Septin 2 interacts with dengue virus replication complex proteins and participates in virus replication in mosquito cells.

Septins are a family of GTP-binding proteins identified in insects and mammals. Septins are components of the cytoskeleton and participate in cytokinesis, chromosomal segregation, intracellular vesicular traffic, and response to pathogens. Human septin 6 was identified as necessary for hepatitis C virus replication. Information about host factors necessary for flavivirus replication in mosquitoes is scarce. Thus, the role of septins in the replicative cycle of dengue virus in Aedes spp. derived cells was investigated. Through bioinformatic analysis, sequences of septin-like proteins were identified. Infected mosquito cells showed increased expression of Sep2. Colocalization analysis, proximity ligation and immunoprecipitation assays indicated that Sep2 interacts with proteins E, NS3 and NS5, but not NS1. Immunoelectron microscopy evidenced the presence of AalSep2 in replicative complexes. Finally, silencing of Sep2 expression resulted in a significant decrease in virus progeny, indicating that Sep2 is a host factor participating in dengue virus replication in mosquito cells.

Development of the indirect flight muscles of Aedes aegypti, a main arbovirus vector.

BACKGROUND: Flying is an essential function for mosquitoes, required for mating and, in the case of females, to get a blood meal and consequently function as a vector. Flight depends on the action of the indirect flight muscles (IFMs), which power the wings beat. No description of the development of IFMs in mosquitoes, including Aedes aegypti, is available. METHODS: A. aegypti thoraces of larvae 3 and larvae 4 (L3 and L4) instars were analyzed using histochemistry and bright field microscopy. IFM primordia from L3 and L4 and IFMs from pupal and adult stages were dissected and processed to detect F-actin labelling with phalloidin-rhodamine or TRITC, or to immunodetection of myosin and tubulin using specific antibodies, these samples were analyzed by confocal microscopy. Other samples were studied using transmission electron microscopy. RESULTS: At L3-L4, IFM primordia for dorsal-longitudinal muscles (DLM) and dorsal-ventral muscles (DVM) were identified in the expected locations in the thoracic region: three primordia per hemithorax corresponding to DLM with anterior to posterior orientation were present. Other three primordia per hemithorax, corresponding to DVM, had lateral position and dorsal to ventral orientation. During L3 to L4 myoblast fusion led to syncytial myotubes formation, followed by myotendon junctions (MTJ) creation, myofibrils assembly and sarcomere maturation. The formation of Z-discs and M-line during sarcomere maturation was observed in pupal stage and, the structure reached in teneral insects a classical myosin thick, and actin thin filaments arranged in a hexagonal lattice structure. CONCLUSIONS: A general description of A. aegypti IFM development is presented, from the myoblast fusion at L3 to form myotubes, to sarcomere maturation at adult stage. Several differences during IFM development were observed between A. aegypti (Nematoceran) and Drosophila melanogaster (Brachyceran) and, similitudes with Chironomus sp. were observed as this insect is a Nematoceran, which is taxonomically closer to A. aegypti and share the same number of larval stages.

Participation of 14-3-3ε and 14-3-3ζ proteins in the phagocytosis, component of cellular immune response, in Aedes mosquito cell lines.

BACKGROUND: Better knowledge of the innate immune system of insects will improve our understanding of mosquitoes as potential vectors of diverse pathogens. The ubiquitously expressed 14-3-3 protein family is evolutionarily conserved from yeast to mammals, and at least two isoforms of 14-3-3, the ε and ζ, have been identified in insects. These proteins have been shown to participate in both humoral and cellular immune responses in Drosophila. As mosquitoes of the genus Aedes are the primary vectors for arboviruses, causing several diseases such as dengue fever, yellow fever, Zika and chikungunya fevers, cell lines derived from these mosquitoes, Aag-2 from Aedes aegypti and C6/36 HT from Aedes albopictus, are currently used to study the insect immune system. Here, we investigated the role of 14-3-3 proteins (ε and ζ isoform) in phagocytosis, the main cellular immune responses executed by the insects, using Aedes spp. cell lines. RESULTS: We evaluated the mRNA and protein expression of 14-3-3ε and 14-3-3ζ in C6/36 HT and Aag-2 cells, and demonstrated that both proteins were localised in the cytoplasm. Further, in C6/36 HT cells treated with a 14-3-3 specific inhibitor we observed a notable modification of cell morphology with filopodia-like structure caused through cytoskeleton reorganisation (co-localization of 14-3-3 proteins with F-actin), more importantly the decrease in Salmonella typhimurium, Staphylococcus aureus and E. coli phagocytosis and reduction in phagolysosome formation. Additionally, silencing of 14-3-3ε and 14-3-3ζ expression by mean of specific DsiRNA confirmed the decreased phagocytosis and phagolysosome formation of pHrodo labelled E. coli and S. aureus bacteria by Aag-2 cells. CONCLUSION: The 14-3-3ε and 14-3-3ζ proteins modulate cytoskeletal remodelling, and are essential for phagocytosis of Gram-positive and Gram-negative bacteria in Aedes spp. cell lines.

Lys48 ubiquitination during the intraerythrocytic cycle of the rodent malaria parasite, Plasmodium chabaudi.

Ubiquitination tags proteins for different functions within the cell. One of the most abundant and studied ubiquitin modification is the Lys48 polyubiquitin chain that modifies proteins for their destruction by proteasome. In Plasmodium is proposed that post-translational regulation is fundamental for parasite development during its complex life-cycle; thus, the objective of this work was to analyze the ubiquitination during Plasmodium chabaudi intraerythrocytic stages. Ubiquitinated proteins were detected during intraerythrocytic stages of Plasmodium chabaudi by immunofluorescent microscopy, bidimensional electrophoresis (2-DE) combined with immunoblotting and mass spectrometry. All the studied stages presented protein ubiquitination and Lys48 polyubiquitination with more abundance during the schizont stage. Three ubiquitinated proteins were identified for rings, five for trophozoites and twenty for schizonts. Only proteins detected with a specific anti- Lys48 polyubiquitin antibody were selected for Mass Spectrometry analysis and two of these identified proteins were selected in order to detect the specific amino acid residues where ubiquitin is placed. Ubiquitinated proteins during the ring and trophozoite stages were related with the invasion process and in schizont proteins were related with nucleic acid metabolism, glycolysis and protein biosynthesis. Most of the ubiquitin detection was during the schizont stage and the Lys48 polyubiquitination during this stage was related to proteins that are expected to be abundant during the trophozoite stage. The evidence that these Lys48 polyubiquitinated proteins are tagged for destruction by the proteasome complex suggests that this type of post-translational modification is important in the regulation of protein abundance during the life-cycle and may also contribute to the parasite cell-cycle progression.

IDENTIFICATION AND EXPRESSION ANALYSIS OF TWO 14-3-3 PROTEINS IN THE MOSQUITO Aedes aegypti, AN IMPORTANT ARBOVIRUSES VECTOR.

The 14-3-3 proteins are evolutionarily conserved acidic proteins that form a family with several isoforms in many cell types of plants and animals. In invertebrates, including dipteran and lepidopteran insects, only two isoforms have been reported. 14-3-3 proteins are scaffold molecules that form homo- or heterodimeric complexes, acting as molecular adaptors mediating phosphorylation-dependent interactions with signaling molecules involved in immunity, cell differentiation, cell cycle, proliferation, apoptosis, and cancer. Here, we describe the presence of two isoforms of 14-3-3 in the mosquito Aedes aegypti, the main vector of dengue, yellow fever, chikungunya, and zika viruses. Both isoforms have the conserved characteristics of the family: two protein signatures (PS1 and PS2), an annexin domain, three serine residues, targets for phosphorylation (positions 58, 184, and 233), necessary for their function, and nine alpha helix-forming segments. By sequence alignment and phylogenetic analysis, we found that the molecules correspond to Ɛ and ζ isoforms (Aeae14-3-3ε and Aeae14-3-3ζ). The messengers and protein products were present in all stages of the mosquito life cycle and all the tissues analyzed, with a small predominance of Aeae14-3-3ζ except in the midgut and ovaries of adult females. The 14-3-3 proteins in female midgut epithelial cells were located in the cytoplasm. Our results may provide insights to further investigate the functions of these proteins in mosquitoes.

Protein phosphorylation during Plasmodium berghei gametogenesis.

Plasmodium gametogenesis within the mosquito midgut is a complex differentiation process involving signaling mediated by phosphorylation, which modulate metabolic routes and protein synthesis required to complete this development. However, the mechanisms leading to gametogenesis activation are poorly understood. We analyzed protein phosphorylation during Plasmodium berghei gametogenesis in vitro in serum-free medium using bidimensional electrophoresis (2-DE) combined with immunoblotting (IB) and antibodies specific to phosphorylated serine, threonine and tyrosine. Approximately 75 protein exhibited phosphorylation changes, of which 23 were identified by mass spectrometry. These included components of the cytoskeleton, heat shock proteins, and proteins involved in DNA synthesis and signaling pathways among others. Novel phosphorylation events support a role for these proteins during gametogenesis. The phosphorylation sites of six of the identified proteins, HSP70, WD40 repeat protein msi1, enolase, actin-1 and two isoforms of large subunit of ribonucleoside reductase were investigated using TiO2 phosphopeptides enrichment and tandem mass spectrometry. In addition, transient exposure to hydroxyurea, an inhibitor of ribonucleoside reductase, impaired male gametocytes exflagellation in a dose-dependent manner, and provides a resource for functional studies.

Proteomic analysis of rat cartilage: the identification of differentially expressed proteins in the early stages of osteoarthritis.

BACKGROUND: Osteoarthritis (OA) is a chronic degenerative disease of the articular cartilage, and its diagnosis is based on symptoms and radiological signs that are only present in the late stages of the disease. Due to the limitations in diagnosing OA before the onset of symptoms, such as pain, little is known about the molecular mechanisms involved in the pathogenesis of OA. Experimental OA models are often used to study the kinetics of the progression of this disease. In this report, we conducted a proteomic study of osteoarthritic cartilage during the early stages of OA using an experimental rat model. RESULTS: Ten proteins that are differentially expressed under early OA conditions were identified by 2-DE and MALDI-TOF/MS. These proteins mediated many processes, such as glycolysis and energy production (Nme2 and Pnp), cartilage matrix (Col2a1), transcription and protein synthesis (Eef1a1 and DJ-1), signal transduction (CaM and Pebp1), transport (Alb and Hba1), and latexin (Lxn). In addition, changes in Lxn expression in early OA were observed and validated by western blot and immunofluorescence analysis. CONCLUSIONS: The proteins that we identified indicate that energy metabolism, cartilage matrix remodelling, and protective cellular mechanisms are associated with early OA. In addition, latexin expression during the early stages of OA could be implicated in cartilage repair.

Differentiation of Entamoeba histolytica: a possible role for enolase.

The study of the encystation process of Entamoeba histolytica has been hampered by the lack of experimental means of inducing mature cysts in vitro. Previously we have found that cytoplasmic vesicles similar to the encystation vesicles of Entamoeba invadens are present in E. histolytica trophozoites only in amebas recovered from experimental amebic liver abscesses. Here we report that a monoclonal antibody (B4F2) that recognizes the cyst wall of E. invadens also identifies a 48 kDa protein in vesicles of E. histolytica trophozoites recovered from hepatic lesions. This protein is less expressed in trophozoites continuously cultured in axenical conditions. As previously reported for E. invadens, the B4F2 specific antigen was identified as enolase in liver-recovered E. histolytica, by two-dimensional electrophoresis, Western blot and mass spectrometry. In addition, the E. histolytica enolase mRNA was detected by RT PCR. The antigen was localized by immunoelectron microscopy in cytoplasmic vesicles of liver-recovered amebas. The B4F2 antibody also recognized the wall of mature E. histolytica cysts obtained from human samples. These results suggest that the enolase-containing vesicles are produced by E. histolytica amebas, when placed in the unfavorable liver environment that could be interpreted as an attempt to initiate the encystation process.

Entamoeba invadens, encystation process and enolase.

The reptilian parasite Entamoeba invadens is accepted as a model for the study of the Entamoeba encystation process. Here we describe the production and characterization of a mAb (B4F2), generated against a component of the E. invadens cyst wall. This mAb specifically recognizes a 48-kDa protein present in cytoplasmic vesicles of cells encysting for 24 h. In mature cysts (96 h), the antigen was detected on the cyst surface. By two-dimensional electrophoresis and mass spectrometry analysis, the B4F2 specific antigen was identified as enolase. Levels of enolase mRNA were increased in encysting cells and the B4F2 mAb was found to inhibit cyst formation. Therefore, these results strongly suggest a new role for enolase in E. invadens encystation, and the B4F2 mAb will be useful tool to study its role in the differentiation process.