ABSTRACT
ATP-independent chaperones are widespread across all domains of life and serve as the first line of defense during protein unfolding stresses. One of the known crucial chaperones for bacterial survival in a hostile environment (e.g., heat and oxidative stress) is the highly conserved, redox-regulated ATP-independent bacterial chaperone Hsp33. Using a bioinformatic analysis, we describe novel eukaryotic homologs of Hsp33 identified in eukaryotic pathogens belonging to the kinetoplastids, a family responsible for lethal human diseases such as Chagas disease as caused by Trypanosoma cruzi, African sleeping sickness caused by Trypanosoma brucei spp., and leishmaniasis pathologies delivered by various Leishmania species. During their pathogenic life cycle, kinetoplastids need to cope with elevated temperatures and oxidative stress, the same conditions which convert Hsp33 into a powerful chaperone in bacteria, thus preventing aggregation of a wide range of misfolded proteins. Here, we focused on a functional characterization of the Hsp33 homolog in one of the members of the kinetoplastid family, T. brucei, (Tb927.6.2630), which we have named TrypOx. RNAi silencing of TrypOx led to a significant decrease in the survival of T. brucei under mild oxidative stress conditions, implying a protective role of TrypOx during the Trypanosomes growth. We then adopted a proteomics-driven approach to investigate the role of TrypOx in defining the oxidative stress response. Depletion of TrypOx significantly altered the abundance of proteins mediating redox homeostasis, linking TrypOx with the antioxidant system. Using biochemical approaches, we identified the redox-switch domain of TrypOx, showing its modularity and oxidation-dependent structural plasticity. Kinetoplastid parasites such as T. brucei need to cope with high levels of oxidants produced by the innate immune system, such that parasite-specific antioxidant proteins like TrypOx - which are depleted in mammals - are highly promising candidates for drug targeting.
ABSTRACT
BACKGROUND: Visceral Leishmaniasis (VL) is a disseminated protozoan infection caused by Leishmania donovani parasites which affects almost half a million persons annually. Most of these are from the Indian sub-continent, East Africa and Brazil. Our study was designed to elucidate the role of symptomatic and asymptomatic Leishmania donovani infected persons in the epidemiology of VL in Northern Ethiopia. METHODS: The efficacy of quantitative real-time kinetoplast DNA/PCR (qRT-kDNA PCR) for detecting Leishmania donovani in dried-blood samples was assessed in volunteers living in an endemic focus. RESULTS: Of 4,757 samples, 680 (14.3%) were found positive for Leishmania k-DNA but most of those (69%) had less than 10 parasites/ml of blood. Samples were re-tested using identical protocols and only 59.3% of the samples with 10 parasite/ml or less were qRT-kDNA PCR positive the second time. Furthermore, 10.8% of the PCR negative samples were positive in the second test. Most samples with higher parasitemias remained positive upon re-examination (55/59 =93%). We also compared three different methods for DNA preparation. Phenol-chloroform was more efficient than sodium hydroxide or potassium acetate. DNA sequencing of ITS1 PCR products showed that 20/22 samples were Leishmania donovani while two had ITS1 sequences homologous to Leishmania major. CONCLUSIONS: Although qRT-kDNA PCR is a highly sensitive test, the dependability of low positives remains questionable. It is crucial to correlate between PCR parasitemia and infectivity to sand flies. While optimal sensitivity is achieved by targeting k-DNA, it is important to validate the causative species of VL by DNA sequencing.
