Using Caenorhabditis elegans to produce functional secretory proteins of parasitic nematodes.

The expression of antigens in their immunologically-active form remains a challenge, both in the analysis of regulatory pathways exploited by parasitic nematodes or in the development of vaccines. Despite the success of native proteins to induce protective immunity, recombinant proteins expressed in bacteria, yeast or insect cells offer only limited protective capacities, presumably due to incorrect folding or missing complex posttranslational modifications. The present study investigates the feasibility of using the free-living nematode Caenorhabditis elegans as an alternative expression system for proteins found in the secretome of parasitic nematodes. Exemplified by the expression of the extracellular superoxide dismutase from Haemonchus contortus (HcSODe) and the extracellular and glycosylated glutathione S-transferase from the filarial parasite Onchocerca volvulus (OvGST1), we continue our efforts to improve production and purification of recombinant proteins expressed in C. elegans. We demonstrate that sufficient quantities of functional proteins can be expressed in C. elegans for subsequent immunological and biochemical studies.

Multiple roles of filopodial dynamics in particle capture and phagocytosis and phenotypes of Cdc42 and Myo10 deletion.

Macrophage filopodia, finger-like membrane protrusions, were first implicated in phagocytosis more than 100 years ago, but little is still known about the involvement of these actin-dependent structures in particle clearance. Using spinning disk confocal microscopy to image filopodial dynamics in mouse resident Lifeact-EGFP macrophages, we show that filopodia, or filopodia-like structures, support pathogen clearance by multiple means. Filopodia supported the phagocytic uptake of bacterial (Escherichia coli) particles by (i) capturing along the filopodial shaft and surfing toward the cell body, the most common mode of capture; (ii) capturing via the tip followed by retraction; (iii) combinations of surfing and retraction; or (iv) sweeping actions. In addition, filopodia supported the uptake of zymosan (Saccharomyces cerevisiae) particles by (i) providing fixation, (ii) capturing at the tip and filopodia-guided actin anterograde flow with phagocytic cup formation, and (iii) the rapid growth of new protrusions. To explore the role of filopodia-inducing Cdc42, we generated myeloid-restricted Cdc42 knock-out mice. Cdc42-deficient macrophages exhibited rapid phagocytic cup kinetics, but reduced particle clearance, which could be explained by the marked rounded-up morphology of these cells. Macrophages lacking Myo10, thought to act downstream of Cdc42, had normal morphology, motility, and phagocytic cup formation, but displayed markedly reduced filopodia formation. In conclusion, live-cell imaging revealed multiple mechanisms involving macrophage filopodia in particle capture and engulfment. Cdc42 is not critical for filopodia or phagocytic cup formation, but plays a key role in driving macrophage lamellipodial spreading.