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Ítem Acceso Abierto First report of canine protothecosis caused by Prototheca wickerhamii in Argentina. Brief literature review(Elsevier, 2025-02-15) Ramadán, Silvana; Bulacio, Lucía; Dalmaso, Hernán; Sepúlveda, Graciela; Sortino, Maximiliano Andrés; Fay, Fabián; Misto, Claudia; Salvador, María Fernanda; Etchecopaz, Alejandro; Cuestas, María Luján; https://orcid.org/0000-0002-9973-1318Protothecosis is an infectious disease caused by microalgae of the genus Prototheca. Prototheca can be found in soil and water and transiently colonize animals. Cutaneous protothecosis can involve not only the skin but also the underlying subcutaneous tissue and lymph nodes. This can lead to clinical signs and a microscopic tissue image that closely resembles a fungal infection. Treatment typically involves antifungal medications. We report the first case of fatal disseminated protothecosis caused by Prototheca wickerhamii in the city of Rosario, Argentina in a 5-year-old female poodle dog. The dog exhibited ocular signs of uveitis and lymphadenitis. To reach a clinical and etiological diagnosis, imaging studies, routine laboratory tests and serological tests were performed. A mycological analysis was conducted on the material obtained by puncturing three lymph nodes. Additionally, morphological, metabolic, and molecular analyses were performed. Antifungal susceptibility testing was also conducted using broth microdilution and diffusion methods. Phenotypic, metabolic, and sequencing techniques identified the isolated organism as P. wickerhamii. This isolate displayed susceptibility to amphotericin, variable susceptibility to itraconazole and voriconazole, and resistance to fluconazole and caspofungin. The frequent presence of pets in our homes highlights the need for a more comprehensive diagnostic approach. This is important because, from a public health perspective, dogs could serve as indicators of algal presence in the environments they frequently share with humans.Ítem Acceso Abierto Efecto de la respuesta inmune celular y humoral en parejas infértiles y su relación con infecciones genitales(INIESTARES S. A., 2017-09-28) Brufman, Adriana Silvia; Colombo, Laura G.R.; Streiger, Esteban; Pusillico, RocíoOBJETIVO: El propósito de este estudio fue determinar la prevalencia de los diferentes microorganismos aislados del tracto urogenital de hombres infértiles y evaluar si existen diferencias en los parámetros seminales según la presencia o ausencia de infecciones genitales. MÉTODOS: Se analizaron en forma retrospectiva los parámetros del semen en 280 muestras de hombres infértiles de acuerdo a los criterios de la Organización Mundial de la Salud (OMS 2010). El análisis microbiológico se realizó según la modificación del método de Stamey-Meares propuesta por Santoianni et al. RESULTADOS: Los estudios microbiológicos mostraron ausencia de microorganismos o presencia de colonizantes habituales no jerarquizables en el 67,86% (GRUPO 1) de las muestras, y presencia de al menos un patógeno o patógeno potencial en 32,14% del total (GRUPO 2). No se observaron diferencias significativas en volumen de eyaculado (p=0,353), valor de pH (p=0,801), movilidad (p>0,30), concentración de ácido cítrico (p=0,383) y viscosidad (p=0,948) entre los dos grupos. El recuento relativo de espermatozoides en los pacientes infectados fue significativamente menor que en aquellos que no presentaban patógenos (p=0,05). Se evaluó además el índice de teratozoospermia (IT). Las muestras de pacientes con infección presentaron valores de IT mayores (p<0,0001). CONCLUSIONES: Un tercio de la población estudiada presentó infecciones genitales. En base a nuestros resultados, consideramos fundamental la realización de un espermocultivo en las primeras etapas del estudio del paciente infértil para contribuir a un adecuado tratamiento de la pareja con fallas reproductivas.Ítem Acceso Abierto The professional identity of STEM faculty as instructors of course-based research experiences(Frontiers Media, 2024-10-24) Hanauer, David I.; Alvey, Richard; An, Ping; Bancroft, Christa; Butela, Kristen; Coleman, Sean; Clase, Kari L.; Collins, Parks; Mussi, María AlejandraThe professional identity of scientists has historically been cultivated to value research over teaching, which can undermine initiatives that aim to reform science education. Course-Based Research Experiences (CRE) and the inclusive Research and Education Communities (iREC) are two successful and impactful reform efforts that integrate research and teaching. The aim of this study is to explicate the professional identity of instructors who implement a CRE within an established iREC and to explore how this identity contributes to the success of these programs. 97 CRE instructors from the Science Education Alliance (SEA) iREC participated in a 2-year, multi-stage, qualitative research project that involved weekly reflective journaling, autoethnographic description, small group evaluation and writing, and large-scale community checking. The resulting description of professional identity consisted of shared values (inclusivity, student success, community membership, ownership/agency, science, overcoming failure, and persistence), specified roles (mentor, advocate, scientist, educator, motivator, collaborator, community builder, learner, evaluator and project manager) and a stated sense of self (dedicated, resilient, pride in students, multiskilled, valued, community member, responsible and overworked). Analysis of individual reflective diary entries revealed how a professional identity underpinned and facilitated the ways in which faculty addressed challenges that arose and worked toward the success of every student. It is the self-concept of the professional identity of the instructor in the context of the CRE classroom that directed the extended commitment and effort that these instructors evidently put into their work with students, which facilitated student engagement, student persistence, and their collective scientific output. The study concludes that a professional identity of STEM faculty in the context of a CRE and iREC combines being a researcher and educator, and that this integrated identity is central for current initiatives aimed at transforming undergraduate STEM education.Ítem Acceso Abierto Models of classroom assessment for course-based research experiences(Frontiers Media, 2023-11-28) Hanauer, David I.; Zhang, Tong; Graham, Mark J.; Adams, Sandra D.; Ahumada Santos, Yesmi Patricia; Mussi, María Alejandra; Diacovich, LautaroCourse-based research pedagogy involves positioning students as contributors to authentic research projects as part of an engaging educational experience that promotes their learning and persistence in science. To develop a model for assessing and grading students engaged in this type of learning experience, the assessment aims and practices of a community of experienced course-based research instructors were collected and analyzed. This approach defines four aims of course-based research assessment—(1) Assessing Laboratory Work and Scientific Thinking; (2) Evaluating Mastery of Concepts, Quantitative Thinking and Skills; (3) Appraising Forms of Scientific Communication; and (4) Metacognition of Learning—along with a set of practices for each aim. These aims and practices of assessment were then integrated with previously developed models of coursebased research instruction to reveal an assessment program in which instructors provide extensive feedback to support productive student engagement in research while grading those aspects of research that are necessary for the student to succeed. Assessment conducted in this way delicately balances the need to facilitate students’ ongoing research with the requirement of a final grade without undercutting the important aims of a CRE education.Ítem Acceso Abierto Designer TALEs enable discovery of cell death-inducer genes(Oxford University Press, 2024-09-09) Roeschlin, Roxana Andrea; Azad, Sepideh M.; Grove, René P.; Chuan, Ana; García, Lucila; Niñoles, Regina; Uviedo, Facundo; Villalobos, Liara; Massimino, Maria E.; Marano, María Rosa; Boch, Jens; Gadea, José; https://orcid.org/0000-0001-6121-7930; https://orcid.org/0009-0001-2033-3430; https://orcid.org/0009-0005-3778-9900; https://orcid.org/0009-0008-0589-9867; https://orcid.org/0000-0002-1625-422X; https://orcid.org/0000-0002-7862-9509; https://orcid.org/0000-0003-0437-589X; https://orcid.org/0000-0001-8399-0107; https://orcid.org/0009-0000-9611-0316; https://orcid.org/0000-0001-6600-8177; https://orcid.org/0000-0001-5883-7506; https://orcid.org/0000-0002-3612-7914Transcription activator-like effectors (TALEs) in plant-pathogenic Xanthomonas bacteria activate expression of plant genes and support infection or cause a resistance response. PthA4AT is a TALE with a particularly short DNA-binding domain harboring only 7.5 repeats which triggers cell death in Nicotiana benthamiana; however, the genetic basis for this remains unknown. To identify possible target genes of PthA4AT that mediate cell death in N. benthamiana, we exploited the modularity of TALEs to stepwise enhance their specificity and reduce potential target sites. Substitutions of individual repeats suggested that PthA4AT-dependent cell death is sequence specific. Stepwise addition of repeats to the C-terminal or N-terminal end of the repeat region narrowed the sequence requirements in promoters of target genes. Transcriptome profiling and in silico target prediction allowed the isolation of two cell death inducer genes, which encode a patatin-like protein and a bifunctional monodehydroascorbate reductase/carbonic anhydrase protein. These two proteins are not linked to known TALE-dependent resistance genes. Our results show that the aberrant expression of different endogenous plant genes can cause a cell death reaction, which supports the hypothesis that TALE-dependent executor resistance genes can originate from various plant processes. Our strategy further demonstrates the use of TALEs to scan genomes for genes triggering cell death and other relevant phenotypes.Ítem Acceso Abierto Genetic and phenotypic changes related to the development of mec-independent oxacillin non-susceptibility in ST8 Staphylococcus aureus recovered after antibiotic therapy in a patient with bacteremia(MDPI, 2024-06-13) Di Gregorio, Sabrina; Weltman, Gabriela; Fabbri, Carolina; Fernández, Silvina; Zárate, Soledad; Smayevsky, Jorgelina; Power, Pablo; Campos, Josefina; Llarrull, Leticia Irene; Mollerach, Marta; https://orcid.org/0000-0003-3787-3318; https://orcid.org/0000-0002-7051-9954; https://orcid.org/0000-0002-5679-4343; https://orcid.org/0000-0002-5735-8471The mec-independent oxacillin non-susceptible S. aureus (MIONSA) strains represent a great clinical challenge, as they are not easily detected and can lead to treatment failure. However, the responsible molecular mechanisms are still very little understood. Here, we studied four clinical ST8-MSSA-t024 isolates recovered during the course of antibiotic treatment from a patient suffering successive episodes of bacteremia. The first isolates (SAMS1, SAMS2, and SAMS3) were susceptible to cefoxitin and oxacillin. The last one (SA2) was susceptible to cefoxitin, resistant to oxacillin, lacked mec genes, and had reduced susceptibility to teicoplanin. SA2 showed higher β-lactamase activity than SAMS1. However, β-lactamase hyperproduction could not be linked to oxacillin resistance as it was not inhibited by clavulanic acid, and no genetic changes that could account for its hyperproduction were found. Importantly, we hereby report the in vivo acquisition and coexistence of different adaptive mutations in genes associated with peptidoglycan synthesis (pbp2, rodA, stp1, yjbH, and yvqF/vraT), which is possibly related with the development of oxacillin resistance and reduced susceptibility to teicoplanin in SA2. Using three-dimensional models and PBP binding assays, we demonstrated the high contribution of the SA2 PBP2 Ala450Asp mutation to the observed oxacillin resistance phenotype. Our results should be considered as a warning for physicians and microbiologists in the region, as MIONSA detection and treatment represent an important clinical challenge.Ítem Acceso Abierto Identification of novel bromodomain inhibitors of Trypanosoma cruzi bromodomain factor 2 (TcBDF2) using a fluorescence polarization-based high-throughput assay(American Society for Microbiology, 2024-06-19) Tavernelli, Luis Emilio; Alonso, Victoria Lucía; Peña, Imanol; Rodríguez Araya, Elvio; Manarin, Romina; Cantizani, Juan; Martin, Julio; Salamanca, Juan; Bamborough, Paul; Calderón, Felix; Gabarro, Raquel; Serra, Esteban Carlos; http://orcid.org/0000-0001-5986-7459Bromodomains are structural folds present in all eukaryotic cells that bind to other proteins recognizing acetylated lysines. Most proteins with bromodomains are part of nuclear complexes that interact with acetylated histone residues and regulate DNA replication, transcription, and repair through chromatin structure remodeling. Bromodomain inhibitors are small molecules that bind to the hydrophobic pocket of bromodomains, interfering with the interaction with acetylated histones. Using a fluorescent probe, we have developed an assay to select inhibitors of the bromodomain factor 2 of Trypanosoma cruzi (TcBDF2) using fluorescence polarization. Initially, a library of 28,251 compounds was screened in an endpoint assay. The top 350-ranked compounds were further analyzed in a dose-response assay. From this analysis, seven compounds were obtained that had not been previously characterized as bromodomain inhibitors. Although these compounds did not exhibit significant trypanocidal activity, all showed bona fide interaction with TcBDF2 with dissociation constants between 1 and 3 µM validating these assays to search for bromodomain inhibitors.Ítem Acceso Abierto 1,3,4-oxadiazoles as inhibitors of the atypical member of the BET family bromodomain factor 3 from Trypanosoma cruzi (TcBDF3)(Frontiers, 2024-10-01) Alonso, Victoria Lucía; Escalante, Andrea Marta; Rodríguez Araya, Elvio; Frattini, Gianfranco; Tavernelli, Luis Emilio; Moreno, Diego M.; Furlán, Ricardo Luis Eugenio; Serra, Esteban CarlosChagas disease, caused by the protozoan parasite Trypanosoma cruzi, affects millions globally, with increasing urban cases outside of Latin America. Treatment is based on two compounds, namely, benznidazole (BZ) and nifurtimox, but chronic cases pose several challenges. Targeting lysine acetylation, particularly bromodomain-containing proteins, shows promise as a novel antiparasitic target. Our research focuses on TcBDF3, a cytoplasmic protein, which is crucial for parasite differentiation that recognizes acetylated alpha-tubulin. In our previous study, A1B4 was identified as a high-affinity binder of TcBDF3, showing significant trypanocidal activity with low host toxicity in vitro. In this report, the binding of TcBDF3 to A1B4 was validated using differential scanning fluorescence, fluorescence polarization, and molecular modeling, confirming its specific interaction. Additionally, two new 1,3,4-oxadiazoles derived from A1B4 were identified, which exhibited improved trypanocide activity and cytotoxicity profiles. Furthermore, TcBDF3 was classified for the first time as an atypical divergent member of the bromodomain extraterminal family found in protists and plants. These results make TcBDF3 a unique target due to its localization and known functions not shared with higher eukaryotes, which holds promise for Chagas disease treatment.Ítem Acceso Abierto Integration of BrfS into the biofilm-controlling cascade promotes sessile Salmonella growth at low temperatures(Elsevier, 2025-01-21) Tulin, Gonzalo; Méndez, Andrea A. E.; Figueroa, Nicolás; Smith, Carol; Folmer, María P.; Serra, Diego Omar; Wade, Joseph T.; Checa, Susana Karina; Soncini, Fernando C.Biofilm formation is stimulated by different stress-related physiological and environmental conditions. In Salmonella and Escherichia coli, curli fibers and phosphoethanolamine-cellulose are the major extracellular components of biofilms. The production of both is under the control of CsgD, a transcriptional regulator whose expression is modulated by a number of factors responding to different signals. The atypical MerR-like regulator MlrA is key in the activation of csgD transcription in both Salmonella and E. coli. Recently, MlrB, a SPI-2-encoded MlrA-like regulator that counteracts MlrA by repressing csgD transcription and biofilm formation inside mac rophages was identified. Here, we characterize STM1266, a Salmonella-specific MlrA-like regulator, recently renamed BrfS. In contrast to mlrA, brfS transcription increases in minimal growth media and at 20 ◦C, a temperature not commonly tested in laboratories. Under these conditions, as well as in salt-limited rich medium, deletion or overexpression of brfS affects extracellular matrix production. Using transcriptomics, we uncovered genes under BrfS control relevant for biofilm formation such as csgB and bapA. Transcriptional analysis of these genes in mutants lacking brfS, csgD or both, indicates that BrfS controls curli biosynthesis both in a CsgDdependent and independent manner. By contrast, at low temperatures, bapA transcription depends only on BrfS, and neither deletion of csgD nor of mlrA modify its expression. Based on these results, we propose that BrfS contributes to Salmonella persistence in the environment, where the pathogen encounters low temperatures and nutrient limitation.Ítem Acceso Abierto Genetic tool development in marine protists: emerging model organisms for experimental cell biology(Nature Research, 2020-04-15) Najle, Sebastián R.; Faktorová, Drahomíra; Nisbet, R. Ellen R.; Fernández Robledo, José A.; Casacuberta, Elena; Sudek, Lisa; Allen, Andrew E.Diverse microbial ecosystems underpin life in the sea. Among these microbes are many unicellular eukaryotes that span the diversity of the eukaryotic tree of life. However, genetic tractability has been limited to a few species, which do not represent eukaryotic diversity or environmentally relevant taxa. Here, we report on the development of genetic tools in a range of protists primarily from marine environments. We present evidence for foreign DNA delivery and expression in 13 species never before transformed and for advancement of tools for eight other species, as well as potential reasons for why transformation of yet another 17 species tested was not achieved. Our resource in genetic manipulation will provide insights into the ancestral eukaryotic lifeforms, general eukaryote cell biology, protein diversification and the evolution of cellular pathways.Ítem Acceso Abierto An updated examination of the perception of barriers for pharmacogenomics implementation and the usefulness of drug/gene pairs in Latin America and the Caribbean(Frontiers Media, 2023-05-11) Salas Hernández, Aimeé; Galleguillos, Macarena; Carrasco, Matías; López Cortés, Andrés; Redal, María Ana; Fonseca Mendoza, Dora; Perez, Germán Roberto; RELIVAFPharmacogenomics (PGx) is considered an emergent field in developing countries. Research on PGx in the Latin American and the Caribbean (LAC) region remains scarce, with limited information in some populations. Thus, extrapolations are complicated, especially in mixed populations. In this paper, we reviewed and analyzed pharmacogenomic knowledge among the LAC scientific and clinical community and examined barriers to clinical application. We performed a search for publications and clinical trials in the field worldwide and evaluated the contribution of LAC. Next, we conducted a regional structured survey that evaluated a list of 14 potential barriers to the clinical implementation of biomarkers based on their importance. In addition, a paired list of 54 genes/drugs was analyzed to determine an association between biomarkers and response to genomic medicine. This survey was compared to a previous survey performed in 2014 to assess progress in the region. The search results indicated that Latin American and Caribbean countries have contributed 3.44% of the total publications and 2.45% of the PGx-related clinical trials worldwide thus far. A total of 106 professionals from 17 countries answered the survey. Six major groups of barriers were identified. Despite the region’s continuous efforts in the last decade, the primary barrier to PGx implementation in LAC remains the same, the “need for guidelines, processes, and protocols for the clinical application of pharmacogenetics/pharmacogenomics”. Cost-effectiveness issues are considered critical factors in the region. Items related to the reluctance of clinicians are currently less relevant. Based on the survey results, the highest ranked (96%–99%) gene/drug pairs perceived as important were CYP2D6/tamoxifen, CYP3A5/tacrolimus, CYP2D6/opioids, DPYD/ fluoropyrimidines, TMPT/thiopurines, CYP2D6/tricyclic antidepressants, CYP2C19/ tricyclic antidepressants, NUDT15/thiopurines, CYP2B6/efavirenz, and CYP2C19/ clopidogrel. In conclusion, although the global contribution of LAC countries remains low in the PGx field, a relevant improvement has been observed in the region. The perception of the usefulness of PGx tests in biomedical community has drastically changed, raising awareness among physicians, which suggests a promising future in the clinical applications of PGx in LAC.Ítem Acceso Abierto Bacterial and plant natriuretic peptides improve plant defence responses against pathogens(BSPP and Wiley, 2018-03-14) Ficarra, Florencia Andrea; Grandellis, Carolina; Garavaglia, Betiana Soledad; Gottig, Natalia; Ottado, JorgelinaPlant natriuretic peptides (PNPs) have been implicated in the regulation of ions and water homeostasis, and their participation in the plant immune response has also been proposed. Xanthomonas citri ssp. citri contains a gene encoding a PNP-like protein (XacPNP) which has no homologues in other bacteria. XacPNP mimics its Arabidopsis thaliana homologue AtPNP-A by modifying host responses to create favourable conditions for pathogen survival. However, the ability of XacPNP to induce plant defence responses has not been investigated. In order to study further the role of XacPNP in vivo, A. thaliana lines over-expressing XacPNP, lines over-expressing AtPNP-A and AtPNP-A-deficient plants were generated. Plants over-expressing XacPNP or AtPNP-A showed larger stomatal aperture and were more resistant to saline or oxidative stress than were PNP-deficient lines. In order to study further the role of PNP in biotic stress responses, A. thaliana leaves were infiltrated with pure recombinant XacPNP, and showed enhanced expression of genes related to the defence response and a higher resistance to pathogen infections. Moreover, AtPNP-A expression increased in A. thaliana on Pseudomonas syringae pv. tomato (Pst) infection. This evidence led us to analyse the responses of the transgenic plants to pathogens. Plants over-expressing XacPNP or AtPNP-A were more resistant to Pst infection than control plants, whereas PNP-deficient plants were more susceptible and showed a stronger hypersensitive response when challenged with non-host bacteria. Therefore, XacPNP, acquired by horizontal gene transfer, is able to mimic PNP functions, even with an increase in plant defence responses.Ítem Acceso Abierto The stringent response plays a key role in Bacillus subtilis survival of fatty acid starvation(Wiley, 2017-02-07) Pulschen, André A.; Sastre, Diego Emiliano; Machinandiarena, Federico; Crotta Asis, Agostina; Albanesi, Daniela; De Mendoza, Diego; Gueiros-Filho, Frederico J.The stringent response is a universal adaptive mechanism to protect bacteria from nutritional and environmental stresses. The role of the stringent response during lipid starvation has been studied only in Gram-negative bacteria. Here, we report that the stringent response also plays a crucial role in the adaptation of the model Gram-positive Bacillus subtilis to fatty acid starvation. B. subtilis lacking all three (p)ppGpp-synthetases (RelBs, RelP and RelQ) or bearing a RelBs variant that no longer synthesizes (p)ppGpp suffer extreme loss of viability on lipid starvation. Loss of viability is paralleled by perturbation of membrane integrity and function, with collapse of membrane potential as the likely cause of death. Although no increment of (p)ppGpp could be detected in lipid starved B. subtilis, we observed a substantial increase in the GTP/ATP ratio of strains incapable of synthesizing (p)ppGpp. Artificially lowering GTP with decoyinine rescued viability of such strains, confirming observations that low intracellular GTP is important for survival of nutritional stresses. Altogether, our results show that activation of the stringent response by lipid starvation is a broadly conserved response of bacteria and that a key role of (p)ppGpp is to couple biosynthetic processes that become detrimental if uncoordinated.Ítem Acceso Abierto Unravelling the lipoyl-relay of exogenous lipoate utilization in Bacillus subtilis(Wiley, 2019-05-19) Rasetto, Natalí B.; Lavatelli, Antonela; Martin, Natalia; Mansilla, María Cecilia; https://orcid.org/0000-0002-1444-8661Lipoate is an essential cofactor for key enzymes of oxidative and one-carbon metabolism. It is covalently attached to E2 subunits of dehydrogenase complexes and GcvH, the H subunit of the glycine cleavage system. Bacillus subtilis possess two protein lipoylation pathways: biosynthesis and scavenging. The former requires octanoylation of GcvH, insertion of sulfur atoms and amidotransfer of the lipoate to E2s, catalyzed by LipL. Lipoate scavenging is mediated by a lipoyl protein ligase (LplJ) that catalyzes a classical two-step ATP-dependent reaction. Although these pathways were thought to be redundant, a ∆lipL mutant, in which the endogenous lipoylation pathway of E2 subunits is blocked, showed growth defects in minimal media even when supplemented with lipoate and despite the presence of a functional LplJ. In this study, we demonstrate that LipL is essential to modify E2 subunits of branched chain ketoacid and pyruvate dehydrogenases during lipoate scavenging. The crucial role of LipL during lipoate utilization relies on the strict substrate specificity of LplJ, determined by charge complementarity between the ligase and the lipoylable subunits. This new lipoyl-relay required for lipoate scavenging highlights the relevance of the amidotransferase as a valid target for the design of new antimicrobial agents among Gram-positive pathogens.Ítem Acceso Abierto Transfection of Capsaspora owczarzaki, a close unicellular relative of animals(The Company of Biologists, 2018) Parra Acero, Helena; Ros Rocher, Núria; Perez Posada, Alberto; Kozyczkowska, Aleksandra; Sánchez Pons, Núria; Nakata, Azusa; Suga, Hiroshi; Najle, Sebastián R.; Ruiz Trillo, Iñaki; https://orcid.org/0000-0003-0897-0186; https://orcid.org/0000-0001-6547-5304How animals emerged from their unicellular ancestor remains a major evolutionary question. New genome data from the closest unicellular relatives of animals have provided important insights into the evolution of animal multicellularity. We know that the unicellular ancestor of animals had an unexpectedly complex genetic repertoire, including many genes that are key to animal development and multicellularity. Thus, assessing the function of these genes among unicellular relatives of animals is key to understanding how they were co-opted at the onset of the Metazoa. However, such analyses have been hampered by the lack of genetic tools. Progress has been made in choanoflagellates and teretosporeans, two of the three lineages closely related to animals, whereas no tools are yet available for functional analysis in the third lineage: the filastereans. Importantly, filastereans have a striking repertoire of genes involved in transcriptional regulation and other developmental processes. Here, we describe a reliable transfection method for the filasterean Capsaspora owczarzaki. We also provide a set of constructs for visualising subcellular structures in live cells. These tools convert Capsaspora into a unique experimentally tractable organism to use to investigate the origin and evolution of animal multicellularity.Ítem Embargo Light modulates important pathogenic determinants and virulence in ESKAPE pathogens Acinetobacter baumannii, Pseudomonas aeruginosa, and Staphylococcus aureus(American Society for Microbiology, 2021-02-08) Tuttobene, Marisel Romina; Pérez, J. F.; Pavesi, Estefanía S.; Pérez Mora, Bárbara; Biancotti, Daiana; Cribb, Pamela; Altilio, Matías; Müller, Gabriela Leticia; Gramajo, Hugo Cesar; Tamagno, G.; Ramírez, María Soledad; Diacovich, Lautaro; Mussi, María Alejandra; https://orcid.org/0000-0002-9904-7890; https://orcid.org/0000-0002-3339-0100; https://orcid.org/0000-0002-4168-3624; Rabinovich, Gabriel: provide HaCaT cells; Voyich, Jovanka: provide the hla and complemented hla mutantLight sensing has been extensively characterized in the human pathogen Acinetobacter baumannii at environmental temperatures. However, the influence of light on the physiology and pathogenicity of human bacterial pathogens at temperatures found in warm-blooded hosts is still poorly understand. In this work, we show that Staphylococcus aureus, Acinetobacter baumannii, and Pseudomonas aeruginosa (ESKAPE) priority pathogens, which have been recognized by the WHO and the CDC as critical, can also sense and respond to light at temperatures found in human hosts. Most interestingly, in these pathogens, light modulates important pathogenicity determinants as well as virulence in an epithelial infection model, which could have implications in human infections. In fact, we found that alpha-toxin-dependent hemolysis, motility, and growth under iron-deprived conditions are modulated by light in S. aureus. Light also regulates persistence, metabolism, and the ability to kill competitors in some of these microorganisms. Finally, light exerts a profound effect on the virulence of these pathogens in an epithelial infection model, although the response is not the same in the different species; virulence was enhanced by light in A. baumannii and S. aureus, while in A. nosocomialis and P. aeruginosa it was reduced. Neither the BlsA photoreceptor nor the type VI secretion system (T6SS) is involved in virulence modulation by light in A. baumannii. Overall, this fundamental knowledge highlights the potential use of light to control pathogen virulence, either directly or by manipulating the light regulatory switch toward the lowest virulence/persistence configuration. IMPORTANCE: Pathogenic bacteria are microorganisms capable of producing disease. Dangerous bacterial pathogens, such as Staphylococcus aureus, Pseudomonas aeruginosa, and Acinetobacter baumannii, are responsible for serious intrahospital and community infections in humans. Therapeutics is often complicated due to resistance to multiple antibiotics, rendering them ineffective. In this work, we show that these pathogens sense natural light and respond to it by modulating aspects related to their ability to cause disease; in the presence of light, some of them become more aggressive, while others show an opposite response. Overall, we provide new understanding on the behavior of these pathogens, which could contribute to the control of infections caused by them. Since the response is distributed in diverse pathogens, this notion could prove a general concept.Ítem Acceso Abierto Osteomielitis por Scedosporium spp., a propósito de un caso(Asociación Argentina de Microbiología (AAM), 2019-06-13) Colombo, Laura G.R.; Gregorini, Eduardo R.; Dalmaso, Hernán; Podestá, María. V.; Luque, Alicia Graciela; Truccolo, Paula; Lerman Tenenbaum, DamiánScedosporium es un hongo de distribución mundial que se encuentra en el suelo y en aguas contaminadas. Raramente afecta tejido óseo y puede hacerlo por inoculación directa a través de traumatismos. Se presenta el caso clínico de un paciente de 54 años con antecedente de accidente acuático y fractura expuesta de tibia-peroné de ambos miembros inferiores, con diagnóstico de osteomielitis crónica bacteriana tratada con antibióticos de amplio espectro por 120 días. Luego de ocho meses iniciado el cuadro, se aísla Scedosporium spp. en colección de miembro afectado; por tal motivo, el paciente recibe terapia con voriconazol asociado a terbinafina.Ítem Embargo Enterococcus faecalis uses a phosphotransferase system permease and a host colonization-related ABC transporter for maltodextrin uptake(American Society for Microbiology, 2017-04-11) Sauvageot, Nicolas; Mokhtari, Abdelhamid; Joyet, Philippe; Budin Verneuil, Aurélie; Blancato, Víctor Sebastián; Repizo, Guillermo Daniel; Henry, Céline; Pikis, Andreas; Thompson, John; Magni, Christian; Hartke, Axel; Deutscher, Josef; Fernández, María: provide plasmid pAGEntMaltodextrin is a mixture of maltooligosaccharides, which are produced by the degradation of starch or glycogen. They are mostly composed of α-1,4- and some α-1,6-linked glucose residues. Genes presumed to code for the Enterococcus faecalis maltodextrin transporter were induced during enterococcal infection. We therefore carried out a detailed study of maltodextrin transport in this organism. Depending on their length (3 to 7 glucose residues), E. faecalis takes up maltodextrins either via MalT, a maltose-specific permease of the phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS), or the ATP binding cassette (ABC) transporter MdxEFG-MsmX. Maltotriose, the smallest maltodextrin, is primarily transported by the PTS permease. A malT mutant therefore exhibits significantly reduced growth on maltose and maltotriose. The residual uptake of the trisaccharide is catalyzed by the ABC transporter, because a malT mdxF double mutant no longer grows on maltotriose. The trisaccharide arrives as maltotriose-6″-P in the cell. MapP, which dephosphorylates maltose-6′-P, also releases Pi from maltotriose-6″-P. Maltotetraose and longer maltodextrins are mainly (or exclusively) taken up via the ABC transporter, because inactivation of the membrane protein MdxF prevents growth on maltotetraose and longer maltodextrins up to at least maltoheptaose. E. faecalis also utilizes panose and isopanose, and we show for the first time, to our knowledge, that in contrast to maltotriose, its two isomers are primarily transported via the ABC transporter. We confirm that maltodextrin utilization via MdxEFG-MsmX affects the colonization capacity of E. faecalis, because inactivation of mdxF significantly reduced enterococcal colonization and/or survival in kidneys and liver of mice after intraperitoneal infection. IMPORTANCE: Infections by enterococci, which are major health care-associated pathogens, are difficult to treat due to their increasing resistance to clinically relevant antibiotics, and new strategies are urgently needed. A largely unexplored aspect is how these pathogens proliferate and which substrates they use in order to grow inside infected hosts. The use of maltodextrins as a source of carbon and energy was studied in Enterococcus faecalis and linked to its virulence. Our results demonstrate that E. faecalis can efficiently use glycogen degradation products. We show here that depending on the length of the maltodextrins, one of two different transporters is used: the maltose-PTS transporter MalT, or the MdxEFG-MsmX ABC transporter. MdxEFG-MsmX takes up longer maltodextrins as well as complex molecules, such as panose and isopanose.Ítem Acceso Abierto Functional characterization of the first lipoyl-relay pathwayfrom a parasitic protozoan(Wiley, 2022-06) Scattolini, Albertina; Lavatelli, Antonela; Vacchina, Paola; Lambruschi, Daniel Andrés; Mansilla, María Cecilia; Uttaro, Antonio Domingo; https://orcid.org/0000-0001-7421-2039; https://orcid.org/0000-0001-7164-213X; https://orcid.org/0000-0001-5866-3657; https://orcid.org/0000-0001-5787-5711; https://orcid.org/0000-0002-1444-8661Lipoic acid (LA) is a sulfur-containing cofactor covalently attached to key enzymes of central metabolism in prokaryotes and eukaryotes. LA can be acquired by scavenging, mediated by a lipoate ligase, or de novo synthesized by a pathway requiring an octanoyltransferase and a lipoate synthase. A more complex pathway, referred to as “lipoyl-relay”, requires two additional proteins, GcvH, the glycine cleavage system H subunit, and an amidotransferase. This route was described so far in Bacillus subtilis and related Gram-positive bacteria, Saccharomyces cerevisiae, Homo sapiens, and Caenorhabditis elegans. Using collections of S. cerevisiae and B. subtilis mutants, defective in LA metabolism, we gathered evidence that allows us to propose for the first time that lipoyl-relay pathways are also present in parasitic protozoa. By a reverse genetic approach, we assigned octanoyltransferase and amidotransferase activity to the products of Tb927.11.9390 (TblipT) and Tb927.8.630 (TblipL) genes of Trypanosoma brucei, respectively. The B. subtilis model allowed us to identify the parasite amidotransferase as the target of lipoate analogs like 8-bromo-octanoic acid, explaining the complete loss of protein lipoylation and growth impairment caused by this compound in T. cruzi. This model could be instrumental for the screening of selective and more efficient chemotherapies against trypanosomiases.Ítem Acceso Abierto On the offensive: the role of outer membrane vesicles in the successful dissemination of New Delhi Metallo-β-lactamase (NDM-1)(American Society for Microbiology, 2021-09-28) Martínez, Melina María Belén; Bonomo, Robert A.; Vila, Alejandro J.; Maffía, Paulo César; González, Lisandro Javier; https://orcid.org/0000-0002-4007-8721; https://orcid.org/0000-0002-3299-894X; https://orcid.org/0000-0002-7978-3233; https://orcid.org/0000-0001-7423-2646; https://orcid.org/0000-0002-0575-1810The emergence and worldwide dissemination of carbapenemase-producing Gram-negative bacteria are a major public health threat. Metallo-β-lactamases (MBLs) represent the largest family of carbapenemases. Regrettably, these resistance determinants are spreading worldwide. Among them, the New Delhi metallo-β-lactamase (NDM-1) is experiencing the fastest and largest geographical spread. NDM-1 β-lactamase is anchored to the bacterial outer membrane, while most MBLs are soluble, periplasmic enzymes. This unique cellular localization favors the selective secretion of active NDM-1 into outer membrane vesicles (OMVs). Here, we advance the idea that NDM-containing vesicles serve as vehicles for the local dissemination of NDM-1. We show that OMVs with NDM-1 can protect a carbapenem-susceptible strain of Escherichia coli upon treatment with meropenem in a Galleria mellonella infection model. Survival curves of G. mellonella revealed that vesicle encapsulation enhances the action of NDM-1, prolonging and favoring bacterial protection against meropenem inside the larva hemolymph. We also demonstrate that E. coli cells expressing NDM-1 protect a susceptible Pseudomonas aeruginosa strain within the larvae in the presence of meropenem. By using E. coli variants engineered to secrete variable amounts of NDM-1, we demonstrate that the protective effect correlates with the amount of NDM-1 secreted into vesicles. We conclude that secretion of NDM-1 into OMVs contributes to the survival of otherwise susceptible nearby bacteria at infection sites. These results disclose that OMVs play a role in the establishment of bacterial communities, in addition to traditional horizontal gene transfer mechanisms. IMPORTANCE: Resistance to carbapenems, last-resort antibiotics, is spreading worldwide, raising great concern. NDM-1 is one of the most potent and widely disseminated carbapenem-hydrolyzing enzymes spread among many bacteria and is secreted to the extracellular medium within outer membrane vesicles. We show that vesicles carrying NDM-1 can protect carbapenem-susceptible strains of E. coli and P. aeruginosa upon treatment with meropenem in a live infection model. These vesicles act as nanoparticles that encapsulate and transport NDM-1, prolonging and favoring its action against meropenem inside a living organism. Secretion of NDM-1 into vesicles contributes to the survival of otherwise susceptible nearby bacteria at infection sites. We propose that vesicles play a role in the establishment of bacterial communities and the dissemination of antibiotic resistance, in addition to traditional horizontal gene transfer mechanisms.