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1.
Front Microbiol ; 13: 1063287, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36726566

RESUMO

Burkholderia pseudomallei (Bp), causing a highly fatal disease called melioidosis, is a facultative intracellular pathogen that attaches and invades a variety of cell types. We previously identified BP1026B_I0091 as a surface attachment protein (Sap1) and an essential virulence factor, contributing to Bp pathogenesis in vitro and in vivo. The expression of sap1 is regulated at different stages of Bp intracellular lifecycle by unidentified regulator(s). Here, we identified SapR (BP1026B_II1046) as a transcriptional regulator that activates sap1, using a high-throughput transposon mutagenesis screen in combination with Tn-Seq. Consistent with phenotypes of the Δsap1 mutant, the ΔsapR activator mutant exhibited a significant reduction in Bp attachment to the host cell, leading to subsequent decreased intracellular replication. RNA-Seq analysis further revealed that SapR regulates sap1. The regulation of sap1 by SapR was confirmed quantitatively by qRT-PCR, which also validated the RNA-Seq data. SapR globally regulates genes associated with the bacterial membrane in response to diverse environments, and some of the genes regulated by SapR are virulence factors that are required for Bp intracellular infection (e.g., type III and type VI secretion systems). This study has identified the complex SapR regulatory network and its importance as an activator of an essential Sap1 attachment factor.

2.
Nat Commun ; 12(1): 1907, 2021 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-33772012

RESUMO

Prokaryotic cell transcriptomics has been limited to mixed or sub-population dynamics and individual cells within heterogeneous populations, which has hampered further understanding of spatiotemporal and stage-specific processes of prokaryotic cells within complex environments. Here we develop a 'TRANSITomic' approach to profile transcriptomes of single Burkholderia pseudomallei cells as they transit through host cell infection at defined stages, yielding pathophysiological insights. We find that B. pseudomallei transits through host cells during infection in three observable stages: vacuole entry; cytoplasmic escape and replication; and membrane protrusion, promoting cell-to-cell spread. The B. pseudomallei 'TRANSITome' reveals dynamic gene-expression flux during transit in host cells and identifies genes that are required for pathogenesis. We find several hypothetical proteins and assign them to virulence mechanisms, including attachment, cytoskeletal modulation, and autophagy evasion. The B. pseudomallei 'TRANSITome' provides prokaryotic single-cell transcriptomics information enabling high-resolution understanding of host-pathogen interactions.


Assuntos
Proteínas de Bactérias/genética , Burkholderia pseudomallei/genética , Biologia Computacional/métodos , Perfilação da Expressão Gênica/métodos , Genes Bacterianos/genética , Fatores de Virulência/genética , Animais , Burkholderia pseudomallei/citologia , Burkholderia pseudomallei/patogenicidade , Linhagem Celular Tumoral , Membrana Celular/microbiologia , Citoplasma/microbiologia , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , Melioidose/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Células RAW 264.7 , Análise de Célula Única/métodos , Vacúolos/microbiologia , Virulência/genética
3.
Sci Rep ; 8(1): 12422, 2018 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-30127446

RESUMO

Natural competency requires uptake of exogenous DNA from the environment and the integration of that DNA into recipient bacteria can be used for DNA-repair or genetic diversification. The Burkholderia genus is unique in that only some of the species and strains are naturally competent. We identified and characterized two genes, comE and crp, from naturally competent B. pseudomallei 1026b that play a role in DNA uptake and catabolism. Single-copies of rhamnose-inducible comE and crp genes were integrated into a Tn7 attachment-site in non-naturally competent Burkholderia including pathogens B. pseudomallei K96243, B. cenocepacia K56-2, and B. mallei ATCC23344. Strains expressing comE or crp were assayed for their ability to uptake and catabolize DNA. ComE and Crp allowed non-naturally competent Burkholderia species to catabolize DNA, uptake exogenous gfp DNA and express GFP. Furthermore, we used synthetic comE and crp to expand the utility of the λ-red recombineering system for genetic manipulation of non-competent Burkholderia species. A newly constructed vector, pKaKa4, was used to mutate the aspartate semialdehyde dehydrogenase (asd) gene in four B. mallei strains, leading to the complete attenuation of these tier-1 select-agents. These strains have been excluded from select-agent regulations and will be of great interest to the field.


Assuntos
Burkholderia pseudomallei/genética , Genes Bacterianos/genética , Animais , Aspartato-Semialdeído Desidrogenase/genética , Linhagem Celular , Reparo do DNA/genética , DNA Bacteriano/genética , Técnicas Genéticas , Vetores Genéticos/genética , Camundongos , Camundongos Endogâmicos BALB C , Células RAW 264.7
4.
Mol Microbiol ; 109(3): 401-414, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29995308

RESUMO

Gene regulation network in Pseudomonas aeruginosa is complex. With a relatively large genome (6.2 Mb), there is a significant portion of genes that are proven or predicted to be transcriptional regulators. Many of these regulators have been shown to play important roles in biofilm formation and maintenance. In this study, we present a novel transcriptional regulator, PA1226, which modulates biofilm formation and virulence in P. aeruginosa. Mutation in the gene encoding this regulator abolished the ability of P. aeruginosa to produce biofilms in vitro, without any effect on the planktonic growth. This regulator is also essential for the in vivo fitness and pathogenesis in both Drosophila melanogaster and BALB/c mouse lung infection models. Transcriptome analysis revealed that PA1226 regulates many essential virulence genes/pathways, including those involved in alginate, pili, and LPS biosynthesis. Genes/operons directly regulated by PA1226 and potential binding sequences were identified via ChIP-seq. Attempts to confirm the binding sequences by electrophoretic mobility shift assay led to the discovery of a co-regulator, PA1413, via co-immunoprecipitation assay. PA1226 and PA1413 were shown to bind collaboratively to the promoter regions of their regulons. A model is proposed, summarizing our finding on this novel dual-regulation system.


Assuntos
Biofilmes/crescimento & desenvolvimento , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/fisiologia , Pseudomonas aeruginosa/patogenicidade , Fatores de Transcrição/metabolismo , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Modelos Animais de Doenças , Drosophila melanogaster , Perfilação da Expressão Gênica , Humanos , Pneumopatias/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pseudomonas aeruginosa/genética , Fatores de Transcrição/genética , Virulência
5.
Sci Rep ; 8(1): 1173, 2018 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-29352173

RESUMO

Maize chlorotic mottle virus has been rapidly spreading around the globe over the past decade. The interactions of maize chlorotic mottle virus with Potyviridae viruses causes an aggressive synergistic viral condition - maize lethal necrosis, which can cause total yield loss. Maize production in sub-Saharan Africa, where it is the most important cereal, is threatened by the arrival of maize lethal necrosis. We obtained maize chlorotic mottle virus genome sequences from across East Africa and for the first time from Ecuador and Hawaii, and constructed a phylogeny which highlights the similarity of Chinese to African isolates, and Ecuadorian to Hawaiian isolates. We used a measure of clustering, the adjusted Rand index, to extract region-specific SNPs and coding variation that can be used for diagnostics. The population genetics analysis we performed shows that the majority of sequence diversity is partitioned between populations, with diversity extremely low within China and East Africa.


Assuntos
Gammaherpesvirinae/fisiologia , Doenças das Plantas/virologia , Sequência de Bases , Biologia Computacional/métodos , Gammaherpesvirinae/isolamento & purificação , Variação Genética , Genoma Viral , Genótipo , Geografia Médica , Sequenciamento de Nucleotídeos em Larga Escala , Filogenia , Polimorfismo de Nucleotídeo Único
6.
Mol Microbiol ; 106(6): 976-985, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29030956

RESUMO

Bacterial cooperative associations and dynamics in biofilm microenvironments are of special interest in recent years. Knowledge of localized gene-expression and corresponding bacterial behaviors within the biofilm architecture at a global scale has been limited, due to a lack of robust technology to study limited number of cells in stratified layers of biofilms. With our recent pioneering developments in single bacterial cell transcriptomic analysis technology, we generated herein an unprecedented spatial transcriptome map of the mature in vitro Pseudomonas aeruginosa biofilm model, revealing contemporaneous yet altered bacterial behaviors at different layers within the biofilm architecture (i.e., surface, middle and interior of the biofilm). Many genes encoding unknown functions were highly expressed at the biofilm-solid interphase, exposing a critical gap in the knowledge of their activities that may be unique to this interior niche. Several genes of unknown functions are critical for biofilm formation. The in vivo importance of these unknown proteins was validated in invertebrate (fruit fly) and vertebrate (mouse) models. We envisage the future value of this report to the community, in aiding the further pathophysiological understanding of P. aeruginosa biofilms. Our approach will open doors to the study of bacterial functional genomics of different species in numerous settings.


Assuntos
Biofilmes/crescimento & desenvolvimento , Regulação Bacteriana da Expressão Gênica , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidade , Animais , Proteínas de Bactérias/genética , Drosophila/genética , Camundongos , Mutação , Transcriptoma , Fatores de Virulência/genética
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