Opinion: What do rescue experiments with heterologous proteins tell us and what not?

Abstract

The recent progress in sequencing technology allowed the compilation of gene lists for a large number of organisms, though many of these organisms are hardly experimentally tractable when compared with well-established model organisms. One popular approach to further characterize genes identified in a poorly tractable organism is to express these genes in a model organism, and then ask what the protein does in this system or if the gene is capable of replacing the homologous endogenous one when the latter is mutated. While this is a valid approach for certain questions, I argue that the results of such experiments are frequently wrongly interpreted. If, for example, a gene from a parasitic nematode is capable of replacing its homologous gene in the model nematode Caenorhabditis elegans, it is often concluded that the gene is most likely involved in the same biological process in its own organism as the C. elegans gene is in C. elegans. This conclusion is not valid. All this experiment tells us is that the chemical properties of the parasite protein are similar enough to the ones of the C. elegans protein that it can perform the function of the C. elegans protein in C. elegans. Here I discuss this misconception and illustrate it using the analog of similar electric switches (components) controlling various devices (processes).

Genomic landscape of drug response reveals mediators of anthelmintic resistance

“…The role of cky-1 in ivermectin resistance is supported by the following: (1) genetic differentiation between susceptible and resistant strains around this locus relative to genome-wide variation that is replicated in geographically and genetically diverse strains here and elsewhere ( Baltrušis et al., 2022 ; Doyle et al., 2019a ; Rezansoff et al., 2016 ; Sallé et al., 2019 ), (2) the presence of non-synonymous variants that are highly differentiated before and after treatment, (3) increased gene expression of cky-1 in resistant strains relative to a susceptible strain (supported by genome-wide RNA-seq [ Laing et al., 2022 ]), and (4) knockdown of the C. elegans ortholog leading to hypersensitivity to ivermectin. We acknowledge that overexpression of cky-1 in C. elegans does not recapitulate the high levels of ivermectin resistance seen in H. contortus or, for example, by concurrent mutation of glutamate-gated chloride channels in C. elegans ( Dent et al., 2000 ); while this may argue against cky-1 as a universal mediator of resistance, it likely reflects the challenge of using a heterologous expression system in which there is an assumption that the biology (and, therefore, response to treatment) is concordant between the free-living and parasitic species, and/or may reflect the multigenic nature of ivermectin resistance in different species ( Evans et al., 2021 ; Streit, 2021 ; Wit et al., 2021 ). Given the lack of an obvious causal non-synonymous variant, we hypothesize that a non-coding variant that influences the expression of cky-1 is under selection in resistant strains of H. contortus ; however, such variants are difficult to validate without genotype and transcriptional phenotype data from a large number of individual worms.…”

Genomic landscape of drug response reveals novel mediators of anthelmintic resistance

“…The role of cky-1 in ivermectin resistance is supported by: (i) genetic differentiation between susceptible and resistant strains around this locus relative to genome-wide variation that is replicated in geographically and genetically diverse strains here and elsewhere (25,34,62), (ii) the presence of non-synonymous variants that are highly differentiated before and after treatment, (iii) increased gene expression of cky-1 in resistant strains relative to susceptible strains (supported by genome-wide RNA-seq (58)), (iv) knockdown of the C. elegans ortholog leading to hypersensitivity to ivermectin, and (v) partial induction of resistance in C. elegans by overexpression of cky-1. We acknowledge that overexpression of cky-1 in C. elegans does not recapitulate the high levels of ivermectin resistance seen in H. contortus or, for example, by concurrent mutation of glutamate-gated chloride channels in C. elegans (21); while this may argue against cky-1 as a universal mediator of resistance, it likely reflects the challenge of using a heterologous expression system in which there is an assumption that the biology (and, therefore, response to treatment) is concordant between the free-living and parasitic species, and/or may reflect the multigenic nature of ivermectin resistance in different species (63)(64)(65). Given the lack of an obvious causal non-synonymous variant, we hypothesise that a non-coding variant that influences the expression of cky-1 is under selection in resistant strains of H. contortus; however, such variants are difficult to validate without genotype and transcriptional phenotype data from a large number of individual worms.…”