First report of environmental isolation of Cryptococcus and Cryptococcus-like yeasts from Boyacá, Colombia.

The Cryptococcus genus comprises more than 100 species, of which C. neoformans and C. gattii are the leading cause of cryptococcosis. The distribution of C. gattii and C. neoformans species complexes has been extensively studied and widely reported globally. Other species such as Naganishia albida, Papiliotrema laurentii, and Papiliotrema flavescens have been reported as pathogenic yeasts. Since there are no reports of environmental isolation in the Boyacá region (Colombia), this study aimed to isolate and characterize Cryptococcus and Cryptococcus-like yeasts from pigeon feces, Eucalyptus, and olive trees distributed in the municipalities of Tunja and Ricaute Alto. The environmental data was recovered, and the isolations obtained were identified by microscopy, biochemical test, MALDI-TOF MS, URA5-RFLP, and sequencing of the ITS and LSU loci. For the 93 pigeon dropping samples collected in Tunja, 23 yielded to C. neoformans, 3 to N. globosa, 2 N. albida and 1 to P. laurentii. Of the 1188 samples collected from olive trees, 17 (1.43%) positive samples were identified as C. gattii species complex (4), C. neoformans species complex (2), P. laurentii (3), N. albida (2), N. globosa (5) and P. flavescens (1). Likewise, specimens of C. neoformans presented molecular type VNI and molecular type VNII; for C. gattii the molecular types found were VGIII and one VGIV by URA5-RFLP but VGIII by MALDI-TOF and sequencing of the ITS and LSU. Therefore, it can be concluded that the species of Cryptococcus, Naganishia and Papiliotrema genera, are present in the environment of Boyacá, and show a predilection for climate conditions that are typical of this region.

Isolation and characterization of wood-decomposing basidiomycetes from the Andean Forest in Boyacá, Colombia.

This study explores the biotechnological potential of lignocellulolytic fungi collected in an oak forest. Fungal collections were obtained from natural reserves located in Boyacá-Colombia, ranging from 2700 to 3000 m.a.s.l. Twenty-three strains were isolated on malt agar, molecular characterization was performed, and ligninolytic and cellulolytic enzymatic activities were screened. Several white-rot fungi of biotechnological importance were identified as follows: Trametes sp., Trametes versicolor, Trametes villosa, Pycnoporus sanguineus, Bjerkandera adjusta, Lentinula boryana, Panus conchatus, Antrodia neotropica, Brunneoporus malicola, Laetiporus gilbertsonii, Stereum sp., Ganoderma sp., and Dichomitus sp. The strains T. versicolor 0554 and 0583, T. villosa 0562, and B. adusta 0556 showed the highest response in the qualitative enzymatic assays. These strains were used to determine their ability to decolorate the dyes aniline blue and Congo red, and it was found that T. villosa 0562 reached a level of decolorization close to 90% after 48 h of submerged culture. The fungal strains obtained here could offer alternatives to develop a process to accomplish sustainable development objectives.

The Combination of Iron and Copper Increases Pathogenicity and Induces Proteins Related to the Main Virulence Factors in Clinical Isolates of Cryptococcus neoformans var. grubii.

In fungi, metals are associated with the expression of virulence factors. However, it is unclear whether the uptake of metals affects their pathogenicity. This study aimed to evaluate the effect of iron/copper in modulating pathogenicity and proteomic response in two clinical isolates of C. neoformans with high and low pathogenicity. METHODS: In both isolates, the effect of 50 µM iron and 500 µM copper on pathogenicity, capsule induction, and melanin production was evaluated. We then performed a quantitative proteomic analysis of cytoplasmic extracts exposed to that combination. Finally, the effect on pathogenicity by iron and copper was evaluated in eight additional isolates. RESULTS: In both isolates, the combination of iron and copper increased pathogenicity, capsule size, and melanin production. Regarding proteomic data, proteins with increased levels after iron and copper exposure were related to biological processes such as cell stress, vesicular traffic (Ap1, Vps35), cell wall structure (Och1, Ccr4, Gsk3), melanin biosynthesis (Hem15, Mln2), DNA repair (Chk1), protein transport (Mms2), SUMOylation (Uba2), and mitochondrial transport (Atm1). Increased pathogenicity by exposure to metal combination was also confirmed in 90% of the eight isolates. CONCLUSIONS: The combination of these metals enhances pathogenicity and increases the abundance of proteins related to the main virulence factors.

Estrategias para la evaluación de extractos de polifenoles en modelos in vitro de cáncer de vías digestivas / Strategies for Evaluation of Polyphenol Extracts on in vitro Models of Digestive Tract Cancer / Estratégias para a avaliação de extratos de polifenóis em modelos in vitro de câncer do sistema digestivo

Introducción: Los polifenoles son compuestos que se encuentran naturalmente en alimentos como frutas, verduras, té, vino y chocolates, a los que se les atribuyen beneficios a la salud humana por su capacidad antioxidante. El cáncer de las vías digestivas se encuentra entre la tercera y quinta causas de muerte para la población, por lo que se ha incrementado el interés por realizar los estudios dirigidos a encontrar compuestos polifenólicos que ayuden en su prevención o tratamiento. Objetivo: Identificar las estrategias disponibles para la evaluación de polifenoles en células de cáncer de vías digestivas. Metodología: Búsqueda de literatura en bases de datos como Ovid, Pubmed, Science Direct y Elsevier Journal. Se seleccionaron artículos en los cuales se reporta el efecto biológico de los polifenoles sobre líneas celulares de cáncer de vías digestivas publicados entre 2012 y 2022. Resultados: Varios estudios han reportado el uso de un buen número de líneas celulares como modelos in vitropara estudios de polifenoles en cáncer y han resaltado las líneas AGS y HT-29, además de técnicas para la caracterización de los polifenoles, como el ensayo 2,2-Difenil-I-Picril Hidrazilo (DPPH). Sin embargo, para evaluar el efecto biológico se identifican diversas pruebas que deben analizarse antes de su implementación. Conclusiones: En la literatura se identifica que existen varias alternativas y estrategias para la evaluación de extrac-tos vegetales en cultivos in vitro de cáncer de vías digestivas; no obstante, antes de pasar al diseño experimental, deben tenerse en cuenta una serie de consideraciones para garantizar la utilidad de los resultados.

Introduction: Polyphenols are compounds naturally found in foods such as fruits, vegetables, tea, wine and chocolates, and it was attributed with benefits to human health due to their antioxidant capacity. Cancer of the digestive tract is between the third and fifth cause of death for the population, increasing the interest in carrying out studies aimed at finding polyphenolic compounds that help in their prevention or treatment. Objective: Identify the available strategies for the evaluation of polyphenols in digestive tract cancer cells. Method: A literature search was performed in databases such Ovid, Pubmed, Science Direct and Elsevier Journal and selected articles reporting the biological effect of polyphenols on digestive tract cancer cell lines, published between 2012 and 2022. Results: Currently studies report the use of a good number of cell lines as in vitro models for poly-phenol studies in cancer highlighting the AGS and HT-29 lines, in addition to techniques for the characterization of polyphenols such as the α, α-diphenyl-ß-picrylhydrazyl DPPH assay, however, to evaluate the biological effect various tests are identified that should be analyzed before implemen-tation. Conclusions: The literature identifies that there are many alternatives and strategies for the evaluation of plant extracts in in vitro cultures of digestive tract cancer, however, before moving on to the experimental design, a number of considerations should be taken into account to ensure the usability of the results

Introdução: Os polifenóis são compostos encontrados naturalmente em alimentos como frutas, legumes, chá, vinho e chocolates, aos quais são atribuídos benefícios para a saúde humana devido à sua capacidade antioxidante. O câncer do sistema digestivo está entre a terceira e a quinta principais causas de morte na população, o que levou a um interesse crescente em estudos destinados a encon-trar compostos polifenólicos que ajudem a prevenir ou tratar esse tipo de câncer. Objetivo: Identificar as estratégias disponíveis para a avaliação dos polifenóis nas células cancerosas do sistema digestivo. Metodologia: Pesquisa bibliográfica em bases de dados como Ovid, Pubmed, Science Direct e Elsevier Journal. Foram selecionados artigos que relatam o efeito biológico dos polifenóis em linhas celulares de câncer do sistema digestivo, publicados entre 2012 e 2022. Resultados: Vários estudos relataram a utilização de várias linhas celulares como modelos in vitro para estudos de polifenóis no câncer destacando as linhas AGS e HT-29, bem como técnicas para a ca-racterização de polifenóis, como o ensaio 2,2-Difenil-I-Picril Hidrazil (DPPH). No entanto, para avaliar o efeito biológico, são identificados vários testes que devem ser analisados antes da sua aplicação. Conclusões: A literatura identifica que existem várias alternativas e estratégias para a avaliação de extratos de plantas em culturas in vitro de câncer do sistema digestivo; no entanto, antes de passar à concepção experimental, é necessário ter em conta uma série de considerações para garantir a uti-lidade dos resultados

Environmental Status of Cryptococcus neoformans and Cryptococcus gattii in Colombia.

The genus Cryptococcus comprises more than 80 species, including C. neoformans and C. gattii, which are pathogenic to humans, mainly affecting the central nervous system. The two species differ in geographic distribution and environmental niche. C. neoformans has a worldwide distribution and is often isolated from bird droppings. On the contrary, C. gattii is reported in tropical and subtropical regions and is associated with Eucalyptus species. This review aims to describe the distribution of environmental isolates of the Cryptococcus neoformans species complex and the Cryptococcus gattii species complex in Colombia. A systematic investigation was carried out using different databases, excluding studies of clinical isolates reported in the country. The complex of the species of C. gattii is recovered mainly from trees of the genus Eucalyptus spp., while the complex of the species of C. neoformans is recovered mainly from avian excrement, primarily Columba livia (pigeons) excrement. In addition, greater positivity was found at high levels of relative humidity. Likewise, an association was observed between the presence of the fungus in places with little insolation and cold or temperate temperatures compared to regions with high temperatures.

Microbiota and skin defense peptides may facilitate coexistence of two sympatric Andean frog species with a lethal pathogen.

Management of hyper-virulent generalist pathogens is an emergent global challenge, yet for most disease systems we lack a basic understanding as to why some host species suffer mass mortalities, while others resist epizootics. We studied two sympatric species of frogs from the Colombian Andes, which coexist with the amphibian pathogen Batrachochytrium dendrobatidis (Bd), to understand why some species did not succumb to the infection. We found high Bd prevalence in juveniles for both species, yet infection intensities remained low. We also found that bacterial community composition and host defense peptides are specific to amphibian life stages. We detected abundant Bd-inhibitory skin bacteria across life stages and Bd-inhibitory defense peptides post-metamorphosis in both species. Bd-inhibitory bacteria were proportionally more abundant in adults of both species than in earlier developmental stages. We tested for activity of peptides against the skin microbiota and found that in general peptides did not negatively affect bacterial growth and in some instances facilitated growth. Our results suggest that symbiotic bacteria and antimicrobial peptides may be co-selected for, and that together they contribute to the ability of Andean amphibian species to coexist with the global pandemic lineage of Bd.

Genotypic Diversity Is Independent of Pathogenicity in Colombian Strains of Cryptococcus neoformans and Cryptococcus gattii in Galleria mellonella.

Cryptococcosis is a potentially fatal opportunistic mycosis that affects the lungs and central nervous system. It has been suggested that certain strains of C. neoformans/C. gattii may have the potential to be more virulent according to the molecular type. This study aims to investigate the association between virulence in the G. mellonella model and genotypic diversity of Colombian clinical and environmental isolates of C. neoformans/C. gattii. A total of 33 clinical and 12 environmental isolates were selected according to their geographical origin and sequence types (STs). Pathogenicity was determined using the G. mellonella model, and the cell and capsular size before and after inoculation was determined. For C. neoformans, virulence in G. mellonella revealed that death occurred on average on day 6 (p < 0.05) and that ST5C, 6C, 25C and 71C were the most virulent. In C. gattii, death occurred at 7.3 days (p < 0.05), and ST47C, 58C, 75A and 106C were the most virulent. Capsular size increased for both species after passage in G. mellonella. In conclusion, the pathogenicity of Cryptococcus strains in the G. mellonella invertebrate model is independent of molecular type or pathogenicity factor, even within the same ST, but it is possible to find variable degrees of pathogenicity.

Construction of Mycobacterium tuberculosis cdd knockout and evaluation of invasion and growth in macrophages.

Cytidine deaminase (MtCDA), encoded by cdd gene (Rv3315c), is the only enzyme identified in nucleotide biosynthesis pathway of Mycobacterium tuberculosis that is able to recycle cytidine and deoxycytidine. An M. tuberculosis knockout strain for cdd gene was obtained by allelic replacement. Evaluation of mRNA expression validated cdd deletion and showed the absence of polar effect. MudPIT LC-MS/MS data indicated thymidine phosphorylase expression was decreased in knockout and complemented strains. The cdd disruption does not affect M. tuberculosis growth both in Mid- dlebrook 7H9 and in RAW 264.7 cells, which indicates that cdd is not important for macrophage invasion and virulence.

Characterisation of iunH gene knockout strain from Mycobacterium tuberculosis

BACKGROUND Tuberculosis (TB) is an infectious disease caused mainly by the bacillus Mycobacterium tuberculosis. The better understanding of important metabolic pathways from M. tuberculosis can contribute to the development of novel therapeutic and prophylactic strategies to combat TB. Nucleoside hydrolase (MtIAGU-NH), encoded by iunH gene (Rv3393), is an enzyme from purine salvage pathway in M. tuberculosis. MtIAGU-NH accepts inosine, adenosine, guanosine, and uridine as substrates, which may point to a pivotal metabolic role. OBJECTIVES Our aim was to construct a M. tuberculosis knockout strain for iunH gene, to evaluate in vitro growth and the effect of iunH deletion in M. tuberculosis in non-activated and activated macrophages models of infection. METHODS A M. tuberculosis knockout strain for iunH gene was obtained by allelic replacement, using pPR27xylE plasmid. The complemented strain was constructed by the transformation of the knockout strain with pNIP40::iunH. MtIAGU-NH expression was analysed by Western blot and LC-MS/MS. In vitro growth was evaluated in Sauton’s medium. Bacterial load of non-activated and interferon-γ activated RAW 264.7 cells infected with knockout strain was compared with wild-type and complemented strains. FINDINGS Western blot and LC-MS/MS validated iunH deletion at protein level. The iunH knockout led to a delay in M. tuberculosis growth kinetics in Sauton’s medium during log phase, but did not affect bases and nucleosides pool in vitro. No significant difference in bacterial load of knockout strain was observed when compared with both wild-type and complemented strains after infection of non-activated and interferon-γ activated RAW 264.7 cells. MAIN CONCLUSION The disruption of iunH gene does not influence M. tuberculosis growth in both non-activated and activated RAW 264.7 cells, which show that iunH gene is not important for macrophage invasion and virulence. Our results indicated that MtIAGU-NH is not a target for drug development.

Characterisation of iunH gene knockout strain from Mycobacterium tuberculosis.

BACKGROUND: Tuberculosis (TB) is an infectious disease caused mainly by the bacillus Mycobacterium tuberculosis. The better understanding of important metabolic pathways from M. tuberculosis can contribute to the development of novel therapeutic and prophylactic strategies to combat TB. Nucleoside hydrolase (MtIAGU-NH), encoded by iunH gene (Rv3393), is an enzyme from purine salvage pathway in M. tuberculosis. MtIAGU-NH accepts inosine, adenosine, guanosine, and uridine as substrates, which may point to a pivotal metabolic role. OBJECTIVES: Our aim was to construct a M. tuberculosis knockout strain for iunH gene, to evaluate in vitro growth and the effect of iunH deletion in M. tuberculosis in non-activated and activated macrophages models of infection. METHODS: A M. tuberculosis knockout strain for iunH gene was obtained by allelic replacement, using pPR27xylE plasmid. The complemented strain was constructed by the transformation of the knockout strain with pNIP40::iunH. MtIAGU-NH expression was analysed by Western blot and LC-MS/MS. In vitro growth was evaluated in Sauton's medium. Bacterial load of non-activated and interferon-γ activated RAW 264.7 cells infected with knockout strain was compared with wild-type and complemented strains. FINDINGS: Western blot and LC-MS/MS validated iunH deletion at protein level. The iunH knockout led to a delay in M. tuberculosis growth kinetics in Sauton's medium during log phase, but did not affect bases and nucleosides pool in vitro. No significant difference in bacterial load of knockout strain was observed when compared with both wild-type and complemented strains after infection of non-activated and interferon-γ activated RAW 264.7 cells. MAIN CONCLUSION: The disruption of iunH gene does not influence M. tuberculosis growth in both non-activated and activated RAW 264.7 cells, which show that iunH gene is not important for macrophage invasion and virulence. Our results indicated that MtIAGU-NH is not a target for drug development.

Biochemical characterization of recombinant nucleoside hydrolase from Mycobacterium tuberculosis H37Rv.

Tuberculosis (TB) is a major global health threat. There is a need for the development of more efficient drugs for the sterilization of the disease's causative agent, Mycobacterium tuberculosis (MTB). A more comprehensive understanding of the bacilli's nucleotide metabolic pathways could aid in the development of new anti-mycobacterial drugs. Here we describe expression and purification of recombinant iunH-encoded nucleoside hydrolase from MTB (MtIAGU-NH). Glutaraldehyde cross-linking results indicate that MtIAGU-NH predominates as a monomer, presenting varied oligomeric states depending upon binding of ligands. Steady-state kinetics results show that MtIAGU-NH has broad substrate specificity, accepting inosine, adenosine, guanosine, and uridine as substrates. Inosine and adenosine displayed positive homotropic cooperativity kinetics, whereas guanosine and uridine displayed hyperbolic saturation curves. Measurements of kinetics of ribose binding to MtIAGU-NH by fluorescence spectroscopy suggest two pre-existing forms of enzyme prior to ligand association. The intracellular concentrations of inosine, uridine, hypoxanthine, and uracil were determined and thermodynamic parameters estimated. Thermodynamic activation parameters (Ea, ΔG(#), ΔS(#), ΔH(#)) for MtIAGU-NH-catalyzed chemical reaction are presented. Results from mass spectrometry, isothermal titration calorimetry (ITC), pH-rate profile experiment, multiple sequence alignment, and molecular docking experiments are also presented. These data should contribute to our understanding of the biological role played by MtIAGU-NH.

Combining molecular dynamics and docking simulations of the cytidine deaminase from Mycobacterium tuberculosis H37Rv.

Cytidine Deaminase (CD) is an evolutionarily conserved enzyme that participates in the pyrimidine salvage pathway recycling cytidine and deoxycytidine into uridine and deoxyuridine, respectively. Here, our goal is to apply computational techniques in the pursuit of potential inhibitors of Mycobacterium tuberculosis CD (MtCDA) enzyme activity. Molecular docking simulation was applied to find the possible hit compounds. Molecular dynamics simulations were also carried out to investigate the physically relevant motions involved in the protein-ligand recognition process, aiming at providing estimates for free energy of binding. The proposed approach was capable of identifying a potential inhibitor, which was experimentally confirmed by IC(50) evaluation. Our findings open up the possibility to extend this protocol to different databases in order to find new potential inhibitors for promising targets based on a rational drug design process.