Abstract
BackgroundBetter 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.ResultsWe 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.ConclusionThe 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.
Highlights
Better knowledge of the innate immune system of insects will improve our understanding of mosquitoes as potential vectors of diverse pathogens
We investigated the role of the 14-3-3 isoforms in phagocytosis of Gram-positive and Gram-negative bacteria in the two cell-lines Aag-2 derived from Ae. aegypti and C6/36 HT from Ae. albopictus
Aedes spp. cells express 14-3-3ε and 14-3-3ζ proteins We first evaluated the gene expression of Aeae14-3-3ε and Aeae14-3-3ζ in cell lines derived from Ae. aegypti and Ae. albopictus
Summary
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. Phagocytosis by haemocytes is the main innate immune process to eliminate invading bacteria in insects [1,2,3,4] This process is initiated with the recognition of the foreign particle, followed by remodelling of the phagocytic cell cytoskeleton and plasma membrane invagination resulting in internalisation of particles into a phagosome [5]. Seven 14-3-3 isotypes (α/β, γ, τ/θ, ε, η, σ, ζ/δ), encoded by individual genes, have been identified in mammals [10] and two (ε and ζ) in insects, such as Drosophila, Bombyx mori and Aedes aegypti [11,12,13]. 14-3-3 proteins are scaffolding proteins that interact with many protein partners to regulate signalling pathways and control cytoskeleton remodelling through the binding of actin molecules, the essential element in phagocytosis [3, 14,15,16]
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