Genetic depletion of the RNA helicase DDX3 leads to impaired elongation of translating ribosomes triggering co-translational quality control of newly synthesized polypeptides.

DDX3 is a multifaceted RNA helicase of the DEAD-box family that plays central roles in all aspects of RNA metabolism including translation initiation. Here, we provide evidence that the Leishmania DDX3 ortholog functions in post-initiation steps of translation. We show that genetic depletion of DDX3 slows down ribosome movement resulting in elongation-stalled ribosomes, impaired translation elongation and decreased de novo protein synthesis. We also demonstrate that the essential ribosome recycling factor Rli1/ABCE1 and termination factors eRF3 and GTPBP1 are less recruited to ribosomes upon DDX3 loss, suggesting that arrested ribosomes may be inefficiently dissociated and recycled. Furthermore, we show that prolonged ribosome stalling triggers co-translational ubiquitination of nascent polypeptide chains and a higher recruitment of E3 ubiquitin ligases and proteasome components to ribosomes of DDX3 knockout cells, which further supports that ribosomes are not elongating optimally. Impaired elongation of translating ribosomes also results in the accumulation of cytoplasmic protein aggregates, which implies that defects in translation overwhelm the normal quality controls. The partial recovery of translation by overexpressing Hsp70 supports this possibility. Collectively, these results suggest an important novel contribution of DDX3 to optimal elongation of translating ribosomes by preventing prolonged translation stalls and stimulating recycling of arrested ribosomes.

New insights in the mode of action of anti-leishmanial drugs by using chemical mutagenesis screens coupled to next-generation sequencing.

Leishmania parasites are responsible for a range of clinical manifestations ranging from self-resolving cutaneous sores to life-threatening diseases. The management of leishmaniasis is complicated in part by the scarcity of treatment options but also by the emerging or established resistance to available drugs. A major driver of resistance in Leishmania is the amplification of resistance genes taking advantage of the highly repetitive genomic landscape of the parasite. The recent advent of whole genome gain of function screens gave new momentum to the study of such resistance mechanisms, leading to the identification of novel resistance factors and drug targets against approved drugs, which include antimony (SbIII), miltefosine (MIL), paromomycin (PMM), and amphotericin B. However, these screens do not pinpoint single nucleotide variations (SNVs), an important contributor of drug resistance. To fill the gap, our recent study describes the optimization of chemical mutagenesis coupled to next generation sequencing, an approach called Mut-seq, as a way to explore networks of drug resistance genes in organisms with a diploid to mosaic aneuploid genome like Leishmania. Our Mut-seq screen revealed associations between genes linked with lipid metabolism and resistance to MIL, and highlighted the role of a protein kinase in translation leading to resistance to PMM.

Coupling chemical mutagenesis to next generation sequencing for the identification of drug resistance mutations in Leishmania.

Current genome-wide screens allow system-wide study of drug resistance but detecting small nucleotide variants (SNVs) is challenging. Here, we use chemical mutagenesis, drug selection and next generation sequencing to characterize miltefosine and paromomycin resistant clones of the parasite Leishmania. We highlight several genes involved in drug resistance by sequencing the genomes of 41 resistant clones and by concentrating on recurrent SNVs. We associate genes linked to lipid metabolism or to ribosome/translation functions with miltefosine or paromomycin resistance, respectively. We prove by allelic replacement and CRISPR-Cas9 gene-editing that the essential protein kinase CDPK1 is crucial for paromomycin resistance. We have linked CDPK1 in translation by functional interactome analysis, and provide evidence that CDPK1 contributes to antimonial resistance in the parasite. This screen is powerful in exploring networks of drug resistance in an organism with diploid to mosaic aneuploid genome, hence widening the scope of its applicability.

DDX3 DEAD-box RNA helicase plays a central role in mitochondrial protein quality control in Leishmania.

DDX3 is a highly conserved member of ATP-dependent DEAD-box RNA helicases with multiple functions in RNA metabolism and cellular signaling. Here, we describe a novel function for DDX3 in regulating the mitochondrial stress response in the parasitic protozoan Leishmania. We show that genetic inactivation of DDX3 leads to the accumulation of mitochondrial reactive oxygen species (ROS) associated with a defect in hydrogen peroxide detoxification. Upon stress, ROS production is greatly enhanced, causing mitochondrial membrane potential loss, mitochondrial fragmentation, and cell death. Importantly, this phenotype is exacerbated upon oxidative stress in parasites forced to use the mitochondrial oxidative respiratory machinery. Furthermore, we show that in the absence of DDX3, levels of major components of the unfolded protein response as well as of polyubiquitinated proteins increase in the parasite, particularly in the mitochondrion, as an indicator of mitochondrial protein damage. Consistent with these findings, immunoprecipitation and mass-spectrometry studies revealed potential interactions of DDX3 with key components of the cellular stress response, particularly the antioxidant response, the unfolded protein response, and the AAA-ATPase p97/VCP/Cdc48, which is essential in mitochondrial protein quality control by driving proteosomal degradation of polyubiquitinated proteins. Complementation studies using DDX3 deletion mutants lacking conserved motifs within the helicase core support that binding of DDX3 to ATP is essential for DDX3's function in mitochondrial proteostasis. As a result of the inability of DDX3-depleted Leishmania to recover from ROS damage and to survive various stresses in the host macrophage, parasite intracellular development was impaired. Collectively, these observations support a central role for the Leishmania DDX3 homolog in preventing ROS-mediated damage and in maintaining mitochondrial protein quality control.