全部 标题 作者
关键词 摘要

OALib Journal期刊
ISSN: 2333-9721
费用:99美元

查看量下载量

相关文章

更多...
PLOS ONE  2012 

Exploration of Chlamydial Type III Secretion System Reconstitution in Escherichia coli

DOI: 10.1371/journal.pone.0050833

Full-Text   Cite this paper   Add to My Lib

Abstract:

Background Type III secretion system is a virulent factor for many pathogens, and is thought to play multiple roles in the development cycle and pathogenesis of chlamydia, an important human pathogen. However, due to the obligate intracellular parasitical nature of chlamydiae and a lack of convenient genetic methodology for the organisms, very limited approaches are available to study the chlamydial type III secretion system. In this study, we explored the reconstitution of a chlamydial type III secretion in Escherichia coli. Results We successfully cloned all 6 genomic DNA clusters of the chlamydial type III secretion system into three bacterial plasmids. 5 of the 6 clusters were found to direct mRNA synthesis from their own promoters in Escherichia coli transformed with the three plasmids. Cluster 5 failed to express mRNA using its own promoters. However, fusion of cluster 5 to cluster 6 resulted in the expression of cluster 5 mRNA. Although only two of the type III secretion system proteins were detected transformed E. coli due to limited antibody availability, type III secretion system-like structures were detected in ultrathin sections in a small proportion of transformed E. coli. Conclusions We have successfully generated E. coli expressing all genes of the chlamydial type III secretion system. This serves as a foundation for optimal expression and assembly of the recombinant chlamydial type III secretion system, which may be extremely useful for the characterization of the chlamydial type III secretion system and for studying its role in chlamydial pathogenicity.

References

[1]  Galan JE, Wolf-Watz H (2006) Protein delivery into eukaryotic cells by type III secretion machines. Nature 444: 567–573.
[2]  Cordes FS, Komoriya K, Larquet E, Yang S, Egelman EH, et al. (2003) Helical structure of the needle of the type III secretion system of Shigella flexneri. J Biol Chem 278: 17103–17107.
[3]  Mota LJ, Cornelis GR (2005) The bacterial injection kit: type III secretion systems. Ann Med 37: 234–249.
[4]  Cornelis GR (2006) The type III secretion injectisome. Nat Rev Microbiol 4: 811–825.
[5]  Troisfontaines P, Cornelis GR (2005) Type III secretion: more systems than you think. Physiology (Bethesda) 20: 326–339.
[6]  Ghosh P (2004) Process of protein transport by the type III secretion system. Microbiol Mol Biol Rev 68: 771–795.
[7]  Viboud GI, Bliska JB (2005) Yersinia outer proteins: role in modulation of host cell signaling responses and pathogenesis. Annu Rev Microbiol 59: 69–89.
[8]  Ginocchio C, Pace J, Galan JE (1992) Identification and Molecular Characterization of a Salmonella typhimurium Gene Involved in Triggering the Internalization of Salmonellae into Cultured Epithelial Cells. Proceedings of the National Academy of Sciences 89: 5976–5980.
[9]  Brumell JH, Tang P, Zaharik ML, Finlay BB (2002) Disruption of the Salmonella-containing vacuole leads to increased replication of Salmonella enterica serovar typhimurium in the cytosol of epithelial cells. Infect Immun 70: 3264–3270.
[10]  Schachter J (1999) Infection and disease epidemiology. p.139–169 In R. S. Stephens (Ed.),. Chlamydia Intracellular Biology, Pathogenesis, ASM Press, Washington DC.
[11]  Campbell LA, Kuo CC (2004) Chlamydia pneumoniae–an infectious risk factor for atherosclerosis? Nat Rev Microbiol 2: 23–32.
[12]  Moulder JW (1991) Interaction of chlamydiae and host cells in vitro. Microbiol Rev 55: 143–190.
[13]  Hybiske K, Stephens RS (2007) Mechanisms of Chlamydia trachomatis Entry into Nonphagocytic Cells. Infection and immunity 75: 3925–3934.
[14]  Hybiske K, Stephens RS (2007) Mechanisms of host cell exit by the intracellular bacterium Chlamydia. Proceedings of the National Academy of Sciences 104: 11430–11435.
[15]  Matsumoto A (1982) Electron microscopic observations of surface projections on Chlamydia psittaci reticulate bodies. J Bacteriol 150: 358–364.
[16]  Gregory WW, Gardner M, Byrne GI, Moulder JW (1979) Arrays of hemispheric surface projections on Chlamydia psittaci and Chlamydia trachomatis observed by scanning electron microscopy. J Bacteriol 138: 241–244.
[17]  Peters J, Wilson DP, Myers G, Timms P, Bavoil PM (2007) Type III secretion a la Chlamydia. Trends Microbiol
[18]  Fields KA (2007) The Chlamydia Type III Secretion System. In: Bavoil P, Wyrick P, editors. Chlamydia Genomics and Pathogenesis: Herizon Bioscience. pp. 219–233.
[19]  Jewett TJ, Fischer ER, Mead DJ, Hackstadt T (2006) Chlamydial TARP is a bacterial nucleator of actin. PNAS %R 101073/pnas0603044103 103: 15599–15604.
[20]  Clifton DR, Dooley CA, Grieshaber SS, Carabeo RA, Fields KA, et al. (2005) Tyrosine phosphorylation of the chlamydial effector protein Tarp is species specific and not required for recruitment of actin. Infect Immun 73: 3860–3868.
[21]  Clifton DR, Fields KA, Grieshaber SS, Dooley CA, Fischer ER, et al. (2004) A chlamydial type III translocated protein is tyrosine-phosphorylated at the site of entry and associated with recruitment of actin. Proc Natl Acad Sci U S A 101: 10166–10171.
[22]  Dehoux P, Flores R, Dauga C, Zhong G, Subtil A (2011) Multi-genome identification and characterization of chlamydiae-specific type III secretion substrates: the Inc proteins. BMC Genomics 12: 109.
[23]  Chellas-Gery B, Wolf K, Tisoncik J, Hackstadt T, Fields KA (2011) Biochemical and localization analyses of putative type III secretion translocator proteins CopB and CopB2 of Chlamydia trachomatis reveal significant distinctions. Infection and immunity 79: 3036–3045.
[24]  Muschiol S, Bailey L, Gylfe A, Sundin C, Hultenby K, et al. (2006) A small-molecule inhibitor of type III secretion inhibits different stages of the infectious cycle of Chlamydia trachomatis. Proc Natl Acad Sci U S A 103: 14566–14571.
[25]  Subtil A, Parsot C, Dautry-Varsat A (2001) Secretion of predicted Inc proteins of Chlamydia pneumoniae by a heterologous type III machinery. Mol Microbiol 39: 792–800.
[26]  Mital J, Miller NJ, Fischer ER, Hackstadt T (2010) Specific chlamydial inclusion membrane proteins associate with active Src family kinases in microdomains that interact with the host microtubule network. Cellular microbiology 12: 1235–1249.
[27]  Qi M, Lei L, Gong S, Liu Q, DeLisa MP, et al. (2011) Chlamydia trachomatis secretion of an immunodominant hypothetical protein (CT795) into host cell cytoplasm. Journal of bacteriology 193: 2498–2509.
[28]  Wolf K, Betts HJ, Chellas-Gery B, Hower S, Linton CN, et al. (2006) Treatment of Chlamydia trachomatis with a small molecule inhibitor of the Yersinia type III secretion system disrupts progression of the chlamydial developmental cycle. Mol Microbiol 61: 1543–1555.
[29]  Bailey L, Gylfe A, Sundin C, Muschiol S, Elofsson M, et al. (2007) Small molecule inhibitors of type III secretion in Yersinia block the Chlamydia pneumoniae infection cycle. FEBS Lett 581: 587–595.
[30]  Fields KA, Hackstadt T (2000) Evidence for the secretion of Chlamydia trachomatis CopN by a type III secretion mechanism. Mol Microbiol 38: 1048–1060.
[31]  Subtil A, Blocker A, Dautry-Varsat A (2000) Type III secretion system in Chlamydia species: identified members and candidates. Microbes Infect 2: 367–369.
[32]  Ho TD, Starnbach MN (2005) The Salmonella enterica serovar typhimurium-encoded type III secretion systems can translocate Chlamydia trachomatis proteins into the cytosol of host cells. Infect Immun 73: 905–911.
[33]  Slepenkin A, Enquist PA, Hagglund U, de la Maza LM, Elofsson M, et al. (2007) Reversal of the antichlamydial activity of putative type III secretion inhibitors by iron. Infect Immun 75: 3478–3489.
[34]  Bao X, Pachikara ND, Oey CB, Balakrishnan A, Westblade LF, et al. (2011) Non-coding nucleotides and amino acids near the active site regulate peptide deformylase expression and inhibitor susceptibility in Chlamydia trachomatis. Microbiology 157: 2569–2581.
[35]  Balakrishnan A, Patel B, Sieber SA, Chen D, Pachikara N, et al. (2006) Metalloprotease inhibitors GM6001 and TAPI-0 inhibit the obligate intracellular human pathogen Chlamydia trachomatis by targeting peptide deformylase of the bacterium. J Biol Chem 281: 16691–16699.
[36]  Li X, Perez L, Pan Z, Fan H (2007) The transmembrane domain of TACE regulates protein ectodomain shedding. Cell Res 17: 985–998.
[37]  Caldwell HD, Kromhout J, Schachter J (1981) Purification and partial characterization of the major outer membrane protein of Chlamydia trachomatis. Infect Immun 31: 1161–1176.
[38]  Jamison WP, Hackstadt T (2008) Induction of type III secretion by cell-free Chlamydia trachomatis elementary bodies. Microbial pathogenesis 45: 435–440.
[39]  Hefty PS, Stephens RS (2007) Chlamydial type III secretion system is encoded on ten operons preceded by sigma 70-like promoter elements. J Bacteriol 189: 198–206.
[40]  Rodgers AK, Budrys NM, Gong S, Wang J, Holden A, et al. (2011) Genome-wide identification of Chlamydia trachomatis antigens associated with tubal factor infertility. Fertility and Sterility 96: 715–721.
[41]  Bao X, Nickels BE, Fan H (2012) Chlamydia trachomatis protein GrgA activates transcription by contacting the nonconserved region of σ66. Proceedings of the National Academy of Sciences 109: 16870–16875.
[42]  Gopal V, Chatterji D (1997) Mutations in the 1.1 Subdomain of Escherichia coli Sigma Factor σ70 and Disruption of its Overall Structure. European Journal of Biochemistry 244: 613–618.
[43]  Nordfelth R, Kauppi AM, Norberg HA, Wolf-Watz H, Elofsson M (2005) Small-Molecule Inhibitors Specifically Targeting Type III Secretion. Infect Immun 73: 3104–3114.
[44]  Lei L, Qi M, Budrys N, Schenken R, Zhong G (2011) Localization of Chlamydia trachomatis hypothetical protein CT311 in host cell cytoplasm. Microbial pathogenesis 51: 101–109.

Full-Text

Contact Us

service@oalib.com

QQ:3279437679

WhatsApp +8615387084133