Trafficking of porin-deficient Salmonella typhimurium mutants inside HeLa cells: ompR and envZ mutants are defective for the formation of Salmonella-induced filaments - PubMed (original) (raw)

Trafficking of porin-deficient Salmonella typhimurium mutants inside HeLa cells: ompR and envZ mutants are defective for the formation of Salmonella-induced filaments

S D Mills et al. Infect Immun. 1998 Apr.

Abstract

Outer membrane porin genes of Salmonella typhimurium, including ompC, ompF, and tppB, are regulated by the products of ompB, a two-component regulatory locus encoding OmpR and EnvZ. S. typhimurium ompR mutants are attenuated in mice, but to date no one has studied the intracellular trafficking of S. typhimurium porin-deficient mutants. In this study, isogenic transposon mutants of S. typhimurium with insertions in ompR, envZ, ompF, ompC, ompD, osmZ, and tppB were compared with wild-type SL1344 for trafficking in the human epithelial cell line HeLa. We found that ompR and envZ mutants were reduced or completely inhibited for the formation of Salmonella-induced filaments (Sifs). This result was confirmed with an ompB deletion mutant. Sifs are tubular structures containing lysosomal glycoprotein which are induced specifically by intracellular Salmonella. Genetic analysis showed that the ompR mutation could be complemented in trans by cloned ompR to restore its ability to induce Sifs. In contrast, mutations in the known ompR-regulated genes ompF, ompC, and tppB (as well as the ompR-independent porin gene, ompD) had no effect on Sif formation relative to that of wild-type SL1344, thus indicating that OmpR does not exert its role on these genes to induce Sif formation. The omp mutants studied were able to invade and replicate in HeLa cells at levels comparable to those in wild-type SL1344. We conclude that OmpR and EnvZ appear to regulate Sif formation triggered by intracellular S. typhimurium.

PubMed Disclaimer

Figures

FIG. 1

_omp_B (ompR and envZ) mutants are defective or highly reduced for Sif formation. Induction of Sif formation in HeLa cells by the Salmonella strains listed in Table 1 was determined at 6 h postinvasion. This graph represents results from one of four experiments where cells (100 for each strain) infected by each S. typhimurium strain tested were evaluated for Sif formation at 6 h postinvasion. Values are given as the percentage of infected cells containing Sifs. The dashed line provides a reference for Sif induction by wild-type SL1344.

FIG. 2

Micrograph illustrating immunofluorescence labeling of Sifs in HeLa cells infected with wild-type SL1344 and porin-deficient mutants at 6 h postinvasion. Shown is a typical Sif (stained with anti-lgp monoclonal antibody) (A) induced by wild-type SL1344. Induction of Sif formation was defective for ompR mutants ARD3 (ompR::Mu dJ) (B) and CJD359 (ompR::Tn_10_) (D), a situation similar to what was previously shown for J1-3 (sifA::Tn_10_ dCm) (F) (33). Sif formation was restored to ARD3 (ompR::Mu dJ) (C) complemented in trans with the cloned ompB locus in pSWLOMP. Triple porin-deficient mutant BRD409 (ompC::Tn_10_, ompF::Mu d1-8, tppB::Mu dJ) induced Sifs similarly to wild-type SL1344 (E).

FIG. 3

Kinetics of Sif formation in HeLa cells. SL1344 ompR mutants were defective for Sif formation in HeLa cells over a time course of 1 to 8 h postinvasion, compared with wild-type SL1344 and J1-3 (sifA::Tn_10_ dCm). In these experiments, J1-3 (sifA::Tn_10_ dCm), ARD3 (ompR::Mu dJ), and ARD3 (pWSK29; cloning vector) did not induce any Sifs, while CJD359 (ompR::Tn_10_) induced the formation of Sifs at a low frequency. ARD3 complemented in trans with the cloned ompB locus (pSWLOMP) exhibited Sif formation kinetics similar to those of wild-type SL1344. This graph shows results from one of three experiments where 100 infected cells for each S. typhimurium strain were evaluated for Sif formation at 1, 2, 4, 6, and 8 h postinvasion. Results are given as percent infected cells containing Sifs.

FIG. 4

Invasion and replication in HeLa cells are not affected in ompR mutants. This time course replication experiment shows that the ompR mutants (ARD3 and CJD359) are able to replicate as well as, if not better than, wild-type SL1344. Values are mean numbers of CFU recovered from three wells of HeLa cells (5 × 104 cells each) infected with S. typhimurium at 1, 2, 4, 6, and 8 h postinvasion. This graph depicts results from one of three representative experiments.

References

1. Benson N R, Goldman B S. Rapid mapping in Salmonella typhimurium with Mud-P22 prophages. J Bacteriol. 1992;174:1673–1681. - PMC - PubMed
1. Bernardini M L, Fontaine A, Sansonetti P J. The two-component regulatory system OmpR-EnvZ controls the virulence of Shigella flexneri. J Bacteriol. 1990;172:6274–6281. - PMC - PubMed
1. Chatfield S, Dorman C J, Hayward C, Dougan G. Role of ompR-dependent genes in Salmonella typhimurium virulence: mutants deficient in both OmpC and OmpF are attenuated in vivo. Infect Immun. 1991;59:449–452. - PMC - PubMed
1. Chen L M, Kaniga K, Galán J E. Salmonella spp. are cytotoxic for cultured macrophages. Mol Microbiol. 1996;21:1101–1115. - PubMed
1. Dorman C J, Chatfield S, Higgins C F, Hayward C, Dougan G. Characterization of porin and ompR mutants of a virulent strain of Salmonella typhimurium: ompR mutants are attenuated in vivo. Infect Immun. 1989;57:2136–2140. - PMC - PubMed

Trafficking of porin-deficient Salmonella typhimurium mutants inside HeLa cells: ompR and envZ mutants are defective for the formation of Salmonella-induced filaments - PubMed (original) (raw)