[논문]Screening of Genes Expressed In Vivo During Interaction Between Chicken and Campylobacter jejuni

Hu, Yuanqing; Huang, Jinlin; Jiao, Xin-An

doi:10.4014/jmb.1308.08092

[국내논문] Screening of Genes Expressed In Vivo During Interaction Between Chicken and Campylobacter jejuni 원문보기

Journal of microbiology and biotechnology, v.24 no.2, 2014년, pp.217 - 224

Hu, Yuanqing (Jiangsu Key Laboratory of Zoonosis, Yangzhou University) , Huang, Jinlin (Jiangsu Key Laboratory of Zoonosis, Yangzhou University) , Jiao, Xin-An (Jiangsu Key Laboratory of Zoonosis, Yangzhou University)

Abstract ▼ AI-Helper

Chicken are considered as the most important source of human infection by Campylobacter jejuni, which primarily arises from contaminated poultry meats. However, the genes expressed in vivo of the interaction between chicken and C. jejuni have not been screened. In this regard, in vivo-induced antigen technology (IVIAT) was applied to identify expressed genes in vivo during interaction between chicken and C. jejuni, a prevalent foodborne pathogen worldwide. Chicken sera were obtained by inoculating C. jejuni NCTC 11168 into Leghorn chickens through oral and intramuscular administration. Pooled chicken sera, adsorbed against in vitro-grown cultures of C. jejuni, were used to screen the inducible expression library of genomic proteins from sequenced C. jejuni NCTC 11168. Finally, 28 unique genes expressed in vivo were successfully identified after secondary and tertiary screenings with IVIAT. The genes were implicated in metabolism, molecular biosynthesis, genetic information processing, transport, regulation and other processes, in addition to Cj0092, with unknown function. Several potential virulence-associated genes were found to be expressed in vivo, including chuA, flgS, cheA, rplA, and Cj0190c. We selected four genes with different functions to compare their expression levels in vivo and in vitro using real-time RT-PCR. The results indicated that these selected genes were significantly upregulated in vivo but not in vitro. In short, the expressed genes in vivo may act as potential virulence-associated genes, the protein encoded by which may be meaningful vaccine candidate antigens for campylobacteriosis. IVIAT provides an important and efficient strategy for understanding the interaction mechanisms between Campylobacter and hosts.

주제어

AI 본문요약
AI-Helper

* AI 자동 식별 결과로 적합하지 않은 문장이 있을 수 있으니, 이용에 유의하시기 바랍니다.

가설 설정

However, DNA repair is essential for its survival in response to significant environmental stress. We hypothesize that these DNA repair genes containing nine in vivo-associated factors may be significant when C. jejuni lives in human or chicken tissues.

제안 방법

jejuni. In this study, IVIAT was employed to investigate the virulence-associated genes in vivo. IVIAT is a rigorous method that can identify virulence-associated antigens, which are expressed instantaneously in vivo when hosts are infected by pathogenic bacteria [29].
In this study, a genomic expression library was constructed using sequenced C. jejuni NCTC 11168, and in vivo-induced antigens were identified from chicken inoculation antisera after being thoroughly adsorbed by cultures of C. jejuni in vitro. These screen of genes expressed in vivo may contribute to understanding the interaction between hosts and microbes, and several antigens are candidate vaccine sites and diagnostic markers.
To construct the genomic expression library of C. jejuni strain NCTC 11168, its genomic DNA was extracted (DNeasy Blood Tissue Kit; Qiagen, Germany) and then partially and randomly digested by Sau3A 1 (Promega, USA). These fragments, ranging from 0.
To evaluate the in vivo expression of C. jejuni genes identified using the IVIAT method, we selected four genes according to their functional category to compare the expression levels in vivo and in vitro using real-time RT-PCR. An in vitro DOC plate culturing method was used for analyzing the RNA transcription, mimicking in vivo conditions.

대상 데이터

Gene Cj0402 was used to normalize these samples because this housekeeping gene is expressed both on DOC and CCDA plates [25]. Duplicate reactions were performed, and three biological replicates were used for each sample. Threshold cycle values were determined using 7500 software ver.
jejuni and DE3 (BL21) whole cells and lysates. In this study, an expression library was generated from 52,700 clones, equating to 4 times the size of the NCTC 11168 genome. We selected several genes, including chuA, flgS, and Cj0190c, through IVIAT that were found to have products that were upregulated in the C.
The C. jejuni NCTC 11168 library in E. coli, represented by a total of approximately 52,700 clones, was screened using extensively adsorbed, pooled immune chicken serum. Indeed, 207 immunoreactive clones were identified after primary screening.

성능/효과

The real-time RT-PCR analysis was performed using a Gene Amp 7500 thermocycler (Applied Biosystems, CA, USA) with the following PCR parameters: 2 min at 50℃, followed by 40 cycles of denaturation at 95℃ for 30 sec and annealing at 60℃ for 34 sec. Four IVI genes (primers are shown in Table 1) were selected, and their transcription levels in the NCTC 11168 strain cultured on DOC plates were compared with those on CCDA plates. Gene Cj0402 was used to normalize these samples because this housekeeping gene is expressed both on DOC and CCDA plates [25].
The real-time PCR results showed that these four genes were drastically upregulated under in vivo-like conditions (>1-fold), while the other five genes were not (Fig. 3).

참고문헌 (42)

Ang CW, Dijkstra JR, de Klerk MA, Endtz HP, van Doorn PA, Jacobs BC, et al. 2010. Host factors determine anti-GM1 response following oral challenge of chickens with Guillain- Barre syndrome derived Campylobacter jejuni strain GB11. PLoS One 5: e9820.

상세보기
Barrero-Tobon AM, Hendrixson DR. 2012. Identification and analysis of flagellar coexpressed determinants (Feds) of Campylobacter jejuni involved in colonization. Mol. Microbiol. 84: 352-369.

상세보기
Cordwell SJ, Len AC, Touma RG, Scott NE, Falconer L, Jones D, et al. 2008. Identification of membrane-associated proteins from Campylobacter jejuni strains using complementary proteomics technologies. Proteomics 8: 122-139.

상세보기
Dasti JI, Tareen AM, Lugert R, Zautner AE, Gross U. 2010. Campylobacter jejuni: a brief overview on pathogenicityassociated factors and disease-mediating mechanisms. Int. J. Med. Microbiol. 300: 205-211.

상세보기
Day CJ, Tiralongo J, Hartnell RD, Logue CA, Wilson JC, von Itzstein M, Korolik V. 2009. Differential carbohydrate recognition by Campylobacter jejuni strain 11168: influences of temperature and growth conditions. PLoS One 4: e4927.

상세보기
Deb DK, Dahiya P, Srivastava KK, Srivastava R, Srivastava BS. 2002. Selective identification of new therapeutic targets of Mycobacterium tuberculosis by IVIAT approach. Tuberculosis 82: 175-182.

상세보기
French N, Barrigas M, Brown P, Ribiero P, Williams N, Leatherbarrow H, et al. 2005. Spatial epidemiology and natural population structure of Campylobacter jejuni colonizing a farmland ecosystem. Environ. Microbiol. 7: 1116-1126.

상세보기
Gaasbeek EJ, van der Wal FJ, van Putten JP, de Boer P, van der Graaf-van Bloois L, de Boer AG, et al. 2009. Functional characterization of excision repair and RecA-dependent recombinational DNA repair in Campylobacter jejuni. J. Bacteriol. 191: 3785-3793.

상세보기
Gu H, Zhu H, Lu C. 2009. Use of in vivo-induced antigen technology (IVIAT) for the identification of Streptococcus suis serotype 2 in vivo-induced bacterial protein antigens. BMC Microbiol. 9: 201.

상세보기
Handfield M, Brady LJ, Progulske-Fox A, Hillman JD. 2000. IVIAT: a novel method to identify microbial genes expressed specifically during human infections. Trends Microbiol. 8: 336-339.

상세보기
Hang L, John M, Asaduzzaman M, Bridges EA, Vanderspurt C, Kirn TJ, et al. 2003. Use of in vivo-induced antigen technology (IVIAT) to identify genes uniquely expressed during human infection with Vibrio cholerae. Proc. Natl. Acad. Sci. USA 100: 8508-8513.

상세보기
Harris JB, Baresch-Bernal A, Rollins SM, Alam A, LaRocque RC, Bikowski M, et al. 2006. Identification of in vivo-induced bacterial protein antigens during human infection with Salmonella enterica serovar Typhi. Infect. Immun. 74: 5161- 5168.

상세보기
Hartley-Tassell LE, Shewell LK, Day CJ, Wilson JC, Sandhu R, Ketley JM, Korolik V. 2010. Identification and characterization of the aspartate chemosensory receptor of Campylobacter jejuni. Mol. Microbiol. 75: 710-730.

상세보기
Hofreuter D, Novik V, Galan JE. 2008. Metabolic diversity in Campylobacter jejuni enhances specific tissue colonization. Cell Host Microbe 4: 425-433.

상세보기
Hu Y, Cong Y, Li S, Rao X, Wang G, Hu F. 2009. Identification of in vivo induced protein antigens of Salmonella enterica serovar Typhi during human infection. Sci. China C Life Sci. 52: 942-948.

상세보기
Hu Y, Shang Y, Huang J, Wang Y, Ren F, Jiao Y, et al. 2013. A novel immunoproteomics method for identifying in vivoinduced Campylobacter jejuni antigens using pre-adsorbed sera from infected patients. BBA Gen. Subjects 1830: 5229- 5235.

상세보기
Ibrahim GF, Fleet GH, Lyons MJ, Walker RA. 1985. Method for the isolation of highly purified Salmonella flagellins. J. Clin. Microbiol. 22: 1040-1044.

상세보기
Jeon B, Itoh K, Misawa N, Ryu S. 2003. Effects of quorum sensing on flaA transcription and autoagglutination in Campylobacter jejuni. Microbiol. Immunol. 47: 833-839.

상세보기
John M, Kudva IT, Griffin RW, Dodson AW, McManus B, Krastins B, et al. 2005. Use of in vivo-induced antigen technology for identification of Escherichia coli O157:H7 proteins expressed during human infection. Infect. Immun. 73: 2665-2679.

상세보기
Joslin SN, Hendrixson DR. 2009. Activation of the Campylobacter jejuni FlgSR two-component system is linked to the flagellar export apparatus. J. Bacteriol. 191: 2656-2667.

상세보기
Leach S, Harvey P, Wali R. 1997. Changes with growth rate in the membrane lipid composition of and amino acid utilization by continuous cultures of Campylobacter jejuni. J. Appl. Microbiol. 82: 631-640.

상세보기
Lertsethtakarn P, Ottemann KM, Hendrixson DR. 2011. Motility and chemotaxis in Campylobacter and Helicobacter. Annu. Rev. Microbiol. 65: 389-410.

상세보기
Li Q, Hu Y, Chen J, Liu Z, Han J, Sun L, Jiao X. 2013. Identification of Salmonella enterica serovar Pullorum antigenic determinants expressed in vivo. Infect. Immun. 81: 3119-3127.

상세보기
Lowry JE, Goodridge L, Vernati G, Fluegel AM, Edwards WH, Andrews GP. 2010. Identification of Brucella abortus genes in elk (Cervus elaphus) using in vivo-induced antigen technology (IVIAT) reveals novel markers of infection. Vet. Microbiol. 142: 367-372.

상세보기
Malik-Kale P, Parker CT, Konkel ME. 2008. Culture of Campylobacter jejuni with sodium deoxycholate induces virulence gene expression. J. Bacteriol. 190: 2286-2297.

상세보기
Parkhill J, Wren BW, Mungall K, Ketley JM, Churcher C, Basham D, et al. 2000. The genome sequence of the foodborne pathogen Campylobacter jejuni reveals hypervariable sequences. Nature 403: 665-668.

상세보기
Pearson B, Pin C, Wright J, I'Anson K, Humphrey T, Wells J. 2003. Comparative genome analysis of Campylobacter jejuni using whole genome DNA microarrays. FEBS Lett. 554: 224-230.

상세보기
Poli VFS, Thorsen L, Olesen I, Wik MT, Jespersen L. 2012. Differentiation of the virulence potential of Campylobacter jejuni strains by use of gene transcription analysis and a Caco-2 assay. Int. J. Food Microbiol. 155: 60-68.

상세보기
Rollins SM, Peppercorn A, Hang L, Hillman JD, Calderwood SB, Handfield M, Ryan ET. 2005. In vivo induced antigen technology (IVIAT). Cell Microbiol. 7: 1-9.
Rollins SM, Peppercorn A, Young JS, Drysdale M, Baresch A, Bikowski MV, et al. 2008. Application of in vivo induced antigen technology (IVIAT) to Bacillus anthracis. PLoS One 3: e1824.

상세보기
Salim KY, Cvitkovitch DG, Chang P, Bast DJ, Handfield M, Hillman JD, de Azavedo JC. 2005. Identification of group A Streptococcus antigenic determinants upregulated in vivo. Infect. Immun. 73: 6026-6038.

상세보기
Smith HK, Shepherd M, Monk C, Green J, Poole RK. 2011. The NO-responsive hemoglobins of Campylobacter jejuni: concerted responses of two globins to NO and evidence in vitro for globin regulation by the transcription factor NssR. Nitric Oxide 25: 234-241.

상세보기
Sztajer H, Lemme A, Vilchez R, Schulz S, Geffers R, Ying Yin Yip C, et al. 2008. Autoinducer-2-regulated genes in Streptococcus mutans UA159 and global metabolic effect of the luxS mutation. J. Bacteriol. 190: 401-415.

상세보기
Velayudhan J, Kelly D. 2002. Analysis of gluconeogenic and anaplerotic enzymes in Campylobacter jejuni: an essential role for phosphoenolpyruvate carboxykinase. Microbiology 148: 685-694.

상세보기
Vucic S, Kiernan MC, Cornblath DR. 2009. Guillain-Barre syndrome: an update. J. Clin. Neurosci. 16: 733-741.

상세보기
Woodall CA, Jones MA, Barrow PA, Hinds J, Marsden GL, Kelly DJ, et al. 2005. Campylobacter jejuni gene expression in the chick cecum: evidence for adaptation to a low-oxygen environment. Infect. Immun. 73: 5278-5285.

상세보기
Wosten MM, Wagenaar JA, van Putten JP. 2004. The FlgS/ FlgR two-component signal transduction system regulates the fla regulon in Campylobacter jejuni. J. Biol. Chem. 279: 16214-16222.

상세보기
Xie L, Zhang S, Yao W. 2011. Construction of cheA insertion mutant of Campylobacter jejuni and the effect of its adhesion on mice jejunum. Acta Microbiologica Sinica 51: 208-213.

상세보기
Young K T, D av is LM, D irita VJ. 2007. Campylobacter jejuni: molecular biology and pathogenesis. Nat. Rev. Microbiol. 5: 665-679.

상세보기
Zhang H, Fan H, Lu C. 2010. Identification of a novel virulence-related gene in Streptococcus suis type 2 strains. Curr. Microbiol. 61: 494-499.

상세보기
Zilbauer M, Dorrell N, Wren BW, Bajaj-Elliott M. 2008. Campylobacter jejuni-mediated disease pathogenesis: an update. Trans. R. Soc. Trop. Med. Hyg. 102: 123-129.

상세보기
Zou YX, Mo ZL, Hao B, Ye XH, Guo DS, Zhang PJ. 2010. Screening of genes expressed in vivo after infection by Vibrio anguillarum M3. Lett. Appl. Microbiol. 51: 564-569.

상세보기

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].

내보내기 구분	파일저장 인쇄 메일전송
구성항목	기본정보 상세정보 관리번호, 논문명, 저널/프로시딩명, 저자 , 발행년, 권, 호, 시작페이지, 끝페이지, 발행기관 관리번호, 논문명, 대등논문명, 저자 , 저널/프로시딩명, 발행기관, 발행년, 발행언어, 권, 호, 시작페이지, 끝페이지, ISBN, ISSN, 주제분야, 키워드, 초록(한글), 초록(영문), 저자(소속기관)
저장형식	Text(ASCII format) Excel format RefWorks Direct Export RIS format (for Reference Manager, ProCite, EndNote), Scholar's Aids, Mendeley
메일정보	받는사람 (필수) @ 보내는사람 (선택) @ 제목 내용 KISTI 검색결과 이메일 서비스
안내	총 건의 자료가 검색되었습니다. 다운받으실 자료의 인덱스를 입력하세요. (1-10,000) 검색결과의 순서대로 최대 10,000건 까지 다운로드가 가능합니다. 데이타가 많을 경우 속도가 느려질 수 있습니다.(최대 2~3분 소요) 다운로드 파일은 UTF-8 형태로 저장됩니다. 파일의 내용이 제대로 보이지 않을실 때는 웹브라우저 상단의 보기 -> 인코딩 -> 자동선택 여부를 확인하십시오. ~ Text(ASCII format) Excel format

연합인증