Serine proteases profiles of Leishmania (Viannia) braziliensis clinical isolates with distinct susceptibilities to antimony.

Glucantime (SbV) is the first-line treatment against American Tegumentary Leishmaniasis. Resistance cases to this drug have been reported and related to host characteristics and parasite phenotypes. In this study, 12 Leishmania (Viannia) braziliensis isolates from patients that presented clinical cure (Responders-R) and relapse or therapeutic failure (Non-responders-NR) after treatment with antimony, were analyzed. These parasites were assessed by in vitro susceptibility to SbIII and SbV, serine proteases activity measured with substrate (z-FR-AMC) and specific inhibitors (TLCK, AEBSF and PMSF). In vitro susceptibility of axenic amastigotes to SbIII showed a significant difference between R and NR groups. The protease assays showed that TLCK inhibited almost 100% of activity in both axenic amastigotes and promastigotes while AEBSF inhibited around 70%, and PMSF showed lower inhibition of some isolates. Principal component and clustering analysis performed with these data yielded one homogeneous cluster with only NR isolates and three heterogeneous clusters with R and NR isolates. Additionally, differential expression of subtilisins (LbrM.13.0860 and LbrM.28.2570) and TXNPx (LbrM.15.1080) was evaluated in promastigotes and axenic amastigotes from both groups. The results showed a higher expression of LbrM.13.0860 and LbrM.15.1080 genes in axenic amastigotes, while LbrM.28.2570 gene had the lowest expression in all isolates, regardless of the parasite form. The data presented here show a phenotypic heterogeneity among the parasites, suggesting that exploration of in vitro phenotypes based on SbIII and serine proteases profiles can aid in the characterization of L. (V.) braziliensis clinical isolates.

M. bovis BCG Moreau N-Terminal Loss Leads to a Less Stable Dodecin With Lower Flavin Binding Capacity.

Tuberculosis still remains a concerning health problem worldwide. Its etiologic agent, Mycobacterium tuberculosis, continues to be the focus of research to unravel new prophylactic and therapeutic strategies against this disease. The only vaccine in use against tuberculosis is based on the in vitro attenuated strain, M. bovis BCG. Dodecin is a dodecameric complex important for flavin homeostasis in Archea and Eubacteria, and the M. tuberculosis protein is described as thermo- and halostable. M. bovis BCG Moreau, the Brazilian vaccine strain, has a single nucleotide polymorphism in the dodecin start codon, leading to a predicted loss of seven amino acids at the protein N-terminal end. In this work we aimed to characterize the effect of this mutation in the BCG Moreau protein features. Our recombinant protein assays show that the predicted BCG homolog is less thermostable than M.tb's but maintains its dodecamerization ability, although with a lower riboflavin-binding capacity. These data are corroborated by structural analysis after comparative modeling, showing that the predicted BCG dodecin complex has a lower interaction energy among its monomers and also a distinct electrostatic surface near the flavin binding pocket. However, western blotting assays with the native proteins were unable to detect significant differences between the BCG Moreau and M.tb orthologs, indicating that other factors may be modulating protein structure/function in the bacterial context.

Evidence for Disruption of Mg2+ Pair as a Resistance Mechanism Against HIV-1 Integrase Strand Transfer Inhibitors.

HIV-1 integrase is the enzyme responsible for integrating the viral DNA into the host genome and is one of the main targets for antiretroviral therapy; however, there are documented cases of resistance against all the currently used integrase strand transfer inhibitors (INSTIs). While some resistance-related mutations occur near the inhibitor's binding site, the mutation N155H occurs on the opposite side of the drug-interacting Mg2+ ions, thus, not interacting directly with the drug molecules and currently lacking an explanation for its resistance mechanism. Moreover, mutation N155H and the resistance-related mutation Q148H are mutually exclusive for unknown reasons. In the present study, we use molecular dynamics simulations to understand the impact of the N155H mutation in the HIV-1 integrase structure and dynamics, when alone or in combination with Q148H. Our findings suggest that the Mg2+ ions of the active site adopt different orientations in each of the mutants, causing the catalytic triad residues involved in the ion coordination to adapt their side-chain configurations, completely changing the INSTIs binding site. The change in the ion coordination also seems to affect the flexibility of the terminal viral DNA nucleotide near the active site, potentially impairing the induced-fit mechanism of the drugs. The explanations obtained from our simulations corroborate previous hypotheses drawn from crystallographic studies. The proposed resistance mechanism can also explain the resistance caused by other mutations that take place in the same region of the integrase and help uncover the structural details of other HIV-1 resistance mechanisms.

Raltegravir-Induced Adaptations of the HIV-1 Integrase: Analysis of Structure, Variability, and Mutation Co-occurrence.

The human immunodeficiency virus type 1 (HIV-1) has several proteins of therapeutic importance, many of which are currently used as drug targets in antiretroviral therapy. Among these proteins is the integrase, which is responsible for the integration of the viral DNA into the host genome - a crucial step for HIV-1 replication. Given the importance of this protein in the replication process, three integrase inhibitors are currently used as an option for antiretroviral therapy: Raltegravir, Elvitegravir, and Dolutegravir. However, the crescent emergence of mutations that cause resistance to these drugs has become a worldwide health problem. In this study, we compared the variability of each position of the HIV-1 integrase sequence in clinical isolates of Raltegravir-treated and drug-naïve patients by calculating their Shannon entropies. A co-occurrence network was created to explore how mutations co-occur in patients treated with Raltegravir. Then, by building tridimensional models of the HIV-1 integrase intasomes, we investigated the relationship between variability, architecture, and co-occurrence. We observed that positions bearing some of the major resistance pathways are highly conserved among non-treated patients and variable among the treated ones. The residues involved in the three main resistance-related mutations could be identified in the same group when the positions were clustered according to their entropies. Analysis of the integrase architecture showed that the high-entropy residues S119, T124, and T125, are in contact with the host DNA, and their variations may have impacts in the protein-DNA recognition. The co-occurrence network revealed that the major resistance pathways N155H and Q148HR share more mutations with each other than with the Y143R pathway, this observation corroborates the fact that the N155H pathway is most commonly converted into Q148HRK than into Y143RCH pathway in patients' isolates. The network and the structure analysis also support the hypothesis that the resistance-related E138K mutation may be a mechanism to compensate for mutations in neighbor lysine residues to maintain DNA binding. The present study reveals patterns by which the HIV-1 integrase adapts during Raltegravir therapy. This information can be useful to comprehend the impacts of the drug in the enzyme, as well as help planning new therapeutic approaches.