides fragilis and Porphyromonas gingivalis, were detectable using MinION, RNA-Sequencing and 16S rRNA amplicon sequencing data. CONCLUSIONS: Long-read sequences generated using MinION sequencing can compensate for low numbers of reads for bacterial classification. MinION sequencing can discriminate between bacterial strains and plasmids and shows potential as a cost-effective tool for rapid microbiome sequencing in a clinical setting.


Assuntos
Bactérias , Neoplasias Colorretais/microbiologia , Microbioma Gastrointestinal , Metagenoma , RNA Bacteriano/genética , RNA Ribossômico 16S/genética , Bactérias/classificação , Bactérias/genética , Neoplasias Colorretais/genética , Feminino , Humanos , Masculino , Análise de Sequência de RNA
15.
PLoS Negl Trop Dis ; 14(5): e0008325, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32453754

RESUMO

Leprosy urgently needs a precise and early diagnostic tool. The sensitivity of the direct (bacilli staining, Mycobacterium leprae DNA) and indirect (antibody levels, T cell assays) diagnostics methods vary based on the clinical form. Recently, PCR-based M. leprae DNA detection has been shown to differentially diagnose leprosy from other dermatological conditions. However, accuracy can still be improved, especially for use with less invasive clinical samples. We tested different commercial DNA extraction kits: DNeasy Blood & Tissue, QIAamp DNA Microbiome, Maxwell 16 DNA Purification, PowerSoil DNA Isolation; as well as in-house phenol-chloroform and Trizol/FastPrep methods. Extraction was performed on M. leprae-infected mouse footpads and different clinical samples of leprosy patients (skin biopsies and scrapings, lesion, oral and nasal swabs, body hair, blood on FTA cards, peripheral whole blood). We observed that the Microbiome kit was able to enrich for mycobacterial DNA, most likely due the enzymatic digestion cocktail along with mechanical disruption involved in this method. Consequently, we had a significant increase in sensitivity in skin biopsies from paucibacillary leprosy patients using a duplex qPCR targeting 16S rRNA (M. leprae) and 18S rRNA (mammal) in the StepOnePlus system. Our data showed that the presence of M. leprae DNA was best detected in skin biopsies and skin scrapings, independent of the extraction method or the clinical form. For multibacillary patients, detection of M. leprae DNA in nasal swabs indicates the possibility of having a much less invasive sample that can be used for the purposes of DNA sequencing for relapse analysis and drug resistance monitoring. Overall, DNA extracted with the Microbiome kit presented the best bacilli detection rate for paucibacillary cases, indicating that investments in extraction methods with mechanical and DNA digestion should be made.


Assuntos
DNA Bacteriano/isolamento & purificação , Mycobacterium leprae/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Animais , DNA Bacteriano/genética , Humanos , Camundongos , Mycobacterium leprae/genética , RNA Bacteriano/genética , RNA Ribossômico 16S/genética , Sensibilidade e Especificidade
16.
PLoS One ; 15(5): e0228479, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32413030

RESUMO

Terminator is a DNA sequence that gives the RNA polymerase the transcriptional termination signal. Identifying terminators correctly can optimize the genome annotation, more importantly, it has considerable application value in disease diagnosis and therapies. However, accurate prediction methods are deficient and in urgent need. Therefore, we proposed a prediction method "iterb-PPse" for terminators by incorporating 47 nucleotide properties into PseKNC-Ⅰ and PseKNC-Ⅱ and utilizing Extreme Gradient Boosting to predict terminators based on Escherichia coli and Bacillus subtilis. Combing with the preceding methods, we employed three new feature extraction methods K-pwm, Base-content, Nucleotidepro to formulate raw samples. The two-step method was applied to select features. When identifying terminators based on optimized features, we compared five single models as well as 16 ensemble models. As a result, the accuracy of our method on benchmark dataset achieved 99.88%, higher than the existing state-of-the-art predictor iTerm-PseKNC in 100 times five-fold cross-validation test. Its prediction accuracy for two independent datasets reached 94.24% and 99.45% respectively. For the convenience of users, we developed a software on the basis of "iterb-PPse" with the same name. The open software and source code of "iterb-PPse" are available at https://github.com/Sarahyouzi/iterb-PPse.


Assuntos
Análise de Sequência de DNA/métodos , Software , Regiões Terminadoras Genéticas , Bacillus subtilis , DNA Bacteriano/química , DNA Bacteriano/genética , Escherichia coli , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator Rho/metabolismo , Terminação da Transcrição Genética
17.
RNA ; 26(9): 1160-1169, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32414857

RESUMO

DEAD-box proteins (DBPs) are RNA remodeling factors associated with RNA helicase activity that are found in nearly all organisms. Despite extensive studies on the mechanisms used by DBPs to regulate RNA function, very little is known about how DBPs themselves are regulated. In this work, we have analyzed the expression and regulation of DeaD/CsdA, the largest of the DBPs in Escherichia coli (E. coli). We show that deaD transcription initiates 838 nt upstream of the start of the coding region. We have also found that DeaD is autoregulated through a negative feedback mechanism that operates both at the level of deaD mRNA stability and Rho-dependent transcription termination, and this regulation is dependent upon its mRNA 5' untranslated region (5' UTR). These findings suggest that DeaD might be regulating the conformation of its own mRNA through its RNA helicase activity to facilitate ribonuclease and Rho access to its 5' UTR.


Assuntos
RNA Helicases DEAD-box/genética , Escherichia coli/genética , Homeostase/genética , Estabilidade de RNA/genética , RNA Mensageiro/genética , Transcrição Genética/genética , Regiões 5' não Traduzidas/genética , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/genética , RNA Bacteriano/genética
18.
J Vis Exp ; (158)2020 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-32420999

RESUMO

Tuberculosis is caused by Mycobacterium tuberculosis (Mtb), a pathogen classified by the United Nations (UN) as a dangerous category B biological substance. For the sake of the workers' safety, handling of all samples presumed to carry Mtb must be conducted in a containment level (CL) 3 laboratory. The TB molecular bacterial load assay (TB-MBLA) test is a reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) test that quantifies Mtb bacillary load using primers and dual-labelled probes for 16S rRNA. We describe the use of heat inactivation to render TB samples noninfectious while preserving RNA for the TB-MBLA. A 1 mL aliquot of the sputum sample in tightly closed 15 mL centrifuge tubes is boiled for 20 min at either 80 °C, 85 °C, or 95 °C to inactivate Mtb bacilli. Cultivation of the heat inactivated and control (live) samples for 42 days confirmed the death of TB. The inactivated sample is then spiked with 100 µL of the extraction control and RNA is extracted following the standard RNA isolation procedure. No growth was observed in the cultures of heat treated samples. The isolated RNA is subjected to real-time RT-qPCR, which amplifies a specific target in the Mtb 16S rRNA gene, yielding results in the form of quantification cycles (Cq). A standard curve is used to translate Cq into bacterial load, or estimated colony forming units per mL (eCFU/mL). There is an inverse relationship between Cq and the bacterial load of a sample. The limitation is that heat inactivation lyses some cells, exposing the RNA to RNases that cause a loss of <1 log10eCFU/mL (i.e., <10 CFU/mL). Further studies will determine the proportion of very low burden patients that cause false negative results due to heat inactivation.


Assuntos
Carga Bacteriana/métodos , Mycobacterium tuberculosis/isolamento & purificação , RNA Bacteriano/análise , Escarro/microbiologia , Tuberculose/diagnóstico , Carga Bacteriana/instrumentação , Carga Bacteriana/normas , Humanos , Mycobacterium tuberculosis/genética , RNA Bacteriano/genética , RNA Ribossômico 16S/genética , Reação em Cadeia da Polimerase em Tempo Real , Manejo de Espécimes , Tuberculose/microbiologia
19.
Stroke ; 51(6): 1844-1854, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32404038

RESUMO

Background and Purpose- Commensal gut bacteria have a profound impact on stroke pathophysiology. Here, we investigated whether modification of the microbiota influences acute and long-term outcome in mice subjected to stroke. Methods- C57BL/6 male mice received a cocktail of antibiotics or single antibiotic. After 4 weeks, fecal bacterial density of the 16S rRNA gene was quantitated by qPCR, and phylogenetic classification was obtained by 16S rRNA gene sequencing. Infarct volume and hemispheric volume loss were measured 3 days and 5 weeks after middle cerebral artery occlusion, respectively. Neurological deficits were tested by the Tape Test and the open field test. Results- Mice treated with a cocktail of antibiotics displayed a significant reduction of the infarct volume in the acute phase of stroke. The neuroprotective effect was abolished in mice recolonized with a wild-type microbiota. Single antibiotic treatment with either ampicillin or vancomycin, but not neomycin, was sufficient to reduce the infarct volume and improved motorsensory function 3 days after stroke. This neuroprotective effect was correlated with a specific microbial population rather than the total bacterial density. In particular, random forest analysis trained for the severity of the brain damage revealed that Bacteroidetes S24.7 and the enzymatic pathway for aromatic metabolism discriminate between large versus small infarct size. Additionally, the microbiota signature in the ampicillin-treated mice was associated with a reduced gut inflammation, long-term favorable outcome shown by an amelioration of the stereotypic behavior, and a reduction of brain tissue loss in comparison to control and was predictive of a regulation of short-chain fatty acids and tryptophan pathways. Conclusions- The findings highlight the importance of the intestinal microbiota in short- and long-term outcomes of ischemic stroke and raises the possibility that targeted modification of the microbiome associated with specific microbial enzymatic pathways may provide a preventive strategy in patients at high risk for stroke. Visual Overview- An online visual overview is available for this article.


Assuntos
Bactérias/crescimento & desenvolvimento , Isquemia Encefálica , Microbioma Gastrointestinal , Doença Aguda , Animais , Bactérias/classificação , Bactérias/genética , Isquemia Encefálica/microbiologia , Isquemia Encefálica/prevenção & controle , Masculino , Camundongos , RNA Bacteriano/genética , RNA Ribossômico 16S/genética , Acidente Vascular Cerebral/microbiologia , Acidente Vascular Cerebral/prevenção & controle
20.
BMC Infect Dis ; 20(1): 366, 2020 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-32448208

RESUMO

BACKGROUND: Kosakonia cowanii, formerly known as Enterobacter cowanii, is a Gram-negative bacillus belonging to the order Enterobacterales. The species is usually recognized as a plant pathogen and has only anecdotally been encountered as a human pathogen. Here we describe the rare case of a K. cowanii infection presenting as an acute cholecystitis and provide a review of available literature. Evident difficulties in species identification by biochemical profiling suggests that potentially, K. cowanii might represent an underestimated human pathogen. CASE PRESENTATION: A 61-year old immunocompromised man presented to the hospital with fever and pain in the upper right abdomen. Sonography revealed an inflamed gall bladder and several gall stones. A cholecystectomy proved diagnosis of an acute cholecystitis with a partial necrosis of the gall bladder. Surgical specimen grew pure cultures of Gram-negative rods unambiguously identified as K. cowanii by MALDI-TOF, 16S-rRNA analysis and whole genome sequencing. CONCLUSIONS: Reporting cases of Kosakonia species can shed light on the prevalence and clinical importance of this rare cause of human infection. Our case is the first to describe an infection without prior traumatic inoculation of the pathogen from its usual habitat, a plant, to the patient. This raises the question of the route of infections as well as the pathogen's ability to colonize the human gut.


Assuntos
Colecistite Aguda/diagnóstico , Colecistite Aguda/microbiologia , Infecções por Enterobacteriaceae/diagnóstico , Enterobacteriaceae/genética , Doenças Raras/diagnóstico , Doenças Raras/microbiologia , Colecistectomia , Infecções por Enterobacteriaceae/microbiologia , Vesícula Biliar/patologia , Cálculos Biliares/cirurgia , Humanos , Hospedeiro Imunocomprometido , Masculino , Pessoa de Meia-Idade , Necrose , RNA Bacteriano/genética , RNA Ribossômico 16S/genética , Resultado do Tratamento , Sequenciamento Completo do Genoma
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