Defects in type III secretion correlate with internalization of Pseudomonas aeruginosa by epithelial cells - PubMed (original) (raw)

Defects in type III secretion correlate with internalization of Pseudomonas aeruginosa by epithelial cells

A R Hauser et al. Infect Immun. 1998 Apr.

Abstract

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.

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Figures

FIG. 1

FIG. 1

Internalization of strain PA103 and mutants. Assays were performed as described in the text. Each value represents the mean CFU of internalized bacteria per well. Although 27 of 33 tested mutants were not taken up by MDCK cells (e.g., mutant 8), six mutants, P, 16, 1, Q, M, and N, did demonstrate statistically significant internalization. Assays were performed a minimum of three times. Error bars represent standard errors of the means.

FIG. 2

FIG. 2

Analysis of secreted proteins of strain PA103 and mutants (Mut). Samples were prepared as described in the text and electrophoresed on SDS-polyacrylamide gels. The locations of bands corresponding to PepA, PepB, PepD, and ExoT are indicated. (A) Proteins present in concentrated culture supernatants as visualized by silver staining. Lane 6 is an example of a broth supernatant of mutant P with undetectable amounts of PepA, PepB, and PepD, but results varied as explained in the text. Molecular weight markers (MWM) are indicated on the right side of the panel. (B) Immunoblot analysis of proteins present in concentrated culture supernatants by using a combination of polyclonal antisera against PepA, PepB, PepD, and ExoT. The band in lane 1 indicated by an asterisk was noted occasionally in various samples and is thought to represent hybridization to a breakdown product of ExoT. (C) Electrophoretic analysis of broth protein aggregates of strain PA103. Samples were prepared and electrophoresed as described in the text. Bands were visualized by silver staining. The locations of bands corresponding to the PepA 73-, 71-, and 69-kDa triplet and to PepB, PepD, and ExoT are indicated.

FIG. 3

FIG. 3

Deduced peptide sequence comparisons of PepB with YopB (A) and PepD with YopD (B). Underlining represents sequences obtained by amino-terminal peptide sequencing of secreted PepB and PepD. Alignments were performed by the method of Needleman and Wunsch (31). |, identity; :, residues with comparison values greater than or equal to the average positive nonidentical comparison value in the scoring matrix; ., residues with comparison values greater than or equal to 1.

FIG. 4

FIG. 4

Model of the interaction between the PA103 type III secretion system and epithelial cells. In wild-type bacteria, the transcriptional activator ExsA allows expression of type III secretion genes. These genes encode PepB, PepD, and effector proteins such as a cytotoxin(s) and a putative factor that inhibits bacterial internalization by epithelial cells, AIF. The cytotoxin and AIF are transported out of the bacterium by the secretion apparatus, which consists of PscJ and other proteins. The cytotoxin and AIF are then translocated by the PepB-PepD complex into the cytoplasmic compartments of epithelial cells, where they act to kill the cells and inhibit bacterial internalization, respectively. Thus, wild-type PA103 has a cytotoxic and noninternalized phenotype. Potential defects present in the mutants discussed in this study are numbered as follows. (1) An ExsA mutant is unable to transcribe type III secretion genes and therefore synthesizes neither the cytotoxin nor AIF (although our data is consistent also with AIF regulation being independent of ExsA). This mutant is noncytotoxic and internalized. (2) Mutant 8 is defective in production of the putative cytotoxin but does secrete AIF. It therefore does not kill epithelial cells, but delivery of AIF to the host cell cytoplasmic compartment prevents bacterial internalization. (3) Mutant N is defective in production of PscJ, a component of the bacterial type III secretion apparatus, and can secrete neither the cytotoxin nor AIF. It is thus noncytotoxic and internalized. (4) Mutant 1 is defective in secretion of PepB and PepD and is therefore unable to translocate the cytotoxin and AIF into epithelial cells. This mutant is also noncytotoxic and internalized by epithelial cells.

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