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1.
FEMS Microbes ; 2: xtab010, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-37334228

RESUMO

Staphylococcus aureus (SA) is the most common bacterial species in chronic wounds. However, there is a lack of understanding of how SA secretions affect the cell biology during the healing process. We studied the effects of biofilm-secretions from SA strain SA29213 on 3T3 fibroblasts. SA29213 is a chronic wound isolate and widely used as a reference strain. We used a series of concentrations of biofilm-conditioned media (BCM) and found 100% BCM is lethal within 10 h. Cells survived in ≤75% BCM but the rate of closure in scratch wound assays was reduced. Treatment with 75% and 50% BCM caused fibroblasts to change shape and develop dendrite like processes. Prolonged treatment with 75% and 50% BCM reduced cell proliferation and increased the 4n deoxyribonucleic acid cell population with cell cycle arrest. There was also an elevation in the senescence marker beta galactosidase and the number of multinucleated cells. Shorter treatments with 75% and 50% SA BCM caused an increase in cell-cell adhesion and a redistribution of ß-catenin from the cell membrane to the cytoplasm along with a change in the appearance and decrease in size of ZO-1, vinculin and paxillin structures. Fibroblasts in the edge of chronic wounds exposed to the secretions of SA may suffer similar effects such as induction of senescence, reduced proliferation and migration, which may contribute to the delayed healing of these chronic infected wounds.

2.
mSphere ; 4(3)2019 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-31243080

RESUMO

Brucella, the agent causing brucellosis, is a major zoonotic pathogen with worldwide distribution. Brucella resides and replicates inside infected host cells in membrane-bound compartments called Brucella-containing vacuoles (BCVs). Following uptake, Brucella resides in endosomal BCVs (eBCVs) that gradually mature from early to late endosomal features. Through a poorly understood process that is key to the intracellular lifestyle of Brucella, the eBCV escapes fusion with lysosomes by transitioning to the replicative BCV (rBCV), a replicative niche directly connected to the endoplasmic reticulum (ER). Despite the notion that this complex intracellular lifestyle must depend on a multitude of host factors, a holistic view on which of these components control Brucella cell entry, trafficking, and replication is still missing. Here we used a systematic cell-based small interfering RNA (siRNA) knockdown screen in HeLa cells infected with Brucella abortus and identified 425 components of the human infectome for Brucella infection. These include multiple components of pathways involved in central processes such as the cell cycle, actin cytoskeleton dynamics, or vesicular trafficking. Using assays for pathogen entry, knockdown complementation, and colocalization at single-cell resolution, we identified the requirement of the VPS retromer for Brucella to escape the lysosomal degradative pathway and to establish its intracellular replicative niche. We thus validated the VPS retromer as a novel host factor critical for Brucella intracellular trafficking. Further, our genomewide data shed light on the interplay between central host processes and the biogenesis of the Brucella replicative niche.IMPORTANCE With >300,000 new cases of human brucellosis annually, Brucella is regarded as one of the most important zoonotic bacterial pathogens worldwide. The agent causing brucellosis resides inside host cells within vacuoles termed Brucella-containing vacuoles (BCVs). Although a few host components required to escape the degradative lysosomal pathway and to establish the ER-derived replicative BCV (rBCV) have already been identified, the global understanding of this highly coordinated process is still partial, and many factors remain unknown. To gain deeper insight into these fundamental questions, we performed a genomewide RNA interference (RNAi) screen aiming at discovering novel host factors involved in the Brucella intracellular cycle. We identified 425 host proteins that contribute to Brucella cellular entry, intracellular trafficking, and replication. Together, this study sheds light on previously unknown host pathways required for the Brucella infection cycle and highlights the VPS retromer components as critical factors for the establishment of the Brucella intracellular replicative niche.


Assuntos
Brucella abortus/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Interações Hospedeiro-Patógeno , RNA Interferente Pequeno , Vacúolos/microbiologia , Brucella abortus/fisiologia , Replicação do DNA , Retículo Endoplasmático/microbiologia , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Técnicas de Silenciamento de Genes , Genoma Bacteriano , Células HeLa , Ensaios de Triagem em Larga Escala , Humanos
3.
J Cell Sci ; 131(4)2018 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-29361547

RESUMO

Entry of the facultative intracellular pathogen Brucella into host cells results in the formation of endosomal Brucella-containing vacuoles (eBCVs) that initially traffic along the endocytic pathway. eBCV acidification triggers the expression of a type IV secretion system that translocates bacterial effector proteins into host cells. This interferes with lysosomal fusion of eBCVs and supports their maturation to replicative Brucella-containing vacuoles (rBCVs). Bacteria replicate in rBCVs to large numbers, eventually occupying most of the cytoplasmic volume. As rBCV membranes tightly wrap each individual bacterium, they are constantly being expanded and remodeled during exponential bacterial growth. rBCVs are known to carry endoplasmic reticulum (ER) markers; however, the relationship of the vacuole to the genuine ER has remained elusive. Here, we have reconstructed the 3-dimensional ultrastructure of rBCVs and associated ER by correlative structured illumination microscopy (SIM) and focused ion beam/scanning electron microscopic tomography (FIB/SEM). Studying B. abortus-infected HeLa cells and trophoblasts derived from B. melitensis-infected mice, we demonstrate that rBCVs are complex and interconnected compartments that are continuous with neighboring ER cisternae, thus supporting a model that rBCVs are extensions of genuine ER.


Assuntos
Brucella abortus/ultraestrutura , Brucella melitensis/ultraestrutura , Retículo Endoplasmático/ultraestrutura , Vacúolos/ultraestrutura , Animais , Brucella abortus/patogenicidade , Brucella melitensis/patogenicidade , Citoplasma/microbiologia , Retículo Endoplasmático/microbiologia , Células HeLa , Interações Hospedeiro-Patógeno/genética , Humanos , Camundongos , Microscopia Eletrônica de Varredura , Trofoblastos/microbiologia , Trofoblastos/ultraestrutura , Sistemas de Secreção Tipo IV/ultraestrutura , Vacúolos/microbiologia
4.
J Cell Biol ; 211(4): 913-31, 2015 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-26598622

RESUMO

Methods enabling the delivery of proteins into eukaryotic cells are essential to address protein functions. Here we propose broad applications to cell biology for a protein delivery tool based on bacterial type III secretion (T3S). We show that bacterial, viral, and human proteins, fused to the N-terminal fragment of the Yersinia enterocolitica T3S substrate YopE, are effectively delivered into target cells in a fast and controllable manner via the injectisome of extracellular bacteria. This method enables functional interaction studies by the simultaneous injection of multiple proteins and allows the targeting of proteins to different subcellular locations by use of nanobody-fusion proteins. After delivery, proteins can be freed from the YopE fragment by a T3S-translocated viral protease or fusion to ubiquitin and cleavage by endogenous ubiquitin proteases. Finally, we show that this delivery tool is suitable to inject proteins in living animals and combine it with phosphoproteomics to characterize the systems-level impact of proapoptotic human truncated BID on the cellular network.


Assuntos
Sistemas de Secreção Tipo III/farmacologia , Células 3T3 , Animais , Apoptose , Proteínas Reguladoras de Apoptose/fisiologia , Proteínas da Membrana Bacteriana Externa/metabolismo , Permeabilidade da Membrana Celular , Sistemas de Liberação de Medicamentos , Células HeLa , Humanos , Camundongos , Dados de Sequência Molecular , Fosforilação , Processamento de Proteína Pós-Traducional , Transporte Proteico , Proteoma/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Peixe-Zebra
6.
Genome Biol ; 16: 220, 2015 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-26445817

RESUMO

Small interfering RNAs (siRNAs) exhibit strong off-target effects, which confound the gene-level interpretation of RNA interference screens and thus limit their utility for functional genomics studies. Here, we present gespeR, a statistical model for reconstructing individual, gene-specific phenotypes. Using 115,878 siRNAs, single and pooled, from three companies in three pathogen infection screens, we demonstrate that deconvolution of image-based phenotypes substantially improves the reproducibility between independent siRNA sets targeting the same genes. Genes selected and prioritized by gespeR are validated and shown to constitute biologically relevant components of pathogen entry mechanisms and TGF-ß signaling. gespeR is available as a Bioconductor R-package.


Assuntos
Técnicas de Silenciamento de Genes , Modelos Estatísticos , Interferência de RNA , Software , Bartonella henselae/genética , Brucella abortus/genética , Células HeLa , Humanos , Fenótipo , RNA Interferente Pequeno , Salmonella typhimurium/genética , Transdução de Sinais , Fator de Crescimento Transformador beta/fisiologia
7.
BMC Genomics ; 15: 1162, 2014 Dec 22.
Artigo em Inglês | MEDLINE | ID: mdl-25534632

RESUMO

BACKGROUND: Large-scale RNAi screening has become an important technology for identifying genes involved in biological processes of interest. However, the quality of large-scale RNAi screening is often deteriorated by off-targets effects. In order to find statistically significant effector genes for pathogen entry, we systematically analyzed entry pathways in human host cells for eight pathogens using image-based kinome-wide siRNA screens with siRNAs from three vendors. We propose a Parallel Mixed Model (PMM) approach that simultaneously analyzes several non-identical screens performed with the same RNAi libraries. RESULTS: We show that PMM gains statistical power for hit detection due to parallel screening. PMM allows incorporating siRNA weights that can be assigned according to available information on RNAi quality. Moreover, PMM is able to estimate a sharedness score that can be used to focus follow-up efforts on generic or specific gene regulators. By fitting a PMM model to our data, we found several novel hit genes for most of the pathogens studied. CONCLUSIONS: Our results show parallel RNAi screening can improve the results of individual screens. This is currently particularly interesting when large-scale parallel datasets are becoming more and more publicly available. Our comprehensive siRNA dataset provides a public, freely available resource for further statistical and biological analyses in the high-content, high-throughput siRNA screening field.


Assuntos
Genômica/métodos , Interferência de RNA , RNA Interferente Pequeno/genética , Linhagem Celular , Biblioteca Gênica , Genômica/normas , Ensaios de Triagem em Larga Escala , Interações Hospedeiro-Patógeno/genética , Humanos , Curva ROC , Reprodutibilidade dos Testes
8.
Proc Natl Acad Sci U S A ; 111(12): 4548-53, 2014 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-24616511

RESUMO

Systematic genetic perturbation screening in human cells remains technically challenging. Typically, large libraries of chemically synthesized siRNA oligonucleotides are used, each designed to degrade a specific cellular mRNA via the RNA interference (RNAi) mechanism. Here, we report on data from three genome-wide siRNA screens, conducted to uncover host factors required for infection of human cells by two bacterial and one viral pathogen. We find that the majority of phenotypic effects of siRNAs are unrelated to the intended "on-target" mechanism, defined by full complementarity of the 21-nt siRNA sequence to a target mRNA. Instead, phenotypes are largely dictated by "off-target" effects resulting from partial complementarity of siRNAs to multiple mRNAs via the "seed" region (i.e., nucleotides 2-8), reminiscent of the way specificity is determined for endogenous microRNAs. Quantitative analysis enabled the prediction of seeds that strongly and specifically block infection, independent of the intended on-target effect. This prediction was confirmed experimentally by designing oligos that do not have any on-target sequence match at all, yet can strongly reproduce the predicted phenotypes. Our results suggest that published RNAi screens have primarily, and unintentionally, screened the sequence space of microRNA seeds instead of the intended on-target space of protein-coding genes. This helps to explain why previously published RNAi screens have exhibited relatively little overlap. Our analysis suggests a possible way of identifying "seed reagents" for controlling phenotypes of interest and establishes a general strategy for extracting valuable untapped information from past and future RNAi screens.


Assuntos
Brucella abortus/efeitos dos fármacos , Bunyaviridae/efeitos dos fármacos , MicroRNAs/genética , Oligonucleotídeos/farmacologia , Interferência de RNA , Salmonella typhimurium/efeitos dos fármacos , Sequência de Bases , Brucella abortus/genética , Bunyaviridae/genética , Genes Bacterianos , Células HeLa , Humanos , RNA Interferente Pequeno/genética , Salmonella typhimurium/genética
9.
Cell Cycle ; 11(10): 1929-37, 2012 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-22544326

RESUMO

The actin cross-linking protein, α-actinin, plays a crucial role in mediating furrow ingression during cytokinesis. However, the mechanism by which its dynamics are regulated during this process is poorly understood. Here we have investigated the role of calcium sensitivity of α-actinin in the regulation of its dynamics by generating a functional calcium-insensitive mutant (EFM). GFP-tagged EFM (EFM-GFP) localized to the equatorial regions during cell division. However, the maximal equatorial accumulation of EFM-GFP was significantly smaller in comparison to α-actinin-GFP when it was expressed in normal cells and cells depleted of endogenous α-actinin. No apparent defects in cytokinesis were observed in these cells. However, F-actin levels at the equator were significantly reduced in cells expressing EFM-GFP as compared with α-actinin-GFP at furrow initiation but were recovered during furrow ingression. These results suggest that calcium sensitivity of α-actinin is required for its equatorial accumulation that is crucial for the initial equatorial actin assembly but is dispensable for cytokinesis. Equatorial RhoA localization was not affected by EFM-GFP overexpression, suggesting that equatorial actin assembly is predominantly driven by the RhoA-dependent mechanism. Our observations shed new light on the role and regulation of the accumulation of pre-existing actin filaments in equatorial actin assembly during cytokinesis.


Assuntos
Actinina/metabolismo , Cálcio/metabolismo , Citocinese/fisiologia , Actinina/antagonistas & inibidores , Actinina/genética , Actinas/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Mutação , Interferência de RNA , RNA Interferente Pequeno , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo
10.
Ann N Y Acad Sci ; 1205: 17-22, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20840248

RESUMO

Umbilical cord blood (UCB) is a valuable alternative source of hematopoietic stem cells (HSCs). It has unique advantages of easy procurement, absence of risk to donors, low risk of transmitting infections, immediate availability, greater tolerance of human leukocyte antigen (HLA) disparity, and lower incidence of inducing severe graft-versus-host disease (GVHD). In the last several years, these features of UCB permit the field of UCB transplantation (UCBT) to move at a faster pace for both children and adults with malignancies and nonmalignancies. However, new strategies and novel developments are expected to improve engraftment and reconstitution, and to enable in utero transplantation for early therapy, as well as to allow the therapy for a wide spectrum of human diseases.


Assuntos
Transplante de Células-Tronco de Sangue do Cordão Umbilical/tendências , Sangue Fetal/citologia , Células-Tronco Hematopoéticas/fisiologia , Adulto , Animais , Transplante de Células-Tronco de Sangue do Cordão Umbilical/métodos , Transplante de Células-Tronco de Sangue do Cordão Umbilical/estatística & dados numéricos , Sangue Fetal/fisiologia , Neoplasias Hematológicas/terapia , Humanos , Síndromes de Imunodeficiência/terapia , Doenças Metabólicas/terapia
11.
Exp Cell Res ; 316(12): 1925-34, 2010 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-20230817

RESUMO

alpha-Actinin is a rod-shaped actin cross-linking protein composed of actin binding domain, spectrin-like repeats of the central rod domain and the EF-hand domain. Cytokinesis in mammalian cells involves remodeling of equatorial actin filaments (F-actin) mediated by alpha-actinin. However, it remains unknown how alpha-actinin interacts with F-actin at the cleavage furrow. To address this question, we have conducted functional analysis of the mutant that either lacks the ability to cross-link F-actin (ABD) or to bind to F-actin (DeltaABD). We found that equatorial localization of alpha-actinin requires both its F-actin binding and cross-linking activities. Unexpectedly, we also found that overexpression of DeltaABD-GFP but not ABD-GFP frequently caused accelerated cytokinesis and ectopic furrowing similar to those observed in cells depleted of alpha-actinin. Immunofluorescence revealed that overexpression of DeltaABD-GFP caused displacement of endogenous alpha-actinin and a decrease in the density of F-actin throughout the entire cortex. Biochemical experiments showed that DeltaABD was able to form heterodimers with endogenous alpha-actinin. These results suggest that the central rod spectrin-like repeats of alpha-actinin is sufficient for its dimerization in vivo. Our findings uncover previously unappreciated functions of the alpha-actinin domains in a cell.


Assuntos
Actinina/metabolismo , Actinas/metabolismo , Citocinese/fisiologia , Citoesqueleto/metabolismo , Actinina/química , Actinina/genética , Actinas/química , Animais , Sítios de Ligação , Células Cultivadas , Imunofluorescência , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Estrutura Terciária de Proteína , Ratos
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