Helicobacter pylori, the causative agent of type B gastritis, peptic ulcers, gastric adenocarcinoma and MALT lymphoma, uses the Cag type IV secretion system to induce a strong proinflammatory response in the gastric mucosa and to inject its effector protein CagA into gastric cells. CagA translocation results in altered host cell gene expression profiles and cytoskeletal rearrangements, and it is considered as a major bacterial virulence trait. Recently, it has been shown that binding of the type IV secretion apparatus to integrin receptors on target cells is a crucial step in the translocation process. Several bacterial proteins, including the Cag-specific components CagL and CagI, have been involved in this interaction. Here, we have examined the localization and interactions of CagI in the bacterial cell. Since the cagI gene overlaps and is co-transcribed with the cagL gene, the role of CagI for type IV secretion system function has been difficult to assess, and conflicting results have been reported regarding its involvement in the proinflammatory response. Using a marker-free gene deletion approach and genetic complementation, we show now that CagI is an essential component of the Cag type IV secretion apparatus for both CagA translocation and interleukin-8 induction. CagI is distributed over soluble and membrane-associated pools and seems to be partly surface-exposed. Deletion of several genes encoding essential Cag components has an impact on protein levels of CagI and CagL, suggesting that both proteins require partial assembly of the secretion apparatus. Finally, we show by co-immunoprecipitation that CagI and CagL interact with each other. Taken together, our results indicate that CagI and CagL form a functional complex which is formed at a late stage of secretion apparatus assembly.
References
[1]
Suerbaum S, Michetti P (2002) Helicobacter pylori infection. N Engl J Med 347: 1175–1186.
[2]
Peek RMJ, Blaser MJ (2002) Helicobacter pylori and gastrointestinal tract adenocarcinomas. Nat Rev Cancer 2: 28–37.
[3]
Amieva MR, El-Omar EM (2008) Host-bacterial interactions in Helicobacter pylori infection. Gastroenterol 134: 306–323.
[4]
Basso D, Zambon CF, Letley DP, Stranges A, Marchet A, et al. (2008) Clinical relevance of Helicobacter pylori cagA and vacA gene polymorphisms. Gastroenterol 135: 91–99.
[5]
Rieder G, Merchant JL, Haas R (2005) Helicobacter pylori cag-Type IV Secretion System Facilitates Corpus Colonization to Induce Precancerous Conditions in Mongolian Gerbils. Gastroenterol 128: 1229–1242.
[6]
Ohnishi N, Yuasa H, Tanaka S, Sawa H, Miura M, et al. (2008) Transgenic expression of Helicobacter pylori CagA induces gastrointestinal and hematopoietic neoplasms in mouse. Proc Natl Acad Sci U S A 105: 1003–1008.
[7]
Hatakeyama M (2008) SagA of CagA in Helicobacter pylori pathogenesis. Curr Opin Microbiol 11: 30–37.
[8]
Fischer W (2011) Assembly and molecular mode of action of the Helicobacter pylori Cag type IV secretion apparatus. FEBS J 278: 1203–1212.
[9]
Terradot L, Waksman G (2011) Architecture of the Helicobacter pylori Cag-type IV secretion system. FEBS J 278: 1213–1222.
[10]
Rohde M, Püls J, Buhrdorf R, Fischer W, Haas R (2003) A novel sheathed surface organelle of the Helicobacter pylori cag type IV secretion system. Mol Microbiol 49: 219–234.
[11]
Tanaka J, Suzuki T, Mimuro H, Sasakawa C (2003) Structural definition on the surface of Helicobacter pylori type IV secretion apparatus. Cell Microbiol 5: 395–404.
[12]
Andrzejewska J, Lee SK, Olbermann P, Lotzing N, Katzowitsch E, et al. (2006) Characterization of the pilin ortholog of the Helicobacter pylori type IV cag pathogenicity apparatus, a surface-associated protein expressed during infection. J Bacteriol 188: 5865–5877.
[13]
Kwok T, Zabler D, Urman S, Rohde M, Hartig R, et al. (2007) Helicobacter exploits integrin for type IV secretion and kinase activation. Nature 449: 862–866.
[14]
Jiménez-Soto LF, Kutter S, Sewald X, Ertl C, Weiss E, et al. (2009) Helicobacter pylori type IV secretion apparatus exploits β1 integrin in a novel RGD-independent manner. PLoS Pathog 5: e1000684.
[15]
Backert S, Fronzes R, Waksman G (2008) VirB2 and VirB5 proteins: specialized adhesins in bacterial type-IV secretion systems? Trends Microbiol 16: 409–413.
[16]
Fischer W, Püls J, Buhrdorf R, Gebert B, Odenbreit S, et al. (2001) Systematic mutagenesis of the Helicobacter pylori cag pathogenicity island: essential genes for CagA translocation in host cells and induction of interleukin-8. Mol Microbiol 42:1337–1348. Erratum. Mol. (2003).Microbiol. 47, 1759.
[17]
Censini S, Lange C, Xiang Z, Crabtree JE, Ghiara P, et al. (1996) cag, a pathogenicity island of Helicobacter pylori, encodes type I-specific and disease-associated virulence factors. Proc Natl Acad Sci USA 93: 14648–14653.
[18]
Selbach M, Moese S, Meyer TF, Backert S (2002) Functional analysis of the Helicobacter pylori cag pathogenicity island reveals both VirD4-CagA-dependent and VirD4-CagA-independent mechanisms. Infect Immun 70: 665–671.
[19]
Sharma CM, Hoffmann S, Darfeuille F, Reignier J, Findeiss S, et al. (2010) The primary transcriptome of the major human pathogen Helicobacter pylori. Nature 464: 250–255.
[20]
Shaffer CL, Gaddy JA, Loh JT, Johnson EM, Hill S, et al. (2011) Helicobacter pylori Exploits a Unique Repertoire of Type IV Secretion System Components for Pilus Assembly at the Bacteria-Host Cell Interface. PLoS Pathog 7: e1002237.
[21]
Dailidiene D, Dailide G, Kersulyte D, Berg DE (2006) Contraselectable streptomycin susceptibility determinant for genetic manipulation and analysis of Helicobacter pylori. Appl Environ Microbiol 72: 5908–5914.
[22]
Fernandez D, Spudich GM, Zhou XR, Christie PJ (1996) The Agrobacterium tumefaciens VirB7 lipoprotein is required for stabilization of VirB proteins during assembly of the T-complex transport apparatus. J Bacteriol 178: 3168–3176.
[23]
Hapfelmeier S, Domke N, Zambryski PC, Baron C (2000) VirB6 is required for stabilization of VirB5 and VirB3 and formation of VirB7 homodimers in Agrobacterium tumefaciens. J Bacteriol 182: 4505–4511.
[24]
Kutter S, Buhrdorf R, Haas J, Schneider-Brachert W, Haas R, et al. (2008) Protein subassemblies of the Helicobacter pylori Cag type IV secretion system revealed by localization and interaction studies. J Bacteriol 190: 2161–2171.
[25]
Jurik A, Hau？er E, Kutter S, Pattis I, Pra？l S, et al. (2010) The coupling protein Cagβ and its interaction partner CagZ are required for type IV secretion of the Helicobacter pylori CagA protein. Infect Immun 78: 5244–5251.
[26]
Pattis I, Weiss E, Laugks R, Haas R, Fischer W (2007) The Helicobacter pylori CagF protein is a type IV secretion chaperone-like molecule that binds close to the C-terminal secretion signal of the CagA effector protein. Microbiology 153: 2896–2909.
[27]
Cascales E, Christie PJ (2003) The versatile bacterial type IV secretion systems. Nat Rev Microbiol 1: 137–149.
[28]
Alvarez-Martinez CE, Christie PJ (2009) Biological diversity of prokaryotic type IV secretion systems. Microbiol Mol Biol Rev 73: 775–808.
[29]
Pinto-Santini DM, Salama NR (2009) Cag3 is a novel essential component of the Helicobacter pylori Cag type IV secretion system outer membrane subcomplex. J Bacteriol 191: 7343–7352.
[30]
Cendron L, Couturier M, Angelini A, Barison N, Stein M, et al. (2009) The Helicobacter pylori CagD (HP0545, Cag24) protein is essential for CagA translocation and maximal induction of interleukin-8 secretion. J Mol Biol 386: 204–217.
[31]
Busler VJ, Torres VJ, McClain MS, Tirado O, Friedman DB, et al. (2006) Protein-Protein Interactions among Helicobacter pylori Cag Proteins. J Bacteriol 188: 4787–4800.
[32]
Sambrook J, Russell DW (2001) Molecular cloning. A laboratory manual. 3rd ed. Cold Spring Harbor Laboratory Press.
[33]
Odenbreit S, Püls J, Sedlmaier B, Gerland E, Fischer W, et al. (2000) Translocation of Helicobacter pylori CagA into gastric epithelial cells by type IV secretion. Science 287: 1497–1500.
[34]
Haas R, Meyer TF, van Putten JPM (1993) Aflagellated mutants of Helicobacter pylori generated by genetic transformation of naturally competent strains using transposon shuttle mutagenesis. Mol Microbiol 8: 753–760.
[35]
Fischer W, Windhager L, Rohrer S, Zeiller M, Karnholz A, et al. (2010) Strain-specific genes of Helicobacter pylori: genome evolution driven by a novel type IV secretion system and genomic island transfer. Nucleic Acids Res 38: 6089–6101.
[36]
Hohlfeld S, Pattis I, Püls J, Plano GV, Haas R, et al. (2006) A C-terminal secretion signal is necessary, but not sufficient for type IV secretion of the Helicobacter pylori CagA protein. Mol Microbiol 59: 1624–1637.
[37]
Püls J, Fischer W, Haas R (2002) Activation of Helicobacter pylori CagA by tyrosine phosphorylation is essential for dephosphorylation of host cell proteins in gastric epithelial cells. Mol Microbiol 43: 961–969.
[38]
Fischer W, Haas R (2004) The RecA protein of Helicobacter pylori requires a posttranslational modification for full activity. J Bacteriol 186: 777–784.
[39]
Wessel D, Flügge U (1984) A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal Biochem 138: 141–143.
