Showing papers by "María Isabel Colombo published in 2015"

Infectious bursal disease virus uptake involves macropinocytosis and trafficking to early endosomes in a Rab5-dependent manner.

Abstract: Infectious bursal disease virus (IBDV) internalization is sparsely known in terms of molecular components of the pathway involved. To describe the cell biological features of IBDV endocytosis, we employed perturbants of endocytic pathways such as pharmacological inhibitors and overexpression of dominant-negative mutants. Internalization analysis was performed quantifying infected cells by immunofluorescence and Western blot detection of the viral protein VP3 at 12 h post-infection reinforced by the analysis of the capsid protein VP2 localization after virus uptake at 1 h post-infection. We compared IBDV infection to the internalization of well-established ligands with defined endocytic pathways: transferrin, cholera-toxin subunit B and dextran. To describe virus endocytosis at the morphological level, we performed ultrastructural studies of viral internalization kinetics in control and actin dynamics-blocked cells. Our results indicate that IBDV endocytic internalization was clathrin- and dynamin-independent, and that IBDV uses macropinocytosis as the primary entry mechanism. After uptake, virus traffics to early endosomes and requires exposure to the low endocytic pH as well as a functional endocytic pathway to complete its replication cycle. Moreover, our results indicate that the GTPase Rab5 is crucial for IBDV entry supporting the participation of the early endosomal pathway in IBDV internalization and infection of susceptible cells.

Coxiella burnetii Phagocytosis Is Regulated by GTPases of the Rho Family and the RhoA Effectors mDia1 and ROCK.

Abstract: The GTPases belonging to the Rho family control the actin cytoskeleton rearrangements needed for particle internalization during phagocytosis. ROCK and mDia1 are downstream effectors of RhoA, a GTPase involved in that process. Coxiella burnetii, the etiologic agent of Q fever, is internalized by the host´s cells in an actin-dependent manner. Nevertheless, the molecular mechanism involved in this process has been poorly characterized. This work analyzes the role of different GTPases of the Rho family and some downstream effectors in the internalization of C. burnetii by phagocytic and non-phagocytic cells. The internalization of C. burnetii into HeLa and RAW cells was significantly inhibited when the cells were treated with Clostridium difficile Toxin B which irreversibly inactivates members of the Rho family. In addition, the internalization was reduced in HeLa cells that overexpressed the dominant negative mutants of RhoA, Rac1 or Cdc42 or that were knocked down for the Rho GTPases. The pharmacological inhibition or the knocking down of ROCK diminished bacterium internalization. Moreover, C. burnetii was less efficiently internalized in HeLa cells overexpressing mDia1-N1, a dominant negative mutant of mDia1, while the overexpression of the constitutively active mutant mDia1-ΔN3 increased bacteria uptake. Interestingly, when HeLa and RAW cells were infected, RhoA, Rac1 and mDia1 were recruited to membrane cell fractions. Our results suggest that the GTPases of the Rho family play an important role in C. burnetii phagocytosis in both HeLa and RAW cells. Additionally, we present evidence that ROCK and mDia1, which are downstream effectors of RhoA, are involved in that process.

Autophagy response: manipulating the mTOR-controlled machinery by amino acids and pathogens.

Abstract: Macroautophagy is a self-degradative process that normally maintains cellular homeostasis via a lysosomal pathway. It is induced by different stress signals, including nutrients and growth factors’ restriction as well as pathogen invasions. These stimuli are modulated by the serine/threonine protein kinase mammalian target of rapamycin (mTOR) which control not only autophagy but also protein translation and gene expression. This review focuses on the important role of mTOR as a master regulator of cell growth and the autophagy pathway. Here, we have discussed the role of intracellular amino acid availability and intracellular pH in the redistribution of autophagic structures, which may contribute to mammalian target of rapamycin complex 1 (mTORC1) activity regulation. We have also discussed that mTORC1 complex and components of the autophagy machinery are localized at the lysosomal surface, representing a fascinating mechanism to control the metabolism, cellular clearance and also to restrain invading intracellular pathogens.

Intracellular Pathogen Invasion of the Host Cells: Role of α-Hemolysin- Induced Autophagy

Abstract: Staphylococcus aureus causes a wide range of diseases in humans, from local infection to life-threatening systemic infection, both in health care facilities and the community Classically it has been considered an extracellular pathogen, but cumulative evidence indicates that it invades cells and replicates intracellularly, leading to staphylococcal persistence and chronic disease It has been proven that S aureus is able to induce an autophagic response which allows bacteria replication and subsequent escape from autophagosomes into the cytoplasm, leading also to cell death One of the key features of S aureus infection is the production of a series of virulence factors, including secreted enzymes and toxins It has been shown that the pore-forming toxin α-hemolysin (Hla) injures epithelial cells by interacting with its receptor, the zinc-dependent metalloprotease ADAM10 In addition, we have demonstrated that Hla is a secreted factor that participates in the activation of the autophagic pathway Once internalized, the toxin is able to activate the autophagic pathway through a PI3K/Beclin-1-independent form Recently we have reported that EPAC and Rap2b, through calpain activation, are the proteins involved in the regulation of Hla-induced autophagy This chapter focuses on a pathogen that not only induces an autophagic response in the host cell, localizing in LC3 decorated compartments, but that also seems to benefit from that fate