<i>Helicobacter pylori</i> flagellin: TLR5 evasion and fusion-based conversion into a TLR5 agonist

Kim, Jee-Hyeon,Namgung, Byeol,Jeon, Ye Ji,Song, Wan Seok,Lee, Jeonghyun,Kang, Seung Goo,Yoon, Sung-il Elsevier 2018 Biochemical and biophysical research communication Vol.505 No.3

<P><B>Abstract</B></P> <P> <I>Helicobacter pylori</I> is a flagellated bacterium of the Epsilonproteobacteria class that causes peptic ulcers. Flagellin is a primary structural protein that assembles into the flagellar filament. Flagellins from bacteria that belong to the Gammaproteobacteria and Firmicutes groups are detected by Toll-like receptor 5 (TLR5) in the host, triggering the innate immune response, and thus have been studied for the development of vaccines against diverse infections through fusion with protein antigens. However, <I>H. pylori</I> flagellin (hFlg) does not stimulate TLR5, allowing <I>H. pylori</I> to evade TLR5-mediated immune surveillance. The unresponsiveness of TLR5 to hFlg, along with the tendency of the hFlg protein to precipitate, limits the utility of hFlg for <I>H. pylori</I> vaccine development. Here, we report a soluble hFlg derivative protein that activates TLR5. We performed expression and purification screens with full-length and fragment hFlg proteins and identified the hypervariable domains as the soluble part of hFlg. The hypervariable domains of hFlg were engineered into a TLR5 agonist through fusion with the TLR5-activating <I>Bacillus subtilis</I> flagellin. Furthermore, based on comparative sequence and mutation analyses, we reveal that hFlg evolved to evade TLR5 detection by modifying residues that correspond to a TLR5-activation hot spot.</P> <P><B>Highlights</B></P> <P> <UL> <LI> <I>Helicobacter</I> flagellin (hFlg) evades TLR5 by modifying a TLR5-activation hot spot. </LI> <LI> The hypervariable domains of hFlg were identified as the soluble part of hFlg. </LI> <LI> hFlg was engineered to activate TLR5 through fusion with <I>Bacillus</I> flagellin. </LI> </UL> </P>

Candida tropicalis 괄고정 담체를 적용한 Airlift Loop Bioreactor에서의 복합 휘발성유기화합물 제거

남궁형규(Hyeong Kyu Namgung),하정협(Jeong Hyub Ha),황선진(Sun Jin Hwang),송지현(Ji Hyeon Song) 大韓環境工學會 2009 대한환경공학회지 Vol.31 No.8

본 연구는 휘발성유기화합물질의 분해능력을 가진 yeast인 Candida tropicalis를 이용하여, 대표적인 휘발성 유기화합물질인 톨루엔과 MEK의 제거효율을 향상시키기 위하여 수행되었다. 반응기는 가스상으로 유입되는 톨루엔과 MEK의 물질전달 능력을 향상시키기 위하여 airlift loop 형태로 선택하였고, yeast 미생물의 효과적인 포괄고정화를 위해 분말활성탄(PAC)과 알지네이트(Alginate), PEG로 고분자 담체를 형성하였다. Airlift loop bioreactor의 물질전달성능을 평가하기 위한 실험을 수행하였으며, 기체체류시간 15초에서 담체를 첨가하지 않은 액상의 톨루엔 물질전달계수(KLa) 값이 1.29 min-1이었으나, 고분자 담체를 첨가한 경우 톨루엔의 KLa는 4.07 min-1로 증가하였다. 따라서 고분자담체를 적용하는 것이 기상으로 유입되는 휘발성유기화합물의 물질전달을 향상시키는 것으로 확인되었다. 이러한 airlift loop bioreactor와 yeast 포괄고정 담체를 적용하여 체류시간을 60초, 30초, 15초에서 유입부하에 변화를 주며 실험을 진행한 결과, 톨루엔 5, 10, 19, 37 g/m3/hr, MEK 4.5, 8.9, 17.8, 35.1 g/m3/hr의 유입부하 변화에도 전체 80% 이상의 안정적인 처리효율을 나타내었다. 또한 airlift loop bioreactor의 분해능을 확인하기 위하여 유입부하를 단기간 변화시켜 주며 실험한 결과, 톨루엔과 MEK의 최대분해능은 각각 70.4 g/m3/hr, 56.4 g/m3/hr로 확인되었다. This research was performed to improve removal efficiency of toluene and methyl ethyl ketone (MEK) using Candida tropicalis, one of the yeast species. An airlift loop bioreactor (ALB) was employed to enhance the capability of mass transfer for toluene and MEK from the gas phase to the liquid, microbial phase. Polymer gel media made from PAC, alginate and PEG was applied for the effective immobilization of the yeast strain on the polymer gel media. The experimental results indicated that the mass transfer coefficient of toluene without polymer gel media was 1.29 min-1 at a gas retention time of 15 sec, whereas the K(L)a value for toluene was increased to 4.07 min-1 by adding the media, confirming the enhanced mass transfer of volatile organic compounds between the gas and liquid phases. The removal efficiency of toluene and MEK by using yeast-immobilized polymer gel media in the ALB was greater than 80% at different pollutant loading rates (5, 10, 19 and 37 g/m3/hr for toluene, 4.5, 8.9, 17.8 and 35.1 g/m3/hr for MEK). In addition, an elimination capacity test conducted by changing inlet loading rates stepwise demonstrated that maximum elimination capacities for toluene and MEK were 70.4 and 56.4 g/m3/hr, respectively.

효모 Candida tropicalis 고정화 담체를 이용한 Airlift 미생물반응기의 톨루엔 제거 및 미생물 성장

남궁형규 ( Hyeong Kyu Namgung ),송지현 ( Ji Hyeon Song ),정미영 ( Mi Young Jung ),황선진 ( Sun Jin Hwan ) 대한상하수도학회 2009 상하수도학회지 Vol.23 No.2

미생물 포괄고정화 담체를 이용한 이단 바이오필터에서의 오염부하량 동적 부하변동시 복합악취 제거효율 변화특성

남궁형규(Hyeong Kyu Namgung),신승규(Seung Kyu Shin),황선진(Sun Jin Hwang),송지현(Ji Hyeon Song) 大韓環境工學會 2010 대한환경공학회지 Vol.32 No.12

본 연구에서는 휘발성 유기화합물의 분해능력을 가진 Candida tropicalis와 황화수소 분해능력을 가진 황산화균을 적용하여 이단 바이오필터를 운전하였으며, 각각의 미생물은 스폰지형 담체에 포괄 고정시켜 사용되었다. 이단 바이오필터에는 벤젠, 톨루엔, 자일렌, 암모니아, 황화수소 등의 복합악취를 유입하며 분해 특성을 파악하였고, 특히 오염부하량 단계변동에 대한 바이오필터의 분해능을 확인하였다. 오염부하량 단계변동은 총유입부하량 단계변동(total EC test), 개별 악취물질 유입부하량 단계변동(individual chemical EC test), 간헐 동적부하 변동(2 days off & 3 days on) 순으로 수행되었다. 총유입부하량 단계변동 실험결과 TVOC와 암모니아, 황화수소의 최대분해능은 각각 61, 5.2, 9.1 g/m3/hr로 확인되었으며, 개별악취물질 유입부하량 단계변동시에는 휘발성 유기화합물질 상호간의 분해능 간섭이 일어나는 것을 확인할 수 있었다. 벤젠 부하량 변동시 톨루엔과 p-자이렌 모두 제거효율에 영향을 받았으며, 톨루엔의 부하량 단계변동시에는 벤젠과 p-자이렌이 각각 30%와 25% 이상의 제거효율의 하락이 일어나는 것을 확인할 수 있었다. 하지만 무기악취와 유기악취간의 상호 분해능 간섭은 일어나지 않는 것을 확인할 수 있었다. 2일간 악취물질의 유입을 중단한 후 다시 악취물질의 유입을 재개하였을 때, 3일 이내에 95% 이상의 악취물질 제거능 회복율을 보였다. 이러한 부하변동 실험결과로 미생물 포괄고정 담체를 적용한 이단 바이오필터가 유·무기 악취의 동시제거에 많은 장점을 가지고 있음을 확인할 수 있었다. A two-stage biofilter was constructed and utilized to determine the removal efficiency when treating dynamic loading of a mixture of odorous compounds including benzene, toluene, p-xylene, ammonia and hydrogen sulfide. A yeast strain, Candida tropicalis, and a sulfur oxidizing bacterial (SOB) strain, Acidithiobacillus caldus sp., were immobilized in polyurethane media and packed in the two-stage biofilter. The experiment of dynamic loading variation was composed of (1) stepwise loading variation of all the odorous compounds (total EC test), (2) stepwise loading variation of each odorous compound, and (3) intermittent loading variation with 2-day-off and 3-day-on. The total EC test showed that the maximum elimination capacity was 61 g/m3/hr for total VOCs, and 5.2 and 9.1 g/m3/hr for ammonia and hydrogen, respectively. In addition, the inhibition between VOCs was observed when the loading of each individual VOC was varied. Especially the stepwise increase in toluene loading resulted in decreases of benzene and p-xylene removal efficiencies about 30% and 25%, respectively. However, the inhibition between organic and inorganic compounds was not observed. The intermittent loading variation with 2-day-off and 3-day-on showed that greater than 95% of the overall removal efficiency was restored in two days after the loading resumed. Consequently, the two-stage biofilter packed with immobilized microorganisms showed advantages over conventional biofilters for the simultaneous treatment of the mixture of organic and inorganic odorous compounds.

암모니아 가스 제거를 위한 포괄고정화 담체 개발

정미영 ( Mi Young Jeong ),남궁형규 ( Hyeong Kyu Namgung ),송지현 ( Ji Hyeon Song ),황선진 ( Sun Jin Hwang ) 한국물환경학회 2009 한국물환경학회지 Vol.25 No.2

Diagnostic value of peripheral blood immune profiling in colorectal cancer

Joungbum Choi,Hyung Gun Maeng,Su Jin Lee,Young Joo Kim,Da Woon Kim,Ha Na Lee,Ji Hyeon Namgung,Hyun-Mee Oh,Tae Joo Kim,Ji Eun Jeong,Sang Jean Park,Yong Man Choi,Yong Won Kang,Seo Gue Yoon,Jong Kyun Lee 대한외과학회 2018 Annals of Surgical Treatment and Research(ASRT) Vol.94 No.6

Purpose: Little is known about the clinical value of peripheral blood immune profiling. Here, we aimed to identify colorectal cancer (CRC)-related peripheral blood immune cells and develop liquid biopsy-based immune profiling models for CRC diagnosis. Methods: Peripheral blood from 131 preoperative patients with CRC and 174 healthy controls was analyzed by flow cytometry and automated hematology. CRC-related immune factors were identified by comparing the mean values of immune cell percentages and counts. Subsequently, CRC diagnostic algorithms were constructed using binary logistic regression. Results: Significant differences were observed in percentages and counts of white blood cells, lymphocytes, neutrophils, regulatory T cells, and myeloid-derived suppressor cells (MDSCs) of patients and controls. The neutrophil/lymphocyte and Th1/Th2 ratios were also significantly different. Likewise, the percentages and counts of peripheral blood programed death 1, cytotoxic T lymphocyte antigen 4, B- and T-lymphocyte attenuator, and lymphocyte activation gene-3 were higher in patients with CRC. The binary logistic regression model included 12 variables, age, CD3<SUP><SUP>+</SUP></SUP>%, NK%, CD4<SUP>+</SUP>CD279<SUP>+</SUP>%, CD4<SUP>+</SUP>CD25<SUP>+</SUP>%, CD4<SUP>+</SUP>CD152<SUP>+</SUP>%, CD3<SUP>+</SUP>CD366<SUP>+</SUP>%, CD3<SUP>+</SUP>CD272<SUP>+</SUP>%, CD3<SUP>+</SUP>CD223<SUP>+</SUP>%, CD158b<SUP>-</SUP>CD314<SUP>+</SUP>CD3<SUP>-</SUP>CD56<SUP>+</SUP>%, Th2%, and MDSCs cells/μL, for the prediction of cancer. Results of retrospective and prospective evaluation of the area under the curve, sensitivity, and specificity were 0.980 and 0.940, 91.53% and 85.80%, and 93.50% and 86.20%, respectively. Conclusion: Peripheral blood immune profiling may be valuable in evaluating the immunity of CRC patients. Our liquid biopsy-based immune diagnostic method and its algorithms may serve as a novel tool for CRC diagnosis. Future largescale studies are needed for better characterization of its diagnostic value and potential for clinical application.