Predominantly Pro-Inflammatory Phenotype with Mixed M1/M2 Polarization of Peripheral Blood Classical Monocytes and Monocyte-Derived Macrophages among Patients with Excessive Ethanol Intake.

Excessive alcohol consumption impairs the immune system, induces oxidative stress, and triggers the activation of peripheral blood (PB) monocytes, thereby contributing to alcoholic liver disease (ALD). We analyzed the M1/M2 phenotypes of circulating classical monocytes and macrophage-derived monocytes (MDMs) in excessive alcohol drinkers (EADs). PB samples from 20 EADs and 22 healthy controls were collected for isolation of CD14+ monocytes and short-term culture with LPS/IFNγ, IL4/IL13, or without stimulation. These conditions were also used to polarize MDMs into M1, M2, or M0 phenotypes. Cytokine production was assessed in the blood and culture supernatants. M1/M2-related markers were analyzed using mRNA expression and surface marker detection. Additionally, the miRNA profile of CD14+ monocytes was analyzed. PB samples from EADs exhibited increased levels of pro-inflammatory cytokines. Following short-term culture, unstimulated blood samples from EADs showed higher levels of soluble TNF-α and IL-8, whereas monocytes expressed increased levels of surface TNF-α and elevated mRNA expression of pro-inflammatory cytokines and inducible nitric oxide synthase. MDMs from EADs showed higher levels of TNF-α and CD206 surface markers and increased IL-10 production. LPS/IFNγ induced higher mRNA expression of Nrf2 only in the controls. miRNA analysis revealed a distinctive miRNA profile that is potentially associated with liver carcinogenesis and ALD through inflammation and oxidative stress. This study confirms the predominantly pro-inflammatory profile of PB monocytes among EADs and suggests immune exhaustion features in MDMs.

A Trypanosoma cruzi phosphoglycerate kinase isoform with a Per-Arnt-Sim domain acts as a possible sensor for intracellular conditions.

Per-ARNT-Sim (PAS) domains constitute a family of domains present in a wide variety of prokaryotic and eukaryotic organisms. They form part of the structure of various proteins involved in diverse cellular processes. Regulation of enzymatic activity and adaptation to environmental conditions, by binding small ligands, are the main functions attributed to PAS-containing proteins. Recently, genes for a diverse set of proteins with a PAS domain were identified in the genomes of several protists belonging to the group of kinetoplastids, however, until now few of these proteins have been characterized. In this work, we characterize a phosphoglycerate kinase containing a PAS domain present in Trypanosoma cruzi (TcPAS-PGK). This PGK isoform is an active enzyme of 58 kDa with a PAS domain located at its N-terminal end. We identified the protein's localization within glycosomes of the epimastigote form of the parasite by differential centrifugation and selective permeabilization of its membranes with digitonin, as well as in an enriched mitochondrial fraction. Heterologous expression systems were developed for the protein with the N-terminal PAS domain (PAS-PGKc) and without it (PAS-PGKt), and the substrate affinities of both forms of the protein were determined. The enzyme does not exhibit standard Michaelis-Menten kinetics. When evaluating the dependence of the specific activity of the recombinant PAS-PGK on the concentration of its substrates 3-phosphoglycerate (3PGA) and ATP, two peaks of maximal activity were found for the complete enzyme with the PAS domain and a single peak for the enzyme without the domain. Km values measured for 3PGA were 219 ± 26 and 8.8 ± 1.3 µM, and for ATP 291 ± 15 and 38 ± 2.2 µM, for the first peak of PAS-PGKc and for PAS-PGKt, respectively, whereas for the second PAS-PGKc peak values of approximately 1.1-1.2 mM were estimated for both substrates. Both recombinant proteins show inhibition by high concentrations of their substrates, ATP and 3PGA. The presence of hemin and FAD exerts a stimulatory effect on PAS-PGKc, increasing the specific activity by up to 55%. This stimulation is not observed in the absence of the PAS domain. It strongly suggests that the PAS domain has an important function in vivo in T. cruzi in the modulation of the catalytic activity of this PGK isoform. In addition, the PAS-PGK through its PAS and PGK domains could act as a sensor for intracellular conditions in the parasite to adjust its intermediary metabolism.

Antimicrobial Peptides (AMPs): Potential Therapeutic Strategy against Trypanosomiases?

Trypanosomiases are a group of tropical diseases that have devastating health and socio-economic effects worldwide. In humans, these diseases are caused by the pathogenic kinetoplastids Trypanosoma brucei, causing African trypanosomiasis or sleeping sickness, and Trypanosoma cruzi, causing American trypanosomiasis or Chagas disease. Currently, these diseases lack effective treatment. This is attributed to the high toxicity and limited trypanocidal activity of registered drugs, as well as resistance development and difficulties in their administration. All this has prompted the search for new compounds that can serve as the basis for the development of treatment of these diseases. Antimicrobial peptides (AMPs) are small peptides synthesized by both prokaryotes and (unicellular and multicellular) eukaryotes, where they fulfill functions related to competition strategy with other organisms and immune defense. These AMPs can bind and induce perturbation in cell membranes, leading to permeation of molecules, alteration of morphology, disruption of cellular homeostasis, and activation of cell death. These peptides have activity against various pathogenic microorganisms, including parasitic protists. Therefore, they are being considered for new therapeutic strategies to treat some parasitic diseases. In this review, we analyze AMPs as therapeutic alternatives for the treatment of trypanosomiases, emphasizing their possible application as possible candidates for the development of future natural anti-trypanosome drugs.

Phosphoglycerate kinase: structural aspects and functions, with special emphasis on the enzyme from Kinetoplastea.

Phosphoglycerate kinase (PGK) is a glycolytic enzyme that is well conserved among the three domains of life. PGK is usually a monomeric enzyme of about 45 kDa that catalyses one of the two ATP-producing reactions in the glycolytic pathway, through the conversion of 1,3-bisphosphoglycerate (1,3BPGA) to 3-phosphoglycerate (3PGA). It also participates in gluconeogenesis, catalysing the opposite reaction to produce 1,3BPGA and ADP. Like most other glycolytic enzymes, PGK has also been catalogued as a moonlighting protein, due to its involvement in different functions not associated with energy metabolism, which include pathogenesis, interaction with nucleic acids, tumorigenesis progression, cell death and viral replication. In this review, we have highlighted the overall aspects of this enzyme, such as its structure, reaction kinetics, activity regulation and possible moonlighting functions in different protistan organisms, especially both free-living and parasitic Kinetoplastea. Our analysis of the genomes of different kinetoplastids revealed the presence of open-reading frames (ORFs) for multiple PGK isoforms in several species. Some of these ORFs code for unusually large PGKs. The products appear to contain additional structural domains fused to the PGK domain. A striking aspect is that some of these PGK isoforms are predicted to be catalytically inactive enzymes or 'dead' enzymes. The roles of PGKs in kinetoplastid parasites are analysed, and the apparent significance of the PGK gene duplication that gave rise to the different isoforms and their expression in Trypanosoma cruzi is discussed.

Antibiotic- and heavy-metal resistance in bacteria isolated from deep subsurface in El Callao region, Venezuela / Resistencia a antibioticos y metales pesados en bacterias aisladas de subsuelo en la región El Callao, Venezuela

Se investigó el efecto de la contaminación con mercurio (Hg) en las comunidades bacterianas del subsuelo profundo en la región de El Callao (Estado Bolívar, Venezuela). Se estudiaron comunidades bacterianas de dos niveles de profundidad (-288 m y -388 m) en una mina de oro con el propósito de describir las características más relevantes de las bacterias indígenas cultivables que colonizaban esta mina. Se evaluaron los patrones de resistencia a antibióticos y metales pesados, presencia del gen merA y plásmidos en aislados resistentes. Se encontró una elevada frecuencia de bacterias indígenas resistentes al Hg y otros metales pesados. De 76 aislados Hg-resistentes probados 73.7 % fueron adicionalmente resistentes a ampicilina; 86.8 % a cloranfenicol; 67.1 % a tetraciclina; 56.6 % a estreptomicina y 51.3 % a kanamicina. Además, se encontró que 40.74 % (-328 m) y 26.53 % (-388 m) de las bacterias Hg-resistentes fueron simultáneamente resistentes tanto a cuatro como a cinco de estos antibióticos. Se detectó la presencia de plásmidos de alto y bajo peso molecular y, a pesar de que los aislados mostraban resistencia a compuestos mercuriales, la presencia del gen merA fue detectada solo en 71.05 % de los cepas. Estos resultados sugieren que la exposición a Hg podría ser una presión selectiva en la proliferación de bacterias resistentes a antibióticos y promover el mantenimiento y propagación de estos genes de resistencia. Sin embargo, la existencia de tales resistencias a estas profundidades podría también apoyar la idea de que la resistencia a antibióticos en estas bacterias es natural y tiene un origen más antiguo que su exposición al Hg.

The effect of contamination with mercury (Hg) in the deep subsurface bacterial communities in the region of El Callao (Bolívar State, Venezuela) was investigated. Bacterial communities from two deep levels (-288 m and -388 m) in a gold mine were studied with the aim of describe the most relevant features of their colonizing indigenous culturable bacteria. Antibiotic and heavy metals resistance patterns, presence of the merA gene and plasmids in resistant isolates were evaluated. A high frequency of resistant indigenous bacteria to Hg and other heavy metals was found. From 76 Hg-resistant isolates tested 73.7 % were, in addition, resistant to ampicillin, 86.8% to chloramphenicol, 67.1 % for tetracycline, 56.6 % streptomycin, and 51.3 % kanamycin. Furthermore, it was found that 40.74 % (-328 mm) and 26.53 % (-388 m) of Hg-resistant bacteria were simultaneously resistant to both four and five of these antibiotics. The presence of low and high molecular weight plasmids was detected and, despite that isolated showed resistance to mercurial compounds, the presence of the gene merA was detected only in 71.05 % of strains. These results suggest that exposure to Hg could be a selective pressure on the proliferation of antibiotic-resistant bacteria and promote the preservation and propagation of these resistance genes. However, the existence of such resistances to these depths could also support the idea that antibiotic resistance in these bacteria is natural and has a more ancient origin than their exposure to Hg.