Otimizaçao da produçao de biomassa e astaxantina pela levedura Phaffia rhodozyma, utilizando processo descontínuo alimentado

Orientadora: Tania Maria Bordin BonfimCo-orientadora: Iara Maria P. MachadoDissertaçao(mestrado)- Universidade Federal do Paraná. Setor de Ciencias da Saúde. Curso de Pós-Graduaçao em Ciencias FarmaceuticasInclui bibliografiaResumo: Astaxantína é um carotenòide amplamente distribuído na natureza, sendo encontrado como principal pigmento em alguns crustáceos (camarão e lagosta), peixes (truta e salmão), pássaros (íbis e flamingo) e microorganismos (a levedura Phaffia rhodozyma e a alga Haematococcus pluvialis). É utilizada principalmente na criação em cativeiro de trutas e salmões, proporcionando a pigmentação característica destas espécies e aumentando a qualidade e aceitação de seus produtos no mercado. Em função do crescente mercado mundial, do elevado custo da astaxantina produzida sinteticamente e da necessidade de se obter astaxantina a partir de fontes naturais, em escala industrial, a um baixo custo e elevada produtividade, buscou-se a otimização da produção de biomassa e astaxantina pela levedura Phaffia rhodozyma, utilizando processos fermentativos do tipo descontinuo alimentado e matérias-primas de baixo custo (caldo de cana-de-açúcar e uréia) como substratos. Obteve-se um processo, otimizado por processo descontino alimentado estendido, com produtividades em biomassa e astaxantina 4,55 e 4,73 vezes maiores do que as produtividades obtidas pelo processo fermentativo descontinuo, respectivamente. As elevadas produtividades obtidas neste processo otimizado são capazes de competir em produtividade com os processos encontrados na literatura e despertar interesse na produção industrial, em função da associação de processos de elevada produtividade em biomassa e astaxantina com meios de fermentação de baixo custo. Paiavras-cnave: matérias-primas de baixo custo; otimização; Phaffia rhodozyma-, processos descontínuo alimentado.Abstract: Astaxanthin is a carotenoid widely distributed in nature, being found as the main pigment in some crustaceans (shrimp and lobster), fish (trout and salmon), birds (flamingo and scarlet ibis; ana microorganisms (the yeast Phaffia rhodozyma and the algae Haematococcus pluvialis). It is mainly used in the trout and salmon fanning, providing the characteristic pigmentation of these fish and increasing the quality and consumer acceptance in the marketplace. Because of the increasing worldwide market and the hign cost of sinthetic astaxanthin, the need of astaxanthin obtained from natural sources, in scaled-up processes, at a low cost and high productivity, we looked for the optimization of the biomass and astaxanthin production by the yeast Phaffia rhodozyma, using fedbatch fermentation processes and low cost raw materials (sugar cane juice and urea) as substrates. It was obtained a process, optimized by fed-batch fermentation, with a biomass and astaxanthin productivity 4.55 and 4.73-fold the productivities obtained by the batch process fermentation, respectively. The high productivities obtained in this optimized process are able to compete in productivity with the processes found in the literature and arouse interest to the industrial production, because of the association of high biomass and astaxanthin productivity processes with iow cost fermentation medium. Key-words: fed-batch processes; low cost matenais; optimization; Phaffia rhodozyma

Functional heterogeneity of the UpaH autotransporter protein from uropathogenic Escherichia coli

Uropathogenic Escherichia coli (UPEC) are responsible for the majority of urinary tract infections(UTI). To cause UTI, UPEC must adhere to epithelial cells of the urinary tract and overcome the shear flow forces of urine. This function is primarily mediated by fimbrial adhesins, which mediate specific attachment to host cell receptors. Another group of adhesins that contribute to UPEC mediated UTI are autotransporter (AT) proteins. AT proteins possess a range of virulence properties such as adherence, aggregation, invasion and biofilm formation. One recently characterized AT protein of UPEC is UpaH, a large AIDA-I type AT protein that contributes to biofilm formation and bladder colonization. In this study, we have characterized a series of naturally occurring variants of UpaH. We demonstrate that extensive sequence variation exists within the passenger-encoding domain of UpaH variants from different UPEC strains. This sequence variation is associated with functional heterogeneity with respect to the ability of UpaH to mediate biofilm formation. In contrast, all of the UpaH variants examined retained a conserved ability to mediate binding to extracellular matrix (ECM) proteins. Bioinformatic analysis of the UpaH passenger domain identified a conserved region (UpaHCR) and hydrophobic region (UpaHHR). Deletion of these domains reduced biofilm formation but not binding to ECM proteins. Despite variation in upaH sequence, the transcription of upaH was repressed by a conserved mechanism involving the global regulator H-NS, and mutation of the hns gene relieved this repression. Overall, our findings shed new light on the regulation and function of the UpaH AT protein

A flow cytometry-based assay to determine the ability of anti-Streptococcus pyogenes antibodies to mediate monocytic phagocytosis in human sera.

Streptococcus pyogenes, commonly referred to as Group A Streptococcus (Strep A), causes a spectrum of diseases, with the potential to progress into life-threatening illnesses and autoimmune complications. The escalating threat of antimicrobial resistance, stemming from the prevalent reliance on antibiotic therapies to manage Strep A infections, underscores the critical need for the development of disease control strategies centred around vaccination. Phagocytes play a critical role in controlling Strep A infections, and phagocytosis-replicating assays are essential for vaccine development. Traditionally, such assays have employed whole-blood killing or opsonophagocytic methods using HL-60 cells as neutrophil surrogates. However, assays mimicking Fcγ receptors- phagocytosis in clinical contexts are lacking. Therefore, here we introduce a flow cytometry-based method employing undifferentiated THP-1 cells as monocytic/macrophage model to swiftly evaluate the ability of human sera to induce phagocytosis of Strep A. We extensively characterize the assay's precision, linearity, and quantification limit, ensuring robustness. By testing human pooled serum, the assay proved to be suitable for the comparison of human sera's phagocytic capability against Strep A. This method offers a valuable complementary assay for clinical studies, addressing the gap in assessing FcγR-mediated phagocytosis. By facilitating efficient evaluation of Strep A -phagocyte interactions, it may contribute to elucidating the mechanisms required for the prevention of infections and inform the development of future vaccines and therapeutic advancements against Strep A infections

Proteomics Characterization of Outer Membrane Vesicles from the Extraintestinal Pathogenic Escherichia coli ΔtolR IHE3034 Mutant

Extraintestinal pathogenic Escherichia coli are the cause of a diverse spectrum of invasive infections in humans and animals, leading to urinary tract infections, meningitis, or septicemia. In this study, we focused our attention on the identification of the outer membrane proteins of the pathogen in consideration of their important biological role and of their use as potential targets for prophylactic and therapeutic interventions. To this aim, we generated a DeltatolR mutant of the pathogenic IHE3034 strain that spontaneously released a large quantity of outer membrane vesicles in the culture supernatant. The vesicles were analyzed by two-dimensional electrophoresis coupled to mass spectrometry. The analysis led to the identification of 100 proteins, most of which are localized to the outer membrane and periplasmic compartments. Interestingly based on the genome sequences available in the current public database, seven of the identified proteins appear to be specific for pathogenic E. coli and enteric bacteria and therefore are potential targets for vaccine and drug development. Finally we demonstrated that the cytolethal distending toxin, a toxin exclusively produced by pathogenic bacteria, is released in association with the vesicles, supporting the recently proposed role of bacterial vesicles in toxin delivery to host cells. Overall, our data demonstrated that outer membrane vesicles represent an ideal tool to study Gram-negative periplasm and outer membrane compartments and to shed light on new mechanisms of bacterial pathogenesis

Otimizaçao da produçao de biomassa e astaxantina pela levedura Phaffia rhodozyma, utilizando processo descontínuo alimentado

Orientadora: Tania Maria Bordin BonfimCo-orientadora: Iara Maria P. MachadoDissertaçao(mestrado)- Universidade Federal do Paraná. Setor de Ciencias da Saúde. Curso de Pós-Graduaçao em Ciencias FarmaceuticasInclui bibliografiaResumo: Astaxantína é um carotenòide amplamente distribuído na natureza, sendo encontrado como principal pigmento em alguns crustáceos (camarão e lagosta), peixes (truta e salmão), pássaros (íbis e flamingo) e microorganismos (a levedura Phaffia rhodozyma e a alga Haematococcus pluvialis). É utilizada principalmente na criação em cativeiro de trutas e salmões, proporcionando a pigmentação característica destas espécies e aumentando a qualidade e aceitação de seus produtos no mercado. Em função do crescente mercado mundial, do elevado custo da astaxantina produzida sinteticamente e da necessidade de se obter astaxantina a partir de fontes naturais, em escala industrial, a um baixo custo e elevada produtividade, buscou-se a otimização da produção de biomassa e astaxantina pela levedura Phaffia rhodozyma, utilizando processos fermentativos do tipo descontinuo alimentado e matérias-primas de baixo custo (caldo de cana-de-açúcar e uréia) como substratos. Obteve-se um processo, otimizado por processo descontino alimentado estendido, com produtividades em biomassa e astaxantina 4,55 e 4,73 vezes maiores do que as produtividades obtidas pelo processo fermentativo descontinuo, respectivamente. As elevadas produtividades obtidas neste processo otimizado são capazes de competir em produtividade com os processos encontrados na literatura e despertar interesse na produção industrial, em função da associação de processos de elevada produtividade em biomassa e astaxantina com meios de fermentação de baixo custo. Paiavras-cnave: matérias-primas de baixo custo; otimização; Phaffia rhodozyma-, processos descontínuo alimentado.Abstract: Astaxanthin is a carotenoid widely distributed in nature, being found as the main pigment in some crustaceans (shrimp and lobster), fish (trout and salmon), birds (flamingo and scarlet ibis; ana microorganisms (the yeast Phaffia rhodozyma and the algae Haematococcus pluvialis). It is mainly used in the trout and salmon fanning, providing the characteristic pigmentation of these fish and increasing the quality and consumer acceptance in the marketplace. Because of the increasing worldwide market and the hign cost of sinthetic astaxanthin, the need of astaxanthin obtained from natural sources, in scaled-up processes, at a low cost and high productivity, we looked for the optimization of the biomass and astaxanthin production by the yeast Phaffia rhodozyma, using fedbatch fermentation processes and low cost raw materials (sugar cane juice and urea) as substrates. It was obtained a process, optimized by fed-batch fermentation, with a biomass and astaxanthin productivity 4.55 and 4.73-fold the productivities obtained by the batch process fermentation, respectively. The high productivities obtained in this optimized process are able to compete in productivity with the processes found in the literature and arouse interest to the industrial production, because of the association of high biomass and astaxanthin productivity processes with iow cost fermentation medium. Key-words: fed-batch processes; low cost matenais; optimization; Phaffia rhodozyma

Comparative analysis of the uropathogenic Escherichia coli surface proteome by tandem mass-spectrometry of artificially induced outer membrane vesicles

Uropathogenic Escherichia coli (UPEC) are the major cause of urinary tract infections. For successful colonisation of the urinary tract, UPEC employ multiple surface-exposed or secreted virulence factors, including adhesins and iron uptake systems. Whilst individual UPEC strains and their virulence factors have been the focus of extensive research, there have been no outer membrane (OM) proteomic studies based on large clinical UPEC collections, primarily due to limitations of traditional methods. In this study, a high-throughput method based on tandem mass-spectrometry of EDTA heat-induced outer membrane vesicles (OMVs) was developed for the characterisation of the UPEC surface-associated proteome. The method was applied to compare the OM proteome of fifty-four UPEC isolates, resulting in the identification of 8789 proteins, consisting of 619 unique proteins, which were subsequently interrogated for their subcellular origin, prevalence and homology to characterised virulence factors. Multiple distinct virulence-associated proteins were identified, including two novel putative iron uptake proteins, an uncharacterised type of chaperone-usher fimbriae and various highly prevalent hypothetical proteins. Our results give fundamental insight into the physiology of UPEC and provide a framework for understanding the composition of the UPEC OM proteome

Comparative proteomics of uropathogenic Escherichia coli during growth in human urine identify UCA-like (UCL) fimbriae as an adherence factor involved in biofilm formation and binding to uroepithelial cells

Uropathogenic Escherichia coli (UPEC) are the primary cause of urinary tract infection (UTI) in humans. For the successful colonisation of the human urinary tract, UPEC employ a diverse collection of secreted or surface-exposed virulence factors including toxins, iron acquisition systems and adhesins. In this study, a comparative proteomic approach was utilised to define the UPEC pan and core surface proteome following growth in pooled human urine. Identified proteins were investigated for subcellular origin, prevalence and homology to characterised virulence factors. Fourteen core surface proteins were identified, as well as eleven iron uptake receptor proteins and four distinct fimbrial types, including type 1, P, F1C/S and a previously uncharacterised fimbrial type, designated UCA-like (UCL) fimbriae in this study. These pathogenicity island (PAI)-associated fimbriae are related to UCA fimbriae of Proteus mirabilis, associated with UPEC and exclusively found in members of the E. coli B2 and D phylogroup. We further demonstrated that UCL fimbriae promote significant biofilm formation on abiotic surfaces and mediate specific attachment to exfoliated human uroepithelial cells. Combined, this study has defined the surface proteomic profiles and core surface proteome of UPEC during growth in human urine and identified a new type of fimbriae that may contribute to UTI

Otimização da produção de biomassa e astaxantina pela levedura Phaffia rhodozyma

A combinação de processos descontínuo alimentado e matérias-primas de baixo custo (caldo de cana-de-açúcar e uréia) foi estudada a fim de otimizar a produção de biomassa e astaxantina pela levedura Phaffia rhodozyma ATCC 24202. No processo otimizado, produtividades em biomassa e astaxantina de 0,327 g/L/h e 0,124 mg/L/h foram obtidas, respectivamente. Comparadas com o processo descontínuo estudado, verificou-se aumento de 4,55 vezes na produtividade em biomassa e 4,73 vezes na produtividade em astaxantina.The combination of fed-batch processes and low cost substrates (sugar cane juice and urea) was studied in view of the optimization of biomass and astaxanthin production by the yeast Phaffia rhodozyma ATCC 24202. In the optimized process, a biomass and astaxanthin productivity of 0.327 g/l/h and 0.124 mg/l/h was achieved, respectively. Compared to the batch process studied, an increase of approximately 4.55-fold in the biomass productivity and 4.73-fold in the astaxanthin productivity was found

Optimization of biomass and astaxanthin production by the yeast Phaffia rhodozyma Otimização da produção de biomassa e astaxantina pela levedura Phaffia rhodozyma

The combination of fed-batch processes and low cost substrates (sugar cane juice and urea) was studied in view of the optimization of biomass and astaxanthin production by the yeast Phaffia rhodozyma ATCC 24202. In the optimized process, a biomass and astaxanthin productivity of 0.327 g/l/h and 0.124 mg/l/h was achieved, respectively. Compared to the batch process studied, an increase of approximately 4.55-fold in the biomass productivity and 4.73-fold in the astaxanthin productivity was found.<br>A combinação de processos descontínuo alimentado e matérias-primas de baixo custo (caldo de cana-de-açúcar e uréia) foi estudada a fim de otimizar a produção de biomassa e astaxantina pela levedura Phaffia rhodozyma ATCC 24202. No processo otimizado, produtividades em biomassa e astaxantina de 0,327 g/L/h e 0,124 mg/L/h foram obtidas, respectivamente. Comparadas com o processo descontínuo estudado, verificou-se aumento de 4,55 vezes na produtividade em biomassa e 4,73 vezes na produtividade em astaxantina