High-level secretion of recombinant monomeric murine and human single-chain Fv antibodies from Drosophila S2 cells

Single-chain variable fragment (scFvs) antibodies are small polypeptides (∼26 kD) containing the heavy (VH) and light (VL) immunoglobulin domains of a parent antibody connected by a flexible linker. In addition to being frequently used in diagnostics and therapy for an increasing number of human diseases, scFvs are important tools for structural biology as crystallization chaperones. Although scFvs can be expressed in many different organisms, the expression level of an scFv strongly depends on its particular amino acid sequence. We report here a system allowing for easy and efficient cloning of (i) scFvs selected by phage display and (ii) individual heavy and light chain sequences from hybridoma cDNA into expression plasmids engineered for secretion of the recombinant fragment produced in Drosophila S2 cells. We validated the method by producing five scFvs derived from human and murine parent antibodies directed against various antigens. The production yields varied between 5 and 12 mg monomeric scFv per liter of supernatant, indicating a relative independence on the individual sequences. The recombinant scFvs bound their cognate antigen with high affinity, comparable with the parent antibodies. The suitability of the produced recombinant fragments for structural studies was demonstrated by crystallization and structure determination of one of the produced scFvs, derived from a broadly neutralizing antibody against the major glycoprotein E2 of the hepatitis C virus. Structural comparison with the Protein Data Bank revealed the typical spatial organization of VH and VL domains, further validating the here-reported expression system

Inhibition of Amebic Cysteine Proteases Blocks Amebic Trogocytosis but Not Phagocytosis

BackgroundEntamoeba histolytica kills human cells by ingesting fragments of live cells until the cell eventually dies, a process termed amebic trogocytosis. In a previous study, we showed that acidified amebic lysosomes are required for both amebic trogocytosis and phagocytosis, as well as cell killing.MethodsAmebic cysteine proteases (CPs) were inhibited using an irreversible inhibitor, E-64d.ResultsInterfering with amebic CPs decreased amebic trogocytosis and amebic cytotoxicity but did not impair phagocytosis.ConclusionsWe show that amebic CPs are required for amebic trogocytosis and cell killing but not phagocytosis. These data suggest that amebic CPs play a distinct role in amebic trogocytosis and cell killing

Inhibition of Amebic Lysosomal Acidification Blocks Amebic Trogocytosis and Cell Killing

Entamoeba histolytica ingests fragments of live host cells in a nibbling-like process termed amebic trogocytosis. Amebic trogocytosis is required for cell killing and contributes to tissue invasion, which is a hallmark of invasive amebic colitis. Work done prior to the discovery of amebic trogocytosis showed that acid vesicles are required for amebic cytotoxicity. In the present study, we show that acidified lysosomes are required for amebic trogocytosis and cell killing. Interference with lysosome acidification using ammonium chloride, a weak base, or concanamycin A, a vacuolar H+ ATPase inhibitor, decreased amebic trogocytosis and amebic cytotoxicity. Our data suggest that the inhibitors do not impair the ingestion of an initial fragment but rather block continued trogocytosis and the ingestion of multiple fragments. The acidification inhibitors also decreased phagocytosis, but not fluid-phase endocytosis. These data suggest that amebic lysosomes play a crucial role in amebic trogocytosis, phagocytosis, and cell killing.IMPORTANCEE. histolytica is a protozoan parasite that is prevalent in low-income countries, where it causes potentially fatal diarrhea, dysentery, and liver abscesses. Tissue destruction is a hallmark of invasive E. histolytica infection. The parasite is highly cytotoxic to a wide range of human cells, and parasite cytotoxic activity is likely to drive tissue destruction. E. histolytica is able to kill human cells through amebic trogocytosis. This process also contributes to tissue invasion. Trogocytosis has been observed in other organisms; however, little is known about the mechanism in any system. We show that interference with lysosomal acidification impairs amebic trogocytosis, phagocytosis, and cell killing, indicating that amebic lysosomes are critically important for these processes

Genetic diversity of Cryptosporidium hominis in a Bangladeshi community as revealed by whole-genome sequencing

We studied the genetic diversity of Cryptosporidium hominis infections in slum-dwelling infants from Dhaka over a 2-year period. Cryptosporidium hominis infections were common during the monsoon, and were genetically diverse as measured by gp60 genotyping and whole-genome resequencing. Recombination in the parasite was evidenced by the decay of linkage disequilibrium in the genome over <300 bp. Regions of the genome with high levels of polymorphism were also identified. Yet to be determined is if genomic diversity is responsible in part for the high rate of reinfection, seasonality, and varied clinical presentations of cryptosporidiosis in this population