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Infect Immun, April 1998, p. 1413-1420, Vol. 66, No. 4
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Defects in Type III Secretion Correlate with Internalization of Pseudomonas aeruginosa by Epithelial Cells

Alan R. Hauser,1 Suzanne Fleiszig,2 Pil Jung Kang,1,dagger Keith Mostov,3,4 and Joanne N. Engel1,*

Department of Medicine,1 Department of Anatomy,3 and Department of Biochemistry and Biophysics,4 University of California, San Francisco, San Francisco, California 94143, and School of Optometry, University of California, Berkeley, Berkeley, California 947202

Received 27 August 1997/Returned for modification 7 November 1997/Accepted 6 January 1998

Previous characterization of Pseudomonas aeruginosa clinical isolates has demonstrated an inverse correlation between cytotoxicity and internalization by epithelial cells. To further investigate this relationship, we tested PA103, a cytotoxic P. aeruginosa strain, and 33 isogenic noncytotoxic transposon mutants for internalization by Madin-Darby canine kidney cells. The majority of the mutants were not internalized, demonstrating that an inverse correlation between cytotoxicity and bacterial uptake by epithelial cells is not absolute. Six of the noncytotoxic mutants, however, demonstrated measurable levels of internalization by standard aminoglycoside exclusion assays even though internalization of wild-type strain PA103 was not detectable. All six had evidence of protein secretion defects involving two proteins, a 40-kDa protein and a 32-kDa protein. These proteins, designated PepB (for Pseudomonas exoprotein B) and PepD, respectively, each had characteristics of type III transported proteins. In addition, nucleotide sequencing studies demonstrated that PepB and PepD are homologs of YopB and YopD, respectively, type III secreted proteins of Yersinia spp. necessary for the translocation of effector molecules into the cytoplasmic compartment of eukaryotic cells. Thus, while many mutations in PA103 result in loss of cytotoxicity without an appreciable increase in internalization, defects in transport of type III secretion proteins PepB and PepD correlate with both loss of cytotoxicity and gain of internalization. These results are consistent with type III secretion of an inhibitor of internalization that requires PepB and PepD for translocation into the host cell.

* Corresponding author. Mailing address: Division of Infectious Disease, Box 0654, University of California, San Francisco, CA 94143-0654. Phone: (415) 476-7355. Fax: (415) 476-9364. E-mail: Joanne_Engel{at}quickmail.ucsf.edu.

dagger Present address: Department of Microbiology and Immunology, Ohio State University, Columbus, OH 43210.




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