Transgenesis and neuronal ablation in parasitic nematodes: revolutionary new tools to dissect host-parasite interactions

Abstract

Ease of experimental gene transfer into viral and prokaryotic pathogens has made transgenesis a powerful tool for investigating the interactions of these pathogens with the host immune system. Recent advances have made this approach feasible for more complex protozoan parasites. By contrast, the lack of a system for heritable transgenesis in parasitic nematodes has hampered progress toward understanding the development of nematode-specific cellular responses. Recently, however, significant strides towards such a system have been made in several parasitic nematodes, and the possible applications of these in immunological research should now be contemplated. In addition, methods for targeted cell ablation have been successfully adapted from Caenorhabditis elegans methodology and applied to studies of neurobiology and behaviour in Strongyloides stercoralis. Together, these new technical developments offer exciting new tools to interrogate multiple aspects of the host-parasite interaction following nematode infection.

Figure 1

Transgenesis in Strongyloides stercoralis. (a) Diagrammatic representation of the syncytial ovary as microinjection site for DNA transfer. (b–i) F1 progeny of free-living female S. stercoralis microinjected as shown in panel (a) with reporter transgene constructs, all containing the Ss era-1 3′ UTR as a terminator. (b,c) DIC and fluorescence images, respectively, of a first-stage larva (L1) expressing gfp under the Ss act-2 promoter; note body wall localization (Scale bar in (b) = 50 µm). (d,e) DIC and fluorescence images, respectively, of an L1 expressing gfp under the Ss gpa-3 promoter; note localization in ampidial neurones (Scale bar in (d) = 10 µm). (f,g) DIC and fluorescence images, respectively, of an L1 expressing gfp under the Ss rps-21 promoter; note ubiquitous pattern of expression (Scale bar in (f) = 50 µm). (h,i) DIC and fluorescence images, respectively, of a parasitic female expressing the Ss rps-21 reporter, recovered from the gerbil intestine at necropsy (Scale bar in (h) = 200 µm).

Figure 2

Selecting transgenic S. stercoralis L3i using the COPAS Biosorter. Sorting Ss act-2::gfp transformants based on GFP fluorescence from a mixed population of GFP-expressing and non-GFP-expressing parasites. Intensity of GFP fluorescence in units of green peak height is plotted against time of flight (TOF), a measure of worm size. Typically, setting the sorting gate at a green peak height of 600 as shown yields a population of worms highly enriched for transformants while retaining parasites with lower levels of fluorescence, possibly indicating a lower transgene copy number, more optimal for heritable transgenesis. The outlier in time of flight (arrow) may indicate two or more worms passing the detector in tandem.

Figure 3

Microlaser ablation of chemosensory neurones in S. stercoralis: a possible means of attenuating or restricting parasite development to early events that are key to initiation of the immune response. (a) Diagrammatic representation of amphidial cell bodies in the head of an L1 of S. stercoralis based on computer-generated three-dimensional reconstruction of electron micrographic sections From Ashton et al. (98), reproduced with permission. (b,c) Cell bodies of ampidial neurones before and after, respectively, microlaser ablation of ASI and ASF (upper and lower red arrow heads, respectively). Un-operated neurone ASG is highlighted (white arrow) for a point of reference. Images courtesy of G. A. Schad. (d) Ingestion of FITC from medium by S. stercoralis L3i following transfer to culture under host-like conditions, an indicator of resumption of development. (e) Effect of laser ablation of ASJ chemosensory neurones on resumption of feeding. Controls consisted of un-operated worms and worms in which an unrelated neurone pair, ASK was ablated. Images in panels b and c are from Ashton et al. (102); reproduced with permission.