Characteristics of Mycobacterium tuberculosis PtpA interaction and activity on the alpha subunit of human mitochondrial trifunctional protein, a key enzyme of lipid metabolism.

During Mycobacterium tuberculosis (Mtb) infection, the virulence factor PtpA belonging to the protein tyrosine phosphatase family is delivered into the cytosol of the macrophage. PtpA interacts with numerous eukaryotic proteins modulating phagosome maturation, innate immune response, apoptosis, and potentially host-lipid metabolism, as previously reported by our group. In vitro, the human trifunctional protein enzyme (hTFP) is a bona fide PtpA substrate, a key enzyme of mitochondrial ß-oxidation of long-chain fatty acids, containing two alpha and two beta subunits arranged in a tetramer structure. Interestingly, it has been described that the alpha subunit of hTFP (ECHA, hTFPα) is no longer detected in mitochondria during macrophage infection with the virulent Mtb H37Rv. To better understand if PtpA could be the bacterial factor responsible for this effect, in the present work, we studied in-depth the PtpA activity and interaction with hTFPα. With this aim, we performed docking and in vitro dephosphorylation assays defining the P-Tyr-271 as the potential target of mycobacterial PtpA, a residue located in the helix-10 of hTFPα, previously described as relevant for its mitochondrial membrane localization and activity. Phylogenetic analysis showed that Tyr-271 is absent in TFPα of bacteria and is present in more complex eukaryotic organisms. These results suggest that this residue is a specific PtpA target, and its phosphorylation state is a way of regulating its subcellular localization. We also showed that phosphorylation of Tyr-271 can be catalyzed by Jak kinase. In addition, we found by molecular dynamics that PtpA and hTFPα form a stable protein complex through the PtpA active site, and we determined the dissociation equilibrium constant. Finally, a detailed study of PtpA interaction with ubiquitin, a reported PtpA activator, showed that additional factors are required to explain a ubiquitin-mediated activation of PtpA. Altogether, our results provide further evidence supporting that PtpA could be the bacterial factor that dephosphorylates hTFPα during infection, potentially affecting its mitochondrial localization or ß-oxidation activity.

A combined approach of rolling-circle amplification-single site restriction endonuclease digestion followed by next generation sequencing to characterize the whole genome and intra-host variants of human Torque teno virus.

Torque Teno Virus (TTV) was initially associated with post-transfusion hepatitis, but growing evidence of its ubiquity in humans is compatible to no apparent clinical significance. TTV is a small non-enveloped virus with a circular single-negative-stranded DNA genome, belonging to the Anelloviridae family. Currently, TTVs are divided in seven phylogenetic groups and are further classified into 21 species. Studies about diversity of TTV in different conditions are receiving increasing interest and in this sense, sequencing of whole genomes for better genetic characterization becomes even more important. Since its discovery in 1997, few TTV complete genomes have been reported worldwide. This is probably due, among other reasons, to the great genetic heterogeneity among TTV strains that prevents its amplification and sequencing by conventional PCR and cloning methods. In addition, although metagenomics approach is useful in these cases, it remains a challenging tool for viromic analysis. With the aim of contributing to the expansion of the TTV whole genomes dataset and to study intra-host variants, we employed a methodology that combined a rolling-circle amplification approach followed by EcoRI digestion, generating a DNA fragment of ∼4Kb consistent with TTV genome length which was sequenced by Illumina next generation sequencing. A genogroup 3 full-length consensus TTV genome was obtained and co-infection with other species (at least those with a single EcoRI cleavage site) was not identified. Additionally, bioinformatics analysis allowed to identify the spectrum of TTV intra-host variants which provides evidence of a complex evolution dynamics of these DNA circular viruses, similarly to what occurs with RNA viruses.

An update on genetic analysis of porcine reproductive and respiratory syndrome virus type 2 (PRRSV-2) in South America: identification of ORF5 sequences of lineage 1A, 1C and 1G.

Data regarding PRRSV-2 in South America are scant and a coordinated criterion for molecular characterization is needed. A phylogenetic analysis was performed using a dataset of 76 ORF5 sequences from South America, and results showed the identification of lineage 5 in the early 2000s and the predominance of lineage 1 at least since 2013. Lineage 1 sequences were further classified into sub-lineages according to a recent molecular characterization study of PRRSV-2 in United States. Our results revealed the recent identification in Uruguay of PRRSV-2 ORF5 sequences of lineage 1 sub-lineage C. Two additional sub-lineages were identified in South America, 1G in Chile and 1A in Peru. Continuous updating the molecular epidemiology of circulating viruses with coordinated investigations among countries is required to control and prevent the emergence of genetic variants of PRRSV-2.

Naturally occurring NS3 resistance-associated variants in hepatitis C virus genotype 1: Their relevance for developing countries.

Hepatitis C virus (HCV) is a major cause of global morbidity and mortality, with an estimated 130-150 million infected individuals worldwide. HCV is a leading cause of chronic liver diseases including cirrhosis and hepatocellular carcinoma. Current treatment options in developing countries involve pegylated interferon-α and ribavirin as dual therapy or in combination with one or more direct-acting antiviral agents (DAA). The emergence of resistance-associated variants (RAVs) after treatment reveals the great variability of this virus leading to a great difficulty in developing effective antiviral strategies. Baseline RAVs detected in DAA treatment-naïve HCV-infected patients could be of great importance for clinical management and outcome prediction. Although the frequency of naturally occurring HCV NS3 protease inhibitor mutations has been addressed in many countries, there are only a few reports on their prevalence in South America. In this study, we investigated the presence of RAVs in the HCV NS3 serine protease region by analysing a cohort of Uruguayan patients with chronic hepatitis C who had not been treated with any DAAs and compare them with the results found for other South American countries. The results of these studies revealed that naturally occurring mutations conferring resistance to NS3 inhibitors exist in a substantial proportion of Uruguayan treatment-naïve patients infected with HCV genotype 1 enrolled in these studies. The identification of these baseline RAVs could be of great importance for patients' management and outcome prediction in developing countries.