[논문]Zerumbone 처리 헬리코박터 파이로리균의 전사체 분석 비교

우현준; 양지영; 김사현

doi:10.48022/mbl.2202.02009

Zerumbone 처리 헬리코박터 파이로리균의 전사체 분석 비교
Comparative Transcriptome Analysis of Zerumbone-Treated Helicobacter pylori 원문보기

Microbiology and biotechnology letters = 한국미생물·생명공학회지, v.50 no.2, 2022년, pp.301 - 309

우현준 (세명대학교 임상병리학과) , 양지영 (농촌진흥청 기능성작물부) , 김사현 (세명대학교 임상병리학과)

초록
AI-Helper

본 연구에서는 제럼본에 처리에 의해 유도된 H. pylori 유전자의 전사적 변화를 분석하였다. NGS를 사용하여 RNA 발현 변화를 분석한 다음 그 결과를 검증하기 위해 RT-PCR을 수행하였다. NGS 분석 결과, 1,632개의 유전자 중 총 23개가 제럼본 처리에 의해 유의하게 발현이 변화된 특이발현 유전자로 분석되었다. DNA 복제와 전사, 병원성 인자 및 T4SS 성분과 관련된 유전자 중 10개는 현저하게 하향 조절되었고 5개는 상향 조절되었다. RT-PCR을 이용하여 유전자의 발현 수준을 재확인하였고 그 결과, 14개 유전자에서 NGS와 동일하게 발현 양상이 변화하였다. RT-PCR은 제럼본 처리에 의해 10개의 유전자(dnaE, dnaQ, rpoA, rpoD, secA, flgE, flhA, virB5, virB8, virB9)의 발현 감소와 4개의 유전자(flaA, flaB, virB4, virD4)의 발현 증가를 보였다. 이러한 본 연구의 결과는 제럼본이 다양한 H. pylori의 병원성과 관련된 인자들을 조절함으로써 H. pylori 감염의 잠재적인 치료제가 될 수 있음을 시사한다.

Abstract ▼ AI-Helper

Helicobacter pylori (H. pylori) establishes infection in the human gastric mucosa for a long time and causes severe gastric diseases such as peptic ulcer and gastric cancer. When H. pylori is exposed to the antibacterial agents or inhibitors, the expression of pathogenic associated genes could be altered. In this study, we analyzed the transcriptional changes of H. pylori genes induced by zerumbone treatment. RNA expression changes were analyzed using next-generation sequencing (NGS), and then reverse transcription-polymerase chain reaction (RT-PCR) was performed to verify the results. As a result of NGS analysis, a total of 23 out of 1,632 genes were differentially expressed by zerumbone treatment. RT-PCR confirmed that zerumbone treatment regulated the expression level of 14 genes. Among the genes associated with DNA replication, transcription, virulence factors and T4SS components, 10 genes (dnaE, dnaQ, rpoA, rpoD, secA, flgE, flhA, virB5, virB8 and virB9) were significantly down-regulated and 4 genes (flaA, flaB, virB4 and virD4) were up-regulated. The results of our current study imply that zerumbone might be a potential therapeutic agent for H. pylori infection by regulating factors related to various H. pylori pathogenicity.

주제어

표/그림 (8)

표 Table 1. List of primer sequences and PCR conditions for RT-PCR.
그림 Fig. 1. Minimum inhibitory concentration of zerumbone against H. pylori. H. pylori (ATCC 49503) was grown in the Mueller-Hinton broth containing the indicated concentration of zerumbone (0, 6.25, 12.5, 25, 50, 100 and 200 μM). After 72 h of incubation, a final optical density of the bacterial suspension was measured at 600 nm wavelength by spectrophotometry. Data are presented as the mean ± SEM. Results from triplicate experiments were analyzed by Student's t-test (***p < 0.001).
그림 Fig. 2. The volcano plot depicting the fold differences in gene expression levels of H. pylori after zerumbone treatment. The volcano plot representation of differential expression analysis of genes in the untreated H. pylori versus zerumbonetreated H. pylori. The x-axis shows log₂ fold changes in expression and the y-axis the log odds of a gene being differentially expressed. Red and blue points mark the genes with significantly increased or decreased expression respectively in untreated H. pylori compared to zerumbone-treated H. pylori.
표 Table 2. Summary of down-regulated genes associated with DNA replication, transcription, virulence factors and T4SS after treatment with zerumbone analyzed by NGS.
그림 Fig. 3. mRNA expression levels of DNA replication-related genes in zerumbone treatment conditions. H. pylori specimens were cultured with indicated concentrations of zerumbone in Mueller-Hinton broth for 72 h and RNA was extracted. (A) Expression of DNA replication machineries (dnaB, dnaE, dnaN, dnaQ, holB and gyrA) in H. pylori treated with zerumbone. Constitutively expressed galE was used as an internal control. (B) Densities of the PCR bands were illustrated as a graph and the data in the bar graphs are presented as mean ± standard error of the mean (SEM) of triplicate experiments were analyzed by Student's t-test (*p < 0.05).
그림 Fig. 4. mRNA expression levels of transcription genes in zerumbone treatment conditions. H. pylori specimens were cultured with indicated concentrations of zerumbone in Mueller- Hinton broth for 72 h and RNA was extracted. (A) Expression of transcription machineries (rpoA, rpoB, rpoD and rpoN) in H. pylori treated with zerumbone. Constitutively expressed galE was used as an internal control. (B) Densities of the PCR bands were illustrated as a graph and the data in the bar graphs are presented as mean ± standard error of the mean (SEM) of triplicate experiments were analyzed by Student's t-test (*p < 0.05 and **p < 0.01).
그림 Fig. 5. mRNA expression levels of virulence factors in zerumbone treatment conditions. H. pylori specimens were cultured with indicated concentrations of zerumbone in Mueller-Hinton broth for 72 h and RNA was extracted. (A) Expression of major virulence factors (cagA, vacA and secA) and flagella components (flaA, flaB, flgE and flhA) in H. pylori treated with zerumbone. Constitutively expressed galE was used as an internal control. (B) Densities of the PCR bands were illustrated as a graph and the data in the bar graphs are presented as mean ± standard error of the mean (SEM) of triplicate experiments were analyzed by Student's t-test (*p < 0.05 and **p < 0.01).
그림 Fig. 6. mRNA expression levels of T4SS components in zerumbone treatment conditions. H. pylori specimens were cultured withindicated concentrations of zerumbone in Mueller-Hinton broth for 72 h and RNA was extracted. (A) Expression of T4SS components(virB2, virB4, virB5, virB6, virB7, virB8, virB9 and virD4) in H. pylori treated with zerumbone. Constitutively expressed galE was used as aninternal control. (B) Densities of the PCR bands were illustrated as a graph and the data in the bar graphs are presented as mean± standard error of the mean (SEM) of triplicate experiments were analyzed by Student’s t-test (*p < 0.05 and **p < 0.01).

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저자의 다른 논문 :

표제어: PCR

동의어: Packet Collision Rate

용어 설명 출처 목록 (6)

용어 설명: PCR은 세균 특이성이 있는 primer를 이용하여 적은 수의 세균이 있을지라도 쉽게 검출할 수 있는 유용한 방법이며, 이를 이용하여 구강 내 치면세균막이나 타액에서 직접 세균을 검출할 수 있게 되었다[8].

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