Clostridium difficile is a Gram-positive bacterium that causes a toxin-mediated disease, typically in individuals whose normal intestinal flora has been compromised by antibiotic therapy. C. difficile is naturally resistant to many antibiotics and produces spores that can withstand harsh environmental conditions and many disinfectants, making the infection difficult to clear and easy to spread. The infection begins when spores from the environment are ingested and germinate upon exposure to taurocholate and glycine in the digestive tract. This germination process is required to initiate infection and thus represents a good target for the development of novel therapeutics. Although spore germination is necessary for disease transmission, the molecular mechanisms regulating this process are poorly understood. Germination relies on sensing a germinant and triggering degradation of the cortex layer of the spore, which is important for spore resistance. Once the cortex is degraded, the spore can undergo outgrowth to a vegetative cell and secrete toxins to cause disease symptoms. There are several discrete steps to the proteolytic cascade that ultimately lead to cortex hydrolysis. First, the pseudoprotease CspC acts as a germinant receptor for the bile salt taurocholate; CspC then relays this signal to the subtilisin-like serine protease, CspB. CspB is required for efficient cleavage and activation of the cortex hydrolase. SleC. Upon proteolytic activation of SleC, cortex hydrolysis can proceed, which allows subsequent outgrowth. To better understand the mechanistic basis of the germination process, we solved the 1.6 Å structure of the required germination protease, CspB, from C. perfringens (a related pathogen). This structure revealed that CspB is comprised of three domains: an associated prodomain, a subtilase domain, and a jellyroll domain. Our work significantly advanced our understanding of the proteolytic cascade that leads to germination; in particular the structure and function of the CspB protease, and the role of its three domains. We have described the four domains of the cortex hydrolase, SleC, and how they contribute to the activity of SleC. We have recently obtained diffraction-quality crystals of the pseudoprotease, CspC, from an organism more closely related to C. difficile, C. bifermentans. Our latest work, focusing on the germination receptor, CspC, has brought us closer to a three-dimensional structure of this protein, which will likely reveal how it binds ligands and functions in germination.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Bacillus cereus est une bactérie sporulante pathogène largement disséminée dans la nature. Les propriétés de résistance de ses spores aux traitements appliqués dans la chaîne de transformation des aliments font de B. cereus un contaminant à l’origine de toxi-infections alimentaires. La température est considérée comme l’un des facteurs environnementaux majeurs influençant la résistance de la spore. La variabilité des propriétés des spores liée à des modifications profondes dans leur structure contribue à une incertitude sur l’efficacité des processus de décontamination. Ce travail de thèse avait pour objectif de caractériser le mécanisme d’assemblage des enveloppes de la spore en fonction de la température de sporulation, en particulier le rôle de la protéine morphogénétique CotE chez B. cereus. La protéine CotE est retrouvée en abondance dans les spores produites à 20°C, une température suboptimale, par rapport à celles produites à 37°C, température optimale de croissance de la souche ATCC14579. La protéine CotE est détectée dans les tuniques et l’exosporium, structures protectrices de la spore. L’observation en microscopie électronique à transmission de spores d’un mutant DcotE révèle un problème d’assemblage de l’exosporium à 37°C et 20°C, mais également un défaut d’assemblage des tuniques à 20°C, ce qui suggère un rôle fondamental de CotE dans la mise en place de ces deux enveloppes, dépendant de la température de sporulation. Par microscopie à fluorescence, nous avons montré la cinétique de production de la protéine CotE au cours de la sporulation ainsi que sa localisation finale dans la spore mature, qui ne sont pas significativement impactées par la température de formation des spores. Nos résultats suggèrent également que la protéine CotE puisse créer le lien maintenant l’exosporium au contact des tuniques et du cortex. Enfin, nous avons montré que la protéine CotE est impliquée dans la germination et la résistance physique et chimique des spores. / Spores of the pathogenic bacterium Bacillus cereus are widespread in the environment and responsible of foodborne poisonings. Spores are a major concern to public health because of high resistance to treatments applied in food processing operations. Sporulation temperature is a main environmental factor that influences spore resistance properties. The variability of the conditions in which spores are formed during the sporulation process deeply modified their structure and consequently the efficiency of decontamination treatments. The aim of this work was to study the mechanism of spore layers assembly as a function of the sporulation temperature, and more precisely the role of the CotE protein in B. cereus. This morphogenetic protein is more detected in spores formed at 20°C, a suboptimal growth temperature than at 37°C, the optimal growth temperature, of the ATCC14579 strain. Observations in transmission electronic microscopy of DcotE spores revealed an assembly default of the spore exosporium at 37°C and 20°C but also of the spore coat at 20°C, suggesting that CotE has a role in the assembly of both layers, depending of the sporulation temperature. By fluorescence microscopy, we evidenced the kinetics of CotE production during sporulation and its final localization in mature spore, which are not dependent on the temperature of spore formation. Our results suggest that CotE could make a link to maintain the exosporium close to coat and cortex structures. Finally we showed that CotE also plays a role in germination and resistance properties of B. cereus spores to physical and chemical treatments.

Há poucos estudos relacionados com a caracterização dos componentes biológicos presentes no material particulado atmosférico, chamados bioaerossóis. Esses componentes biológicos podem assumir uma relação direta com a deflagração de doenças e também estão relacionados com os processos climáticos, em especial como núcleos de formação de gelo. Dentre os bioaerossóis, foram estudados, neste trabalho, os fungos anemófilos, sendo os principais os Basidiomycota, Ascomycota e os Fungos mitospóricos. O objetivo desse trabalho foi estimar a contribuição dos aerossóis fúngicos para a composição biogênica do material particulado atmosférico da Região Metropolitana de São Paulo. Foram realizadas medidas em dois sítios: na Cidade Universitária e no Pico do Jaguará. Para isso, foram caracterizados os tipos fúngicos utilizando-se um microscópio ótico e estimada a massa dos aerossóis de origem fúngica utilizando-se biomarcadores. Foram realizadas coletas durante 2013, 2014 e 2015 com o amostrador Burkard 7-day Recording Sampler, sendo que em 2015 também foram realizadas coletas com filtros de quartzo com o amostrador Airmetrics MiniVol portable Sampler. Foram identificados e obtida a concentração dos tipos fúngicos observados na atmosfera da RMSP, sendo encontrados 39 grupos principais de fungos, sendo os principais os Basidiomycotas. A concentração média total foi de 5736 (± 2459) esporos/m³ por dia. Os Ascomycota, Basidiomycota e os fungos mitospóricos se correlacionaram com as variáveis meteorológicas de forma diferente. Observou-se variação da concentração de esporos durante os diferentes períodos do dia, sendo que a maior concentração de esporos ocorreu na madrugada, possivelmente devido às condições meteorológicas (elevada umidade e temperaturas mais amenas), contudo a concentração dos fungos mitospóricos foi maior durante o período da tarde, principalmente devido aos mecanismos de liberação de esporos. No verão e na primavera foram obtidas as maiores concentrações de Ascósporos e Basidiósporos e no inverno e outono foram obtidas as maiores concentrações dos fungos mitospóricos. Além disso, observou-se uma correlação positiva e significativa entre a concentração de esporos totais e o material particulado inalável (MP10), indicando que ambos são influenciados da mesma forma pelas variáveis meteorológicas e/ou tem uma fonte em comum, como por exemplo a ressuspensão da poeira do solo. O Arabitol, Manitol, Treitol e os esporos de fungos são positivamente correlacionados, indicando que os três açucares podem ser traçadores de esporos de fungos na atmosfera. Utilizando-se um fator de conversão proposto na literatura foi possível estimar que 2% da concentração do MP10 e 8% da concentração do OC estão associados aos esporos de fungos, indicando a importância dos esporos fúngicos. Com o uso de modelos receptores foram observadas 6 fontes para o MP10: queima de biomassa resultante de processos industriais; queima de biomassa resultante da queima da vegetação; aerossóis fúngicos; formação de aerossol secundário; ressuspensão do solo e emissão veicular. Esses resultados constituem um avanço para a pesquisa em bioaerossóis no Brasil, já que muitas dessas análises ainda não haviam sido realizadas em áreas urbanas no Brasil / Although the biogenic components of Particulate Matter (PM) can have a direct relationship with the outbreak of respiratory diseases and can be linked to changes in climate processes there are very few studies related to its characterization in the atmospheric aerosols. Among the bioaerosols, the airborne fungi were studied, and the Basidiomycota, Ascomycota and Mitosporic fungi were the main characterized. The goal of this work was to estimate the contribution of the fungal aerosol to the RMSP atmosphere, with samplings in two sites: Cidade universitária (USP) and Pico do Jaraguá (PJ). For that, the fungi types were characterized using an optical microscope and it was estimated the mass of the fungal aerosol using biomarkers. Sampling was carried out during 2013, 2014 and 2015 with the \"Burkard 7-day Recording Sampler\", and in 2015, filters were sampled with the \"Airmetrics MiniVol portable Sampler. It was identified and determined the concentration of the fungi types observed in the RMSP atmosphere, and were found 39 main groups of fungi. The main group was the Basidiomycota. The mean concentration of the total fungi was 5736 (± 2459) spores/m³ per day. The Ascomycota, Basidiomycota and the mitosporic fungi correlated in different ways with the meteorological variables. A variation of the spores concentration during different times of the day was observed, with the occurrence of the highest concentration of spores at dawn, possibly due to weather conditions (high humidity and cooler temperatures), but the concentration of the mitosporic fungi was higher during the afternoon, mainly due to the spores release mechanisms. In the summer and spring were observed higher concentrations of Ascospores and Basidiospores and in the autumn and winter were obtained higher concentration of Mitospores. Besides that, it was also observed a positive and significant correlation between the fungal spore with the particulate matter, indicating that both are influenced in the same way by weather variables and/or has a common source such as soil resuspension. Arabitol, Mannitol, Threitol and the fungi spores are positive correlated, indicating that these three sugar alcohols can be a tracer for fungi spores in the atmosphere. Using the conversion factor proposed in the literature was possible to estimate that 2% of the PM10 concentration and 8% of the OC concentration it is due to fungi spores, indicating its importance. Using receptor modeling it was identified six sources for PM10, the biomass burning resulting from industrial processes, the biomass burning resulting from vegetation burning, the fungal aerosol, the secondary formation aerosol, the soil resuspension and vehicular emission. These results are a breakthrough for research on bioaerosols in Brazil, since many of this analyzes had never been performed in urban areas in Brazil

Há poucos estudos relacionados com a caracterização dos componentes biológicos presentes no material particulado atmosférico, chamados bioaerossóis. Esses componentes biológicos podem assumir uma relação direta com a deflagração de doenças e também estão relacionados com os processos climáticos, em especial como núcleos de formação de gelo. Dentre os bioaerossóis, foram estudados, neste trabalho, os fungos anemófilos, sendo os principais os Basidiomycota, Ascomycota e os Fungos mitospóricos. O objetivo desse trabalho foi estimar a contribuição dos aerossóis fúngicos para a composição biogênica do material particulado atmosférico da Região Metropolitana de São Paulo. Foram realizadas medidas em dois sítios: na Cidade Universitária e no Pico do Jaguará. Para isso, foram caracterizados os tipos fúngicos utilizando-se um microscópio ótico e estimada a massa dos aerossóis de origem fúngica utilizando-se biomarcadores. Foram realizadas coletas durante 2013, 2014 e 2015 com o amostrador Burkard 7-day Recording Sampler, sendo que em 2015 também foram realizadas coletas com filtros de quartzo com o amostrador Airmetrics MiniVol portable Sampler. Foram identificados e obtida a concentração dos tipos fúngicos observados na atmosfera da RMSP, sendo encontrados 39 grupos principais de fungos, sendo os principais os Basidiomycotas. A concentração média total foi de 5736 (± 2459) esporos/m³ por dia. Os Ascomycota, Basidiomycota e os fungos mitospóricos se correlacionaram com as variáveis meteorológicas de forma diferente. Observou-se variação da concentração de esporos durante os diferentes períodos do dia, sendo que a maior concentração de esporos ocorreu na madrugada, possivelmente devido às condições meteorológicas (elevada umidade e temperaturas mais amenas), contudo a concentração dos fungos mitospóricos foi maior durante o período da tarde, principalmente devido aos mecanismos de liberação de esporos. No verão e na primavera foram obtidas as maiores concentrações de Ascósporos e Basidiósporos e no inverno e outono foram obtidas as maiores concentrações dos fungos mitospóricos. Além disso, observou-se uma correlação positiva e significativa entre a concentração de esporos totais e o material particulado inalável (MP10), indicando que ambos são influenciados da mesma forma pelas variáveis meteorológicas e/ou tem uma fonte em comum, como por exemplo a ressuspensão da poeira do solo. O Arabitol, Manitol, Treitol e os esporos de fungos são positivamente correlacionados, indicando que os três açucares podem ser traçadores de esporos de fungos na atmosfera. Utilizando-se um fator de conversão proposto na literatura foi possível estimar que 2% da concentração do MP10 e 8% da concentração do OC estão associados aos esporos de fungos, indicando a importância dos esporos fúngicos. Com o uso de modelos receptores foram observadas 6 fontes para o MP10: queima de biomassa resultante de processos industriais; queima de biomassa resultante da queima da vegetação; aerossóis fúngicos; formação de aerossol secundário; ressuspensão do solo e emissão veicular. Esses resultados constituem um avanço para a pesquisa em bioaerossóis no Brasil, já que muitas dessas análises ainda não haviam sido realizadas em áreas urbanas no Brasil / Although the biogenic components of Particulate Matter (PM) can have a direct relationship with the outbreak of respiratory diseases and can be linked to changes in climate processes there are very few studies related to its characterization in the atmospheric aerosols. Among the bioaerosols, the airborne fungi were studied, and the Basidiomycota, Ascomycota and Mitosporic fungi were the main characterized. The goal of this work was to estimate the contribution of the fungal aerosol to the RMSP atmosphere, with samplings in two sites: Cidade universitária (USP) and Pico do Jaraguá (PJ). For that, the fungi types were characterized using an optical microscope and it was estimated the mass of the fungal aerosol using biomarkers. Sampling was carried out during 2013, 2014 and 2015 with the \"Burkard 7-day Recording Sampler\", and in 2015, filters were sampled with the \"Airmetrics MiniVol portable Sampler. It was identified and determined the concentration of the fungi types observed in the RMSP atmosphere, and were found 39 main groups of fungi. The main group was the Basidiomycota. The mean concentration of the total fungi was 5736 (± 2459) spores/m³ per day. The Ascomycota, Basidiomycota and the mitosporic fungi correlated in different ways with the meteorological variables. A variation of the spores concentration during different times of the day was observed, with the occurrence of the highest concentration of spores at dawn, possibly due to weather conditions (high humidity and cooler temperatures), but the concentration of the mitosporic fungi was higher during the afternoon, mainly due to the spores release mechanisms. In the summer and spring were observed higher concentrations of Ascospores and Basidiospores and in the autumn and winter were obtained higher concentration of Mitospores. Besides that, it was also observed a positive and significant correlation between the fungal spore with the particulate matter, indicating that both are influenced in the same way by weather variables and/or has a common source such as soil resuspension. Arabitol, Mannitol, Threitol and the fungi spores are positive correlated, indicating that these three sugar alcohols can be a tracer for fungi spores in the atmosphere. Using the conversion factor proposed in the literature was possible to estimate that 2% of the PM10 concentration and 8% of the OC concentration it is due to fungi spores, indicating its importance. Using receptor modeling it was identified six sources for PM10, the biomass burning resulting from industrial processes, the biomass burning resulting from vegetation burning, the fungal aerosol, the secondary formation aerosol, the soil resuspension and vehicular emission. These results are a breakthrough for research on bioaerosols in Brazil, since many of this analyzes had never been performed in urban areas in Brazil

<p>Peat mosses (<i>Sphagnum</i>) are widely distributed and are a major component of mire vegetation and peat throughout the boreal and temperate regions. Most boreal <i>Sphagnum</i> species regularly produce sporophytes, but the ecological role of the spore has been questioned. This study shows that the spores can form a spore bank and have the ability to germinate and contribute to moss establishment whenever suitable conditions occur. The results suggest that spore production is important for explaining the wide distribution and omnipresence of <i>Sphagnum</i> in nutrient-poor wetlands. The results further imply that initial recruitment from spores predominates in <i>Sphagnum</i> after disturbance or formation of suitable habitats.</p><p> A series of experiments showed that addition of phosphorus-containing substrates, such as fresh plant litter or moose dung, resulted in spore establishment on bare, moist peat. A field experiment indicated establishment rates of about 1% of sown, germinable spores on peat with added substrates. Plant litter on moist soil, without a closed cover of bryophytes, is an important safe site for the establishment of <i>Sphagnum</i> spores. The results fit the observed pattern of colonisation by <i>Sphagnum</i> beneath <i>Eriophorum vaginatum</i> tussocks in mires severely disturbed by peat extraction. Successful long-distance dispersal was indicated by the occurrence of several regionally new or rare <i>Sphagnum</i> species in disturbed mires.</p><p>Spore number per sporophyte ranged among <i>Sphagnum</i> species from 18 500 to 240 000, with a trade-off between spore number and spore size. Annual spore production was estimated at 15 million spores per square metre on two investigated mires. Sporophyte production showed a large interannual variation. Sporophyte production was positively related to the amount of precipitation the preceding summer. This was probably because a high water level promoted gametangium formation. Spore dispersal occurred in July and August. The earlier timing of spore dispersal in the more drought-sensitive, hollow-inhabiting sphagna should reduce the risk of sporophytes drying out prematurely during summer droughts.</p><p>Spores kept refrigerated up to 13 years retained high germinability. A field experiment showed that <i>Sphagnum</i> can form a persistent spore bank, with a potential longevity of several decades.</p>

Bacterial spores have remarkable resistance to a variety of harsh conditions, causing spoilage in food industry and becoming the primary bacterial agent in biowarfare and bioterrorism. In this study, inactivation mechanisms of Bacillus amyloliquefaciens (BA) spores by non-thermal plasma (NTP) were investigated by using Fourier-transform infrared spectroscopy (FTIR) as a major tool to exam spores after NTP treatment. Chemometric techniques, such as multivariate classification models based on soft independent modeling of Class Analogy (SIMCA) and Principal Component Analysis (PCA), were employed to identify functional group changes in FTIR spectra. The IR absorbance bands correlated to dipicolinic acid (DPA) decreased after NTP treatment indicating that DPA released and then reacted with reactive species generated by NTP and it was confirmed by nuclear magnetic resonance (NMR). Also IR absorbance bands corresponding to protein structure changed. FTIR combined with UV-Vis spectroscopy was used to monitor spore germination. Large amount of DPA released in a short time when spores germinated at 50°C, showing that DPA released in response to heating. NTP treated spores could germinate with little DPA release due to sub-lethal effects induced by plasma. Also an empirical model based on Weibull distribution was established to describe the spore germination process showing that NTP treated spores exhibited abnormal germination pattern. Inactivation mechanisms of NTP with air as feed gas was compared with high-pressure, wet heat, chemical treatment using chlorine dioxide (CD) and NTP with argon as feed gas. The results showed that few chemical changes in spores after autoclave and high pressure treatments, though protein structure changed. CD and NTP with air as feed gas inactivated spores by oxidation. DPA released after NTP with argon as feed gas treatment and it is possible that UV and charged particles accounts for the inactivation. This study provides in depth insight into the inactivation mechanism of NTP and information for optimizing NTP process.

Peat mosses (Sphagnum) are widely distributed and are a major component of mire vegetation and peat throughout the boreal and temperate regions. Most boreal Sphagnum species regularly produce sporophytes, but the ecological role of the spore has been questioned. This study shows that the spores can form a spore bank and have the ability to germinate and contribute to moss establishment whenever suitable conditions occur. The results suggest that spore production is important for explaining the wide distribution and omnipresence of Sphagnum in nutrient-poor wetlands. The results further imply that initial recruitment from spores predominates in Sphagnum after disturbance or formation of suitable habitats. A series of experiments showed that addition of phosphorus-containing substrates, such as fresh plant litter or moose dung, resulted in spore establishment on bare, moist peat. A field experiment indicated establishment rates of about 1% of sown, germinable spores on peat with added substrates. Plant litter on moist soil, without a closed cover of bryophytes, is an important safe site for the establishment of Sphagnum spores. The results fit the observed pattern of colonisation by Sphagnum beneath Eriophorum vaginatum tussocks in mires severely disturbed by peat extraction. Successful long-distance dispersal was indicated by the occurrence of several regionally new or rare Sphagnum species in disturbed mires. Spore number per sporophyte ranged among Sphagnum species from 18 500 to 240 000, with a trade-off between spore number and spore size. Annual spore production was estimated at 15 million spores per square metre on two investigated mires. Sporophyte production showed a large interannual variation. Sporophyte production was positively related to the amount of precipitation the preceding summer. This was probably because a high water level promoted gametangium formation. Spore dispersal occurred in July and August. The earlier timing of spore dispersal in the more drought-sensitive, hollow-inhabiting sphagna should reduce the risk of sporophytes drying out prematurely during summer droughts. Spores kept refrigerated up to 13 years retained high germinability. A field experiment showed that Sphagnum can form a persistent spore bank, with a potential longevity of several decades.

The chicken eggshell possesses physical and chemical barriers to protect the embryo from pathogens. The avian eggshell cuticle is the outmost layer of the eggshell whose protein constituents remain largely unknown. Since eggs with incomplete or absent cuticle are more susceptible to bacterial contamination, we hypothesize that cuticle protein components play an important role in microbial resistance. In our study, at least 47 proteins were identified by LC/MS/MS in the non-calcified cuticle layer. Similar to Kunitz-like protease inhibitor (also annotated as ovocalyxin-25, OCX-25) and ovocalyxin-32 (OCX-32) were two of most abundant proteins of the cuticle proteins. Some proteins that have antimicrobial activity were also detected in the proteomic results, such as lysozyme C, ovotransferrin, ovocalyxin-32, cystatin, ovoinhibitor. This study represents the first comprehensive report of the cuticle proteome. Since the sequence similarity of the kunitz motif in OCX-25 is similar to that of BPTI, it is predicted that it will have the same trypsin inhibitory and antimicrobial activity against Gram-positive and/or Gram-negative bacteria. In order to test the antimicrobial property and trypsin inhibitor activity of OCX-25, cuticle proteins were extracted by 1N HCl. Antimicrobial activity was monitored using the Bioscreen C instrument; and antimicrobial activity was identified primarily against Staphylococcus aureus. Trypsin inhibitor activity was studied by using a specific trypsin assay, and the assay indicated that the cuticle proteins could inhibit the reaction of trypsin and substrate. Therefore, the current research has provided some insight into the antimicrobial and enzymatic aspects of the cuticle proteins, and its function for egg protection. Eggshell membranes are another important component of the chicken eggshell.Due to its insoluble and stable properties, there are still many questions regarding formation and constituents of the eggshell membranes. The purpose of our study was to identify eggshell membrane proteins, particularly these responsible for its structural features, by examining the transcriptome of the white isthmus during its formation. Bioinformatics tools were applied to analyze the differentially expressed genes as well as their encoded proteins. Some interesting proteins were encoded by the over-expressed genes in the white isthmus during the formation of eggshell membranes, such as Collagen X, and similar to spore coat protein SP75. These proteins may have potential applications. Our study provides a detailed description of the chicken white isthmus transcriptome during formation of the eggshell membranes; it could lead to develop the strategies to improve food safety of the table egg.