Serratia marcescens internalization and replication in human bladder epithelial cells

BACKGROUND: Serratia marcescens, a frequent agent of catheterization-associated bacteriuria, strongly adheres to human bladder epithelial cells in culture. The epithelium normally provides a barrier between lumal organisms and the interstitium; the tight adhesion of bacteria to the epithelial cells can lead to internalization and subsequent lysis. However, internalisation was not shown yet for S. marcescens strains. METHODS: Elektronmicroscopy and the common gentamycin protection assay was used to assess intracellular bacteria. Via site directed mutagenesis, an hemolytic negative isogenic Serratia strain was generated to point out the importance of hemolysin production. RESULTS: We identified an important bacterial factor mediating the internalization of S. marcescens, and lysis of epithelial cells, as the secreted cytolysin ShlA. Microtubule filaments and actin filaments were shown to be involved in internalization. However, cytolysis of eukaryotic cells by ShlA was an interfering factor, and therefore hemolytic-negative mutants were used in subsequent experiments. Isogenic hemolysin-negative mutant strains were still adhesive, but were no longer cytotoxic, did not disrupt the cell culture monolayer, and were no longer internalized by HEp-2 and RT112 bladder epithelial cells under the conditions used for the wild-type strain. After wild-type S. marcescens became intracellular, the infected epithelial cells were lysed by extended vacuolation induced by ShlA. In late stages of vacuolation, highly motile S. marcescens cells were observed in the vacuoles. S. marcescens was also able to replicate in cultured HEp-2 cells, and replication was not dependent on hemolysin production. CONCLUSION: The results reported here showed that the pore-forming toxin ShlA triggers microtubule-dependent invasion and is the main factor inducing lysis of the epithelial cells to release the bacteria, and therefore plays a major role in the development of S. marcescens infections

Activation of Serratia marcescens Hemolysin through a Conformational Change

For Serratia marcescens, secreted hemolysin/cytotoxin is not only secreted but also activated by an outer membrane protein. Excluding posttranslational processing by mass spectrometry, the conformation of active and inactive ShlA derivatives strongly differed in electrophoretic mobilities, gel permeation chromatography, sensitivity to trypsin, circular dichroism, and intrinsic fluorescence. We concluded that ShlB interacts with ShlA during secretion and imposes a conformational change in ShlA to form the active hemolysin

Hemolysis of human erythrocytes in coculture with bacterial cells; (◆)W225; (□)SM001; (■)SM001 pRSC01; (▲)HB101 pRSC01; (△)HB101 pMMB67EH; (○)BL21 pRO3; (x)BL21 pT7-5

<p><b>Copyright information:</b></p><p>Taken from "internalization and replication in human bladder epithelial cells"</p><p>BMC Infectious Diseases 2004;4():16-16.</p><p>Published online 9 Jun 2004</p><p>PMCID:PMC441377.</p><p>Copyright © 2004 Hertle and Schwarz; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.</p

Invasion of different strains in cultured RT112 epithelial cells after 2-h incubation at 37°C at an MOI of 2

<p><b>Copyright information:</b></p><p>Taken from "internalization and replication in human bladder epithelial cells"</p><p>BMC Infectious Diseases 2004;4():16-16.</p><p>Published online 9 Jun 2004</p><p>PMCID:PMC441377.</p><p>Copyright © 2004 Hertle and Schwarz; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.</p

A Cytotoxicity by LDH assay of different strains on RT112 cultured black bars and Hep-2 gray bars epithelial cells after 1-h incubation at 37°C MOI 5

<p><b>Copyright information:</b></p><p>Taken from "internalization and replication in human bladder epithelial cells"</p><p>BMC Infectious Diseases 2004;4():16-16.</p><p>Published online 9 Jun 2004</p><p>PMCID:PMC441377.</p><p>Copyright © 2004 Hertle and Schwarz; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.</p> [E.C.] BL21 pT7-5 served as a negative control