Biology subjects

Betanzos, A.

Publications and source records attributed to Betanzos, A..

5 recordsLinked to original sources

bioRxiv2025

The dengue virus NS1 protein alters Aedes aegypti midgut permeability and favors virus dissemination

The dengue virus is an arbovirus of public health importance transmitted by Aedes aegypti mosquitoes. The NS1 protein is a multifunctional glycoprotein, highly conserved among Orthoflaviviruses, that is secreted from infected cells, circulate in the sera of dengue patients at high concentrations and has been involved with pathogenesis by several mechanisms, including vascular leakage and immune evasion. However, the role of NS1 within the mosquito vector is not fully understood. In this study, we observed that feeding female Aedes aegypti mosquitoes with blood meals supplemented with recombinant NS1 resulted in increased midgut permeability, as evaluated by the diffusion of the non-toxic dye Brilliant Blue FCF into the hemocoel. Histological and transmission electron microscopy analysis revealed epithelial damage and disruption of the midgut cellular junctions. Immunofluorescence assays further demonstrated the delocalization of septate junction proteins essential for epithelial integrity. In addition, metalloproteinase expression in the midgut was also reduced. All these effects were abolished when NS1 was heat-inactivated indicating that they were NS1-dependent. Significantly, virus blood meals containing NS1 resulted in enhanced dissemination of DENV to secondary organs and earlier virus presence in the mosquito salivary glands, in comparison with meals treated with specific NS1 antibodies. Our findings reveal a novel function for NS1 in mosquitoes, and expand the understanding of NS1 functions beyond its human pathophysiological role. In addition, highlight NS1 as a strategic intervention point to reduce transmission efficiency in mosquitoes. ImportanceDengue is the most important virus disease transmitted by mosquitoes to humans. Thus, a deeper understanding of the virus-vector interaction is required for the development of control measures and the mosquito transmission capacity. In this work, we present evidence indicating that the NS1 protein plays a role in the establishment and dissemination of the dengue virus infection in Aedes aegypti mosquitoes. Ingested NS1 was found to disrupt the septate junctions and to inhibit metalloproteinase expression of the midgut, allowing the virus to escape the digestive tract. In addition, NS1 was found to promote virus dissemination into the hemocoel, carcass and salivary glands. These findings uncover new functions for NS1 in the mosquito and highpoint ways to interfere with vector competence.

microbiology↗

bioRxiv2024

FKBP12-BOUND CALCINEURIN CONTROL TMEM16A ACTIVITY

The Ca2+-dependent Cl- channel TMEM16A activity is vital for mammals. Critical functions such as blood pressure, pain, fluid and electrolyte secretion, peristalsis, and electrical activity depend on TMEM16A Cl- fluxes. Ciclosporin A (CsA) and FK506, two immunosuppressants that inhibit calcineurin (CaN), down-regulate TMEM16A function. However, the underlying mechanism remains undetermined. CsA binds to cyclophilin 1, whereas FK506 binds to FKBP12, and both complexes subsequently inhibit CaN, a phosphatase that maintains TMEM16A function. Here, we show that TMEM16A-EGFP and mRFP-FKBP12 colocalize in HEK-AD293 cells stimulated with the Ca2+ ionophore ionomycin, FKBP12 co-immunoprecipitated with TMEM16A expressed in CaN-depleted HEK-AD293 cells, ionomycin favoured TMEM16A and FKBP12 interaction in a bimolecular fluorescence complementation assay in live cells, and TMEM16A currents recorded from CaN-depleted HEK-AD293 cells were insensitive to CsA and FK506. These data support the idea that FKBP12 works as an auxiliary protein of TMEM16A, thus enabling the interaction of CaN under physiological conditions. This heteromerization is essential to sustain TMEM16A activity. On the contrary, disrupting the Ca2+-stimulated CaN-FKBP12-TMEM16A heterotrimer or inhibiting CaN with CsA or FK506 does not preclude TMEM16A activation but decreases activity.

physiology↗

bioRxiv2023

Arpin deficiency increases actomyosin contractility and vascular permeability

Arpin was discovered as an inhibitor of the Arp2/3 complex localized at the lamellipodial tip of fibroblasts, where it regulated migration steering. Recently, we showed that arpin stabilizes the epithelial barrier in an Arp2/3-dependent manner. However, expression and functions of arpin in endothelial cells (EC) have not yet been described. Arpin mRNA and protein are expressed in EC and downregulated by pro-inflammatory cytokines. Arpin depletion in HUVEC causes the formation of actomyosin stress fibers leading to increased permeability in an Arp2/3-independent manner. Instead, inhibitors of ROCK1 and ZIPK, kinases involved in the generation of stress fibers, normalize the loss-of-arpin effects on actin filaments and permeability. Arpin-deficient mice are viable but show a characteristic vascular phenotype in the lung including edema, microhemorrhage and vascular congestion, increased F-actin levels and vascular permeability. Our data show that, apart from being an Arp2/3 inhibitor, arpin is also a regulator of actomyosin contractility and endothelial barrier integrity. SUMMARYThe expression and functions of arpin in endothelial cells are unknown. We show that arpin controls actomyosin contractility and endothelial barrier integrity in an Arp2/3-independent manner via ROCK1/ZIPK. Arpin-deficient mice are viable, but also show increased basal and induced vascular permeability.

cell biology↗

bioRxiv2023

Cytotrophoblast cells are selectively permissive and favor Zika virus, but no other related flavivirus, invasion to the placental stroma

BACKGROUNDZika virus (ZIKV) is highly teratogenic, in contrast with dengue virus (DENV) or the yellow fever virus vaccine (YFV-17D). The mechanisms employed by ZIKV to cross the placenta need to be elucidated. METHODSParallel infections with ZIKV, DENV and YFV-17D were compared in terms of efficiency, activation of mTOR pathways and cytokine secretion profile in human cytotrophoblastic HTR8 cells (CTB), and monocytic U937 cells, differentiated to M2 macrophages (M2-MO). RESULTSIn CTB, ZIKV replication was significantly more efficient than DENV or YFV-17D. In M2-MO, ZIKV replication continued to be more efficient, although differences between strains were reduced. Significantly greater activation of Phospho-S6r and Phospho-AKT/Ser473 fractions in CTB infected with ZIKV than with DENV or YFV-17D, was observed. CTB treated with the mTOR inhibitors rapamycin or AZD8055, showed a 20-fold-reduction in ZIKV yield, versus 5 and 3.5-fold for DENV and YFV-17D, respectively. Finally, we detected that ZIKV infection, but not DENV or YFV-17D, efficiently inhibited the interferon response of CTB cells. CONCLUSIONSThese results suggest that CTB cells are permissive and act favoring ZIKV entry into the placental stroma, over DENV and YFV-17D and that the mTOR complex is a switch that enhances the replication of ZIKV in CTB cells.

microbiology↗

bioRxiv2021

Protein SUMOylation is crucial for phagocytosis in Entamoeba histolytica trophozoites

During phagocytosis, a key event in the virulence of the protozoan Entamoeba histolytica, several molecules in concert contact the target, generate pseudopodia, and internalize and digest the ingested prey. Posttranslational modifications provide proteins the timing and signaling to intervene in these processes. SUMOylation is a posttranslational modification that in several systems grants a fine tuning for protein functions, protein interactions and cellular location, but it has not been studied in E. histolytica. In this paper, we characterized the E. histolytica SUMO gene and its product (EhSUMO) and elucidated the EhSUMO 3D-structure. Furthermore, here we studied the relevance of SUMOylation in phagocytosis, particularly in its association with EhADH (an ALIX family protein) and EhVps32 (a protein of the ESCRT-III complex), both involved in phagocytosis. Our results indicated that EhSUMO has an extended N-terminus that differentiates other SUMO from ubiquitin. It also presents the GG residues at the C-terminus and the {Psi}KXE/D binding motif, both involved in target protein contact. Additionally, E. histolytica genome possesses the enzymes belonging to the SUMOylation-deSUMOylation machineries. Confocal microscopy assays, using -EhSUMO antibodies disclosed a remarkable membrane activity with convoluted and changing structures in trophozoites during erythrophagocytosis. SUMOylated proteins appeared in pseudopodia, phagocytic channels, and around the adhered and ingested erythrocytes. Docking analysis predicted interaction of EhSUMO with EhADH, and immunoprecipitation and immunofluorescence assays revealed that the EhADH-EhSUMO association increased during phagocytosis, whereas the EhVps32-EhSUMO interaction appeared stronger since basal conditions. In EhSUMO knocked down trophozoites, the bizarre membranous structures disappeared, and EhSUMO interaction with EhADH and EhVps32 diminished. Our results evidenced the presence of a SUMO gene in E. histolytica and the SUMOylation relevance during phagocytosis. Authors AbstractPhagocytosis is one of the main functions that Entamoeba histolyitica trophozoites carry out during the invasion to the host. Many proteins are involved in this fascinating event, in which the plasmatic membrane undergoes to multiple and speedy changes. Posttraductional modifications activate proteins in the precise time that they must get involved. SUMOylation, that consists in the non-covalent binding of SUMO protein with target molecules, is one of the main changes suffered by proteins in order to enable them to participate in cellular functions. SUMOylation had not been studied in E. histolytica nor in phagocytosis, and our working hypothesis is that this event is deeply engaged in the ingestion of target molecules and cells. The results of this paper prove the presence of an intronless bona fide EhSUMO gene encoding for a predicted 12.6 kDa protein that is actively involved in phagocytosis. Silencing of the EhSUMO gene affected the rate of phagocytosis and interfered with the EhADH and EhVps32 function, two proteins involved in phagocytosis, strongly supporting the importance of SUMOylation in this event.

microbiology↗