Seminario de Título entregado a la Universidad de Chile en cumplimiento parcial de los requisitos para optar al Título de Ingeniero en Biotecnología Molecular. / Los hongos filamentosos de ambientes extremos son fuentes promisorias de metabolitos secundarios de carácter novedoso. En particular, el género fúngico Pseudogymnoascus, que habita distintos ambientes fríos, entre ellos la Antártida, posee un metabolismo secundario poco explorado. En estudios previos de la cepa fúngica antártica Pseudogymnoascus sp. 131209-E2A-C5II-EB se identificaron los genes codificantes para enzimas policétido sintasa GymB, GymC, GymD y Gym722, de los cuales solo los dos primeros se expresaron fuertemente en las condiciones de cultivo ensayadas. El objetivo de este trabajo fue identificar nuevos genes codificantes para enzimas policétido sintasa en la cepa Pseudogymnoascus sp 131209-E2A-C5II-EB, e identificar las condiciones de cultivo en las cuales se induce su expresión. Para encontrar estas condiciones de cultivo adecuadas se emplearon distintos medios de cultivo (aproximación OSMAC) y el remodelador de cromatina 5-azacitidina. Por último, se realizaron las primeras actividades para, mediante la técnica de ARN de interferencia, identificar el metabolito sintetizado por la ruta de biosíntesis de la que forma parte el gen GymD. En resumen, en este trabajo se identificó un nuevo gen codificante para una enzima policétido sintasa, al cual se denominó Gym36. Por otro lado, mediante la aproximación OSMAC, se identificó que el medio PDB es donde se obtienen mayores niveles de inducción de manera generalizada de los genes estudiados (a excepción de Gym722 que se mantuvo sin expresión). La adición del remodelador de cromatina 5-azacitidina no logró inducir la expresión de los genes estudiados, pero si se observó que la adición de dimetilsulfóxido logró alterar positivamente los niveles de expresión de estos. Finalmente, por electroporación de conidias germinadas, se logró obtener una cepa transformante de la cepa en estudio con el gen GymD atenuado. Como trabajo futuro, se espera que al contar con un mayor número de cepas atenuantes se pueda identificar el metabolito sintetizado por la ruta de biosíntesis de la que forma parte el gen. / Filamentous fungi from extreme environments are a promising source of novel secondary metabolites. In particular, fungal genus Pseudogymnoascus, which inhabits different cold environments, including Antarctica, has a secondary metabolism that has been poorly explored. In previous studies of the Antarctic fungal strain Pseudogymnoascus sp. 131209-E2A-C5II-EB the genes coding for polyketide synthase enzymes GymB, GymC, GymD and Gym722 were identified, of which only the first two were strongly expressed in the tested culture conditions. The objective of this work was to identify new coding genes for polyketide synthase enzymes in the strain Pseudogymnoascus sp 131209-E2A-C5II-EB, and to identify the culture conditions in which their expression is induced. To find these suitable culture conditions, different culture media (OSMAC approach) and the 5-azacitidine chromatin remodeler were used. Finally, the first activities were carried out to identify, through the RNA interference technique, the metabolite synthesized by the biosynthesis pathway to which the GymD gene belongs. In summary, in this work it was identified a new gene coding for a polyketide synthase enzyme, named Gym36. On the other hand, through the OSMAC approach, it was found that the PDB medium is where the highest levels of induction are obtained in a generalized manner of the genes studied (with the exception of Gym722 that remained silent). The addition of the chromatin remodeler 5-azacitidine failed to induce the expression of the genes studied, but it was observed that the addition of dimethylsulfoxide was able to positively alter the expression levels of these. Finally, by electroporation of germinated conidia, it was possible to obtain a transformant strain of the studied strain with the GymD gene attenuated. As future work, it is expected that having a greater number of attenuating strains, will allow the identification of the metabolite synthesized by the biosynthesis pathway of which the gene belongs.

A microscopic fungus Pseudogymnoascus destructans (Ascomycota: Pseudeurotiaceae) causes illness known as white-nose syndrome (WNS) causing death of bats during hibernation. The illness occurs in the North America and in Europe. The fungus is characteristic by asymmetrically curved conidia, by slow growth and growth at low temperatures (below 20 řC). The aim of this study is to clarify properties responsible for unique ecelogy of Pseudogymnoascus destructans by comparison with ecological related or unrelated pathogenic or nonpathogenic fungi. This part includes study of tolerance to physiological stresses and recognition of spectrum of utilizating nutrients (compounds of carbon, nitrogen, phosphorus, sulphur and nutrient supplements). Testing to physiological stresses should help to estimate a potentiality of fungus to spread out of caves. The last aim is to develop a selective isolation medium for P. destructans. Influence of several types of physiological stress (e.g. UVA, UVA with UVB, 25 řC, 30 řC, 37 řC and dryness) was investigated with fluorescent stain propidium iodide (PI) by flow cytometry. The spores of Pseudogymnoascus destructans and three fungi from underground spaces were not viable after 3 weeks at 37 řC. Other stresses did not cause a decreasing of viability or some stresses caused...

Emerging infectious diseases of wildlife are among the greatest threats to biodiversity. Indeed, when pathogens are introduced into naïve host populations, they can impose novel selective pressures that may cause severe host declines or even extinction. However, disease impacts may vary both within and among host species. Thus, one of the key goals for management is to identify factors that drive variation in host susceptibility to infection, as they may improve our understanding of hosts' potential to develop disease resistance and/or tolerance and inform conservation strategies aimed at facilitating host persistence. For instance, Pseudogymnoascus destructans (Pd) - an invasive pathogenic fungus that causes white-nose syndrome (WNS) in hibernating bats - is highly virulent, has killed millions of bats in North America, and continues to spread at an alarming rate. Yet, the continued persistence of bat colonies in contaminated areas despite initial mass mortality events suggests variation in survival among infected individuals. I thus aimed to better understand intraspecific drivers of variation in bat susceptibility to WNS and their implications for population persistence and management in affected areas. Specifically, my objectives were to: 1) evaluate the extent to which variation in hibernaculum microclimate temperature and humidity affects Pd infection severity and disease progression in affected bats during hibernation, 2) compare how bats from colonies that vary in duration of exposure to Pd and from different age classes behaviorally respond to the infection, and examine how these behavioral changes affect host fitness and 3) model the population dynamics of remnant bat populations to assess the likeliness of persistence and the potential effectiveness of management interventions in affected colonies. I addressed these objectives through field research, experimental infection studies, and demographic modeling of the little brown myotis (Myotis lucifugus). In my dissertation, I first provide causal evidence of environmentally-driven variation in pathogen growth and infection severity on bats in the field. Both warmer and more humid microclimates contribute to the severity of the infection by promoting the production of conidia, the erosion of wing tissues, and, therefore, the transmission potential and virulence of Pd. I then document potential mechanistic links between Pd-induced behavioral change and host fitness. Higher infection levels, independent of bats' past exposure to Pd or age class, may cause individuals to groom longer, prolong euthermic arousals, accelerate the depletion of fat reserves, and ultimately increase mortality risk. Finally, I predict that populations will face a high risk of extirpation in the next decade or two if no management action is taken, but that interventions such as environmental control of Pd and hibernaculum microclimate manipulation can prevent short-term population collapse in remnant bat populations. Together, these studies provide key, mechanistic insight into the pathology of WNS and the probability of persistence of affected bat colonies, while highlighting the importance of prioritizing winter habitat preservation and enhancement for the conservation of hibernating bats. / Biology

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Le syndrome du museau blanc (SMB), causé par le champignon Pseudogymnoascus destructans (Pd), a mis en péril les populations de chauves-souris hibernantes en Amérique du Nord. Certaines espèces sont hautement vulnérables à la maladie alors que d’autres espèces semblent être résistantes ou tolérantes à l’infection. Plusieurs facteurs physiologiques et environnementaux peuvent expliquer ces différences. Or avant 2015, peu d’études avaient porté sur le microbiome de la peau en relation avec cette maladie. La présente thèse vise à caractériser le microbiome cutané de chiroptères affectés par le SMB afin d’identifier les facteurs de vulnérabilité ou de résistance à la maladie. L’objectif principal est de déterminer comment le microbiome est affecté par la maladie ainsi que de déterminer si celui-ci à un rôle dans la protection face à l’infection fongique. Au Chapitre 1, nous avons tout d’abord exploré et comparé le microbiote cutané de petites chauves-souris brunes (Myotis lucifugus) non affectées par le SMB avec celui de chauves-souris survivantes au SMB pour tester l’hypothèse selon laquelle le microbiote cutané est modifié par la maladie. Nos résultats montrent que le site d’hibernation influence fortement la composition et la diversité du microbiote cutané. Les sites d’hibernations Pd positifs et négatifs diffèrent significativement en termes de diversité, ainsi qu’en termes de composition du microbiote. La diversité est réduite au sein du microbiote des chauves-souris survivantes au SMB et enrichi en taxons tels que Janthinobacterium, Micrococcaceae, Pseudomonas, Ralstonia et Rhodococcus. Certains de ces taxons sont reconnus pour leur potentiel antifongique et des souches spécifiques de Rhodococcus et de Pseudomonas peuvent inhiber la croissance de Pd. Nos résultats sont cohérents avec l’hypothèse selon laquelle l’infection par Pd modifie le microbiote cutané des chauves-souris survivantes et suggèrent que le microbiote peut jouer un rôle de protection face au SMB. Au Chapitre 2, nous avons étudié le microbiote d’une espèce résistante au champignon Pd en milieu contrôlé avant et après infection afin d’établir la réponse potentielle à la maladie. L’espèce étudiée est la grande chauve-souris brune (Eptesicus fuscus) dont le microbiote cutané pourrait jouer un rôle de protection contre l’infection. Nos résultats montrent que la diversité du microbiote de la grande chauve-souris brune inoculée avec Pd est plus variable dans le temps, tandis que la diversité du microbiote des chauves-souris du groupe contrôle demeure stable. Parmi les taxons les plus abondants, Pseudomonas et Rhodococcus, deux taxons connus pour leur potentiel antifongique contre Pd et d’autres champignons, sont restés stables durant l’expérience. Ainsi, bien que l’inoculation par le champignon Pd ait déstabilisé le microbiote cutané, les bactéries aux propriétés antifongiques n’ont pas été affectées. Cette étude est la première à démontrer le potentiel du microbiote cutané d’une espèce de chauves-souris pour la résistance au SMB. Au Chapitre 3, le microbiome cutané de la petite chauve-souris brune a été évalué en milieu naturel dans le contexte du SMB, à l’aide de la métagénomique, une approche haute résolution pour observer le potentiel fonctionnel du microbiome (métagénome fonctionnel). Nos résultats ont permis d’établir que le temps depuis l’infection a un effet significatif sur le métagénome fonctionnel. En effet, les chauves-souris dans la première année suivant l’infection ont un métagénome fonctionnel perturbé qui subit une perte de diversité fonctionnelle importante. Toutefois, le métagénome fonctionnel revient à une structure et composition similaire d’avant infection après 10 ans. Certaines fonctions détectées suite à l’infection sont associées à des gènes reliés au transport et à l’assimilation de métaux, des facteurs limitants pour la croissance du champignon. Ces gènes pourraient donc avoir un rôle à jouer dans la résistance ou la vulnérabilité à la maladie. Globalement, l’étude du métagénome chez la petite chauve-souris brune indique une vulnérabilité du métagénome fonctionnel au champignon, mais que celui-ci semble se rétablir après 10 ans. Une telle réponse pourrait avoir un impact sur la résilience de M. lucifugus. Cette thèse a permis d’acquérir des connaissances fondamentales sur le microbiome cutané des chauves-souris en hibernation pour mieux comprendre les communautés microbiennes de la peau dans le contexte du SMB. Le microbiome pourrait en effet jouer un rôle dans la vulnérabilité et la résistance des chauves-souris à la maladie, et il est essentiel d’adapter notre façon d’aborder la protection de ces espèces et de leur microbiome. Nous souhaitons que les travaux de cette thèse permettent de sensibiliser les acteurs de la conservation à l’existence et à l’importance potentielle du microbiome pour la santé de son hôte. Cette thèse fait également état de l’avancement des méthodes d’analyses qui permettront d’être de plus en plus précis et d’appliquer les connaissances du microbiome en biologie de la conservation. / White-nose syndrome (WNS) caused by the fungus Pseudogymnoascus destructans (Pd) has put hibernating bat populations at risk in North America. Some species are highly vulnerable to the disease while other species appear to be resistant or tolerant. Several physiological and environmental factors can explain these differences. However, before 2015, few studies have focused on the skin microbiome in relation to this disease. The present thesis aims to characterize the cutaneous microbiome of bats affected by WNS in order to identify the factors of vulnerability or resistance to the disease. The main objective is to determine how the microbiome can protect against the Pd fungus, or conversely how the microbiome is altered by the fungal infection. In Chapter 1, we first explored and compared the skin microbiota of little brown bats (Myotis lucifugus) unaffected by WNS with that of WNS survivors to test the hypothesis that the skin microbiota is modified by the disease. Our results show that the hibernation site strongly influences the composition and diversity of the skin microbiota. The Pd positive and negative sites differ significantly in terms of diversity, as well as in terms of the composition of the microbiota. Diversity is reduced within the microbiota of bats surviving WNS and enriched in taxa such as Janthinobacterium, Micrococcaceae, Pseudomonas, Ralstonia, and Rhodococcus. Some of these taxa are recognized for their antifungal potential and specific strains of Rhodococcus and Pseudomonas may inhibit the growth of Pd. Our results are consistent with the hypothesis that Pd infection modifies the skin microbiota of surviving bats and suggest that the microbiota may play a protective role against WNS. In Chapter 2, we studied in a controlled environment the microbiota of a species that exhibits evidence of resistance with mild WNS symptoms, before and after infection, to establish the potential response to the disease. The species studied is the big brown bat (Eptesicus fuscus), whose skin microbiota could play a protective role against infection. Our results show that the diversity of the microbiota of big brown bats inoculated with Pd is more variable over time, while the diversity of the microbiota of the control bats remains stable. Among the most abundant taxa, Pseudomonas and Rhodococcus, two taxa known for their antifungal potential against Pd and other fungi, remained stable during the experiment. Thus, although inoculation with the Pd fungus destabilized the skin microbiota, bacteria with antifungal properties were not affected. This study is the first to demonstrate the potential of the skin microbiota of a bat species for resistance to WNS. In Chapter 3, the skin microbiome of the little brown bat was evaluated in the natural environment in the context of WNS, using metagenomics, a higher-resolution approach to observe the functional potential of the microbiome (functional metagenome). Our results established that the time since infection has a significant effect on the functional metagenome. Indeed, bats in the first year after infection have a disrupted functional metagenome that undergoes a significant loss of functional diversity. However, the functional metagenome returns to a similar structure and composition to that observed before infection after 10 years. Certain functions detected following infection are associated with genes linked to the transport and assimilation of metals, known limiting factors for the growth of the fungus. These genes could therefore have a role to play in resistance or vulnerability to the disease. Overall, this metagenomics study indicates functional metagenome vulnerability to the fungus, although the original functional metagenome is reestablished after 10 years. Such diversified response could impact M. lucifugus resilence. This thesis provides fundamental knowledge on the skin microbiome of hibernating bats to better understand the microbial communities of the skin in the context of WNS. The microbiome could indeed play a role in the vulnerability and resistance of bats to disease and it is essential to adapt our way of approaching the protection of these species and their microbiomes. We hope that the results of this thesis will raise awareness among conservation stakeholders about the existence and potential importance of the microbiome for the health of its host. This thesis also reports on the advancement of analytical methods that will make it possible to be more and more precise and to apply knowledge of the microbiome in conservation biology.