Serial Knockout of Diphthamide Biosynthesis (DPH) Genes Result in the Secretion of Immuntoxins and Leads to the Enhancement of Pseudomonas Aeruginosa Exotoxin A (PE) Based Immuntoxin



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Diphthamide is a post-translational modification of histidine found on eukaryotic elongation factor 2 (eEF-2), which is generated in a multi-step biosynthetic process by a series of seven diphthamide biosynthesis enzymes (DPH1-7) and is highly conserved among eukaryotes and archaebacteria. Previous studies have shown that inactivation of DPH1, 2, 4, or 5 genes lead to the prevention of ADP ribosylation of diphthamide and resistance to diphtheria toxin (DT) and Pseudomonas exotoxin A (PE). However, the physiological function of diphthamide has remained rather obscure. We have used CRISPR-Cas9 to conduct systemic knockout (KO) of all the seven DPH genes in multiple cell lines and have investigated the long-term effect on cellular survival and protein synthesis in both in vitro and in vivo characterizations. Our data show that, in contrast to early reports, the deletion of DPH1, 2, 4, or 5 has either an immediate or delayed detrimental effect on cell proliferation, survival and protein synthesis. Most interestingly, cells can completely tolerate the deletion of either DPH6 or DPH7 with or without the presence of DT or PE toxin. We also conducted a series of combinatorial KOs of DPH genes and the data are consistent with the notion that loss of any of DPH genes results in the phenotype of the first KO gene in consecutive order. These observations thus shed new light on the distinct functions of these diphthamide-eEF2 conversion enzymes and also lead to the conclusion that only DPH 6 and 7 can be inactivated without affecting cell viability.



Immunotoxin diphthamide