Differences in Charge Distribution in Leishmania tarentolae Leishmanolysin Result in a Reduced Enzymatic Activity.

Leishmania tarentolae is a non-pathogenic trypanosomatid isolated from lizards widely used for heterologous protein expression and extensively studied to understand the pathogenic mechanisms of leishmaniasis. The repertoire of leishmanolysin genes was reported to be expanded in L. tarentolae genome, but no proteolytic activity was detected. Here, we analyzed L. tarentolae leishmanolysin proteins from the genome to the structural levels and evaluated the enzymatic activity of the wild-type and overexpressing mutants of leishmanolysin. A total of 61 leishmanolysin sequences were retrieved from the L. tarentolae genome. Five of them were selected for phylogenetic analysis, and for three of them, we built 3D models based on the crystallographic structure of L. major ortholog. Molecular dynamics simulations of these models disclosed a less negative electrostatic potential compared to the template. Subsequently, L. major LmjF.10.0460 and L. tarentolae LtaP10.0650 leishmanolysins were cloned in a pLEXSY expression system into L. tarentolae. Proteins from the wild-type and the overexpressing parasites were submitted to enzymatic analysis. Our results revealed that L. tarentolae leishmanolysins harbor a weak enzymatic activity about three times less abundant than L. major leishmanolysin. Our findings strongly suggest that the less negative electrostatic potential of L. tarentolae leishmanolysin can be the reason for the reduced proteolytic activity detected in this parasite.

The Essential Role of Cholesterol Metabolism in the Intracellular Survival of Mycobacterium leprae Is Not Coupled to Central Carbon Metabolism and Energy Production.

UNLABELLED: Mycobacterium leprae induces the formation of lipid droplets, which are recruited to pathogen-containing phagosomes in infected macrophages and Schwann cells. Cholesterol is among the lipids with increased abundance in M. leprae-infected cells, and intracellular survival relies on cholesterol accumulation. The present study investigated the capacity of M. leprae to acquire and metabolize cholesterol. In silico analyses showed that oxidation of cholesterol to cholest-4-en-3-one (cholestenone), the first step of cholesterol degradation catalyzed by the enzyme 3ß-hydroxysteroid dehydrogenase (3ß-HSD), is apparently the only portion of the cholesterol catabolic pathway seen in Mycobacterium tuberculosis preserved by M. leprae. Incubation of bacteria with radiolabeled cholesterol confirmed the in silico predictions. Radiorespirometry and lipid analyses performed after incubating M. leprae with [4-(14)C]cholesterol or [26-(14)C]cholesterol showed the inability of this pathogen to metabolize the sterol rings or the side chain of cholesterol as a source of energy and carbon. However, the bacteria avidly incorporated cholesterol and, as expected, converted it to cholestenone both in vitro and in vivo. Our data indicate that M. leprae has lost the capacity to degrade and utilize cholesterol as a nutritional source but retains the enzyme responsible for its oxidation to cholestenone. Thus, the essential role of cholesterol metabolism in the intracellular survival of M. leprae is uncoupled from central carbon metabolism and energy production. Further elucidation of cholesterol metabolism in the host cell during M. leprae infection will establish the mechanism by which this lipid supports M. leprae intracellular survival and will open new avenues for novel leprosy therapies. IMPORTANCE: Our study focused on the obligate intracellular pathogen Mycobacterium leprae and its capacity to metabolize cholesterol. The data make an important contribution for those interested in understanding the mechanisms of mycobacterial pathogenesis, since they indicate that the essential role of cholesterol for M. leprae intracellular survival does not rely on its utilization as a nutritional source. Our findings reinforce the complexity of cholesterol's role in sustaining M. leprae infection. Further elucidation of cholesterol metabolism in the host cell during M. leprae infection will establish the mechanism by which this lipid supports M. leprae intracellular survival and will open new avenues for novel leprosy therapies.

Whole-Genome Sequences of Mycobacterium abscessus subsp. massiliense Isolates from Brazil.

The Mycobacterium abscessus complex comprises multidrug-resistant, opportunistic, and rapidly growing pathogens responsible for severe infections. Here, we report the genome composition of four Mycobacterium abscessus subsp. massiliense isolates from three sources: two from the lung of a cystic fibrosis patient, one from a mammary cyst, and one from a gutter system.

Studying effects of different protonation states of His11 and His102 in ribose-5-phosphate isomerase of Trypanosoma cruzi: an example of cooperative behavior.

The Trypanosoma cruzi ribose-5-phosphate isomerase B (TcRpiB) is a crucial piece in the pentose phosphate pathway and thus is a potential drug target for treatment of Chagas' disease. TcRpiB residues, such as Cys69, Asp45, Glu149 and Pro47, have confirmed their roles in substrate recognition, catalytic reaction and binding site conformation. However, the joint performance of His11 and His102, in the D-ribose-5-phosphate (R5P) in the catalysis is not well understood. In this work, we probed the influence of different protonation states of His11 and His102 on the behavior of the ligand R5P using molecular dynamics simulations, network analysis and thermodynamic integration. Simulations revealed that a protonated His11 combined with a neutral His102 (His11+‒His102) was able to stabilize the ligand R5P in the binding site. Moreover, calculated relative free energy differences showed that when protonated His11 was coupled to a neutral His102 an exergonic process takes place. On the other hand, neutral His11 combined with a protonated His102 (His11‒His102+), sampled conformations that resembled the catalyzed product D-ribulose-5-phosphate (Ru5P). Network analysis also demonstrated some peculiarities for these systems with some negatively correlated nodes in the binding site for His11‒His102+, and exclusive suboptimal paths for His11+‒His102. Therefore, the combined approach presented in this paper proposes two suitable protonation states for the TcRpiB catalytic mechanism, where an extra proton in either histidines might favor R5P binding or influence isomerization reaction to Ru5P. Our results may guide further in silico drug discovery studies. Communicated by Ramaswamy H. Sarma.

Exploring the Potential Role of Moonlighting Function of the Surface-Associated Proteins From Mycobacterium bovis BCG Moreau and Pasteur by Comparative Proteomic.

Surface-associated proteins from Mycobacterium bovis BCG Moreau RDJ are important components of the live Brazilian vaccine against tuberculosis. They are important targets during initial BCG vaccine stimulation and modulation of the host's immune response, especially in the bacterial-host interaction. These proteins might also be involved in cellular communication, chemical response to the environment, pathogenesis processes through mobility, colonization, and adherence to the host cell, therefore performing multiple functions. In this study, the proteomic profile of the surface-associated proteins from M. bovis BCG Moreau was compared to the BCG Pasteur reference strain. The methodology used was 2DE gel electrophoresis combined with mass spectrometry techniques (MALDI-TOF/TOF), leading to the identification of 115 proteins. Of these, 24 proteins showed differential expression between the two BCG strains. Furthermore, 27 proteins previously described as displaying moonlighting function were identified, 8 of these proteins showed variation in abundance comparing BCG Moreau to Pasteur and 2 of them presented two different domain hits. Moonlighting proteins are multifunctional proteins in which two or more biological functions are fulfilled by a single polypeptide chain. Therefore, the identification of such proteins with moonlighting predicted functions can contribute to a better understanding of the molecular mechanisms unleashed by live BCG Moreau RDJ vaccine components.

Mitochondrial disfunction and ROS production are essential for anti-Trypanosoma cruzi activity of ß-lapachone-derived naphthoimidazoles.

Chagas disease is caused by the hemoflagellate protozoa Trypanosoma cruzi and is one of the most important neglected tropical diseases, especially in Latin American countries, where there is an association between low-income populations and mortality. The nitroderivatives used in current chemotherapy are far from ideal and present severe limitations, justifying the continuous search for alternative drugs. Since the1990s, our group has been investigating the trypanocidal activity of natural naphthoquinones and their derivatives, and three naphthoimidazoles (N1, N2 and N3) derived from ß-lapachone were found to be most effective in vitro. Analysis of their mechanism of action via cellular, molecular and proteomic approaches indicates that the parasite mitochondrion contains one of the primary targets of these compounds, trypanothione synthetase (involved in trypanothione production), which is overexpressed after treatment with these compounds. Here, we further evaluated the participation of the mitochondria and reactive oxygen species (ROS) in the anti-T. cruzi action of naphthoimidazoles. Preincubation of epimastigotes and trypomastigotes with antioxidants (α-tocopherol and urate) strongly protected the parasites from the trypanocidal effect of naphthoimidazoles, decreasing the ROS levels produced and reverting the mitochondrial swelling phenotype. The addition of pro-oxidants (menadione and H2O2) before the treatment induced an increase in parasite lysis. Despite the O2 uptake and mitochondrial complex activity being strongly reduced by N1, N2 and N3, urate partially restored the mitochondrial metabolism only in N1-treated parasites. In parallel, MitoTEMPO, a mitochondrial-targeted antioxidant, protected the functionality of the mitochondria in N2- and N3-treated parasites. In addition, the trypanothione reductase activity was remarkably increased after treatment with N1 and N3, and molecular docking demonstrated that these two compounds were positioned in pockets of this enzyme. Based on our findings, the direct impairment of the mitochondrial electron transport chain by N2 and N3 led to an oxidative misbalance, which exacerbated ROS generation and led to parasite death. Although other mechanisms cannot be discounted, mainly in N1-treated parasites, further investigations are required.

AnEnPi: identification and annotation of analogous enzymes.

BACKGROUND: Enzymes are responsible for the catalysis of the biochemical reactions in metabolic pathways. Analogous enzymes are able to catalyze the same reactions, but they present no significant sequence similarity at the primary level, and possibly different tertiary structures as well. They are thought to have arisen as the result of independent evolutionary events. A detailed study of analogous enzymes may reveal new catalytic mechanisms, add information about the origin and evolution of biochemical pathways and disclose potential targets for drug development. RESULTS: In this work, we have constructed and implemented a new approach, AnEnPi (the Analogous Enzyme Pipeline), using a combination of bioinformatics tools like BLAST, HMMer, and in-house scripts, to assist in the identification, annotation, comparison and study of analogous and homologous enzymes. The algorithm for the detection of analogy is based i) on the construction of groups of homologous enzymes and ii) on the identification of cases where a given enzymatic activity is performed by two or more proteins without significant similarity between their primary structures. We applied this approach to a dataset obtained from KEGG Comprising all annotated enzymes, which resulted in the identification of 986 EC classes where putative analogy was detected (40.5% of all EC classes). AnEnPi is of considerable value in the construction of initial datasets that can be further curated, particularly in gene and genome annotation, in studies involving molecular evolution and metabolism and in the identification of new potential drug targets. CONCLUSION: AnEnPi is an efficient tool for detection and annotation of analogous enzymes and other enzymes in whole genomes. It is available for academic use at: http://bioinfo.pdtis.fiocruz.br/AnEnPi/

Structural modeling and docking studies of ribose 5-phosphate isomerase from Leishmania major and Homo sapiens: a comparative analysis for Leishmaniasis treatment.

Leishmaniases are caused by protozoa of the genus Leishmania and are considered the second-highest cause of death worldwide by parasitic infection. The drugs available for treatment in humans are becoming ineffective mainly due to parasite resistance; therefore, it is extremely important to develop a new chemotherapy against these parasites. A crucial aspect of drug design development is the identification and characterization of novel molecular targets. In this work, through an in silico comparative analysis between the genomes of Leishmania major and Homo sapiens, the enzyme ribose 5-phosphate isomerase (R5PI) was indicated as a promising molecular target. R5PI is an important enzyme that acts in the pentose phosphate pathway and catalyzes the interconversion of d-ribose-5-phosphate (R5P) and d-ribulose-5-phosphate (5RP). R5PI activity is found in two analogous groups of enzymes called RpiA (found in H. sapiens) and RpiB (found in L. major). Here, we present the first report of the three-dimensional (3D) structures and active sites of RpiB from L. major (LmRpiB) and RpiA from H. sapiens (HsRpiA). Three-dimensional models were constructed by applying a hybrid methodology that combines comparative and ab initio modeling techniques, and the active site was characterized based on docking studies of the substrates R5P (furanose and ring-opened forms) and 5RP. Our comparative analyses show that these proteins are structural analogs and that distinct residues participate in the interconversion of R5P and 5RP. We propose two distinct reaction mechanisms for the reversible isomerization of R5P to 5RP, which is catalyzed by LmRpiB and HsRpiA. We expect that the present results will be important in guiding future molecular modeling studies to develop new drugs that are specially designed to inhibit the parasitic form of the enzyme without significant effects on the human analog.

ESTs from Seeds to Assist the Selective Breeding of Jatropha curcas L. for Oil and Active Compounds.

We report here on the characterization of a cDNA library from seeds of Jatropha curcas L. at three stages of fruit maturation before yellowing. We sequenced a total of 2200 clones and obtained a set of 931 non-redundant sequences (unigenes) after trimming and quality control, ie, 140 contigs and 791 singlets with PHRED quality ≥10. We found low levels of sequence redundancy and extensive metabolic coverage by homology comparison to GO. After comparison of 5841 non-redundant ESTs from a total of 13193 reads from GenBank with KEGG, we identified tags with nucleotide variations among J. curcas accessions for genes of fatty acid, terpene, alkaloid, quinone and hormone pathways of biosynthesis. More specifically, the expression level of four genes (palmitoyl-acyl carrier protein thioesterase, 3-ketoacyl-CoA thiolase B, lysophosphatidic acid acyltransferase and geranyl pyrophosphate synthase) measured by real-time PCR proved to be significantly different between leaves and fruits. Since the nucleotide polymorphism of these tags is associated to higher level of gene expression in fruits compared to leaves, we propose this approach to speed up the search for quantitative traits in selective breeding of J. curcas. We also discuss its potential utility for the selective breeding of economically important traits in J. curcas.

MamMiBase: a mitochondrial genome database for mammalian phylogenetic studies.

UNLABELLED: MamMiBase, the mammalian mitochondrial genome database, is a relational database of complete mitochondrial genome sequences of mammalian species. The database is useful for phylogenetic analysis, since it allows a ready retrieval of nucleotide and aminoacid individual alignments, in three different formats (NEXUS for PAUP program, for MEGA program and for PHYLIP program) of the 13 protein coding mitochondrial genes. The user may download the sequences that are useful for him/her based on their parameters values, such as sequence length, p-distances, base content, transition transversion ratio, gamma, which are also given by MamMiBase. A simple phylogenetic tree (neighbor-joining tree with Jukes Cantor distance) is also available for download, useful for parameter calculations and other simple tasks. AVAILABILITY: MamMiBase is available at http://www.mammibase.lncc.br