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Background: Cryptococcus neoformans is an opportunistic fungal pathogen that can cause meningitis in immunocompromised individuals. The objective of this work was to study the relationship between the phenotypes and genotypes of isolates of clinical origin from different cities in Colombia. Methods: Genome classification of 29 clinical isolates of C. neoformans var. grubii was performed using multilocus sequence typing (MLST), and genomic sequencing was used to genotype protein-coding genes. Pathogenicity was assessed in a larval model, and melanin production and capsule size were evaluated in vitro and in vivo. Results: Eleven MLST sequence types (STs) were found, the most frequent being ST69 (n = 9), ST2, ST93, and ST377 (each with n = 4). In the 29 isolates, different levels of pigmentation, capsule size and pathogenicity were observed. Isolates classified as highly pathogenic showed a tendency to exhibit larger increases in capsule size. In the analysis of polymorphisms, 48 non-synonymous variants located in the predicted functional domains of 39 genes were found to be associated with capsule size change, melanin, or pathogenicity. Conclusions: No clear patterns were found in the analysis of the phenotype and genotype of Cryptococcus. However, the data suggest that the increase in capsule size is a key variable for the differentiation of pathogenic isolates, regardless of the method used for its induction., Ministerio de Ciencia, Innovación y Universidades (España), Research Vice Presidency of the Pontificia Universidad Javeriana in Bogotá, Colombia, Ministerio de Ciencia, Tecnología e Innovación (Colombia), Depto. de Microbiología y Parasitología, Fac. de Farmacia, TRUE, pub

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., Ministerio de Ciencia e Innovación (MICINN), Ministerio de Ciencia e Innovación (MICINN)/ISCII, Research Vice Presidency of the Pontificia Universidad Javeriana in Bogotá, Colombia, I, Depto. de Microbiología y Parasitología, Fac. de Farmacia, TRUE, pub

Durante el desarrollo de la enfermedad fúngica invasiva (EFI), los metales participan de manera importante en la interacción entre los hongos y las células del hospedero: el hierro y el cobre actúan como cofactores en múltiples reacciones enzimáticas. Estos metales en la célula fúngica se relacionan de manera importante con la expresión de factores de virulencia gobernados por metaloproteasas [1,2]. Por otro lado, en el hospedero, son esenciales en la respuesta eficiente de mecanismos efectores, por ejemplo, en las células del sistema inmunológico. Por lo tanto, el éxito en el establecimiento de la EFI pude depender de la capacidad para competir y adquirir metales del entorno del hospedero [3,4].La levadura ambiental C. neoformans forma parte del complejo de especies que causa la criptococosis, una micosis oportunista adquirida por la inhalación de propágulos fúngicos presentes en el ambiente. Es una infección potencialmente fatal que afecta los pulmones y el sistema nervioso central [5]. Esta levadura produce infecciones frecuentemente en individuos inmunocomprometidos, por ejemplo, en pacientes con SIDA, con trasplante de órganos o con terapias inmunosupresoras..., During invasive fungal disease (IFD) development, metals play an important role in the interaction between fungi and host cells, iron, and copper act as cofactors in multiple enzymatic reactions. These metals in the fungal cell are associated to the expression of virulence factors governed by metalloproteases [1,2]. On the other hand, in the host, they are essential in the efficient response of effector mechanisms, for example, in cells of the immune system. Therefore, the success of IFD establishment may depend on the ability to acquire and compete for metals from the host environment [3,4].The environmental yeast C. neoformans is part of the species complex that causes cryptococcosis, an opportunistic mycosis, acquired by inhalation of fungal propagules present in the environment. It´s a potentially fatal infection that affects the lungs and central nervous system [5]. This yeast frequently affects immunocompromised individuals, for example, in patients with AIDS, organ transplants, or immunosuppressive therapies...

White Band Disease (WBD) is one of the most harmful coral syndromes in the Caribbean, deteriorating the structure of many corals during the eighties and nineties. Since its etiology is not entirely known, the use of techniques aimed a t identifying the associated agents could provide an approach to the composition of the populations involved in the deterioration of A. cervicornis. Therefore, this research's objective was to isolate and characterize bacteria associated with WBD in A. cervicornis from hanging nurseries of Rosario and San Bernardo Corals National Natural Park, Colombia, using MALDI-TO F MS and PCR. The results showed the presence of Bacillus cereus bacteria in individuals with bleaching signs from nurseries and transplant areas on Isla Tesoro and Isla Ceiner. One of these strains sequenced and phylogenetically analyzed was closely related t o Bacillus thuringiensis, with similarities of up to 99%. The presence of B. cereus could indicate a special relationship between this microorganism and WBD. However, it is imperative to carry out more studies on identifying the bacteria associated with this disease to support these findings. © 2022 Latin American and Caribbean Consortium of Engineering Institutions. All rights reserved.

Durante el desarrollo de la enfermedad fúngica invasiva (EFI), los metales participan de manera importante en la interacción entre los hongos y las células del hospedero: el hierro y el cobre actúan como cofactores en múltiples reacciones enzimáticas. Estos metales en la célula fúngica se relacionan de manera importante con la expresión de factores de virulencia gobernados por metaloproteasas [1,2]. Por otro lado, en el hospedero, son esenciales en la respuesta eficiente de mecanismos efectores, por ejemplo, en las células del sistema inmunológico. Por lo tanto, el éxito en el establecimiento de la EFI pude depender de la capacidad para competir y adquirir metales del entorno del hospedero [3,4].La levadura ambiental C. neoformans forma parte del complejo de especies que causa la criptococosis, una micosis oportunista adquirida por la inhalación de propágulos fúngicos presentes en el ambiente. Es una infección potencialmente fatal que afecta los pulmones y el sistema nervioso central [5]. Esta levadura produce infecciones frecuentemente en individuos inmunocomprometidos, por ejemplo, en pacientes con SIDA, con trasplante de órganos o con terapias inmunosupresoras...

Objective. Isolate, identify and molecularly characterize pathogenic Cryptococcus isolates for humans from environmental and clinical samples from the city of Cúcuta. Materials and methods. A total of 1300 samples were collected from 446 trees of 10 different species in 10 public areas of Cúcuta. Concomitantly, clinical isolates of Cryptococcus neoformans were obtained (June 2016-June 2017). The samples were cultivated in Guizottia abysinica seed medium and were then biochemically identified and characterized by PCR fingerpinting and RFLP of the URA5 gene. Results. C. neoformans displayed an environmental prevalence of 4.3% (19 positive individuals), and that of C. gattii was 0.2% (1 positive individual); this yielded a total of 20 Cryptococcus-positive trees and 21 isolates (two from the same individual). Santander Park registered 47.6% of the global prevalence (10/21 isolates), followed by La Victoria Park with 23.8% (5/21 isolates), corresponding to C. neoformans. One C. gattii isolate was collected from a Ficus benjamina tree located in Mercedes Ábrego Park. Genotypic analysis revealed the presence of C. neoformans var. grubii VNI in 85.7% of environmental isolates as well as 100% of clinical isolates. VNII and VGII molecular types represented 9.5% and 4.8% of the environmental isolates, respectively. Conclusions. The longitudinal sampling of previously reported environmental niches of the fungus reveals its presence and suggests that permanent monitoring is required both in the environment and in patients, especially in endemic areas of the city., RESUMEN Objetivo. Aislar, identificar y caracterizar molecularmente aislamientos de Cryptococcus patógenos para humanos a partir de muestras ambientales y clínicas de la ciudad de Cúcuta. Materiales y métodos. Se recolectaron 1300 muestras de 446 árboles de 10 especies diferentes, en 10 zonas públicas de Cúcuta. Concomitantemente, se obtuvieron aislados clínicos de Cryptococcus neoformans (junio de 2016-junio de 2017). Se realizó cultivo en agar semillas de Guizottia abysinica, posterior identificación bioquímica y caracterización genética mediante PCR-huella Digital y RFLP-URA5. Resultados. Se determinó prevalencia ambiental para C. neoformans de 4.3% (19 individuos positivos) y C. gattii de 0.2% (1 individuo positivo), para un total de 21 aislados y 20 árboles positivos. El parque Santander registró el 47.6% de la prevalencia global (10/21 aislados), seguido del parque La Victoria con 23.8% (5/21 aislados), correspondientes a C. neoformans. Se obtuvo un aislado de C. gattii en un individuo Ficus benjamina del parque Mercedes Ábrego. El análisis genotípico reveló presencia de C. neoformans var. grubii VNI en el 85.7% de los aislados ambientales, así como en el 100% de los clínicos, seguido de VNII y VGII en 9.5% y 4.8% de los aislados ambientales, respectivamente. Conclusiones. El muestreo longitudinal de los nichos ambientales previamente reportados del hongo revela su presencia y sugiere que se requiere una vigilancia permanente tanto en el medio ambiente como en los pacientes, especialmente en las zonas endémicas de la ciudad.

Different signal transduction pathways mediated by MAP kinases have been described in Candida albicans. These pathways sense different stimuli and, therefore, elaborate specific responses. Hog1 was identified as the MAPK that is primarily involved in stress response and virulence, while Cek1 was more specific to cell wall biogenesis, mating and biofilm formation. In the present work, mutants defective in both pathways have been characterized under osmotic stress. Both routes are required for a full response against high osmotic challenge, since mutants defective in both pathways displayed aberrant morphology, cell polarity defects and abnormal chitin deposition, which correlate with loss of viability and appearance of apoptotic markers. These alterations occurred in spite of proper Hog1 and Cek1 phosphorylation and increased intra-cellular glycerol accumulation. The relevance of both routes in virulence is shown as ssk1 msb2 sho1 opy2 mutants are avirulent in a mouse systemic model of infection and display reduced virulence in the Galleria mellonella model., Comunidad de Madrid, Ministerio de Economía y Competitividad (MINECO), REIPI, Spanish Network for the Research in Infectious Diseases, ISCIII, Depto. de Microbiología y Parasitología, Fac. de Farmacia, TRUE, pub

The ability to switch from yeast to hyphal growth is essential for virulence in Candida albicans. The cell surface is the initial point of contact between the fungus and the host. In this work, a free-gel proteomic strategy based on tryptic digestion of live yeast and hyphae cells and protein identification using LC-MS/MS methodology was used to identify cell surface proteins. Using this strategy, a total of 943 proteins were identified, of which 438 were in yeast and 928 were in hyphae. Of these proteins, 79 were closely related to the organization and biogenesis of the cell wall, including 28 GPI-anchored proteins, such as Hyr1 and Sod5 which were detected exclusively in hyphae, and Als2 and Sap10which were detected only in yeast. A group of 17 proteins of unknown function were subsequently studied by analysis of the corresponding deletion mutants. We found that four new proteins, Pst3, Tos1, Orf19.3060 and Orf19.5352 are involved in cell wall integrity and in C. albicans' engulfment by macrophages. Moreover, the putative NADH-ubiquinone-related proteins, Ali1, Mci4, Orf19.287 and Orf19.7590, are also involved in osmotic and oxidative resistance, yeast to hypha transition and the ability to damage and invade oral epithelial cells., Ministerio de Economía y Competitividad, Comunidad Autónoma de Madrid, Spanish Network for the Research in Infectious Diseases, PRB2-ISCIII, R01DE017088, Depto. de Microbiología y Parasitología, Fac. de Farmacia, TRUE, pub

Se han venido realizando investigaciones en el campo de la aerobiología y dispersión de hongos filamentosos en ambientes hospitalarios con pacientes que tienen un alto riesgo de desarrollar una enfermedad fúngica invasiva (EFI), con una tasa de mortalidad del 50-80%. El hongo filamentoso mayormente asociado a esta patología es Aspergillus fumigatus. En el año 2015, realizamos un muestreo ambiental con el fin de describir la presentación y dinámica poblacional de hongos filamentosos de importancia clínica, encontrando a A. fumigatus en el 70% de áreas muestreadas, estas cepas fueron analizadas por la tecnología MALDI TOF la cual arrojo como resultado la identificación de cuatro ciados de las cepas encontradas, el 80,4% de estas exhibieron resistencia a voriconazol e itraconazol. Con los resultados de este proyecto se busca ampliar la frontera del conocimiento que se tiene en el impacto real de hongos ambientales en el desarrollo de EFI.

El diagnóstico rápido de las infecciones fúngicas por hongos filamentosos es fundamental para reducir la mortalidad y las complicaciones y dirigir de manera oportuna la terapia antifúngica. La espectrometría de masas conocida como MALDI-TOF, está emergiendo como un método de identificación en microbiología clínica. Sin embargo, la detección de hongos filamentosos por esta técnica aún está en desarrollo y sigue siendo un reto debido a la falta de un protocolo universal de extracción de proteínas. Por tal razón este proyecto tuvo como objetivo estandarizar el protocolo de extracción de proteínas para la identificación de hongos filamentosos patógenos para humanos relacionados con los filos Ascomycota y Mucoromycota por la tecnología MALDI-TOF / MS de Biotyper® - Bruker. Se emplearon dos protocolos para la extracción de proteínas que parten de una primera fase recomendada por el fabricante que inicia por la formación de biomasas en medio líquido Sabouraud. El primer protocolo se llevó a cabo mediante el uso de ácido fórmico al 80% y acetonitrilo al 100% y en el segundo se utilizaron perlas de vidrio, ácido fórmico al 80%, acetonitrilo al 100% y congelación. Con respecto a los resultados de los 22 aislamientos analizados, el 86.37% pudo identificarse con una puntuación superior a 1.7. Los restantes (13,63%) fueron aislamientos complejos de identificar con puntuaciones que oscilaban entre 0 a 1,3. En conclusión en el presente estudio se desarrolló el protocolo 1 el cual fue el método relevante y reproducible para identificar varios hongos filamentosos que pertenecen al filo Ascomycota y Mucoromycota.