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Title Page
Contents
ABSTRACT 9
LIST OF ABBREVIATIONS 15
I. General Introduction 20
II. Review of Related Literature 22
A. The Exoproteome of Lactobacilli 22
i. Covalently-Anchored Proteins 23
ii. Noncovalently-Anchored Proteins 24
iii. Secreted Proteins 25
iv. Moonlighting Proteins 27
B. Related Cell Surface Adhesion Proteins in This Study 29
i. Lectins and Lectin-Like Proteins 29
ii. MucBP-Domain Proteins 29
iii. Rib/Alpha/Esp-like Repeat Proteins 30
C. Current Methods of Studying the Bacterial Cell Surface (Surfaceome) 35
i. Cell Shaving 35
ii. Cell wall digestion and disruption 35
iii. Cell Surface Labeling 36
D. Cell-Surface Mediated Host-microbiota Interactions: Significance of Lactobacillus Probiotic Adhesion 38
i. Probiotic Adhesion, Colonization and Gut Homeostasis 40
ii. Probiotic Adhesion and Protection: Antagonism, Immunomodulation and Pathogen Exclusion 42
III. Data Analysis and Findings 47
Chapter 1. Determining the Nature of Lectin-Like Adhesins of Lactobacillus mucosae LM1 and Lactobacillus johnsonii PF01 47
1.1. Introduction 47
1.2. Materials and Methods 48
1.3. Results and Discussion 52
1.4. Summary and Insights from This Study 64
1.5. References 66
Chapter 2. Comparative Genomic and Exoproteomic Analysis of the Adhesive Swine Isolate Lactobacillus mucosae LM1 69
2.1. Introduction 69
2.2. Materials and Methods 70
2.3. Results and Discussion 79
2.4. Summary and Insights from This Study 128
2.5. References 129
Chapter 3. Genomic Mining and Functional Analysis of a Novel LPxTG Rib Protein from Lactobacillus johnsonii PF01 Using Surface-Displayed Technology 141
3.1. Introduction 141
3.2. Materials and Methods 143
3.3. Results and Discussion 151
3.4. Summary and Insights from This Study 190
3.5. References 192
Chapter 4. Characterization of the Surface-Displayed R4850 in Protection against the Opportunistic Pathogen Streptococcus agalactiae, a Group B Streptocci (GBS) 202
4.1. Introduction 202
4.2. Materials and Methods 204
4.3. Results and Discussion 206
4.4. Summary and Insights from This Study 215
4.5. References 216
IV. General Conclusion 217
V. References 219
VI. Appendices 247
Appendix A. Spearman's correlation analysis of the adhesion of the lactobacilli strains to the different adhesion substrates 247
Appendix B. Cell-surface associated Proteins of L. mucosae LM1 249
Appendix C. Hypergeometric and Bayesian statistics application on identified surface- shaved proteins in LM1 253
Appendix D. Known and putative moonlighting cytoplasmic proteins detected in strain LM1 259
Appendix E. Highlighted Proteins in PPI Network with Highest LFQ Intensities 262
VII. List of Publications 263
Table 1. Genome summary 83
Table 2. Genome statistics of L. mucosae LM1 83
Table 3. Number of genes associated with general COG functional categories 84
Table 4. Summary of putative surface exposed (PSE) proteins in L. mucosae LM1 95
Table 5. Summary of predicted adhesion proteins and notable PSE proteins from the genome sequence of L. mucosae LM1 99
Table 6. Identified surface adhesion factors from the exoproteogenomic analysis of LM1 112
Table 7. Putative cell surface-associating moonlighting proteins in LM1 with potential in mediating host-microbe interactions 121
Table 8. Primers used to amplify the Rib inserts for surface display in L. casei 525 145
Figure 1. Schematic representation of the surface exposed proteins of Gram-positive... 26
Figure 2. Schematic illustration of the association of moonlighting proteins with the cell surface of Lactobacillus. 28
Figure 3. The Alp family of proteins. 33
Figure 4. Summary of conserved InterPro (IPR) domains in putative surface-exposed (PSE) and secreted proteins in lactobacilli. 34
Figure 5. Surface proteomic analysis of Propionibacterium freudenreichii strains using... 37
Figure 6. Schematic representation of the adaptation and probiotic factors studied and... 39
Figure 7. The mucus layers of the human small intestine and colon. 41
Figure 8. Overview of the probiotic mechanisms of action in protection of the gut mucosal... 43
Figure 9. Immunomodulatory functions of probiotic bacteria. 45
Figure 10. Average percent adhesion of L. rhamnosus GG, L. mucosae LM1, and L.... 54
Figure 11. Models explaining possible adhesion mechanisms and interference. 55
Figure 12. Effects of carbohydrates on adhesion of L. rhamnosus GG, L. mucosae LM1, and L. johnsonii PF01 to IPEC-J2 cells. 57
Figure 13. Effects of carbohydrates on the adhesion of L. rhamnosus GG, L. mucosae LM1, L. johnsonii PF01, E. coli K88, and S. enterica... 60
Figure 14. Relationships between CIA distances and probiotic inhibition of pathogen adhesion to mucin/mucus. 63
Figure 15. Systematic exoproteogenomic profiling workflow applied to L. mucosae LM1. 73
Figure 16. SEM showing the aggregation and adhesion capacity of L. mucosae LM1 on the... 82
Figure 17. ANI Dendrogram of closely-related lactobacilli strains to L. mucosae LM1. 86
Figure 18. Glycogen metabolism pathway in L. mucosae LM1. 89
Figure 19. Folate biosynthesis pathways in specific lactobacilli. 91
Figure 20. MUMMER alignment between L. mucosae LM1 and L. mucosae DPC 6426 (left) and L. mucosae LM1 and L. mucosae CRL 573... 93
Figure 21. Graph charts showing 97
Figure 22. Total number of identified raw proteins from the trypsin treated fraction in L.... 104
Figure 23. Proteins identified via trypsin and proteinase K treatments. 105
Figure 24. Enzymatic treatment of the lactobacilli strains L. mucosae LM1, L. johnsonii... 106
Figure 25. Upregulated and downregulated proteins in L. mucosae LM1 in the presence of... 114
Figure 26. Scanning electron microscope image of L. mucosae LM1 cells showing rough... 117
Figure 27. Whole protein-protein (PPI) interaction network constructed using L.... 124
Figure 28. Cytoscape clustering methods (A) MCODE clustering and (B) MCL clustering were used to identify specific protein complexes in... 127
Figure 29. Comparative genomic analysis map of L. johnsonii genomes. 153
Figure 30. Gene organization of the PF01_16770 gene (surface protein Rib). 156
Figure 31. Multiple sequence alignment of the Rib repeat domains. 157
Figure 32. Comparison of Lactobacillus Rib homologs. 158
Figure 33. Gel electrophoresis of the Rib/alpha-like insert sequences after digestion with... 161
Figure 34. IGRIB Insert Sequence. 162
Figure 35. MRIB Insert Sequence. 163
Figure 36. R4850 Insert Sequence. 164
Figure 37. R9LP Insert Sequence. 165
Figure 38. Plasmid construct map of the L. casei 525 PgsA surface display vector. 166
Figure 39. Western blot analysis of L. casei 525 PgsA-Rib fusion proteins. 167
Figure 40. Adhesion of the L. casei 525 Rib recombinants to various substrates. 170
Figure 41. Autoaggregation assay. 171
Figure 42. Determination of the surface physicochemical properties of PgsA only and the... 172
Figure 43. Digestion of R48p PCR amplicons with NdeI and XhoI. 176
Figure 44. Competition Assay of L. casei 525 pgsA-R4850 recombinant with R48p-His6. 177
Figure 45. Effect of IPEC-J2 co-incubation with the Lactobacillus Rib recombinants on IL-10 production. 181
Figure 46. Effect of IPEC-J2 co-incubation with the Lactobacillus Rib recombinants on TNF-α production. 182
Figure 47. Summary of proteins identified from the supernatant and lysed cells. 186
Figure 48. Upregulation and downregulation of ANXA1, ANXA2, and Protein Cluster of... 187
Figure 49. Proposed anti-inflammatory pathways of L. casei 525 surface-displaying R4850. 189
Figure 50. Preliminary check and adhesion analysis of β-hemolytic S. agalactiae (SABA)... 207
Figure 51. Inhibition of GBS adhesion with purified R48p. 209
Figure 52. Protection conferred by surface-displayed Rib components against GBS during... 211
Figure 53. Protection conferred by surface-displayed Rib components against GBS during... 213
초록보기 더보기
Cell surface proteins mediate host-microbe interactions that determines their ability to interact and colonize specific niches. Their ability lies on the presence of specific cell surface-associated proteins that would allow them to adhere and compete with other bacteria. Thus, this study aimed to analyze the surface proteins that can mediate these interactions through both genomic and proteomic approaches, complemented with various in vitro assays, heterologous surface-display and a novel bioinformatic approach utilizing hypergeometric distribution and Bayesian analysis to identify moonlighting proteins that also mediate these interactions. In the study of the swine isolate strains, Lactobacillus mucosae LM1 and L. johnsonii PF01, their adhesive and competitive ability against gut pathogens Escherichia coli K88 and Salmonella Typhimurium have been studied. Both LM1 and PF01 showed good adhesive qualities, with LM1 showing more lectin-like activities compared to PF01 that resulted in better adherence to both mucin and epithelial cells, but that PF01 better matched E. coli K88 glycan specificity, allowing it to better inhibit the pathogen. Further analysis also shows the importance of moonlighting proteins such as GroEL, DnaK and Ef-Tu and a shed protein LBLM1_04890 (adhesin-like protein) in the adhesive, colonization and potential immunomodulatory effects of strain LM1. On the other hand, genome mining of the surface protein Rib shows interesting immunomodulatory capacities of the Rib repeat domain, especially in inducing anti-inflammatory effects, showing significant increases in IL-10 and induction of annexin proteins ANXA1 and ANXA2. The whole surface protein Rib shows specific binding to mucus and epithelial cells with the presence of a bacterial Ig domain and MucBP domains, showing a multi-functional role of this protein in adhesion, immunomodulation and colonization. Its ability to reduce the colonization and adhesion capacity of pathogenic GBS strain Streptococcus agalactiae BAA-1176 is also a significant finding for further optimization and studies of its application. Here, it is further confirmed that both LM1 and PF01 have potential host-microbe interaction abilities that can enhance their capacity as probiotics.
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