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.

Biomarkers of disease severity in patients with visceral leishmaniasis co-infected with HIV.

Visceral leishmaniasis (VL) is caused by the protozoan Leishmania spp, transmitted by sand fly bites. VL is one of the deadliest tropical infection diseases, yet the coinfection with HIV virus drastically increases relapses, treatment failure and mortality. The concomitant action of these two pathogens leads to high cellular activation independently of the progression to AIDS. In addition, microbial translocation and bacterial infections are thought to contribute worsening the clinical picture. Identifying biomarkers associated with disease severity is of interest for clinical management of patients with VL-HIV/AIDS. Thus, we analyzed in the sera several markers including interleukins (IL-1ß, IL-6, IL-8, and IL-17), interferon-γ (IFN- γ), tumor necrosis factor (TNF), lipopolysaccharide (LPS), soluble CD14 (sCD14), macrophage migration inhibitory factor (MIF) and intestinal fatty acid-binding protein (IFABP). These markers were compared with disease severity in 24 patients with VL/HIV presenting different clinical outcomes. Disease severity was defined by the probability of death calculated using a score set system derived by the Kala-Cal® software. Probability of death ranged from 3.7% to 97.9%, with median of 28.8%. Five patients died (20%). At the univariate analysis, disease severity was correlated with TNF, IFN-γ and sCD14. LPS was positively correlated with sCD14 specifically in patients with low CD4+ count (CD4+ T-cell <200 cells/mL). Most importantly, the multivariate analysis including LPS, CD4+count and sCD14 showed that sCD14 was the only independent predictor for disease severity and death. Altogether, our results indicated that sCD14 is a powerful marker of pathogenicity and death for patients with VL-HIV/AIDS.

Human Competence to Transmit Leishmania infantum to Lutzomyia longipalpis and the Influence of Human Immunodeficiency Virus Infection.

Visceral leishmaniasis (VL) caused by Leishmania infantum is a lethal disease transmitted by sand flies. Although, considered a zoonosis with dogs held as the main reservoirs, humans are also sources of infection. Therefore, control policies currently focused on dog culling may need to consider that VL and human immunodeficiency virus (HIV)/VL patients may also be infectious, contributing to transmission. Reservoir competence of patients with VL without and with HIV infection and of persons asymptomatically infected with Leishmania was assessed by xenodiagnosis with the vector Lutzomyia longipalpis. Parasites in sand fly's guts were identified by using optical microscopy and by conventional polymerase chain reaction (PCR). Leishmania infantum blood parasite burden was determined by quantitative PCR. Among the 61 participants, 27 (44%) infected sand flies as seen by microscopy or PCR. When infectiousness was assessed by microscopy, xenodiagnosis was positive in five (25%) patients not infected with HIV, whereas nine (45%) of those harboring HIV were positive. Among the 19 asymptomatic patients four (21%) infected sand flies only demonstrated by PCR. One (50%) asymptomatic patient with HIV had a positive xenodiagnosis by microscopy. 9/372 (2.4%) and 37/398 (9.2%) sand flies were infected when feeding in patients without and with HIV, respectively. Infectiousness was poorly correlated with quantitative PCR. The study shows that asymptomatic humans are capable of transmitting L. infantum, that ill persons with HIV infection are more infectious to sand flies, and that humans are more important reservoirs than previously thought. This fact may be considered when designing control policies for zoonotic VL.