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Molecular characterization of InvE and SicA of the SPI-1 Type III Secretion System in Salmonella enterica serovar Typhimurium

  • 김진석 (대학원 생명과학과 분자유전학전공)

초록/요약

Gram-negative pathogen utilizes the type III secretion systems (T3SSs) to deliver virulence effector proteins into the host cells. T3SS consists of three parts: basal body in membranes, needle for effectors translocation and tip complex. As the T3SS is known for the most complex protein-delivery structure in bacteria, the temporal and precise regulation of protein synthesis and the secretion-order among T3SS substrates must be established. In this study, I focused on two T3SS regulators in Salmonella enterica serovar Typhimurium: a gatekeeper protein InvE and a class II chaperone SicA. Using various bacterial mutant strains and plasmids encoding truncated constructs of InvE, I found that both terminal regions of InvE are required for the secretion of translocases SipB, SipC and SipD, and that InvE has a putative secretion signal in the N-terminus. In addition, it appears that the SicAA44E mutant, with the substitutions at TPR motifs could not chaperone-activity for SipB, whereas it could activate the expression of the sigDE genes by interacting with T3SS activator InvF. These observations suggest that T3SS has evolved the highly elaborate mechanisms to regulate the complex T3SS hierarchies for the successful pathogenesis.

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목차

General abstract ……………………………………… 1

PART I ………………………………………………… 2
Molecular characterization of InvE regulator in the secretion of type III secretion translocases in Salmonella enterica serovar Typhimurium

1. Abstract ……………………………………………… 3
2. Introduction ……………………………………… 5
3. Materials and Methods ………………………… 8
Bacterial strains and growth conditions ………… 8
Construction of S. Typhimurium strains ………… 8
Construction of recombinant plasmids ………… 10
Preparation of whole cell proteins and secreted proteins of S. Typhimurium …………………… 12
Analysis of SipD stability ……………………… 13
Immunoblotting ………………………………… 13
Invasion assay …………………………………… 14
Statistical analysis ……………………………… 15
4. Results ……………………………………………… 20
The C-terminus of InvE is necessary for the secretions of SipB and SipC. ………………………………… 20
The C-terminal coiled-coil domain of InvE is not required for the secretion-specificity of SipB. …………… 24
InvE181-372 allows for the secretion of translocases and effector proteins but fails to fully complement the invasion defect of the invE mutant. …………… 28
Expression of InvE181-372 results in a defect in SipD stability. ……………………………………… 32
The N-terminal polypeptides of InvE are responsible for the regulation of SipB secretion. …………… 36
InvE possesses a putative secretion signal in its N-terminus. …………………………………………… 39
5. Discussion ……………………………………… 44
6. References ……………………………………… 48

PART II …………………………………………… 56
Functional analysis of Type III chaperone SicA carrying mutations at canonical residues of TPR-like motif in Salmonella enterica serovar Typhimurium

1. Abstract ……………………………………………… 57
2. Introduction ……………………………………… 58
3. Materials and Methods ………………………… 60
Bacterial strains and growth conditions ………… 60
Construction of S. Typhimurium strains ………… 60
Plasmid construction ………………………………… 61
Analysis of whole cell and culture supernatant proteins 61
Invasion assays ……………………………………… 62
Total RNA isolation, cDNA synthesis and RT-PCR 63
In-vitro binding assay ………………………………… 63
Statistical analysis …………………………………… 64
4. Results and Discussion ………………………… 67
SicA mutant possessing the mutation in the canonical residues of TPR-like motif is unable to stabilize SipB 67
SicAA44E is able to activate the sigDE operon.…… 71
SicAA44E could interact with InvF. …………… 75
5. Conclusion ……………………………………… 77
6. References ……………………………………… 78

Abstract in Korean ……………………………… 82

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