Benznidazole biotransformation and multiple targets in <i>Trypanosoma</i> cruzi revealed by metabolomics

&lt;b&gt;Background&lt;/b&gt;&lt;p&gt;&lt;/p&gt; The first line treatment for Chagas disease, a neglected tropical disease caused by the protozoan parasite Trypanosoma cruzi, involves administration of benznidazole (Bzn). Bzn is a 2-nitroimidazole pro-drug which requires nitroreduction to become active, although its mode of action is not fully understood. In the present work we used a non-targeted MS-based metabolomics approach to study the metabolic response of T. cruzi to Bzn.&lt;p&gt;&lt;/p&gt; &lt;b&gt;Methodology/Principal findings&lt;/b&gt;&lt;p&gt;&lt;/p&gt; Parasites treated with Bzn were minimally altered compared to untreated trypanosomes, although the redox active thiols trypanothione, homotrypanothione and cysteine were significantly diminished in abundance post-treatment. In addition, multiple Bzn-derived metabolites were detected after treatment. These metabolites included reduction products, fragments and covalent adducts of reduced Bzn linked to each of the major low molecular weight thiols: trypanothione, glutathione, γ-glutamylcysteine, glutathionylspermidine, cysteine and ovothiol A. Bzn products known to be generated in vitro by the unusual trypanosomal nitroreductase, TcNTRI, were found within the parasites, but low molecular weight adducts of glyoxal, a proposed toxic end-product of NTRI Bzn metabolism, were not detected.&lt;p&gt;&lt;/p&gt; &lt;b&gt;Conclusions/significance&lt;/b&gt;&lt;p&gt;&lt;/p&gt; Our data is indicative of a major role of the thiol binding capacity of Bzn reduction products in the mechanism of Bzn toxicity against T. cruzi

Diversity of HIV-1 Subtype B: Implications to the Origin of BF Recombinants

BACKGROUND: The HIV-1 subtype B epidemic in Brazil is peculiar because of the high frequency of isolates having the GWGR tetramer at V3 loop region. It has been suggested that GWGR is a distinct variant and less pathogenic than other subtype B isolates. METHODOLOGY/PRINCIPAL FINDINGS: Ninety-four percent of the HIV-1 subtype B worldwide sequences (7689/8131) obtained from the Los Alamos HIV database contain proline at the tetramer of the V3 loop of the env gene (GPGR) and only 0.74% (60/8131) have tryptophan (GWGR). By contrast, 48.4% (161/333) of subtype B isolates from Brazil have proline, 30.6% (102/333) contain tryptophan and 10.5% (35/333) have phenylalanine (F) at the second position of the V3 loop tip. The proportion of tryptophan and phenylalanine in Brazilian isolates is much higher than in worldwide subtype B sequences (chi-square test, p = 0.0001). The combined proportion of proline, tryptophan and phenylalanine (GPGR+GWGR+GFGR) of Brazilian isolates corresponds to 89% of all amino acids in the V3 loop. Phylogenetic analysis revealed that almost all subtype B isolates in Brazil have a common origin regardless of their motif (GWGR, GPGR, GGGR, etc.) at the V3 tetramer. This shared ancestral origin was also observed in CRF28_BF and CRF29_BF in a genome region (free of recombination) derived from parental subtype B. These results imply that tryptophan substitution (e.g., GWGR-to-GxGR), which was previously associated with the change in the coreceptor usage within the host, also occurs at the population level. CONCLUSIONS/SIGNIFICANCE: Based on the current findings and previous study showing that tryptophan and phenylalanine in the V3 loop are related with coreceptor usage, we propose that tryptophan and phenylalanine in subtype B isolates in Brazil are kept by selective mechanisms due to the distinct coreceptor preferences in target cells of GWGR, GFGR and GFGR viruses

Development of Trypanosoma cruzi in vitro assays to identify compounds suitable for progression in Chagas’ disease drug discovery

Chagas' disease is responsible for significant mortality and morbidity in Latin America. Current treatments display variable efficacy and have adverse side effects, hence more effective, better tolerated drugs are needed. However, recent efforts have proved unsuccessful with failure of the ergosterol biosynthesis inhibitor posaconazole in phase II clinical trials despite promising in vitro and in vivo studies. The lack of translation between laboratory experiments and clinical outcome is a major issue for further drug discovery efforts. Our goal was to identify cell-based assays that could differentiate current nitro-aromatic drugs nifurtimox and benznidazole from posaconazole. Using a panel of T. cruzi strains including the six major lineages (TcI-VI), we found that strain PAH179 (TcV) was markedly less susceptible to posaconazole in vitro. Determination of parasite doubling and cycling times as well as EdU labelling experiments all indicate that this lack of sensitivity is due to the slow doubling and cycling time of strain PAH179. This is in accordance with ergosterol biosynthesis inhibition by posaconazole leading to critically low ergosterol levels only after multiple rounds of division, and is further supported by the lack of effect of posaconazole on the non-replicative trypomastigote form. A washout experiment with prolonged posaconazole treatment showed that, even for more rapidly replicating strains, this compound cannot clear all parasites, indicative of a heterogeneous parasite population in vitro and potentially the presence of quiescent parasites. Benznidazole in contrast was able to kill all parasites. The work presented here shows clear differentiation between the nitro-aromatic drugs and posaconazole in several assays, and suggests that in vitro there may be clinically relevant heterogeneity in the parasite population that can be revealed in long-term washout experiments. Based on these findings we have adjusted our in vitro screening cascade so that only the most promising compounds are progressed to in vivo experiments

The complete genome sequence of Corynebacterium pseudotuberculosis FRC41 isolated from a 12-year-old girl with necrotizing lymphadenitis reveals insights into gene-regulatory networks contributing to virulence

Trost E, Ott L, Schneider J, et al. The complete genome sequence of Corynebacterium pseudotuberculosis FRC41 isolated from a 12-year-old girl with necrotizing lymphadenitis reveals insights into gene-regulatory networks contributing to virulence. BMC Genomics. 2010;11(1): 728