Thermal instability in the toxin catalytic subunit may be a common property of toxins that exit the endoplasmic reticulum (ER) by exploiting the mechanism of ER-associated degradation (ERAD). The Haemophilus ducreyi cytolethal distending toxin (HdCDT) does not utilize ERAD to exit the ER, so we predicted the structural properties of its catalytic subunit (HdCdtB) would differ from other ER-translocating toxins. Here, we document the heat-stable properties of HdCdtB which distinguish it from other ER-translocating toxins. Cell-based assays further suggested that HdCdtB does not unfold before exiting the ER and that it may move directly from the ER lumen to the nucleoplasm. These observations suggest a novel mode of ER exit for HdCdtB.

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Bacterial persistence is considered as one of the primary reason for antibiotic tolerance besides genetically acquired antibiotic resistance. Persisters are the subpopulation of a clonal bacterial population, which can survive environmental extremes and become invulnerable to stresses due to limited metabolic activities and physiological functions. Cognate toxin and antitoxin (TA) pairs, which are transcribed simultaneously from the same or different operons within the bacterial chromosomes or plasmids, play an important role for bacterial survival during stressful growth environments. Pseudomonas aeruginosa PAO1 is one of the most versatile microorganisms in the environment. Despite its ubiquitous presence, no studies have shown the differential expression pattern of its toxin-antitoxins, and persistence related genes. The purpose of the following study is to analyze differential expression of P. aeruginosa PAO1 type II toxin-antitoxins and persistence related genes under different growth conditions and to show how their stoichiometric ratio changes during different growth conditions. Differential expression analysis indicated that the toxins and antitoxin pairs behave differently under different growth conditions. In addition, the genes related to persistence presented relatively consistent differential expression pattern under different growth environment. / Master of Science / Bacterial persistence is one of the main reason for antibiotic tolerance and recurrent infections. Toxin-antitoxin molecules play an important role during bacterial persistence. Change in the expression of toxin, antitoxins, and persistence related genes and the ratio of the toxin to antitoxin mRNA molecules are important for bacterial survival in stressful environments. Pseudomonas aeruginosa PAO1 is one of most ubiquitous bacteria and responsible for recurrent infection in patients with weaker and compromised immunity. This mRNA sequence (RNA-Seq) analysis study of P. aeruginosa PAO1 showed different expression levels of toxin, antitoxin, and persistence related genes in various stressful growth conditions. This expression also showed the different ratios of the toxin to antitoxin mRNA molecules under different stress conditions. These implicate the different hypothetical roles of these toxin and antitoxin molecules in different growth conditions.

O Clostridium perfringens é um microrganismo anaeróbio que está presente no solo e no trato intestinal dos mamíferos. Provoca intoxicação alimentar nos seres humanos, doenças enterotoxêmicas nos animais domésticos e gangrena gasosa em ambos os grupos. O C. perfringens é classificado em cinco tipos (A, B, C, D e E) mediante a produção de quatro toxinas principais (alfa, beta, épsilon e iota). Neste trabalho foi possível padronizar a técnica de PCR para detectar a presença dos genes cpa, cpb e etx a partir de culturas de C. perfringens. A sensibilidade analítica da técnica de PCR a partir de culturas de C. perfringens foi de 2,27 ng/µL para o gene cpa, 22,7 pg/µL para o gene cpb e 22,7 pg/µL para o gene etx. A pesquisa dos genes cpa, cpb e etx partir de 35 amostras de C. perfringens isoladas de bovinos revelou que 16 (45,7%) eram do tipo A; 18 (51,4%) eram do tipo C e 1 (2,9%) era do tipo B. Não foi observada nenhuma amostra do tipo D. A metodologia de PCR revelou-se útil na tipificação de amostras de C. perfringens isoladas de bovinos, contribuindo para o diagnóstico dessa bacteriose neste país, eliminando as dificuldades de tipificação oriundas do alto custo e da indisponibilidade de anti-soros para a tipificação pela reação de soroneutralização e evitando a utilização de animais de laboratório. / Clostridium perfringens is an anaerobic micro-organism that is present in the soil and gastrointestinal tract of mammals. It causes food poisoning in humans, enterotoxemic diseases in domestic animals and gas gangrene in both. C. perfringens is classified into five types (A, B, C, D and E) according to the production of four major toxins (alpha, beta, epsilon and iota). In this trial was possible to standardize the PCR?s technique to detect cpa, cpb and etx genes from cultures of C. perfringens. PCR?s analythical sensibility was 2.27 ng/µL for cpa gene, 22.7 pg/µL for cpb gene and 22.7 pg/µL for etx gene. The research of cpa, cpb and etx genes from 35 samples of C. perfringens isolated from cattle reveals that 16 (45.7%) were classified as type A, 18 (51.4%) as type C and 1 (2.9%) as type B. No sample of type D was observed. PCR?s technique reveals to be usefull to typify samples of C. perfringens isolated from cattle, contributing to diagnose of this bacterial disease in this country and solving typifing problems represented by the high costs of the process and by the lack of antiserum that is required to typify the micro-organism by seroneutralization. PCR?s technique avoid the use of laboratory animals, too.

O Clostridium perfringens é um microrganismo anaeróbio que está presente no solo e no trato intestinal dos mamíferos. Provoca intoxicação alimentar nos seres humanos, doenças enterotoxêmicas nos animais domésticos e gangrena gasosa em ambos os grupos. O C. perfringens é classificado em cinco tipos (A, B, C, D e E) mediante a produção de quatro toxinas principais (alfa, beta, épsilon e iota). Neste trabalho foi possível padronizar a técnica de PCR para detectar a presença dos genes cpa, cpb e etx a partir de culturas de C. perfringens. A sensibilidade analítica da técnica de PCR a partir de culturas de C. perfringens foi de 2,27 ng/µL para o gene cpa, 22,7 pg/µL para o gene cpb e 22,7 pg/µL para o gene etx. A pesquisa dos genes cpa, cpb e etx partir de 35 amostras de C. perfringens isoladas de bovinos revelou que 16 (45,7%) eram do tipo A; 18 (51,4%) eram do tipo C e 1 (2,9%) era do tipo B. Não foi observada nenhuma amostra do tipo D. A metodologia de PCR revelou-se útil na tipificação de amostras de C. perfringens isoladas de bovinos, contribuindo para o diagnóstico dessa bacteriose neste país, eliminando as dificuldades de tipificação oriundas do alto custo e da indisponibilidade de anti-soros para a tipificação pela reação de soroneutralização e evitando a utilização de animais de laboratório. / Clostridium perfringens is an anaerobic micro-organism that is present in the soil and gastrointestinal tract of mammals. It causes food poisoning in humans, enterotoxemic diseases in domestic animals and gas gangrene in both. C. perfringens is classified into five types (A, B, C, D and E) according to the production of four major toxins (alpha, beta, epsilon and iota). In this trial was possible to standardize the PCR?s technique to detect cpa, cpb and etx genes from cultures of C. perfringens. PCR?s analythical sensibility was 2.27 ng/µL for cpa gene, 22.7 pg/µL for cpb gene and 22.7 pg/µL for etx gene. The research of cpa, cpb and etx genes from 35 samples of C. perfringens isolated from cattle reveals that 16 (45.7%) were classified as type A, 18 (51.4%) as type C and 1 (2.9%) as type B. No sample of type D was observed. PCR?s technique reveals to be usefull to typify samples of C. perfringens isolated from cattle, contributing to diagnose of this bacterial disease in this country and solving typifing problems represented by the high costs of the process and by the lack of antiserum that is required to typify the micro-organism by seroneutralization. PCR?s technique avoid the use of laboratory animals, too.

Master of Science in Biomedical Sciences / Department of Diagnostic Medicine/Pathobiology / Natalia Cernicchiaro / David G. Renter / Cattle shed Shiga toxin-producing Escherichia coli (STEC) in their feces. Therefore, cattle pose a risk to contaminate produce, water, and beef products intended for human consumption. The United States Department of Agriculture Food Safety and Inspection Service consider seven STEC serogroups (O26, O45, O103, O111, O121, O145, and O157) as adulterants in raw, non-intact beef products. Contrary to O157, the frequency and distribution of non-O157 serogroups and virulence genes have not been well-established in cattle. Therefore, the objectives of my thesis research were: 1) to appraise and synthesize data from peer-reviewed literature on non-O157 serogroup and virulence gene prevalence, and 2) to determine the prevalence of seven STEC in feedlot cattle feces across seasons. A systematic review and meta-analysis of published literature were conducted to gather, summarize, and interpret the existent data regarding non-O157 serogroup and virulence gene prevalence in cattle. Random-effects meta-analyses were used to obtain pooled non-O157 fecal prevalence estimates for continents worldwide and meta-regression analyses were conducted to evaluate effects of specific factors on between-study heterogeneity. Results indicated that non-O157 serogroup and virulence gene fecal prevalence significantly differed (P < 0.05) by geographic region, with North America yielding the highest pooled prevalence estimate worldwide. While previous research has demonstrated a strong seasonal shedding pattern of STEC O157, data regarding the seasonality of non-O157 STEC shedding in cattle is very limited. A repeated cross-sectional study was conducted to obtain serogroup and virulence gene prevalence data for the seven STEC in pre-harvest cattle feces, in summer and winter. We found that non-O157 serogroups were recovered in fecal samples collected in both seasons but virulence genes, thus STEC, were rarely detected in summer and undetected in winter. In conclusion, non-O157 STEC are present in cattle feces at very low frequencies, but STEC O103 and O157 significantly differed (P < 0.05) between seasons. Overall, the research described in this thesis greatly contributes to the limited body of data regarding non-O157 serogroup and virulence gene distribution in cattle and provides a better understanding of two major risk factors, season and geographic distribution, associated with STEC fecal shedding in cattle.