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1.
Microbiologyopen ; 8(4): e00684, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-29978561

RESUMO

Francisella tularensis is the causative agent of the infectious disease tularemia and is designated a category A bioterrorism agent. The type VI secretion system encoded by the Francisella pathogenicity island (FPI) is necessary for intracellular growth; however, the functions of FPI proteins are largely unknown. In this study, we found that the FPI protein intracellular growth locus E (IglE) showed a unique localization pattern compared to other FPI proteins. Deleting iglE from Francisella tularensis subsp. novicida (F. novicida) decreased intracellular growth. Immunoprecipitation and pull-down assays revealed that IglE was associated with ß-tubulin. Additionally, GFP-fused IglE colocalized with microtubule organizing centers (MTOCs) in 293T cells. The iglE deletion mutant was transferred with dynein toward MTOCs and packed into lysosome-localizing areas. Conversely, the wild-type F. novicida exhibited intracellular growth distant from MTOCs. In addition, IglE expressed in 293T cells colocalized with dynein. These results suggest that IglE helps to prevent dynein- and MTOC-mediated intracellular trafficking in host cells to inhibit the transport of F. novicida toward lysosomes.


Assuntos
Proteínas de Bactérias/metabolismo , Francisella tularensis/patogenicidade , Ilhas Genômicas , Centro Organizador dos Microtúbulos/microbiologia , Tularemia/microbiologia , Proteínas de Bactérias/genética , Linhagem Celular , Dineínas/genética , Dineínas/metabolismo , Francisella tularensis/genética , Francisella tularensis/metabolismo , Humanos , Lisossomos/metabolismo , Lisossomos/microbiologia , Transporte Proteico , Tularemia/metabolismo , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
2.
BMC Microbiol ; 13: 185, 2013 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-23919807

RESUMO

BACKGROUND: The developmental cycle of the obligate intracellular pathogen Chlamydia is dependant on the formation of a unique intracellular niche termed the chlamydial inclusion. The inclusion is a membrane bound vacuole derived from host cytoplasmic membrane and is modified significantly by the insertion of chlamydial proteins. A unique property of the inclusion is its propensity for homotypic fusion. The vast majority of cells infected with multiple chlamydial elementary bodies (EBs) contain only a single mature inclusion. The chlamydial protein IncA is required for fusion, however the host process involved are uncharacterized. RESULTS: Here, through live imaging studies, we determined that the nascent inclusions clustered tightly at the cell microtubule organizing center (MTOC) where they eventually fused to form a single inclusion. We established that factors involved in trafficking were required for efficient fusion as both disruption of the microtubule network and inhibition of microtubule trafficking reduced the efficiency of fusion. Additionally, fusion occurred at multiple sites in the cell and was delayed when the microtubule minus ends were either no longer anchored at a single MTOC or when a cell possessed multiple MTOCs. CONCLUSIONS: The data presented demonstrates that efficient homotypic fusion requires the inclusions to be in close proximity and that this proximity is dependent on chlamydial microtubule trafficking to the minus ends of microtubules.


Assuntos
Infecções por Chlamydia/metabolismo , Chlamydia trachomatis/fisiologia , Corpos de Inclusão/microbiologia , Microtúbulos/microbiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Linhagem Celular , Infecções por Chlamydia/microbiologia , Chlamydia trachomatis/genética , Interações Hospedeiro-Patógeno , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Centro Organizador dos Microtúbulos/microbiologia , Transporte Proteico
3.
Infect Immun ; 75(11): 5105-17, 2007 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17724071

RESUMO

The intracellular survival of the bacterial pathogen Chlamydia trachomatis depends on protein synthesis by the microbe soon after internalization. Pharmacologic inhibition of bacterial translation inhibits early trafficking of the parasitophorous vacuole (inclusion) to the microtubule-organizing center (MTOC) and promotes its fusion with lysosomes, which is normally blocked by Chlamydia. Depletion of cellular tryptophan pools by gamma interferon-inducible indoleamine-2,3-dioxygenase (IDO) is believed to be the major innate immune mechanism controlling C. trachomatis infection in human cells, an action to which the bacteria can respond by converting into a nonreplicating but highly reactivatable persistent state. However, whether severe IDO-mediated tryptophan starvation can be sufficient to fully arrest the chlamydial life cycle and thereby counteract the onset of persistence is unknown. Here we demonstrate that at low exogenous tryptophan concentrations a substantial fraction of C. trachomatis bacteria fail to traffic to the MTOC or to switch into the conventional persistent state in gamma interferon-induced human cells. The organisms stay scattered in the cell periphery, do not retain infectivity, and display only low transcriptional activity. Importantly, the rate at which these aberrant Chlamydia bacteria become reactivated upon replenishment of cellular tryptophan pools is substantially lower. Thus, severe tryptophan depletion in cells with high IDO activity affects chlamydial development more rigorously than previously described.


Assuntos
Chlamydia trachomatis/imunologia , Triptofano/metabolismo , Chlamydia trachomatis/metabolismo , Chlamydia trachomatis/patogenicidade , Citoplasma/microbiologia , Expressão Gênica/imunologia , Células HeLa , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Interferon gama/imunologia , Centro Organizador dos Microtúbulos/microbiologia , RNA Bacteriano/biossíntese , RNA Mensageiro/biossíntese , Transcrição Gênica/imunologia , Virulência
4.
Folia Parasitol (Praha) ; 52(1-2): 103-10, 2005 May.
Artigo em Inglês | MEDLINE | ID: mdl-16004369

RESUMO

The microsporidium Vittaforma corneae develops within the target cell cytoplasm. In the present study, green monkey kidney (E6) cells infected at 30 degrees C, 35 degrees C or 37 degrees C with V. corneae developed enlarged multinucleate structures of up to 200 microm in any horizontal dimension made up either of a single cell or of multiple fused cells. A number of epithelial cell types (SW-480, HT-29, Caco-2 and HCT-8) were infected with V. corneae but did not induce the same highly organized structures, suggesting that for the structure to develop, the host cell must be capable of continued mitosis, and not be differentiated or be detaching from the surface matrix. Live cell imaging of infected E6 cells revealed large, multinucleate infected cells characterized by a central focus from which radiated parasite stages and host cell mitochondria. Immunocytochemistry identifying gamma and alpha tubulin suggested that a single centrally-located microtubule organizing centre governed the distribution of parasite stages and host cell organelles, with mitochondria and parasites being eventually transported towards the periphery of the structure. Whole cell patch clamp analysis of infected cells indicated an average five-fold increase in total membrane capacitance, consistent with an enlarged single cell. Scanning electron microscopy revealed cell-like protrusions around the periphery of the structure with the intervening space being made up of parasites and cell debris. Clearly in the case of V. corneae-infected E6 cells the parasite-host cell relationship involves subverting the host cell cytoskeleton and cell volume control, providing the parasite with the same protected niche as does a xenoma.


Assuntos
Células Gigantes/microbiologia , Células Gigantes/ultraestrutura , Microsporídios/ultraestrutura , Animais , Linhagem Celular , Chlorocebus aethiops , Células Epiteliais/microbiologia , Imuno-Histoquímica , Microscopia Eletrônica de Varredura , Centro Organizador dos Microtúbulos/microbiologia , Técnicas de Patch-Clamp , Temperatura
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