Cytoplasmic Actin Is an Extracellular Insect Immune Factor which Is Secreted upon Immune Challenge and Mediates Phagocytosis and Direct Killing of Bacteria, and Is a Plasmodium Antagonist
(A) Incubation of E. coli and S. aureus with actin (Ac), AgMDL1 (MDL1), actin +AgMDL1 (Ac+MDL1) or the control protein GUS. Untreated bacteria were incubated with PBS. The percentage of phagocytosing cells was calculated as the number of S2 cells containing at least one phagocytized FITC-labeled bacterium compared to the total number of cells in the field. For each experiment at least 16 fields were counted and the data are representative of three independent experiments. Each bar represents the mean ± the standard deviation. Statistical significance was determined using Student’s t-test.(B) Fluorescent microscopy of S2 cells (red) and FITC-labeled E. coli or S. aureus bacteria (green) incubated with the recombinant proteins actin (Ac) or AgMDL1 (MDL1) alone, or both together (Ac+MDL1) or the control protein GUS. Untreated bacteria were incubated with PBS. (C) Viability of E. coli and S. aureus was determined after 1 or 24 hr of incubation with recombinant proteins (Ac, MDL1, Ac+MDL1, GUS) and compared to untreated bacteria. Error bars represent the mean ± the standard deviation. Statistical significance was determined using Student’s t-test. (D) An. gambiae survival rates after silencing of actin, AgMDL1, actin and AgMDL1, or GFP (control) and challenge of female mosquitoes with E. coli (OD 1.5) or S. aureus (OD 0.4) four days later. Three biological experiments were performed and combined and statistical analysis consisted of a log-rank test to determine the overall significance between all groups, followed by pairwise comparisons between GFP and the other three groups. (E) An. gambiae thorax (6 μg), abdomen (6 μg) and midgut (12 μg) tissues along with hemolymph (2 μg) fractions challenged with LPS (100ng) for 4 hr analyzed for the presence of actin 4 days after silencing of actin or GFP (control). (F) Hemolymph extract from adult female An. gambiae probed for the presence of actin after silencing of GFP (control), actin (Ac) or AgMDL1 (MDL1).