Vittoria, Maria (2024) Exploring novel Bacillus spore-forming strains from the human gut: probiotic potential, therapeutic applications, and molecular insights. [Tesi di dottorato]

Preview

Text Vittoria_Maria_36.pdf Download (26MB) | Preview

Collection description

This PhD thesis was conducted in the Biology Department of “Federico II” University of Naples under the supervision of Prof. Ezio Ricca as part of the XXXVI cycle of the Doctorate in Biology. The PhD scholarship is framed within the National Research and Innovation Operational Program 2014-2020 for 'Innovative Doctorates of Industrial Character'. In recent years, understanding of the complex relationship between the human gut microbiota and human health has increased, leading to growing interest in harnessing the therapeutic potential of probiotics. Among the countless microbial inhabitants of the human gut, spore-forming bacteria belonging to the genus Bacillus, have emerged as promising candidates for probiotic applications. These resilient organisms, capable of forming endospores, show remarkable survival under harsh environmental conditions, offering advantages in probiotic formulations and delivery. The quest to identify and characterize novel Bacillus spore-forming organisms from the human gut is an important goal in the field of probiotic research. Not only do these organisms have the potential to provide health benefits, but they also have unique physiological properties that deserve to be explored. The thesis is structured into three chapters and two appendices and undertakes a comprehensive investigation to elucidate the probiotic properties, genomic characteristics and therapeutic applications of novel Bacillus spore-forming strains isolated from the human gut environment. The study emphasizes the significance of exploring new strains as probiotics to identify distinctive properties to meet the increasing demand for non-traditional therapeutic approaches and microbiome- based therapies. In Chapter 1 are presented the results of the initial isolation and characterization of spore-forming bacteria inhabiting the human intestinal environment. According to the general guidelines for the safety evaluation of microorganisms isolated from human microbiota, four strains out of the thirty-two isolates were chosen for their safety and for their beneficial properties e.g. biofilm production, antimicrobial activity, presence of gene clusters potentially encoding novel antimicrobial peptides, antioxidant activity, induction of a cytoprotective stress response in human intestinal cells in vitro. The results were published on July 31, 2023: Probiotics as an Alternative to Antibiotics: Genomic and Physiological Characterization of Aerobic Spore Formers from the Human Intestine. Microorganisms, 11(8), Article 8. Chapter 2 focuses on the in vivo evaluation of the probiotic properties of two intestinal isolate strains, Bacillus subtilis SF106 and Bacillus clausii (recently renamed Alkalihalobacillus clausii) SF174. Probiotic treatments and microbiota- targeted approaches have been shown to be beneficial in several pathologies, including chronic inflammation of the intestine. This chapter reports the results obtained by administering spores of these two strains in a mouse model of chemically induced ulcerative colitis. The spores were found to attenuate overall inflammation and symptoms, showed immunomodulatory trend, improved the integrity of the intestinal barrier and remodeled the gut microbiota in favor of beneficial microbes. The results of this research have been included in an article currently under review. Chapter 3 is dedicated to the study of a human intestinal isolate that differed from all other isolates due to its production of cytoplasmic granules and the presence of spores with unusual curved morphology. The isolate, MV19, was assigned to the species B. cereus sensu stricto. Analysis of growth and sporulation at different temperatures revealed that at the sub-optimal growth temperature of 25 °C sporulation was slow and less efficient but a high total number of fully functional spores was produced. At the optimal growth temperature of 42 °C sporulation was faster and some of the released spore were curved, structurally and functionally defective. This suggested an important adaptation characteristic of these bacteria to environmental conditions. The results of this chapter were published on February 3, 2023: Sporulation efficiency and spore quality in a human intestinal isolate of Bacillus cereus. Research in Microbiology, Volume 174, Issue 6, 2023, 104030, ISSN 0923-2508. Appendix I and Appendix II describe the side experiments conducted during the three years of my PhD, focusing on elucidating the mechanisms governing spore structure and permeability. This adds valuable insight into how Bacillus spores interact with their environment, which is also linked to their probiotic functionality. Appendix I describes a molecular study of protein assembly in the surface layers of spores of the model organism Bacillus subtilis. The study focuses on the protein CotG, an important component of the spore surface. Under canonical conditions, CotG is phosphorylated by the thermolabile kinase CotH and assembles on the surface of the spores; our results showed that, depending on the sporulation temperature, CotG tends to aggregate in the cytoplasm of the mother cell, dragging along other coat proteins. This gives the spores a different structure and consequently different physiological properties. Results were published on March 7, 2022: CotG controls spore surface formation in response to the temperature of growth in Bacillus subtilis. Environmental Microbiology, 24: 2078-2088. Appendix II discusses the ability of CotG protein to regulate spore permeability through its central region of positively charged tandem repeats. These repeats facilitate the interaction and aggregation of CotG with other coat proteins, resulting in a protein layer that controls the permeability of external molecules. The level of permeability of the spore structure appears to be determined by the distance between the N and C termini of the CotG protein. This model can be applied to most spore formers of the Bacillus genus because they all possess CotG-like proteins. These results were published on November 10, 2022: CotG Mediates Spore Surface Permeability in Bacillus subtilis. American Society for Microbiology, mBIO, Volume 13, Issue 6. In conclusion, this PhD thesis presents a comprehensive investigation into the isolation and characterization of endospore-forming Bacillus spp. that contributes to the advancement of knowledge in the field of probiotics and offers potential applications for these bacteria in various healthcare contexts. The overall results shed light on the probiotic potential for human health, but also on the intricate molecular mechanisms that regulate spore structure and function. These results are promising for the development of new probiotic applications and contribute to the broader field of microbiology.

Downloads

Actions (login required)

View Item