• Journal of Microbiology and Biotechnology
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• 제32권11호
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• pp.1396-1405
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• 2022

Cholera remains a major global public health problem, for which oral cholera vaccines (OCVs) being a valuable strategy. Patients, who have recovered from cholera, develop antibody responses against LPS, cholera toxin (CT), toxin-coregulated pilus (TCP) major subunit A (TcpA) and other antigens; thus, these responses are potentially important contributors to immunity against Vibrio cholerae infection. However, assessments of the efficacy of current OCVs, especially inactivated OCVs, have focused primarily on O-antigen-specific antibody responses, suggesting that more sophisticated strategies are required for inactivated OCVs to induce immune responses against TCP, CT, and other antigens. Previously, we have shown that the toxT-139F allele enables V. cholerae strains to produce CT and TCP under simple laboratory culture conditions. Thus, we hypothesized that V. cholerae strains that express TCP via the toxT-139F allele induce TCP-specific antibody responses. As anticipated, V. cholerae strains that expressed TCP through the toxT-139F allele elicited antibody responses against TCP when the inactivated bacteria were delivered via a mouse model. We have further developed TCP-expressing V. cholerae strains that have been used in inactivated OCVs and shown that they effect an antibody response against TcpA in vivo, suggesting that V. cholerae strains with the toxT-139F allele are excellent candidates for cholera vaccines.

• 대한수의학회지
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• 제26권1호
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• pp.97-102
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• 1986

The prevalence of diarrhea caused by enterotoxigenic Escherichia coli was surveyed on 445 calves in 6 farms which were located in the central part of Korea. The incidence of enterotoxigenic Escherichia coli in calves with diarrhea was investigated by detecting the K99 and F41 antigens from the isolated strains of Escherichia coli The incidence of colibacillosis in calves was 23.3%. Of 238 strains of Escherichia coli isolated from calves with diarrhea, 73 strains(30.6%) were proved possessing the K99 antigen by mannose-resistant hemagglutination(MRHA) using horse red blood cells and 79(33.1%) possessing F41 antigens by MRHA using guinea-pig red blood cells. The minca medium, nutrient broth, tryptic soy broth and brain heart infusion were tested for yield of K99 and F41 pili. The production of pili was greatest in minea medium. The best detachment method of the K99 and F41 pili from the cells was heat treatment for 20 minutes at $60^{\circ}C$ and concentration by precipitation with ammonium sulfate. The purified antigens of K99 and F41 were polypeptides with molecular weights of 18,500 and 29,500, respectively by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

• 대한수의학회지
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• 제26권1호
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• pp.103-108
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• 1986

The oil emulsion and alhydrogel pilli vaccines were prepared from a strain(O9: K35, K99, F41) of enterotoxigenic Escherichia coli isolated from calves with diarrhea and their immunogenicity was tested in guinea-pigs, pregnant goats and cows. Serum antibody responses to K99 and F41 antigens in guinea-pigs given experimental oil and gel vaccines peaked at 4 and 6 weeks after vaccinations. At that time, the mean hemagglutination inhibition titers to K99 and F41 antigens in guinea-pigs given oil vaccine were 1:25 and 1:1, 218 and those given gel vaccine were 1:54 and 1:724 respectively. Agglutinin titers in pregnant goats given the oil vaccine were significantly higher(mean 1:2,347) compared to those of control group(mean 1:160). Less than 12.5% of goatlings from vaccinated goats developed scours compared to nearly 100% in control group after oral challenge with enterotoxigenic Escherichia coil within 24 hours after birth. The highest agglutinin titers of cow serum and colostrum and of the serum of calves 48 hours after birth from cows given oil vaccine were 1:256, 1:512 and 1:64 respectively. On the other hand, those titers of serum and colostrum and of the serum of nursing calves from nonvaccinated cows were 1:8, 1:16 and 1:20 respectively. The protective efficacy of the oil emulsion vaccine was 72.1% under field conditions. These results strongly indicated that the vaccine could be applied for protection of diarrhea caused by enterotoxigenic Escherichia coli in calves.

• 생명과학회지
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• 제30권1호
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• pp.88-95
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• 2020

유기용매 내성 세균인 Pseudomonas sp. BCNU 106을 10%(v/v) 톨루엔에 노출시킨 후 8시간 동안 random arbitrarily primed polymerase chain reaction (RAP-PCR)기법을 이용하여 메신져 RNA 발현 레벨을 조사하였다. 총 100개의 상향발현된 발현 산물 중에서 50개의 상보적인 단편들이 반복적으로 재현성있게 발현되는 것으로 확인되어, 이들을 클로닝을 하였으며 나아가 유전자 염기서열을 결정하였다. Blast analysis 결과, 톨루엔은 LysR family transcriptional regulator 그리고 RNA polymerase factor sigma-32같은 전사와 관련된 유전자들의 발현 레벨을 적응적으로 증가시키는 것으로 확인되었다. 그리고 톨루엔 스트레스 존재 하에서 inorganic ion 수송과 대사와 관련된 catalase와 Mn²⁺/Fe²⁺ transporter 유전자의 발현이 증가되었으며, 신호전달과 대사와 기능적으로 관련된 type IV pilus assembly PilZ와 multi-sensor signal transduction histidine kinase 유전자들의 발현 증가도 확인되었다. 한편 톨루엔 노출 후 탄수화물 수송과 대사와 관련된 beta-hexosaminidase 유전자발현 레벨이 증가하였다. 나아가 DNA polymerase III subunit epsilon, DNA-3-methyladenine glycosylase II와 DEAD/DEAH box helicase domain-containing 유전자들과 같은 DNA 복제, 재조합 그리고 수복에 관련성이 있는 유전자들의 발현 레벨 그리고 심지어는 ABC transporter 유전자와 같은 방어 메커니즘에 관련성이 있는 유전자들의 발현 레벨이 적응적으로 증가되는 것으로 밝혀졌다. 특히 10% 톨루엔 존재하에서 ABC transportor, Mn²⁺/Fe²⁺ transporter 및 β-hexosaminidase 유전자에 해당하는 RNA들이 괄목하게 유도되는 것이 확인되었다. 그러므로 유기용매 내성 세균 Pseudomonas sp. BCNU 106이 유기용매에 대하여 내성을 나타내는데 있어서 방어 메커니즘, 세포내 이온 항상성 그리고 바이오 필름 형성이 필수적인 것으로 확인되었다.

• Journal of Microbiology and Biotechnology
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• 제27권11호
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• pp.1971-1982
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• 2017

Piliated Lactobacillus rhamnosus (pLR) strains possess higher adherent capacity than non-piliated strains. The objective of this study was to isolate and characterize probiotic pLR strains in human fecal samples. To this end, mouse polyclonal antiserum (anti-SpaA) against the recombinant pilus protein (SpaA) of L. rhamnosus strain GG (LGG) was prepared and tested for its reactivity and specificity. With the anti-SpaA, a method combining the de Man, Rogosa, and Sharpe (MRS) agar plating separation and colony immunoblotting (CIB) was developed to isolate pLR from 124 human fecal samples. The genetic and phenotypic characteristics of the resultant pLR isolates were compared by randomly amplified polymorphic DNA (RAPD) fingerprinting, and examination of adhesion to Caco-2 cells, hydrophobicity, autoaggregation, and in vitro gastrointestinal tolerance. Anti-SpaA specifically reacted with three pLR strains of 25 test strains, as assessed by western blotting, immunofluorescence flow cytometry, and immunoelectron microscopy (IEM) assays. The optimized MRS agar separation plus anti-SpaA-based CIB procedure could quantitatively detect $2.5{\times}10^3CFU/ml$ of pLR colonies spiked in $10^6CFU/ml$ of background bacteria. Eight pLR strains were identified in 124 human fecal samples, and were confirmed by 16S RNA gene sequencing and IEM identification. RAPD fingerprinting of the pLR strains revealed seven different patterns, of which only two isolates from infants showed the same RAPD profiles with LGG. Strain PLR06 was obtained with high adhesion and autoaggregation activities, hydrophobicity, and gastrointestinal tolerance. Anti-SpaA-based CIB is a rapid and inexpensive method for the preliminary screening of novel adherent L. rhamnosus strains for commercial purposes.

• Journal of Microbiology and Biotechnology
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• 제33권6호
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• pp.736-744
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• 2023

The introduction of the toxT-139F allele triggers the expression of TCP (toxin co-regulated pilus) and CT (cholera toxin) under simple laboratory culture conditions in most Vibrio cholerae strains. Such V. cholerae strains, especially strains that have been used in OCVs (oral cholera vaccines), can induce antibody responses against TCP in animal models. However, CT produced in these V. cholerae strains is secreted into the culture medium. In this study, V. cholerae strains that can express intracellular CTB under the control of the toxT-139F allele have been constructed for potential application in OCVs. First, we constructed a recombinant plasmid directly linking the ctxAB promoter to ctxB without ctxA and confirmed CTB expression from the plasmid in V. cholerae containing the toxT-139F allele. We constructed another recombinant plasmid to express NtrCTB, from which 14 internal amino acids-from the 7^th to the 20^th amino acid-of the leader peptide of CTB have been omitted, and we found that NtrCTB remained in the cells. Based on those results, we constructed V. cholerae strains in which chromosomal ctxAB is replaced by ntrctxB or ntrctxB-dimer. Both NtrCTB and NtrCTB-dimer remained in the bacterial cells, and 60% of the NtrCTB-dimer in the bacterial cells was maintained in a soluble form. To develop improved OCVs, these strains could be tested to see whether they induce immune responses against CTB in animal models.