Streptococcus gordonii SK12 mutant fimbriae의 면역화학적 연구

윤정원,백대일 대한구강보건학회 2003 大韓口腔保健學會誌 Vol.27 No.4

A number of adhesive properties have been associated with various streptococci that colonise on teeth. Streptococcus gordonii is known to attach ability to saliva treated hydroxyapatite, an in vitro model for bacterial interactions with salivary components of the acquired pellicle. Fimbriae on Streptococcus gordonii promotes colonization of a host by mediating bacterial adsorption to specific host tissue receptors. 1. Streptococcus gordonii SK12's SHA adsorption was induced by fimbriae type 1 and type 2. Mutants by the absence of two type fimbriae were lower by 20% in SHA adsorption. 2. Streptococcus gordonii SK12's fimbriae's precipitation was started in 2-4 ug Ig M of anti-Actinomyces viscosus T14V. 3. According to bactericidal assay by PMNs to Streptococcus gordonii SK12 compared with parent strain, M6 mutation was lower by 67% in bactericidal activity; and sialidase treated became 50% lower than untreated. 4. By the crossed immunoelectrophoresis of displayed variation of 32 and 43 kD of glycoprotein on fimbriae of Mutants.

국내 Bordetella pertussis 분리균주에서 Agglutinogen과 Fimbriae 혈청형 변이 분석

정상운,문유미,성화영,강연호,유재연,Jung, Sang-Oun,Moon, Yu-Mi,Sung, Hwa-Young,Kang, Yeon-Ho,Yu, Jae-Yon 한국미생물학회 2008 미생물학회지 Vol.44 No.3

Bordetella pertussis는 유아의 호흡기감염질환인 백일해의 원인균으로 배신도입에 의해 발생률이 크게 낮아졌다. 그러나 최근에 일부 백신접종률이 높은 국가에서 증가된 백일해 발생건수가 보고되고 있으며, 그 추정 원인 중에 하나는 백신주와 유행주 사이의 유전형 흑은 혈청형 변이 이다. 따라서 분리주에 대한 변이 현황을 유전형 혹은 혈청형 분석을 통하여 확인하는 것이 필요하고 국내에서 백일해 발병증가에 대한 가능성을 추정해야 한다. 이에 본 연구에서는 $1999\sim2006$년 사이에 분리된 국내 백일해 분리균주의 변이 양상을 agglutinogen과 fimbriae에 대한 혈청형 조사를 통해 확인하였다. 그 결과 agg 1과 fim 2가 국내 분리주에서 가장 많이 발견되는 혈청형이었고, 두 항원 모두 시대에 따른 혈청형 변이 현상이 확인되었으며, 특히 agglutinogen의 경우는 백신균주(agg 1,2)와는 다른 유형(agg 1)이 증가되는 유형으로 나타났다. 그러나 fimbriae의 경우는 백신균주(fim 2)와 동일한 유형이 증가되어 차이를 나타내었다. 이러한 결과는 백일해 발병증가가 나타나는 국가들에서도 확인되는 현상으로 국내에서의 백일해 발병증가에 대한 보다 정확한 예측을 위해서는 항원결정기 유전자에 대한 변이정도 및 정상인에서의 항체가 분포양상 등에 대한 연구가 필요할 것으로 사료된다. Bordetella pertussis is pathogenic bacteria causing pertussis, a infectious respiratory disease for the infants. The incidence rate of pertussis was significantly decreased after introduction of vaccine. However, increased pertussis cases are recently reported in several countries with high vaccine coverage. One of the inferred reasons is genotype or serotype variation between circulating strains and vaccine strains. Therefore, it is required to confirm the variation status of the isolates by genotype or serotype analysis and the possibility of pertussis outbreak in Korea should be estimated. For this, the serotype variations of the isolates from $1999\sim2006$ were investigated in agglutinogen and fimbriae. As the result, the most frequent serotype in the isolated strains was agglutinogen 1 and fimbriae 2 serotypes. Moreover, serotype transition from vaccine serotypes to non-vaccine serotypes was observed. Especially, the transition pattern of agglutinogen serotype was directed to increase a different type (agg 1) from the vaccine type (agg 1,2). However, in case of fimbriae, the same type (fim 2) with vaccine strain was increased. These results were also observed in other countries with increasing incidence of pertussis. For more predictable results to know increasing possibility of pertussis incidence in Korea, the studies on genetic variations of antigenic determinant genes and prevalence of antibody titer in normal population should be performed in the further.

요로감염환자에서 혈청학적 방법을 이용한 P-pili특이혈중 항체의 조사

이원용(Won-Yong Lee),김종배(Jong-Bae Kim) 대한의생명과학회 1996 Biomedical Science Letters Vol.2 No.1

비뇨기계 병원성 대장균의 중요한 병원성 인자 중의 하나로 인정되고 있는 p-fimbriae의 subtype의 분포를 확인하기 위하여 요로감염증으로 확진된 환자의 혈청을 이용하여 immunoblotting을 실시하였고, 이와 동시에 효소면역 측정법을 실시하여 p-fimbriae특이 항체 보유를 확인하였다. Immunoblotting 결과 우리나라 요로감염증환자에서 높은 빈도로 확인되는 p-fimbriae subtype의 분포는 F7₁34(56.7%), F7₂28(46.7%), F13 30(50%) 등이 높게 나타났으며, 이와 같은 결과는 효소면역측정법에서도 동일하게 나타났다. 그러나 P-pili를 순수분리하지 않고 whole cell을 이용한 효소면역 측정법은 교차반응 때문에 비뇨기 감염증의 혈청학적인 진단에 적합하지 않는 것으로 나타났다. 또 우리나라의 요로감염 환자에게 항체 양성율이 높은 F7₁, F7₂, F13만을 혼합하여 항원으로 이용한 효소면역측정법의 특이도와 민감도가 각각 92.6%, 90%로 나타나, 이와 같은 방법을 임상진단에 응용할 수 있을 것으로 판단되었다. Escherichia coli is one of the most common etiological agents in urinary tract infection. An important virulence factor is the adhesive capacity of E.coli to uroepithelial cell, mediated by bacterial fimbriae. The Adhesion property has been regarded as an important virulence determinant in urinary tract infections. A total of 60 patients, who were diagnosed microbiologically as urinary tract infections, were examined by immunoblotting and enzyme-linked immunosorbent assay(ELISA). Uropathogenic E. coli with recombinant plasmid were positive for mannose resistant hemagglutination (MRHA). For identification of p-fimbriae subtype in uropathogenic E. coli, In the immunoblot analysis, specific bands in the range of p-fimbriae molecular weight of 17KD-22KD were identified. For the distribution of p-fimbriae subtype in the patient sera, 34/60(56.7%) were positive for F7₁, 28/60(46.7%) were positive for F7₂ and 30/60(50%) were positive for F13 with immunoblotting method. similar trends were observed in the enzyme-linked immunosorbent assay. Relatively good specificity(92.6%) and sensitivity(90%) were found in the ELISA test system using mixed antigens of purified F7₁, F7₂ and F13 p-fimbriae, and 60 sera from patients with urinary tract infections. In conclusion The serological tests were convenient method in diagnosis of urinary tract infections. among those ELISA could be recommended in diagnosis of urinary tract infections.

요로감염환자에서 혈청학적 방법을 이용한 P-pili특이혈중 항체의 조사

이원용,김종배 THE KOREAN SOCIETY FOR BIOMEDICAL LABORATORY SCIEN 1996 Journal of biomedical laboratory sciences Vol.2 No.1

비뇨기계 병원성 대장균의 중요한 병원성 인자 중의 하나로 인정되고 있는 p-fimbriae의 subtype의 분포를 확인하기 위하여 요로감염증으로 확진된 환자의 혈청을 이용하여 immunoblotting을 실시하였고, 이와 동시에 효소면역 측정법을 실시하여 p-fimbriae특이 항체 보유를 확인하였다. Immunoblotting 결과 우리나라 요로감염증환자에서 높은 빈도로 확인되는 p-fimbriae subtype의 분포는 F7₁34(56.7%), F7₂28(46.7%), F13 30(50%) 등이 높게 나타났으며, 이와 같은 결과는 효소면역측정법에서도 동일하게 나타났다. 그러나 P-pili를 순수분리하지 않고 whole cell을 이용한 효소면역 측정법은 교차반응 때문에 비뇨기 감염증의 혈청학적인 진단에 적합하지 않는 것으로 나타났다. 또 우리나라의 요로감염 환자에서 항체 양성율이 높은 F7₁, F7₂, F13 만을 혼합하여 항원으로 이용한 요소면역측정법의 특이도와 민감도가 각각 92.6%, 90%로 나타나, 이와 같은 방법을 임상진단에 응용할 수 있은 것으로 판단되었다. Escherichia coli is one of the most common etiological agents in urinary tract infection. An important virulence factor is the adhesive capacity of E.coli to uroepithelial cell, mediated by bacterial fimbriae. The Adhesion property has been regarded as an important virulence determinant in urinary tract infections. A total of 60 patients, who were diagnosed microbiologically as urinary tract infections, were examined by immunoblotting and enzyme-linked immunosorbent assay(ELISA). Uropathogenic E. coli with recombinant plasmid were positive for mannose resistant hemagglutination (MRHA). For identification of p-fimbriae subtype in uropathogenic E. coli, In the immunoblot analysis, specific bands in the range of p-fimbriae molecular weight of 17KD-22KD were identified. For the distribution of p-fimbriae subype in the patient sera, 34/60(56.7%) were positive for F7₁28/60(46.7%) were positive for F7₂and 30/60(50%) were positive for F13 with immunoblotting method. similar trends were observed in the enzyme-linked immunosorbent assay. Relatively good specificity(92.6%) and sensitivity(90%) were found in the ELISA test system using mixed antigens of purified F7₁,F7₂and F13 p-fimbriae, and 60 sera from patients with urinary tract infections. In conclusion The serological tests were convenient method in diagnosis of urinary tract infections. among those ELISA could be recommended in diagnosis of urinary tract infections.

살모넬라가 발현하는 stf 오페론의 조절과 병원성 인자로서의 기능

김삼웅,김영희,강호영,Kim Sam-Woong,Kim Young-Hee,Kang Ho-Young 한국생명과학회 2005 생명과학회지 Vol.15 No.4

stf 오페론은 stfA CDEFG로 구성되며, S. typhimurium과 S. choleraesuis에서는 완전하게 존재한다. 그러나 S. typhi에서는 이 오페론이 결여되어 있고 S. paratyphi A에서는 stfC의 유전자가 돌연변이 되어 있다. 이 섬모는 class 1형태의 섬모로 분류되며, StfD chaperone을 다른 섬모를 구성하는 chaperone들과 비교할 때 각 subunit들의 C-말단 잔기의 분석은 StfD chaperone이 FGS subfamily와 유사한 특성을 보였다. stf 오페론이 lacZYA 유전자와 fusion된 S. typhimurium 돌연변이 균주를 사용하여 MacConkey 고체배지에서 장시간 배양한 후 $Lac^+$ 표현형을 보이는 21 isolate들을 분리하였다. $Lac^+$ 균주들은 34 세대 당 $0.28\~1.75$의 빈도로 발생하였다. 21 isolate들은 구성적으로 stf operon을 발현했지만, 범용의 조절자인 RpoS, OmpR, CpxR등에 의해 조절되지 않았다. Mouse독성 실험에서 S. typhimurium $_X8661$은 야생형인 $_X3761$에 비교하여 6.7배의 약독화를 보였다. The stf (Salmonella typhimurium fimbriae) operon consisting of stfA(CDEFG assumes to encode putative fimbriae. The complete stf operon is existed in S. typhimurium and S. choleraesuis, whereas it is absent in S. typhi. Analyses of the amino acid residues between major subunit StfA of the Stf fimbriae and those of known other fimbriaes suggested that Stf belongs to class I type fimbriae. Through comparison of StfD chaperone with the other fimbrial chaperones, and of C-terminus in subunits of Stf fimbriae, it belongs to FGS (with a short Fl-G1 loop) subfamily. In order to investigate the expression of stf operon, we have constructed a Salmonella strain containing a chromosomal stfA::lacZYA transcriptional fusion, resulting in S. typhimurium $_X8532$. The strain $_X8532$ lacked the expression of \beta-galactosidase$ under normal culture conditions. However, with longer incubation time of the S. typhimurium $_X8532$, we have isolated 21 individual strains exhibiting $Lac^+$ phenotype. $Lac^+$ phenotype was appeared as approximately 0.03 frequency per generation. All isolates expressed lacZ constitutively in the various environmental conditions. Various global regulatory proteins including RpoS, OmpR, and CpxR were not involved in the regulation of the stf operon. A S. typhimurium $_X8661$ mutant lacking stfAC function attenuated 6.7 folds more than that of wild type $_X3761$ in the mouse virulence test, suggesting in the somehow involved in the Salmonella pathogenesis.

Regulatory T-cell vaccination independent of auto-antigen

David W Pascual,Xinghong Yang,Kathryn Holderness,전상무,Massimo Maddaloni,Irina Kochetkova 생화학분자생물학회 2014 Experimental and molecular medicine Vol.46 No.-

To date, efforts to treat autoimmune diseases have primarily focused on the disease symptoms rather than on the cause of the disease. In large part, this is attributed to not knowing the responsible auto-antigens (auto-Ags) for driving the self-reactivity coupled with the poor success of treating autoimmune diseases using oral tolerance methods. Nonetheless, if tolerogenic approaches or methods that stimulate regulatory T (Treg) cells can be devised, these could subdue autoimmune diseases. To forward such efforts, our approach with colonization factor antigen I (CFA/I) fimbriae is to establish bystander immunity to ultimately drive the development of auto-Ag-specific Treg cells. Using an attenuated Salmonella vaccine expressing CFA/I fimbriae, fimbriae-specific Treg cells were induced without compromising the vaccine’s capacity to protect against travelers’diarrhea or salmonellosis. By adapting the vaccine’s anti-inflammatory properties, it was found that it could also dampen experimental inflammatory diseases resembling multiple sclerosis (MS) and rheumatoid arthritis. Because of this bystandereffect, disease-specific Treg cells are eventually induced to resolve disease. Interestingly, this same vaccine could elicit the required Treg cell subset for each disease. For MS-like disease, conventional CD25þ Treg cells are stimulated, but for arthritisCD39þ Treg cells are induced instead. This review article will examine the potential of treating autoimmune diseases without having previous knowledge of the auto-Ag using an innocuous antigen to stimulate Treg cells via the production of transforming growth factor-b and interleukin-10.

Role of Cranberry on Bacterial Adhesion Forces and Implications for Escherichia coli–Uroepithelial Cell Attachment

Paola A. Pinzón-Arango,Yatao Liu,Terri A. Camesano 한국식품영양과학회 2009 Journal of medicinal food Vol.12 No.2

Previous clinical research has suggested that the consumption of cranberry products prevents the adhesion of Escherichia coli to uroepithelial cells by causing changes in bacterial fimbriae. Atomic force microscopy was used to probe the adhesion forces between E. coli (nonfimbriated strain HB101 and the P-fimbriated variant HB101pDC1) and a model surface (silicon nitride), to determine the effect of growth in cranberry products on bacterial adhesion. Bacteria were grown in tryptic soy broth supplemented with either light cranberry juice cocktail (L-CJC) or cranberry proanthocyanidins (PACs). Growth of E. coli HB101pDC1 and HB101 in L-CJC or PACs resulted in a decrease in adhesion forces with increasing number of cultures. In a macroscale bacteria–uroepithelial cell adhesion assay a decrease in bacterial attachment was observed for E. coli HB101pDC1 grown in L-CJC or PACs. This effect was reversible because bacteria that were regrown in cranberry-free medium regained their ability to attach to uroepithelial cells, and their adhesion forces reverted to the values observed in the control condition. Exposure to increasing concentrations of L-CJC resulted in a decrease of bacterial attachment to uroepithelial cells for the P-fimbriated strain after L-CJC treatment (27% by weight) and after PACs treatment (345.8 μg/mL). Cranberry products affect the surface properties, such as fimbriae and lipopolysaccharides, and adhesion of fimbriated and nonfimbriated E. coli. The concentration of cranberry products and the number of cultures the bacteria were exposed to cranberry determines how much the adhesion forces and attachment are altered.

Purification and Antigenic Heterogeneity of Major Fimbrial Proteins of Porphyromonas gingivalis

Lee, Jin-Yong The Official Publication of Korean Academy of Oral 1996 International Journal of Oral Biology Vol.20 No.1

Binding of Porphyromonas gingivalis to pellicle-coated teeth is mediated to a large extent by fimbrillin, a major fimbrial protein. Major fimbrial proteins were purified to homogeneity by gel filtration on a Sepharose CL-6B column using 6 M guanidine HCl from P. gingivalis strains 2561, A7A1-28, and 9-14K-1 which are representatives of different fimbrial groups tentatively defined by N-terminal amino acid sequences of fimbrillin molecules. Polyclonal antibodies were raised against the purified fimbrial proteins and the immunoglobulin G (IgG) fractions were purified by protein Aaffinity chromatography. In immunogold localization using the IgG antibodies under the electron microscope, gold particles were found to be deposited along the fimbrial structures on the whole cells of P. gingivalis, suggesting that the antibodies recognize native forms of the fimbriae. Antigenic heterogeneity of major fimbrial proteins among P. gingivalis strains was examined by immunoblot analysis using the IgG antibodies to the purified fimbrial proteins and as antigens; partially dissociated fimbriae from 21 strains of P. gingivalis. Fimbrial proteins of strains with amino acids Val-Gly-Asp at positions 4 to 6 reacted strongly with antibodies to the fimbrial protein of the strain 2561 (anti-2561)and weakly with anti-A7A1-28. Strains which belong to fimbrial groups with the amino acid residue Glu at position 4, and Asn-Ala-Gly at positions 4 to 6, reacted with anti-A7A1-28. In contrast, stains 9-14K-1 and HG 564 which have N-terminal sequences distinct from all other strains reacted only with anti-9-14K-1. The present study demonstrates antigenic diversity in the major fimbrial proteins of P. gingivalis strains. Most of the strains belonging to the same fimbrial group, based on the N-terminal sequence of fimbrillin, appeared to be antigenically homologous; most of the strains tested appeared to belong to one of three antigenic groups based on major fimbrial proteins.

Role of Cranberry on Bacterial Adhesion Forces and Implications for Escherichia coli-Uroepithelial Cell Attachment

Pinzon-Arango, Paola A.,Liu, Yatao,Camesano, Terri A. The Korean Society of Food Science and Nutrition 2009 Journal of medicinal food Vol.12 No.2

Previous clinical research has suggested that the consumption of cranberry products prevents the adhesion of Escherichia coli to uroepithelial cells by causing changes in bacterial fimbriae. Atomic force microscopy was used to probe the adhesion forces between E. coli (nonfimbriated strain HB101 and the P-fimbriated variant HB101pDC1) and a model surface (silicon nitride), to determine the effect of growth in cranberry products on bacterial adhesion. Bacteria were grown in tryptic soy broth supplemented with either light cranberry juice cocktail (L-CJC) or cranberry proanthocyanidins (PACs). Growth of E. coli HB101pDC1 and HB101 in L-CJC or PACs resulted in a decrease in adhesion forces with increasing number of cultures. In a macroscale bacteria-uroepithelial cell adhesion assay a decrease in bacterial attachment was observed for E. coli HB101pDC1 grown in L-CJC or PACs. This effect was reversible because bacteria that were regrown in cranberry-free medium regained their ability to attach to uroepithelial cells, and their adhesion forces reverted to the values observed in the control condition. Exposure to increasing concentrations of L-CJC resulted in a decrease of bacterial attachment to uroepithelial cells for the P-fimbriated strain after L-CJC treatment (27% by weight) and after PACs treatment ($345.8\;{\mu}g/mL$). Cranberry products affect the surface properties, such as fimbriae and lipopolysaccharides, and adhesion of fimbriated and nonfimbriated E. coli. The concentration of cranberry products and the number of cultures the bacteria were exposed to cranberry determines how much the adhesion forces and attachment are altered.

요로병원성 대장균의 P-fimbriae 보유율

박준현,박숙자 고신대학교 의학부 1991 高神大學校 醫學部 論文集 Vol.7 No.1

저자들은 고신대학 의학부 부속 고신의료원에 내원한 환자들의 뇨에서 분리한 대장균을 69균주를 대상으로 MRHA와 P-fimbriae 특이항체와의 응집유뮤를 관찰함으로서 P-fimbriae 보유유무를 조사하여 다음과 같은 결과를 얻었다. 1. MRHA 양성율은 59%였으며, 신우신염군에서 94%로서 방광염군(45%), 무증상 세균뇨군에 비해 의의있게 높았다. 2. P-fimbriae 보유율은 46%였으며 신우신염군에서 69%로서 무증상세균뇨군(32%)에 비해 의의있게 높았다. 3. P-fimbriae 보유유무에 따른 항균제에 대한 다제내성의 정도는 유익한 차이가 없었다. 이상의 성적을 종합하여 보면 대장균에서 P-fimbriae에 대한 검사는 뇨로병원성 대장균을 검색하는데 유요할 것으로 판단되며, 뇨로 병원성 대장균의 병인학적 연구와 역학적 조사에 유용할 것으로 사료되는 바이다. The prevalence of uropathogenic Escherichia coli bearing P-fimbriae was assessed in 69 patients with urinary tract infection. Male to female ratio was 1:4.8 and patients with 40-60 year old were most prevalent. The presence of P-fimbriae on E. coli was determined by concurence of MRHA positive and agglutination with anti P-fimbriae specific antibody which was made by immunization of rabbit with ER2 strain which is P-fimbriated strain. The 94% (15/16) of E. coli causing pyelonephritis was MRHA positive While among strains causing cystitis and asymptomatic bacteriuria MRHA positive were found 45% and 59% of cases, respectively. And, the occurence of P-fimbriated E. coli was 69% in pyelonephritis while among cystitis and asymtomatic bacteriuria P-fimbriae were found in 45% and 32% of cases, respectively. The antibiotic resistance patterns were not different between P-fimbriated and non-P-fimbriated E. coli. The positive rates of orinithin decarboxylase test was significantly higher in P-fimbriated E. coli than non-P-fimbriated E. coli.