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Chapter 29 : Characterization of a 16-Kilodalton Species-Specific Protein of Promoting Uptake in Amoebae

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Characterization of a 16-Kilodalton Species-Specific Protein of Promoting Uptake in Amoebae, Page 1 of 2

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Abstract:

is the major agent responsible for Legionnaires' disease. To identify proteins that participate in the interaction of and its host, the authors screened a genomic library of strain Corby with anti-Corby antiserum. P16-related proteins and p16 sequences were present exclusively in strains belonging to . Characterization on the protein level was done by screening whole bacteria with MAb 61/1 as dot blot. The mutant showed the expected reaction type-loss of reactivity with the P16-specific monoclonal antibody. By comparing growth on the solid medium it was determined that p16 is not essential for growth of and that genetic manipulations did not generate additional effects that influenced growth or colony morphology. To investigate the importance of P16 for attachment, uptake, and/or intracellular growth in its different host cells, infection assays were performed in differentiated U937 cells and in . The wild-type phenotype was restored in the complemented mutant. From these results the authors speculate that the surface-exposed 16-kDa protein promotes the attachment/initial uptake and might act as an adhesin in the receptor-mediated uptake mechanism in amoebae. Further infection assays with other known natural hosts will show whether the described locus p16 might be responsible for the broad host spectrum of in contrast to the other species of .

Citation: Steudel C, Helbig J, Lück C. 2002. Characterization of a 16-Kilodalton Species-Specific Protein of Promoting Uptake in Amoebae, p 165-169. In Marre R, Abu Kwaik Y, Bartlett C, Cianciotto N, Fields B, Frosch M, Hacker J, Lück P (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555817985.ch29

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Figures

Image of FIGURE 1
FIGURE 1

Comparison of the expression of antigen in extracellular and intracellular cultures analyzed by indirect ELISA with monoclonal antibodies. Expression of antigen was measured for strain Corby cultured in broth (extracellular) and in (intracellular) each for 16 h. The amounts of legionellae used in the indirect ELISA were approximately 5 × 10 per well, as determined by plating broth and intra-amoebal-grown bacteria on buffered charcoal-yeast extract (BYCE) agar. Monoclonal antibodies used for detection were Mab 22/1 specific for Mip, which is expressed constitutively ( ); MAb 39/1 ( ) specific for Hsp60, a protein synthesized primarily under extracellular ( ) conditions; MAb 61/1 specific for P16; and one antibody specific for OmpM ( ), a protein present especially under intracellular conditions. OD 492 nm, optical density at 492 nm.

Citation: Steudel C, Helbig J, Lück C. 2002. Characterization of a 16-Kilodalton Species-Specific Protein of Promoting Uptake in Amoebae, p 165-169. In Marre R, Abu Kwaik Y, Bartlett C, Cianciotto N, Fields B, Frosch M, Hacker J, Lück P (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555817985.ch29
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Image of FIGURE 2
FIGURE 2

Growth of strains in cells: comparison between wild-type Corby, isogeneic knockout mutant CP7, and complemented mutant CP7 (pCS-31). Infection assays were performed with a multiplicity of infection of 100. The number of intracellular bacteria was determined at indicated time points postinfection. Each value represents the mean colony count of six samples and error bars indicate standard deviation. □, wild type; ■, knockout mutant; , complemented mutant.

Citation: Steudel C, Helbig J, Lück C. 2002. Characterization of a 16-Kilodalton Species-Specific Protein of Promoting Uptake in Amoebae, p 165-169. In Marre R, Abu Kwaik Y, Bartlett C, Cianciotto N, Fields B, Frosch M, Hacker J, Lück P (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555817985.ch29
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References

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1. Abu Kwaik, Y.,, and L. L. Pederson. 1996. The use of differential display-PCR to isolate and characterize a Legionella pneumophila locus induced during the intracellular infection of macrophages. Mol. Microbiol. 21:543556.
2. Abu Kwaik, Y.,, and N. C. Engleberg. 1994. Cloning and molecular characterization of a Legionella pneumophila gene induced by intracellular infection and by various in vitro stress conditions. Mol. Microbiol. 13:243251.
3. Abu Kwaik, Y.,, B. L. Eisenstein,, and N. C. Engleberg. 1993. Phenotypic modulation by Legionella pneumophila upon infection of macrophages. Inject. Immun. 61:13201329.
4. Helbig, J. H.,, B. Ludwig,, P. C. Lück,, A. Groh,, W. Witzleb,, and J. Hacker. 1995. Monoclonal antibodies to Legionella Mip proteins recognize genus- and species-specific epitopes. Clin. Diagn. Lab. Immunol. 2:160165.
5. Heuner, K.,, L. Bender-Beck,, B. C. Brand,, P. C. Lück,, K. H. Mann,, R. Marre,, M. Ott,, and J. Hacker. 1995. Cloning and genetic characterization of the flagellum subunit gene (flaA) of Legionella pneumophila serogroup 1. Inject. Immun. 63:24992507.
6. Lück, P.C.,, J. W. Schmitt,, A. Hengerer,, and J. H. Helbig. 1998. Subinhibitory concentrations of antimicrobial agents reduce the uptake of Legionella pneumophila into Acanthamoeba castel-lanii and U937 cells by altering the expression of virulence-associated antigens. Antimkrob. Agents Chemother. 42:28702876.
7. Moffat, J. F.,, and L. S. Tompkins. 1992. A quantitative model of intracellular growth of Legionella pneumophila in Acanthamoeba castellanii. Inject. Immun. 60:296301.
8. Sambrook, J.,, E. F. Fritsch,, and T. Maniatis. 1989. Molecular Cloning: a Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

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