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Type III Secretion Signal of SipB in Salmonella typhimurium and its Application as an Antigen Delivery System

  • 장정임 (Jung-Im Jang, 대학원 생명과학과 분자생물학전공)

초록/요약

Type III secretion system (T3SS) in Gram-negative bacteria is a complex protein secretion apparatus that delivers the virulence proteins into host cells directly. T3SS substrates typically possess two functional requirements for the efficient secretion: the secretion signal within the first 20 amino acids and the chaperone-binding domain within the N-terminal 140 residues. In this study, it was shown that T3SS translocon protein SipB in Salmonella enterica serovar Typhimurium contains the first export signal in the N-terminus, whereas the C-terminus (amino acids 481-490 and 500-593) is required for the secretion through the Salmonella pathogenicity island (SPI)-1 T3SS. In addition, the secretion levels of recombinant SipB proteins that are targeted to the SPI-1 T3SS were dramatically decreased in the invE mutant strain compared with that of the wild-type strain. It suggests that the C-terminus of SipB is necessary for the SPI-1-dependent secretion and that InvE recognizes this region and then leads SipB to the SPI-1 secretion apparatus. The secretion signal of T3SS substrates could be used for the delivery of the heterologous antigens into the host cells. When tetanus toxin fragment C, the antigenic determinant of tetanus toxin in Clostridium tetani, was fused to the first 160 amino acid residues of SipB, this recombinant protein was observed in the culture supernatant and on the bacterial surface. The immunization of mice with attenuated Salmonella strains containing TTFC-delivery plasmid enhanced the antigen-specific immune responses.

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목차

General abstract 1
Part I. The C-terminus of type III translocase SipB is required for the Salmonella pathogenicity island 1-dependent secretion and is regulated by InvE 3
1.1 Abstract 4
1.2 Introduction 5
1.3 Materials and Methods 9
1.3.1 Strains and bacterial cultures 9
1.3.2 Construction of S. Typhimurium mutant strains 15
1.3.3 P22 transduction 17
1.3.4 Plasmids construction 19
1.3.5 Site-directed mutagenesis 21
1.3.6 Preparation of secreted proteins and whole cell lysates for SDS-PAGE 22
1.3.7 Immunoblotting 23
1.3.8 Invasion assays 24
1.3.9 Statistical analysis 25
1.4 Results 26
1.4.1 The C-terminus of SipB is required for SPI-1-dependent secretion. 26
1.4.2 C-terminal amphipathic helix affects SipB secretion. 30
1.4.3 SipB1-500 was secreted via SPI-1 T3SS. 33
1.4.4 Amino acids 481 to 490 of SipB are necessary for the SPI-1-dependent secretion. 36
1.4.5 InvE regulates the secretion of SipB via SPI-1 T3SS. 40
1.5 Discussion 42
1.6 References 45
Part II. Expression and delivery of tetanus toxin fragment C fused to the N-terminal domain of SipB enhances specific immune responses in mice. 53
2.1 Abstract 54
2.2 Introduction 55
2.3 Materials and Methods 58
2.3.1 Bacterial strains and growth conditions 58
2.3.2 Plasmid construction 61
2.3.3 Isolation of proteins secreted by S. Typhimurium 63
2.3.4 Subcellular fractionation 64
2.3.5 Plasmid stability test 65
2.3.6 Invasion assay 66
2.3.7 Purification of the TTFC protein 67
2.3.8 Infection of mice 68
2.3.9 Measurement of the anti-TTFC response 69
2.3.10 Statistical Analysis 70
2.4 Results 71
2.4.1 SipB-mediated secretion of TTFC in S. Typhimurium vaccine strains 71
2.4.2 Subcellular localization of TTFC in S. Typhimurium vaccine strains 75
2.4.3 Antibody responses in mice immunized with S. Typhimurium harboring TTFC delivery plasmids 78
2.4.4 Construction of a medium copy number plasmid containing TTFC delivery cassettes 80
2.4.5 Secretion and outer membrane localization of TTFC encoded by a medium copy plasmid 84
2.4.6 Antibody responses in mice immunized with BRD509 harboring TTFC delivery plasmids 86
2.5 Discussion 89
2.6 References 94
Abstract in Korean 100

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