This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rabin, S. D. P.
Right arrow Articles by Hauser, A. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rabin, S. D. P.
Right arrow Articles by Hauser, A. R.

 Previous Article  |  Next Article 

Infection and Immunity, January 2005, p. 573-582, Vol. 73, No. 1
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.1.573-582.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Functional Regions of the Pseudomonas aeruginosa Cytotoxin ExoU

Shira D. P. Rabin1 and Alan R. Hauser1,2*

Departments of Microbiology/Immunology,1 Medicine, Northwestern University, Chicago, Illinois2

Received 14 July 2004/ Returned for modification 25 August 2004/ Accepted 27 September 2004

ExoU, a potent patatin-like phospholipase, causes rapid cell death following its injection into host cells by the Pseudomonas aeruginosa type III secretion system. To better define regions of ExoU required for cytotoxicity, transposon-based linker insertion mutagenesis followed by site-directed mutagenesis of individual residues was employed by using a Saccharomyces cerevisiae model system. Random insertion of five amino acids identified multiple regions within ExoU that are required for cell killing. Five regions were chosen for further characterization: three corresponded to the oxyanion hole, hydrolase motif, and catalytic aspartate motif of the patatin-like domain within the N-terminal half of ExoU; one corresponded to an uncharacterized part of the patatin-like domain; and one corresponded to a region near the C terminus. Specific individual amino acid substitutions in each of the four N-terminal regions prevented killing of yeast and significantly reduced phospholipase activity. Whereas five amino acid insertions in the fifth region near the C terminus markedly reduced cytotoxicity and phospholipase activity, substitution of individual amino acids did not abolish either activity. To determine whether each of the five identified regions of ExoU was also essential for cytotoxicity in human cells, representative mutant forms of ExoU fused to green fluorescent protein were expressed in HeLa cells. These variants of ExoU were readily visualized and caused minimal cytotoxicity to HeLa cells, while wild-type ExoU fused to green fluorescent protein induced significant cell lysis and no detectable fluorescence. Thus, a minimum of five regions, including one which is well removed from the patatin-like domain, are required for the cytotoxicity and phospholipase activity of ExoU.

* Corresponding author. Mailing address: Department of Microbiology/Immunology, Northwestern University, 303 East Chicago Ave., Searle 6-495, Chicago, IL 60611. Phone: (312) 503-1044. Fax: (312) 503-1339. E-mail: ahauser{at}northwestern.edu.

Editor: J. B. Bliska

Infection and Immunity, January 2005, p. 573-582, Vol. 73, No. 1
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.1.573-582.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Diaz, M. H., Shaver, C. M., King, J. D., Musunuri, S., Kazzaz, J. A., Hauser, A. R. (2008). Pseudomonas aeruginosa Induces Localized Immunosuppression during Pneumonia. Infect. Immun. 76: 4414-4421 [Abstract] [Full Text]  
  • Sutterwala, F. S., Mijares, L. A., Li, L., Ogura, Y., Kazmierczak, B. I., Flavell, R. A. (2007). Immune recognition of Pseudomonas aeruginosa mediated by the IPAF/NLRC4 inflammasome. JEM 204: 3235-3245 [Abstract] [Full Text]  
  • Lee, V. T., Pukatzki, S., Sato, H., Kikawada, E., Kazimirova, A. A., Huang, J., Li, X., Arm, J. P., Frank, D. W., Lory, S. (2007). Pseudolipasin A Is a Specific Inhibitor for Phospholipase A2 Activity of Pseudomonas aeruginosa Cytotoxin ExoU. Infect. Immun. 75: 1089-1098 [Abstract] [Full Text]  
  • Rabin, S. D. P., Veesenmeyer, J. L., Bieging, K. T., Hauser, A. R. (2006). A C-Terminal Domain Targets the Pseudomonas aeruginosa Cytotoxin ExoU to the Plasma Membrane of Host Cells.. Infect. Immun. 74: 2552-2561 [Abstract] [Full Text]  
  • Shaver, C. M., Hauser, A. R. (2006). Interactions between effector proteins of the Pseudomonas aeruginosa type III secretion system do not significantly affect several measures of disease severity in mammals. Microbiology 152: 143-152 [Abstract] [Full Text]  
  • Shohdy, N., Efe, J. A., Emr, S. D., Shuman, H. A. (2005). Pathogen effector protein screening in yeast identifies Legionella factors that interfere with membrane trafficking. Proc. Natl. Acad. Sci. USA 102: 4866-4871 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»