Mimicry of a G protein mutation by pertussis toxin expression in transgenic Caenorhabditis elegans

Abstract

Pathogens produce virulence factors that interact directly with host molecules, but in many cases the host targets are unknown. The genetic and molecular identification of these orphan targets is often not feasible with mammalian experimental models. However, a substantial number of known targets are molecules and pathways that are conserved among eukaryotes, and therefore the use of nonmammalian model hosts to identify orphan targets may prove useful. To demonstrate the feasibility of this approach, we transformed the nematode Caenorhabditis elegans with a gene encoding the catalytic subunit of pertussis toxin (PTX), which in mammals inactivates G(o/i)alpha proteins. Expression of PTX in C. elegans produced phenotypes almost identical to those of a null mutation in the nematode gene encoding G(o/i)alpha. Furthermore, PTX suppressed the phenotype of a constitutively active form of nematode G(o/i)alpha protein. These results indicate that PTX is functional in nematodes and acts specifically on the C. elegans homologue of the mammalian target.

FIG. 1

Expression of PTX in transgenic C. elegans. Immunoblot showing the catalytic subunit of PTX in nematodes without (−) or with (+) heat shock. ptx⁺, nematode strain XA1433 carrying the ptx gene under a heat shock promoter. w.t., wild-type nematode strain N2. S1, 500 pg of purified catalytic subunit S1. Brightness and contrast were adjusted using Adobe Photoshop.

FIG. 2

Phenotypes of PTX expression. Adult nematodes were heat shocked, transferred to NGM agar seeded with E. coli, and photographed. (A) w.t., wild-type strain N2. (B) ptx⁺, strain XA1433, carrying a heat shock inducible ptx gene. (C) Δgoa-1, strain CB363, a goa-1 deletion mutant. Color transparencies were digitally scanned, converted to grayscale, and adjusted slightly for brightness and contrast with Adobe Photoshop.

FIG. 3

Rates of pharyngeal pumping without (−) or with (+) heat shock treatment. w.t., wild-type strain N2. ptx⁺, strain XA1433, carrying a heat shock inducible ptx gene. Δgoa-1, strain CB363 carrying a goa-1 deletion allele, n363. Each cluster of four shows assays on a separate day. Data are mean pumps per minute ± standard deviation for 12 animals.

FIG. 4

Rescue of constitutive goa-1 mutation by PTX expression. Nematodes were heat shocked, transferred to seeded NGM agar, and photographed. (A) Parental strain PS1493, carrying the constitutive goa-1 transgene syIs9. (B) Progeny of PS1493 hermaphrodite and wild-type male. (C) Progeny of PS1493 and a ptx male. Genotypes of relevant integrated transgenes are indicated; marker genotypes are omitted for clarity. Images were processed as described in the legend to Fig. 2.