Abstract
BackgroundSerratia 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.MethodsElektronmicroscopy 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.ResultsWe 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.ConclusionThe 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.
Highlights
Serratia marcescens, a frequent agent of catheterization-associated bacteriuria, strongly adheres to human bladder epithelial cells in culture
Potential virulence factors involved in this pathogenicity are proteases, a nuclease, a lecitinase, and the hemolysin, all of which are secreted by the bacterium
After isolation of hemolytic-negative mutants SM001 and SM011, the genomic recombination region in the shlB/shlA locus was amplified by PCR and sequenced to confirm the mutation
Summary
A frequent agent of catheterization-associated bacteriuria, strongly adheres to human bladder epithelial cells in culture. The renal colonization of this strain was more than five times higher than that of the ShlA-negative recipient strain in an experimental rat pyelonephritis model This hemolysin has been shown to be cytotoxic to epithelial cells in culture [15]. These data imply a major role of the hemolysin in pathogenicity; the experiments have been mostly carried out with the purified hemolysin protein. It has been shown in a Caenorhabditis elegans infection model that a ShlA-negative mutant is no longer pathogenic [20]. There are frequent reports of nosocomial Serratia outbreaks, the molecular mechanisms of S. marcescens pathogenicity in vivo are still poorly understood
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