黃顯宗 東吳大學 微生物學系 腸炎弧菌之研究. pathogenic vibrios v. cholerae and v....
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黃顯宗東吳大學 微生物學系
腸炎弧菌之研究
Pathogenic vibrios
V. cholerae and V. mimicus V. parahaemolyticus V. fluvialis and V. furnissii V. hollisae V. alginolyticus V. vulnificus V. damsela Aeromonas spp. Plesiomonas spp.
Food poisoning outbreaks in Taiwan, 1981-2005
Outbreaks ％
0
10
20
30
40
50
60
70
衛生署資料
Molecular Epidemiology分子流行病學
Pulsed-field Gel Electrophoresis, PFGE
727.5 kb
339.5 kb
48.5 kb
SfiI digestion
Frequency of O3:K6 in Foodborne Poisonings in middle Taiwan, 1995 - 1999
0
10
20
30
40
50
60
70
80
95 96 97 98 99
From C.S.Chiou
%
Typing of New O3:K6
M I5 I6 I7 I8 I1 I1 I4 I2 I3
Typing of Old O3:K6
M R A3 A3 A3 A3 A1 A1 A3 M M A2 A3
Dendrogram for Vibrio parahaemolyticus
New O3:K6
Old O3:K6
DISSIMILARITY 0 5 10 15 20 25 PATTERN A1 A2 A3 A4 A5 A6 A7 B1 B2 B3 B4 B5 C1 C2 C3 C4 C5 C6 D1 D2 D3 D4 J K E1 E2 E3 E4 E5 F1 F2 F3 G1 G2 G3 H1 H2 H3 H4 H5 I1 I2 I3 I4 I5 I6 I7 I8 L1 L2 M N O1 O2 P Q R S
Minimum spanning tree of 174 global strains
Yang’s lab, Beijing
VP882 Phage
MyoviridaeWith plasmid-like genome71 ORFProtelomeraseInverted Repeat SequenceLuxRAlso infect V. vulnificus, V. cholerae
VP882 Phage Genome
Protelomerase LuxR
3 plasmid-like phages: N15, KO2 and PY54
2 plasmid-like phages: VP882 and phiHAP-1
11 bp 11 bp
12 bp12 bp
telO
telO
(c/g)CAT(t/a)(a/c)TACGCGCGTA(t/g)(a/t)ATG(g/c)
CCCATACTATACGTATAGTATGGG
Analysis of binding domains by Weblogo tool
Viable but nonculturable state
活而不長狀態
VBNC state
Culturable: plate count on TSA-3% NaCl Viable: Live/Dead BacLight Count
Resuscitation of VBNC cells
The 4C culture
was shifted to 2
5C Adding catalase
to Plate count m
edium （） , no
effect
MPN no differen
t result
Resuscitation
Resuscitation
2D-PAGE analysis, 2 and 6 weeks
Control RT- 4C 4C
4 7
4 7 4 7
2D-PAGE analysis, 0, 2 and 6 weeks
Expression enhanced in 4C
•Protein Name Accession Number pI MW (Da)
1 (380)a RNA polymerase alpha-subunit [Vibrio sp. R-21432] gi|58416042 4.74 31817
4 (286)DNA-directed RNA polymerase, alpha subunit/40 kD subuni
t [Vibrio vulnificus CMCP6] gi|27360308 4.78 36437
5 (346) 30S ribosomal protein S1 [Vibrio alginolyticus 12G01] gi|91229576 4.89 43063
6 (626) elongation factor Tu [Vibrio alginolyticus 12G01] gi|91227309 4.80 43082
elongation factor Tu [Synechococcus sp. WH 8103] gi|4093215 4.54 19439
7 (664) dihydrolipoamide acetyltransferase [Vibrio alginolyticus 12G01]
gi|91228629 4.73 65178
16 (475)b Peroxiredoxin [Vibrio vulnificus CMCP6] gi|27360037 4.93 22285
17 (361) b glyceraldehyde 3-phosphate dehydrogenase [Vibrio parahaemolyticus RIMD 2210633] gi|28807150 5.26 35204
30 (568) antioxidant, AhpC/Tsa family [Vibrio parahaemolyticus RIMD 2210633]
gi|28805566 5.03 22223
11 (744) a unnamed protein product [Vibrio alginolyticus] gi|48337 5.05 55557
ATP synthase F1, alpha subunit [Vibrio parahaemolyticus RIMD 2210633]
gi|28808097 5.10 56601
12 (134) conserved hypothetical protein [Vibrio parahaemolyticus RIMD 2210633]
gi|28806394 4.97 55542
14 (529)a Translation elongation factor G [Vibrio vulnificus CMCP6] gi|27361244 5.33 36932
15 (627) elongation factor TU [Vibrio parahaemolyticus RIMD 2210633]
gi|28807956 4.80 43125
18 (745) b
phosphoribosylaminoimidazolecarboxamide formyltransferase/IMP cyclohydrolase [Vibrio cholerae O1 biovar eltor str.
N16961]gi|9654686 5.21 57291
AICAR transformylase/IMP cyclohydrolase PurH [Vibrio vulnificus CMCP6]
gi|27360775 5.05 57428
20 (235) c amino acid ABC transporter, periplasmic amino acid-binding protein [Vibrio parahaemolyticus RIMD 2210633] gi|28806611 5.67 36657
2D-PAGE analysis, 2 and 6 weeks
Protein Expression enhanced in 4C Transcription and translation
ATP production
Antioxidation
Amino acid ABC transporter enhanced in
RT-4C
Role of these proteins in the induction an
d maintenance of VBNC state?
Expression of selected genes
Elongation factor
glyceraldehyde 3-phosphate dehydrogenase
AhpC conserved hypothetical protein
ABC transporter
•2,3,4: VBNC induction for 2, 4,6 wk2,3,4: VBNC induction for 2, 4,6 wk•5,6,7:prestarved for 24 h5,6,7:prestarved for 24 h
Hypothesis for VBNC state
Role of Antioxidants
Catalase
AhpC
SOD
……
Cytoskeleton細胞骨架
Change of cell morphology
Change to coccoid shape, entering stationary phase or under starvation/cold...stress
0
0.5
1
1.5
2
2.5
3
log phase early stationary late stationary
Change of cell length
Cytoskeleton of bacteria
Major bacterial cytoskeletons ： MreB (Actin) FtsZ (Tubulin) CreS (Intermediate Filament) MinCDE
MreB
CreS
FtsZ
MinCD
MinE
YFP labeling of MreB
Helical structure of MreB
85% 7% 7% >1%
MreB-YFP labeled V. parahaemolyticus
Exponential phase
MreB-YFP labeled V. parahaemolyticus
Early stationary phase
(A)
(B)
(C)
(D)
MreB-YFP labeled V. parahaemolyticus
Induction of VBNC
Proposed model
進行中的計畫
台灣牡蠣養殖環境中致病性腸炎弧菌的變化
多重環境壓力對腸炎弧菌耐受性的影響與機制
Website ： http://microbiology.scu.edu.tw/wong/ Blog ： http://www.wretch.cc/blog/wonghc70/ Tel ： (886) 2-28819471 ext 6852 Email ： [email protected]
Thanks