Genome-Wide RNAi Screens Identify Genes Required for Ricin and PE Intoxications

Abstract

Protein toxins such as Ricin and Pseudomonas exotoxin (PE) pose major public health challenges. Both toxins depend on host cell machinery for internalization, retrograde trafficking from endosomes to the ER, and translocation to cytosol. Although both toxins follow a similar intracellular route, it is unknown how much they rely on the same genes. Here we conducted two genome-wide RNAi screens identifying genes required for intoxication and demonstrating that requirements are strikingly different between PE and Ricin, with only 13% overlap. Yet factors required by both toxins are present from the endosomes to the ER, and, at the morphological level, the toxins colocalize in multiple structures. Interestingly, Ricin, but not PE, depends on Golgi complex integrity and colocalizes significantly with a medial Golgi marker. Our data are consistent with two intertwined pathways converging and diverging at multiple points and reveal the complexity of retrograde membrane trafficking in mammalian cells.

Whole-genome RNAi screen highlights components of the endoplasmic reticulum/Golgi as a source of resistance to immunotoxin-mediated cytotoxicity

“…A study of ricin and PE toxicity sought gene products that, when silenced, rescued cells from toxin-mediated inhibition of protein synthesis (24). Hits from this assay would be equivalent to our mitigator list with two distinctions: Moreau et al assayed for rescue of protein synthesis inhibition at 8 h, whereas we assayed for protection in a 72-h viability assay, and they used native PE, whereas we used PE-based immunotoxins (24).…”

“…Hits from this assay would be equivalent to our mitigator list with two distinctions: Moreau et al assayed for rescue of protein synthesis inhibition at 8 h, whereas we assayed for protection in a 72-h viability assay, and they used native PE, whereas we used PE-based immunotoxins (24). overlap [∼7% pairwise overlap between the 178 "validated" hits in the Moreau study and the top mitigators presented here (RSA: P < 0.01)].…”

“…In addition, within the cytosol, the diphthamide pathway responsible for the multistep posttranslational modification of EF2, converting histidine 715 to diphthamide, is necessary (22,23). Beyond these components, the total number of constituents is unknown, although it is under study (24). Silencing of genes associated with the immunotoxin pathway would likely change cell sensitivity.…”

Immunotoxins for leukemia

“…The ricin pathway to the cytosol more closely resembles the PE pathway than the DT one ( Figure 1). 15 Toxins are released from antibodies in several ways: by proteases, by disulfide bond reduction, or by exposure to an acidic environment. Protein toxins joined to antibodies via peptide bonds require proteolytic cleavage.…”

Pathogen and Toxin Entry - How Pathogens and Toxins Induce and Harness Endocytotic Mechanisms

“…This indiscriminate binding of ricin also explains why it is so difficult to identify distinct molecular players involved in the intracellular trafficking of ricin, while the retrograde transport of the Shiga toxin, which specifically uses the glycosphingolipid Gb3 as cellular receptor, is currently much better characterised [21,68]. Recently, a genome-wide RNAi screen shed some light on the molecular requirements of ricin intoxication [69]. This study corroborates earlier observations that only a subset of molecular factors that are required for ricin trafficking is also involved in the retrograde transport of other toxins, such as Shiga toxin or Pseudomonas exotoxin, and that several intertwined retrograde transport pathways exist in parallel.…”