HrpA anchors meningococci to the dynein motor and affects the balance between apoptosis and pyroptosis.

BACKGROUND: In Neisseria meningitidis the HrpA/HrpB two-partner secretion system (TPS) was implicated in diverse functions including meningococcal competition, biofilm formation, adherence to epithelial cells, intracellular survival and vacuolar escape. These diverse functions could be attributed to distinct domains of secreted HrpA. METHODS: A yeast two-hybrid screening, in vitro pull-down assay and immunofluorescence microscopy experiments were used to investigate the interaction between HrpA and the dynein light-chain, Tctex-type 1 (DYNLT1). In silico modeling was used to analyze HrpA structure. Western blot analysis was used to investigate apoptotic and pyroptotic markers. RESULTS: The HrpA carboxy-terminal region acts as a manganese-dependent cell lysin, while the results of a yeast two-hybrid screening demonstrated that the HrpA middle region has the ability to bind the dynein light-chain, Tctex-type 1 (DYNLT1). This interaction was confirmed by in vitro pull-down assay and immunofluorescence microscopy experiments showing co-localization of N. meningitidis with DYNLT1 in infected epithelial cells. In silico modeling revealed that the HrpA-M interface interacting with the DYNLT1 has similarity with capsid proteins of neurotropic viruses that interact with the DYNLT1. Indeed, we found that HrpA plays a key role in infection of and meningococcal trafficking within neuronal cells, and is implicated in the modulation of the balance between apoptosis and pyroptosis. CONCLUSIONS: Our findings revealed that N. meningitidis is able to effectively infect and survive in neuronal cells, and that this ability is dependent on HrpA, which establishes a direct protein-protein interaction with DYNLTI in these cells, suggesting that the HrpA interaction with dynein could be fundamental for N. meningitidis spreading inside the neurons. Moreover, we found that the balance between apoptotic and pyroptotic pathways is heavily affected by HrpA.

Yeast Two-Hybrid Assay to Identify Interacting Proteins.

This article describes the general method to perform the classical two-hybrid system. Although it has already been more than 25 years since this technique was developed, it still represents one of the best and most inexpensive, time saving, and straightforward methods to identify and study protein-protein interactions. Indeed, this system can be easily used to identify interacting proteins for a given protein, to check interactions between two known proteins, or to map interacting domains. Most of the interactions revealed using the two-hybrid assay have been proven to be binary direct interactions. Data comparison with other systems, such as mass spectrometry, have demonstrated that this system is at least as reliable. In fact, its use is increasing with time, and at present numerous variants of the yeast two-hybrid assay have been developed, including high-throughput systems that promote the generation of a proteome-scale map of protein-protein interactions in specific system. © 2018 by John Wiley & Sons, Inc.

Modulation of RAB7A Protein Expression Determines Resistance to Cisplatin through Late Endocytic Pathway Impairment and Extracellular Vesicular Secretion.

BACKGROUND: Cisplatin (CDDP) is widely used in treatment of cancer, yet patients often develop resistance with consequent therapeutical failure. In CDDP-resistant cells alterations of endocytosis and lysosomal functionality have been revealed, although their causes and contribution to therapy response are unclear. METHODS: We investigated the role of RAB7A, a key regulator of late endocytic trafficking, in CDDP-resistance by comparing resistant and sensitive cells using western blotting, confocal microscopy and real time PCR. Modulation of RAB7A expression was performed by transfection and RNA interference, while CDDP sensitivity and intracellular accumulation were evaluated by viability assays and chemical approaches, respectively. Also extracellular vesicles were purified and analyzed. Finally, correlations between RAB7A and chemotherapy response was investigated in human patient samples. RESULTS: We demonstrated that down-regulation of RAB7A characterizes the chemoresistant phenotype, and that RAB7A depletion increases CDDP-resistance while RAB7A overexpression decreases it. In addition, increased production of extracellular vesicles is modulated by RAB7A expression levels and correlates with reduction of CDDP intracellular accumulation. CONCLUSIONS: We demonstrated, for the first time, that RAB7A regulates CDDP resistance determining alterations in late endocytic trafficking and drug efflux through extracellular vesicles.

Luminescent CdSe@ZnS nanocrystals embedded in liposomes: a cytotoxicity study in HeLa cells.

The use of fluorescent nanocrystals (NCs) as probes for bioimaging applications has emerged as an advantageous alternative to conventional organic fluorescent dyes. Therefore their toxicological evaluation and intracellular delivery are currently a primary field of research. In this work, hydrophobic and highly fluorescent CdSe@ZnS NCs were encapsulated into the lipid bilayer of liposomes by the micelle-to-vesicle transition (MVT) method. The obtained aqueous NC-liposome suspensions preserved the spectroscopic characteristics of the native NCs. A systematic study of the in vitro toxicological effect on HeLa cells of these red emitting NC-liposomes was then carried out and compared to that of empty liposomes. By using liposomes of different phospholipid composition, we evaluated the effect of the lipid carrier on the cytotoxicity towards HeLa cells. Surprisingly, a cell proliferation and death study along with the MTT test on HeLa cells treated with NC-liposomes have shown that the toxic effects of NCs, at concentrations up to 20 nM, are negligible compared to those of the lipid carrier, especially when this is constituted by the cationic phospholipid DOTAP. In particular, obtained data suggest that DOTAP has a dose- and time-dependent toxic effect on HeLa cells. In contrast, the addition of PEG to the liposomes does not alter significantly the viability of the cells. In addition, the ability of NC-liposomes to penetrate the HeLa cells was assessed by fluorescence and confocal microscopy investigation. Captured images show that NC-liposomes are internalized into cells through the endocytic pathway, enter early endosomes and reach lysosomes in 1 h. Interestingly, red emitting NCs co-localized with endosomes and were positioned at the limiting membrane of the organelles. The overall results suggest that the fluorescent system as a whole, NCs and their carrier, should be considered for the development of fully safe biological applications of CdSe@ZnS NCs, and provide essential indications to define the optimal experimental conditions to use the proposed system as an optical probe for future in vivo experiments.

Micropatterning control of tubular commitment in human adult renal stem cells.

The treatment of renal injury by autologous, patient-specific adult stem cells is still an unmet need. Unsolved issues remain the spatial integration of stem cells into damaged areas of the organ, the commitment in the required cell type and the development of improved bioengineered devices. In this respect, biomaterials and architectures have to be specialized to control stem cell differentiation. Here, we perform an extensive study on micropatterned extracellular matrix proteins, which constitute a simple and non-invasive approach to drive the differentiation of adult renal progenitor/stem cells (ARPCs) from human donors. ARPCs are interfaced with fibronectin (FN) micropatterns, in the absence of exogenous chemicals or cellular reprogramming. We obtain the differentiation towards tubular cells of ARPCs cultured in basal medium conditions, the tubular commitment thus being specifically induced by micropatterned substrates. We characterize the stability of the tubular differentiation as well as the induction of a polarized phenotype in micropatterned ARPCs. Thus, the developed cues, driving the functional commitment of ARPCs, offer a route to recreate the microenvironment of the stem cell niche in vitro, that may serve, in perspective, for the development of ARPC-based bioengineered devices.