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.


CONCLUSIONS
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.
Hertle
Ralf
R
Microbiology/Membranphysiology, University of Tübingen, Germany. ralf.hertle@mikrobio.uni-tuebingen.de
Schwarz
Heinz
H
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001422:Bacterial Adhesion; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics; D002460:Cell Line; D045744:Cell Line, Tumor; D004847:Epithelial Cells / Q000382:microbiology; D005839:Gentamicins / Q000494:pharmacology; D006460:Hemolysin Proteins / Q000096:biosynthesis / Q000235:genetics; D006461:Hemolysis / Q000235:genetics; D006801:Humans; D046529:Microscopy, Electron, Transmission; D016297:Mutagenesis, Site-Directed; D012706:Serratia marcescens / Q000187:drug effects / Q000235:genetics / Q000472:pathogenicity* / Q000502:physiology*; D001743:Urinary Bladder / Q000166:cytology / Q000382:microbiology*; D014617:Vacuoles / Q000382:microbiology; D014774:Virulence
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D005839:Gentamicins; D006460:Hemolysin Proteins; C089911:ShlA protein, Serratia marcescens