| Summary: | 碩士 === 國立中興大學 === 分子生物學研究所 === 105 === 1.Identification of pathogenic Escherichia coli from diarrheal piglets in Taiwan
Piglets after weaning for 1 to 3 weeks, often acute or chronic diarrhea, and gradually dehydration, serious or even death. The survival of piglets, although the intestinal mucosa because of its immune system gradually established without diarrhea, so this piglet slow growth, its feed utilization (Gain/Food intake) is low, known as the postweaning diarrhea (PWD). PWD induced piglet death, medical expenses and survival of the pig''s growth slowdown in the global pig industry, major economic losses. PWD is the most important cause of animal origin of enterotoxigenic E. coli (ETEC), which produce heat labile enterotoxin (LT), heat stable enterotoxin type A (STa), heat stable enterotoxin type B (STb) and enteroaggregative E. coli heat-stable enterotoxin 1 (EAST1). Small number of PWDs were caused by STEAEC (producing Shiga Toxin (Stx) Enteroaggregative E. coli). Therefore, this study analyzes the bacteria collected from the samples of the healthy and diarrhea piglets from 2013, and identified 68 strains of E. coli by biochemical eight tubes. Then use PCR to check this 68 isolates strains have STI, STII, LTI, astA and Stx2e or not, then identified this 68 isolates strains were which pathogen of E. coli. This study found that 20 E. coli has F18 flagellum gene, and also have LT and ST genes, but not have EAST1 gene. The 20 strains of E. coli should be ETECs, which maybe cause PWD.
The 68 isolates strains inhibition activities of DH5α and the plasmid for expression of imm was transformed into DH5α. If isolates strains do not kill two host, is mean this isolates strains do not produce toxin; if it can only kill DH5α host, is mean this isolates strains can produce Col Ib protein; if it can kill two host, is mean this isolates strains can produce other Colicin protein. The result shows, we have 41.2% healthy and 29.4% diarrhea piglets isolates strains can produce Col Ib protein; 17.6% healthy and 54.9% diarrhea piglets isolates strains can produce other Colicin protein. This 54.9% diarrhea piglets isolates strains maybe cause PWD.
From the sensitivity of 68 E. coli to purified Col Ib, show that 10 μg/mL of Col Ib can inhibit 51% diarrhea piglets isolates strains, but just inhibit 35.3% healthy piglets isolates strains. In the future, we will test whether Col Ib can be added to the suckling pig feed to prevent Taiwan pig farms from diarrhea. We will use 10 μg/mL concentration, this concentration can be achieved to inhibit the growth of diarrhea piglets isolates strains, while not affecting the growth of healthy piglets isolates strains.
In this study, we found ETEC (39.2%) of the gene carrying the F18 cilia gene and secreting LT and ST, and not carrying the EAST1 gene, and the strain (54.9%) that produced other Colicins, maybe cause PWD.
2.The role of immunity protein of colicin Ib in Escherichia coli
So far, all E. coli strains in nature that produce colicins also produce a small protein called immune protein specific to the colicin. It is generally believed that the immune protein binds the corresponding colicin in vivo, thus has no toxicity to the bacteria producing the colicin. However, our laboratory has constructed an E. coli strain that produces colicin Ib (Col Ib) but not the corresponding immunity protein (Imm), and the strain grew normally. The purpose of this study is to understand the role of Imm in E. coli.
At first, it was demonstrated that BW25113 or W3110 carrying plasmid with genes for ColIb-C-his (his tag at C terminus of Col Ib) or ColIb-N-his (his tag at N terminus of Col Ib) showed no harm and no difference on bacterial growth after induction of either protein. Induction by mytomycin C or IPTC showed no difference on bacterial growth.
BW25113 carrying plasmid responsible for production of ColIb-N-his through mytomycin C induction was grown in 20 mL or 250 mL medium. After induction, ColIb-N-his could be detected in the cell-free supernatant, but not the cytoplasmic protein DnaK, indicating ColIb-N-his could be secreted into medium by bacteria. So was BW25113 carrying plasmid responsible for production of ColIb-C-his grown in 20 mL medium. But for BW25113 carrying the same plasmid grown in 250 mL medium, both both DnaK and ColIb-C-his could be detected in the supernatant, indicating bacterial lysis occurred under this particular growth condition.
The His-tag hybridization signal and the bacteriocidal activity of the bacterial cell extract was determined with BW25113 carrying ColIb-C-his or ColIb-N-his plasmid and grown in 20 mL or 250 mL medium. After induction by mytomycin C, BW25113 carrying ColIb-C-his plasmid grown in 20 mL medium showed the best activity. The his-tag hybridization signal and the bacteriocidal activity of bacterial cell extract was also checked with W3110 carrying ColIb-C-his plasmid with which ColIb-C-his production was induced by mytomycin C or IPTC. Induction by mytomycin C had higher hybridization signal and the bacteriocidal activity than induction by IPTG.
Quasilysis on bacterial growth was found with W3110 and DH5α, but not BW25113, carrying plasmid with genes for imm-C-his (his tag at C terminus of Imm) after mytomycin C induction. The His tag hybridization signal of the protein extract increased as the induction time increased. However, W3110 carrying the same imm-C-his plasmid but plus Col Ib plasmid (Col Ib without his tag) did not show quasilysis on growth.
By using W3110 carrying both Col Ib-C-his plasmid and Imm-C-his plasmid which either induction by mytomycin C or IPTG, it is demonstrated that the bacteriocidal activity of the cell extract increased by induction of Imm-C-his. Thus, Imm-C-his increased the bacteriocidal activity of Col Ib-C-his in the bacteria.
The bacteriocidal activities of supernatants from W3110 carrying both Col Ib plasmid (Col Ib without his tag) and pACYC184-imm plasmid (Imm without his tag), as well as W3110 carrying both Col Ib plasmid and pACYC184 plasmid, were determined after induction by mytomycin C. The former had higher activity than the later, indicating that Imm could facilitate the secretion of Col Ib from bacteria.
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