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1.
Rev Inst Med Trop Sao Paulo ; 61: e51, 2019 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-31531629

RESUMO

A drug resistance survey involving Mycobacterium tuberculosis isolated from patients of a tertiary Hospital in the Rio de Janeiro city (RJ), Brazil, between the years 1996 and 1998 revealed a high frequency of isoniazid (HR) resistance. These isolates were revisited and genotyped. Patients came from different RJ neighborhoods and municipalities, and 70% were outpatients. Applying the 3' and 5' IS 6110 -RFLP and the Spoligotype genotyping methods, the clonal structure of this population was investigated obtaining a snapshot of past epidemiological events. The 3' clusters were subsequently 5' IS 6110 -RFLP typed. Spoligotyping was analyzed in the SITVIT2 database. Epidemiological relationships were investigated. The major lineage was T (54.4%), and SIT 53/T1 and SIT 535/T1 were the most frequent. The T1 sublineage comprises 12.8% of resistant strains and SIT 535 were assigned for 31.8% of them. Orphan patterns corresponded to 12% and 73.3% and belonged to the T lineage. One pattern was unlisted in the SITVIT2. The 5' IS 6110 -RFLP did not confirm 3/12 of the 3' IS 6110 -RFLP clusters. A combination of all methods decreased the number of clusters to three. Nosocomial transmission was associated with one cluster involving a hospital cupbearer. This event was suspected in a multidrug resistant-TB inpatient caregiver who harbored a mixed infection. The 3' IS 6110 clusters were associated with HR (p=0.046). These genotypic retrospective data may reflect a fraction of more extensive recent transmission in different communities that may be corroborated by the concentration of HR patients, and may serve as a database for further evolutionary and characterization evaluation of circulating strains and together with epidemiological data favors a more effective transmission control.


Assuntos
Elementos de DNA Transponíveis/genética , Farmacorresistência Bacteriana/genética , Mycobacterium tuberculosis/genética , Tuberculose/microbiologia , Adolescente , Adulto , Técnicas de Tipagem Bacteriana , Brasil , DNA Bacteriano/genética , Feminino , Genótipo , Técnicas de Genotipagem , Humanos , Masculino , Mycobacterium tuberculosis/classificação , Mycobacterium tuberculosis/isolamento & purificação , Polimorfismo de Fragmento de Restrição , Estudos Retrospectivos
2.
Genome Biol ; 20(1): 141, 2019 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-31315652

RESUMO

BACKGROUND: The long introns of mammals are pools of evolutionary potential due to the multiplicity of sequences that permit the acquisition of novel exons. However, the permissibility of genes to this type of acquisition and its influence on the evolution of cell regulation is poorly understood. RESULTS: Here, we observe that human genes are highly permissive to the inclusion of novel exonic regions permitting the emergence of novel regulatory features. Our analysis reveals the potential for novel exon acquisition to occur in over 30% of evaluated human genes. Regulatory processes including the rate of splicing efficiency and RNA polymerase II (RNAPII) elongation control this process by modulating the "window of opportunity" for spliceosomal recognition. DNA damage alters this window promoting the inclusion of repeat-derived novel exons that reduce the ribosomal engagement of cell cycle genes. Finally, we demonstrate that the inclusion of novel exons is suppressed in hematological cancer samples and can be reversed by drugs modulating the rate of RNAPII elongation. CONCLUSION: Our work demonstrates that the inclusion of repeat-associated novel intronic regions is a tightly controlled process capable of expanding the regulatory capacity of cells.


Assuntos
Éxons , Regulação da Expressão Gênica , Genoma Humano , Transcriptoma , Dano ao DNA , Elementos de DNA Transponíveis , Genes cdc , Neoplasias Hematológicas/metabolismo , Humanos , Íntrons , Spliceossomos
3.
Nature ; 571(7764): 219-225, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31189177

RESUMO

Conventional CRISPR-Cas systems maintain genomic integrity by leveraging guide RNAs for the nuclease-dependent degradation of mobile genetic elements, including plasmids and viruses. Here we describe a notable inversion of this paradigm, in which bacterial Tn7-like transposons have co-opted nuclease-deficient CRISPR-Cas systems to catalyse RNA-guided integration of mobile genetic elements into the genome. Programmable transposition of Vibrio cholerae Tn6677 in Escherichia coli requires CRISPR- and transposon-associated molecular machineries, including a co-complex between the DNA-targeting complex Cascade and the transposition protein TniQ. Integration of donor DNA occurs in one of two possible orientations at a fixed distance downstream of target DNA sequences, and can accommodate variable length genetic payloads. Deep-sequencing experiments reveal highly specific, genome-wide DNA insertion across dozens of unique target sites. This discovery of a fully programmable, RNA-guided integrase lays the foundation for genomic manipulations that obviate the requirements for double-strand breaks and homology-directed repair.


Assuntos
Sistemas CRISPR-Cas/genética , Elementos de DNA Transponíveis/genética , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Edição de Genes/métodos , Mutagênese Insercional/métodos , RNA Bacteriano/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/metabolismo , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Escherichia coli/genética , Genoma Bacteriano/genética , Integrases/genética , Integrases/metabolismo , Mutagênese Sítio-Dirigida/métodos , RNA Guia/genética , Especificidade por Substrato , Vibrio cholerae/genética
4.
Dokl Biochem Biophys ; 485(1): 95-100, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31201623

RESUMO

This is the first study to investigate the molecular-genetic organization of polytene chromosome interbands located on both molecular and cytological maps of Drosophila genome. The majority of the studied interbands contained one gene with a single transcription initiation site; the remaining interbands contained one gene with several alternative promoters, two or more unidirectional genes, and "head-to-head" arranged genes. In addition, intricately arranged interbands containing three or more genes in both unidirectional and bidirectional orientation were found. Insulator proteins, ORC, P-insertions, DNase I hypersensitive sites, and other open chromatin structures were situated in the promoter region of the genes located in the interbands. This area is critical for the formation of the interband, an open chromatin region in which gene transcription and replication are combined.


Assuntos
Elementos de DNA Transponíveis , Genes Essenciais , Cromossomos Politênicos/genética , Regiões Promotoras Genéticas , Animais , Drosophila melanogaster
5.
BMC Bioinformatics ; 20(1): 354, 2019 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-31234777

RESUMO

BACKGROUND: Helitron is a rolling-circle DNA transposon; it plays an important role in plant evolution. However, Helitron distribution and contribution to evolution at the family level have not been previously investigated. RESULTS: We developed the software easy-to-annotate Helitron (EAHelitron), a Unix-like command line, and used it to identify Helitrons in a wide range of 53 plant genomes (including 13 Brassicaceae species). We determined Helitron density (abundance/Mb) and visualized and examined Helitron distribution patterns. We identified more than 104,653 Helitrons, including many new Helitrons not predicted by other software. Whole genome Helitron density is independent from genome size and shows stability at the species level. Using linear discriminant analysis, de novo genomes (next-generation sequencing) were successfully classified into Arabidopsis thaliana groups. For most Brassicaceae species, Helitron density negatively correlated with gene density, and Helitron distribution patterns were similar to those of A. thaliana. They preferentially inserted into sequence around the centromere and intergenic region. We also associated 13 Helitron polymorphism loci with flowering-time phenotypes in 18 A. thaliana ecotypes. CONCLUSION: EAHelitron is a fast and efficient tool to identify new Helitrons. Whole genome Helitron density can be an informative character for plant classification. Helitron insertion polymorphism could be used in association analysis.


Assuntos
Brassicaceae/genética , Genoma de Planta , Software , Arabidopsis/classificação , Arabidopsis/genética , Brassicaceae/classificação , Elementos de DNA Transponíveis/genética , Análise Discriminante , Evolução Molecular , Sequenciamento de Nucleotídeos em Larga Escala , Filogenia , Análise de Sequência de DNA
6.
Mar Pollut Bull ; 142: 533-536, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31232334

RESUMO

We report the occurrence and genomic features of multidrug-resistant vancomycin-resistant Enterococcus faecium vanA belonging to a novel sequence type (designated ST1336), carrying a Tn1546-like element, in marine brown mussels (Perna perna) from anthropogenically affected coastal waters of the Atlantic coast of Brazil, highlighting a potential source of dissemination for related ecosystems, with additional consequences for seafood safety and quality.


Assuntos
Enterococcus faecium/efeitos dos fármacos , Enterococcus faecium/isolamento & purificação , Perna (Organismo)/microbiologia , Resistência a Vancomicina/genética , Animais , Proteínas de Bactérias/genética , Brasil , Carbono-Oxigênio Ligases/genética , Elementos de DNA Transponíveis , Ecossistema , Enterococcus faecium/genética , Humanos , Testes de Sensibilidade Microbiana
7.
Genome Biol ; 20(1): 120, 2019 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-31186073

RESUMO

BACKGROUND: The three-dimensional (3D) organization of chromosomes is linked to epigenetic regulation and transcriptional activity. However, only few functional features of 3D chromatin architecture have been described to date. The KNOT is a 3D chromatin structure in Arabidopsis, comprising 10 interacting genomic regions termed KNOT ENGAGED ELEMENTs (KEEs). KEEs are enriched in transposable elements and associated small RNAs, suggesting a function in transposon biology. RESULTS: Here, we report the KNOT's involvement in regulating invasive DNA elements. Transgenes can specifically interact with the KNOT, leading to perturbations of 3D nuclear organization, which correlates with the transgene's expression: high KNOT interaction frequencies are associated with transgene silencing. KNOT-linked silencing (KLS) cannot readily be connected to canonical silencing mechanisms, such as RNA-directed DNA methylation and post-transcriptional gene silencing, as both cytosine methylation and small RNA abundance do not correlate with KLS. Furthermore, KLS exhibits paramutation-like behavior, as silenced transgenes can lead to the silencing of active transgenes in trans. CONCLUSION: Transgene silencing can be connected to a specific feature of Arabidopsis 3D nuclear organization, namely the KNOT. KLS likely acts either independent of or prior to canonical silencing mechanisms, such that its characterization not only contributes to our understanding of chromosome folding but also provides valuable insights into how genomes are defended against invasive DNA elements.


Assuntos
Arabidopsis/genética , Inativação Gênica , Genoma de Planta , Conformação de Ácido Nucleico , Transgenes , Elementos de DNA Transponíveis
8.
Nat Commun ; 10(1): 2710, 2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-31221974

RESUMO

In animals and plants, the H3K9me3 and H3K27me3 chromatin silencing marks are deposited by different protein machineries. H3K9me3 is catalyzed by the SET-domain SU(VAR)3-9 enzymes, while H3K27me3 is catalyzed by the SET-domain Enhancer-of-zeste enzymes, which are the catalytic subunits of Polycomb Repressive Complex 2 (PRC2). Here, we show that the Enhancer-of-zeste-like protein Ezl1 from the unicellular eukaryote Paramecium tetraurelia, which exhibits significant sequence and structural similarities with human EZH2, catalyzes methylation of histone H3 in vitro and in vivo with an apparent specificity toward K9 and K27. We find that H3K9me3 and H3K27me3 co-occur at multiple families of transposable elements in an Ezl1-dependent manner. We demonstrate that loss of these histone marks results in global transcriptional hyperactivation of transposable elements with modest effects on protein-coding gene expression. Our study suggests that although often considered functionally distinct, H3K9me3 and H3K27me3 may share a common evolutionary history as well as a common ancestral role in silencing transposable elements.


Assuntos
Elementos de DNA Transponíveis/genética , Inativação Gênica , Histonas/genética , Paramecium tetraurellia/genética , Complexo Repressor Polycomb 2/metabolismo , Metilação de DNA , Processamento de Proteína Pós-Traducional/genética , Ativação Transcricional/genética
9.
Biochemistry (Mosc) ; 84(4): 398-406, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31228931

RESUMO

To identify Yersinia pestis genes involved in the microbe's resistance to cationic antimicrobial peptides, the strategy of random transposon mutagenesis with a Tn5 minitransposon was used, and the library was screened for detecting polymyxin B (PMB) susceptible mutants. The mutation responsible for PMB-sensitive phenotype and the lipopolysaccharide (LPS) structure were characterized for the Y. pestis strain KM218-A3. In this strain the mini-Tn5 was located in an open reading frame with the product homologous to the E. coli protein GmhB (82% identity) functioning as d-glycero-d-manno-heptose-1,7-diphosphate phosphatase. ESI FT ICR mass spectrometry of anions was used to study the structure of the unmodified LPS of Y. pestis KM218-A3, and molecules were revealed with the full-size LPS core or with two types of an incomplete core: consisting of Kdo-Kdo or Ko-Kdo disaccharides and Hep-(Kdo)-Kdo or Hep-(Ko)-Kdo trisaccharides. The performed complementation confirmed that the defect in the biological properties of the mutant strain was caused by inactivation of the gmhB gene. These findings indicated that the gmhB gene product of Y. pestis is essential for production of wild-type LPS resistant to antimicrobial peptides and serum.


Assuntos
Elementos de DNA Transponíveis/genética , Yersinia pestis/metabolismo , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Sequência de Carboidratos , Farmacorresistência Bacteriana/genética , Lipopolissacarídeos/análise , Lipopolissacarídeos/química , Lipopolissacarídeos/metabolismo , Testes de Sensibilidade Microbiana , Mutagênese , Polimixina B/farmacologia , Espectrometria de Massas por Ionização por Electrospray , Yersinia pestis/efeitos dos fármacos , Yersinia pestis/genética
10.
Microbiol Res ; 223-225: 63-71, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31178053

RESUMO

The phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS) catalyzes the translocation of sugar substrates with their concomitant phosphorylation in bacteria. In addition to its intrinsic role in sugar transport and metabolism, numerous recent studies report the versatility of the PTS to interconnect energy and signal transduction in response to sugar availability. In this study, the role of PTS in Salmonella virulence regulation was explored. To decipher the regulatory network coordinated by the PTS during Salmonella infection, a transcriptomic approach was applied to a transposon insertion mutant with defective expression of ptsI and crr, which encode enzyme I and enzyme IIAGlc of the PTS, respectively. There were 114 differentially expressed genes (DEGs) exhibiting two-fold or higher expression changes in the transposon mutant strain, with 13 up-regulated genes versus 101 down-regulated genes. One-third of the DEGs were associated with energy production and carbohydrate/amino acid metabolism pathways, implicating the prominent role of the PTS in carbohydrate transport. With regard to regulation of virulence, the tested mutant decreased the expression of genes associated with quorum sensing, Salmonella pathogenicity islands, flagella, and the PhoPQ regulon. We investigated the possibility of PTS-mediated regulation of virulence determinants identified in the transcriptomic analysis and proposed a regulatory circuit orchestrated by the PTS in Salmonella infection of host cells. These results suggest that Salmonella divergently controls virulence attributes in accordance with the availability of carbohydrates in the environment.


Assuntos
Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Fosfoenolpiruvato/metabolismo , Fosfotransferases/metabolismo , Salmonella/genética , Salmonella/metabolismo , Fatores de Virulência/genética , Transporte Biológico , Elementos de DNA Transponíveis , Flagelos/genética , Perfilação da Expressão Gênica , Proteínas de Membrana/genética , Mutação , Fosforilação , Regulon , Salmonella/patogenicidade , Infecções por Salmonella , Salmonella typhimurium/genética , Transdução de Sinais , Transcriptoma , Sistemas de Secreção Tipo III/genética , Virulência/genética
11.
Vet Microbiol ; 233: 28-38, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31176409

RESUMO

The anti-phagocytic abilities of bacteria often affect bacterial pathogenicity. Here, random mutant library of Streptococcus equi subsp. zooepidemicus (SEZ) was constructed using transposon mutagenesis. After careful screening, 30 transposon mutants with different transposon insertion sites were identified by conducting quantitative phagocytosis and insertion-site confirmation assays, whose anti-phagocytic abilities were significantly reduced relative to the wild-type strain. Insertion sites of 19 strains were monocistronic, including genes coding membrane proteins, transporters, and enzymes with unknown pathological function, such as sadM, adhP, purD, guaA, alpha-galactosidase coding gene, ABC transporter permease coding gene, metallo-beta-lactamase coding gene, and three secreted enzyme coding genes spuZ, slaB, and endoS, as well as known virulence factor coding genes, such as hasA and szM. The insertion sites of another 11 strains were polycistronic. We focused on four monocistronic-mutant strains: MhtpZ, MspuZ, MslaB, and MendoS. The anti-phagocytic abilities of not only the mutants that were precoincubated with the recombinant proteins, but also the complement strains were significantly more pronounced than those of all four corresponding mutants. The polyclonal antiserum against SlaB or EndoS also significantly decreased the anti-phagocytic capacity of wild-type SEZ. All four mutants exhibited significantly decreased viability in whole blood and reduced lethality in mice relative to the wild-type strain. Thus, we identified a variety of new anti-phagocytic factors, particularly multiple SEZ secreted enzymes. These factors are instrumental in the phagocytic resistance of SEZ in the absence of opsonin. Our results provide a framework for further studies of SEZ pathogenesis and relevant vaccine development for novel potential targets.


Assuntos
Genes Bacterianos , Óperon , Fagócitos/microbiologia , Fagocitose , Streptococcus equi/genética , Animais , Elementos de DNA Transponíveis , Feminino , Biblioteca Gênica , Camundongos , Camundongos Endogâmicos ICR , Mutagênese , Mutação , Células RAW 264.7 , Streptococcus equi/patogenicidade , Fatores de Virulência/genética
12.
Plant Cell Rep ; 38(9): 1081-1097, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31134349

RESUMO

KEY MESSAGE: Duplicate POT1 genes must rapidly diverge or be inactivated. Protection of telomeres 1 (POT1) encodes a conserved telomere binding protein implicated in both chromosome end protection and telomere length maintenance. Most organisms harbor a single POT1 gene, but in the few lineages where the POT1 family has expanded, the duplicate genes have diversified. Arabidopsis thaliana bears three POT1-like loci, POT1a, POT1b and POT1c. POT1a retains the ancestral function of telomerase regulation, while POT1b is implicated in chromosome end protection. Here we examine the function and evolution of the third POT1 paralog, POT1c. POT1c is a new gene, unique to A. thaliana, and was derived from a duplication event involving the POT1a locus and a neighboring gene encoding ribosomal protein S17. The duplicate S17 locus (dS17) is highly conserved across A. thaliana accessions, while POT1c is highly divergent, harboring multiple deletions within the gene body and two transposable elements within the promoter. The POT1c locus is transcribed at very low to non-detectable levels under standard growth conditions. In addition, no discernable molecular or developmental defects are associated with plants bearing a CRISPR mutation in the POT1c locus. However, forced expression of POT1c leads to decreased telomerase enzyme activity and shortened telomeres. Evolutionary reconstruction indicates that transposons invaded the POT1c promoter soon after the locus was formed, permanently silencing the gene. Altogether, these findings argue that POT1 dosage is critically important for viability and duplicate gene copies are retained only upon functional divergence.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Dosagem de Genes , Homeostase do Telômero/genética , Proteínas de Ligação a Telômeros/metabolismo , Telômero/genética , Arabidopsis/enzimologia , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Elementos de DNA Transponíveis/genética , Evolução Molecular , Duplicação Gênica , Mutação , Regiões Promotoras Genéticas/genética , Telomerase/genética , Telomerase/metabolismo , Proteínas de Ligação a Telômeros/genética
13.
BMC Genomics ; 20(1): 436, 2019 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-31142281

RESUMO

BACKGROUND: Bacillus cereus sensu lato s.l.) is a group of bacteria displaying close phylogenetic relationships but a high ecological diversity. The three most studied species are Bacillus anthracis, Bacillus cereus sensu stricto and Bacillus thuringiensis. While some species are pathogenic to mammals or associated with food poisoning, Bacillus thuringiensis is a well-known entomopathogenic bacterium used as biopesticide worldwide. B. cereus s.l. also contains a large variety of mobile genetic elements (MGEs). RESULTS: In this study, we detail the occurrence and plasmid vs. chromosome distribution of several MGEs in 102 complete and annotated genomes of B. cereus s.l. These MGEs include 16 Insertion Sequence (IS) families, the Tn3 family, 18 different Bacillus cereus repeats (BCRs) and 30 known group II introns. CONCLUSIONS: Our analysis not only shows the diversity of these MGEs among strains of the same species and between different species within the B. cereus s.l. group, but also highlights the potential impact of these elements on the plasticity of the plasmid pool, and the TEs (Transposable Elements) - species relationship within B. cereus s.l.


Assuntos
Bacillus cereus/genética , Sequências Repetitivas Dispersas , Toxinas Bacterianas/genética , Elementos de DNA Transponíveis , Genoma Bacteriano , Íntrons , Plasmídeos/genética , Sequências Repetitivas de Ácido Nucleico
14.
Huan Jing Ke Xue ; 40(5): 2234-2239, 2019 May 08.
Artigo em Chinês | MEDLINE | ID: mdl-31087861

RESUMO

Microplastics and antibiotic resistance genes (ARGs) are emerging pollutants/contaminants, and are also the research hotspots concerning environmental health in the past few years. To explore the effects of microplastics on ARGs in estuarine sediment, three different microplastics were added to microcosm incubation experiments of sediments. Then, we investigated the persistence, abundance, diversity, and shifts of the ARGs in estuarine sediments by high-throughput quantitative polymerase chain reaction (PCR). The results showed that the microplastics significantly changed the structure of ARGs in the sediments. PVC and PE, which are hard to degrade, had significant effects on the structures and types of ARGs. However, the PVA, which is soluble, reduced the types and persistence of ARGs significantly. The abundance of ARGs in S_PVC, S_PE, and S_PVA were 4.1×109, 8.1×109, and 2.0×109 copies·g-1, respectively. The abundance of ARGs in sediments with added PE almost increased by one order of magnitude, implying that microplastics could significantly increase the abundance of ARGs in sediments. Furthermore, OLS regression analysis showed that ARGs are significantly correlated with transposon and integron, suggesting that mobile genetic elements (MGEs) may promote the transfer and dissemination of ARGs.


Assuntos
Genes Bacterianos , Sedimentos Geológicos/análise , Plásticos/análise , Poluentes da Água/análise , Elementos de DNA Transponíveis , Resistência Microbiana a Medicamentos/genética , Estuários , Integrons
15.
BMC Genomics ; 20(1): 431, 2019 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-31138110

RESUMO

BACKGROUND: BCG is the most widely used vaccine of all time and remains the only licensed vaccine for use against tuberculosis in humans. BCG also protects other species such as cattle against tuberculosis, but due to its incompatibility with current tuberculin testing regimens remains unlicensed. BCG's efficacy relates to its ability to persist in the host for weeks, months or even years after vaccination. It is unclear to what degree this ability to resist the host's immune system is maintained by a dynamic interaction between the vaccine strain and its host as is the case for pathogenic mycobacteria. RESULTS: To investigate this question, we constructed transposon mutant libraries in both BCG Pasteur and BCG Danish strains and inoculated them into bovine lymph nodes. Cattle are well suited to such an assay, as they are naturally susceptible to tuberculosis and are one of the few animal species for which a BCG vaccination program has been proposed. After three weeks, the BCG were recovered and the input and output libraries compared to identify mutants with in vivo fitness defects. Less than 10% of the mutated genes were identified as affecting in vivo fitness, they included genes encoding known mycobacterial virulence functions such as mycobactin synthesis, sugar transport, reductive sulphate assimilation, PDIM synthesis and cholesterol metabolism. Many other attenuating genes had not previously been recognised as having a virulence phenotype. To test these genes, we generated and characterised three knockout mutants that were predicted by transposon mutagenesis to be attenuating in vivo: pyruvate carboxylase, a hypothetical protein (BCG_1063), and a putative cyclopropane-fatty-acyl-phospholipid synthase. The knockout strains survived as well as wild type during in vitro culture and in bovine macrophages, yet demonstrated marked attenuation during passage in bovine lymph nodes confirming that they were indeed involved in persistence of BCG in the host. CONCLUSION: These data show that BCG is far from passive during its interaction with the host, rather it continues to employ its remaining virulence factors, to interact with the host's innate immune system to allow it to persist, a property that is important for its protective efficacy.


Assuntos
Elementos de DNA Transponíveis , Mycobacterium bovis/genética , Animais , Vacina BCG , Bovinos , Colesterol/metabolismo , Biblioteca Gênica , Genes Bacterianos , Aptidão Genética , Mycobacterium bovis/metabolismo , Oxazóis , Açúcares/metabolismo , Sulfatos/metabolismo , Tuberculose Bovina/microbiologia
16.
MBio ; 10(2)2019 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-31040245

RESUMO

Staphylococcus aureus has the ability to cause infections in multiple organ systems, suggesting an ability to rapidly adapt to changing carbon and nitrogen sources. Although there is little information about the nutrients available at specific sites of infection, a mature skin abscess has been characterized as glucose depleted, indicating that peptides and free amino acids are an important source of nutrients for the bacteria. Our studies have found that mutations in enzymes necessary for growth on amino acids, including pyruvate carboxykinase (ΔpckA) and glutamate dehydrogenase (ΔgudB), reduced the ability of the bacteria to proliferate within a skin abscess, suggesting that peptides and free amino acids are important for S. aureus growth. Furthermore, we found that collagen, an abundant host protein that is present throughout a skin abscess, serves as a reservoir of peptides. To liberate peptides from the collagen, we identified that the host protease, MMP-9, as well as the staphylococcal proteases aureolysin and staphopain B function to cleave collagen into peptide fragments that can support S. aureus growth under nutrient-limited conditions. Moreover, the oligopeptide transporter Opp3 is the primary staphylococcal transporter responsible for peptide acquisition. Lastly, we observed that the presence of peptides (3-mer to 7-mer) induces the expression of aureolysin, suggesting that S. aureus has the ability to detect peptides in the environment.IMPORTANCE Staphylococcus aureus has the ability to cause infections in a variety of niches, suggesting a robust metabolic capacity facilitating proliferation under various nutrient conditions. The mature skin abscess is glucose depleted, indicating that peptides and free amino acids are important sources of nutrients for S. aureus Our studies have found that mutations in both pyruvate carboxykinase and glutamate dehydrogenase, enzymes that function in essential gluconeogenesis reactions when amino acids serve as the major carbon source, reduce bacterial burden in a murine skin abscess model. Moreover, peptides liberated from collagen by host protease MMP-9 as well as the staphylococcal protease aureolysin support S. aureus growth in an Opp3-dependent manner under nutrient-limited conditions. Additionally, the presence of peptides induces aureolysin expression. Overall, these studies define one pathway by which S. aureus senses a nutrient-limiting environment and induces factors that function to acquire and utilize carbon from host-derived sources.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Peptídeo Hidrolases/metabolismo , Proteínas/metabolismo , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/crescimento & desenvolvimento , Staphylococcus aureus/metabolismo , Animais , Análise Mutacional de DNA , Elementos de DNA Transponíveis , Modelos Animais de Doenças , Camundongos Endogâmicos C57BL
17.
J Med Microbiol ; 68(6): 874-881, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31116101

RESUMO

PURPOSE: To assess the antibiotic resistance, transposon profiles, serotype distribution and vaccine coverage rates in 110 erythromycin-resistant S. pneumoniae clinical isolates. METHODOLOGY: Erythromycin, clindamycin, tetracycline, chloramphenicol and kanamycin susceptibilities were assessed using the E-test/disc diffusion method. Inducible macrolide resistance was tested using the erythromycin-clindamycin double disc diffusion test. Serogrouping and serotyping were performed using latex particle agglutination and the Quellung reaction, respectively. Drug resistance genes and transposon-specific genes were investigated by PCR. RESULTS: Of the isolates, 93  % were resistant to clindamycin; 81  % were resistant to tetracycline; 76  % were multi-drug-resistant, having resistance to both clindamycin and tetracycline; and 12  % had extended-drug resistance, being resistant to clindamycin, tetracycline, chloramphenicol and kanamycin. The majority of isolates (88.2 %) exhibited the cMLSB phenotype. The association between the cMLSB phenotype and tetracycline resistance was related to transposons Tn2010 (38.2 %), Tn6002 (21.8 %) and Tn3872 (18.2 %). M and iMLSB phenotypes were observed in 7 and 5  % of the isolates, respectively. The most frequent serotype was 19 F (40 %). Among the erythromycin-resistant pneumococci, vaccine coverage rates for the 13-valent pneumococcal conjugate vaccine (PCV-13) and the 23-valent pneumococcal polysaccharide vaccine (PPSV-23) were 76.4 and 79.1  %, respectively, compared to 82.2 and 85.1 % transposon-carrying isolates. CONCLUSIONS: Multi-drug resistance among erythromycin-resistant S. pneumoniae isolates mainly occurs due to the horizontal spread of the Tn916 family of transposons. The majority of the transposon-carrying isolates are covered by 13- and 23-valent pneumococcal vaccines. Since serotype distribution and transposons in S. pneumoniae isolates may change over time, close monitoring is essential.


Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana Múltipla , Eritromicina/farmacologia , Infecções Pneumocócicas/microbiologia , Streptococcus pneumoniae/genética , Cápsulas Bacterianas/imunologia , Elementos de DNA Transponíveis/genética , Genótipo , Humanos , Fenótipo , Infecções Pneumocócicas/tratamento farmacológico , Infecções Pneumocócicas/epidemiologia , Vacinas Pneumocócicas , Sorogrupo , Streptococcus pneumoniae/imunologia , Turquia/epidemiologia
18.
Int J Mol Sci ; 20(8)2019 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-31022896

RESUMO

Psychrobacter sp. DAB_AL32B, originating from Spitsbergen island (Arctic), carries the large plasmid pP32BP2 (54,438 bp). Analysis of the pP32BP2 nucleotide sequence revealed the presence of three predicted phenotypic modules that comprise nearly 30% of the plasmid genome. These modules appear to be involved in fimbriae synthesis via the chaperone-usher pathway (FIM module) and the aerobic and anaerobic metabolism of carnitine (CAR and CAI modules, respectively). The FIM module was found to be functional in diverse hosts since it facilitated the attachment of bacterial cells to abiotic surfaces, enhancing biofilm formation. The CAI module did not show measurable activity in any of the tested strains. Interestingly, the CAR module enabled the enzymatic breakdown of carnitine, but this led to the formation of the toxic by-product trimethylamine, which inhibited bacterial growth. Thus, on the one hand, pP32BP2 can enhance biofilm formation, a highly advantageous feature in cold environments, while on the other, it may prevent bacterial growth under certain environmental conditions. The detrimental effect of harboring pP32BP2 (and its CAR module) seems to be conditional, since this replicon may also confer the ability to use carnitine as an alternative carbon source, although a pathway to utilize trimethylamine is most probably necessary to make this beneficial. Therefore, the phenotype determined by this CAR-containing plasmid depends on the metabolic background of the host strain.


Assuntos
Plasmídeos/genética , Psychrobacter/genética , Regiões Árticas , Aderência Bacteriana , Sequência de Bases , Biofilmes/crescimento & desenvolvimento , Carnitina/metabolismo , Elementos de DNA Transponíveis , Fenótipo , Plasmídeos/metabolismo , Psychrobacter/fisiologia
19.
BMC Genomics ; 20(1): 305, 2019 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-31014230

RESUMO

BACKGROUND: Evolutionary theory indicates that the dynamics of aging in the soma and reproductive tissues may be distinct. This difference arises from the fact that only the germline lineage establishes future generations. In the soma, changes in the landscape of heterochromatin have been proposed to have an important role in aging. This is because redistribution of heterochromatin during aging has been linked to the derepression of transposable elements and an overall loss of somatic gene regulation. A role for changes in the chromatin landscape in the aging of reproductive tissues is less well established. Whether or not epigenetic factors, such as heterochromatin marks, are perturbed in aging reproductive tissues is of interest because, in special cases, epigenetic variation may be heritable. Using mRNA sequencing data from late-stage egg chambers in Drosophila melanogaster, we characterized the landscape of altered gene and transposable element expression in aged reproductive tissues. This allowed us to test the hypothesis that reproductive tissues may differ from somatic tissues in their response to aging. RESULTS: We show that age-related expression changes in late-stage egg chambers tend to occur in genes residing in heterochromatin, particularly on the largely heterochromatic 4th chromosome. However, these expression differences are seen as both decreases and increases during aging, inconsistent with a general loss of heterochromatic silencing. We also identify an increase in expression of the piRNA machinery, suggesting an age-related increased investment in the maintenance of genome stability. We further identify a strong age-related reduction in the expression of mitochondrial transcripts. However, we find no evidence for global TE derepression in reproductive tissues. Rather, the observed effects of aging on TEs are primarily strain and family specific. CONCLUSIONS: These results identify unique responses in somatic versus reproductive tissue with regards to aging. As in somatic tissues, female reproductive tissues show reduced expression of mitochondrial genes. In contrast, the piRNA machinery shows increased expression during aging. Overall, these results also indicate that global loss of TE control observed in other studies may be unique to the soma and sensitive to genetic background and TE family.


Assuntos
Envelhecimento/genética , Elementos de DNA Transponíveis/genética , Drosophila melanogaster/fisiologia , Perfilação da Expressão Gênica , Mitocôndrias/genética , Ovário/fisiologia , RNA Interferente Pequeno/genética , Animais , Drosophila melanogaster/genética , Feminino , Genoma Mitocondrial/genética , Heterocromatina/genética , Óvulo/metabolismo , RNA Mensageiro/genética
20.
Genome Biol Evol ; 11(5): 1487-1500, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-31028389

RESUMO

Phytopathogen genomes are under constant pressure to change, as pathogens are locked in an evolutionary arms race with their hosts, where pathogens evolve effector genes to manipulate their hosts, whereas the hosts evolve immune components to recognize the products of these genes. Colletotrichum higginsianum (Ch), a fungal pathogen with no known sexual morph, infects Brassicaceae plants including Arabidopsis thaliana. Previous studies revealed that Ch differs in its virulence toward various Arabidopsis thaliana ecotypes, indicating the existence of coevolutionary selective pressures. However, between-strain genomic variations in Ch have not been studied. Here, we sequenced and assembled the genome of a Ch strain, resulting in a highly contiguous genome assembly, which was compared with the chromosome-level genome assembly of another strain to identify genomic variations between strains. We found that the two closely related strains vary in terms of large-scale rearrangements, the existence of strain-specific regions, and effector candidate gene sets and that these variations are frequently associated with transposable elements (TEs). Ch has a compartmentalized genome consisting of gene-sparse, TE-dense regions with more effector candidate genes and gene-dense, TE-sparse regions harboring conserved genes. Additionally, analysis of the conservation patterns and syntenic regions of effector candidate genes indicated that the two strains vary in their effector candidate gene sets because of de novo evolution, horizontal gene transfer, or gene loss after divergence. Our results reveal mechanisms for generating genomic diversity in this asexual pathogen, which are important for understanding its adaption to hosts.


Assuntos
Colletotrichum/genética , Elementos de DNA Transponíveis , Genoma Fúngico , Arabidopsis , Colletotrichum/patogenicidade , Genes Essenciais , Variação Genética , Doenças das Plantas , Sintenia , Virulência
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