Characterization of a Cytotoxic Factor in Culture Filtrates of Serratia marcescens

Abstract

Serratia marcescens culture filtrates have been reported to be cytotoxic to mammalian cells. Using biochemical and genetic approaches, we have identified a major source of this cytotoxic activity. Both heat and protease treatments abrogated the cytotoxicity of S. marcescens culture filtrates towards HeLa cells, suggesting the involvement of one or more protein factors. A screen for in vitro cytotoxic activity revealed that S. marcescens mutant strains that are deficient in production of a 56-kDa metalloprotease are significantly less cytotoxic to mammalian cells. Cytotoxicity was significantly reduced when culture filtrates prepared from wild-type strains were pretreated with either EDTA or 1,10-phenanthroline, which are potent inhibitors of the 56-kDa metalloprotease. Furthermore, cytotoxic activity was restored when the same culture filtrates were incubated with zinc divalent cations, which are essential for enzymatic activity of the 56-kDa metalloprotease. Finally, recombinant expression of the S. marcescens 56-kDa metalloprotease conferred a cytotoxic phenotype on the culture filtrates of a nonpathogenic Escherichia coli strain. Collectively, these data suggest that the 56-kDa metalloprotease contributes significantly to the in vitro cytotoxic activity commonly observed in S. marcescens culture filtrates.Serratia marcescens is a gram-negative enteric bacterium that can function as an opportunistic pathogen within immunocompromised hosts (7,14,23,24,41). S. marcescens is a source of nosocomial infections, in part because the organism readily adheres to invasive hospital instrumentation, such as catheters, endoscopes, and intravenous tubing (1,17,27,32,34), and is relatively resistant to standard sterilization and disinfection protocols (3,8,52,53). Resistances to ␤-lactams, cephalosporins, and aminoglycosides have been reported, thereby complicating treatment of S. marcescens nosocomial infections (5,6,16,35,56).Upon introduction into the host, S. marcescens can infect numerous sites, including the urinary (42, 60) and respiratory (2) epithelia, muscle and subcutaneous tissues (17), the kidneys (25, 42), the lungs (13, 47), and also the heart and pericardium (58, 59). In addition, S. marcescens eye infections are common and are a frequent cause of keratitis (4, 29-31, 36, 65). In general, S. marcescens infections induce inflammation and fever, but fatal bacteremia can develop in patients weakened by previous infection, surgery, or immunosuppression (24,59,64,67). Despite numerous reported S. marcescens infections and the emergence of antibiotic-resistant strains (7,14,23,62), the virulence mechanisms of this organism are poorly understood.Carbonell and coworkers reported that S. marcescens culture filtrates exhibited pronounced in vitro cytotoxicity to cultured mammalian cells (11,12). Importantly, cytotoxicity was detected to various extents in all the strains that were tested, regardless of biotype or serotype (12). However, the cytotoxic factor or factors in S. marcescens culture filtrates were not identi...

Pathogenesis of microbial keratitis

“…Vaccination of rabbits with purified protease preparations from S. marcescens exhibited significantly less corneal destruction upon corneal encounter with live bacteria, indicating that proteases are important virulence factors during the development of serratial keratitis [96]. [115] showed that mutant strains of S. marcescens that are deficient in the production of 56 kDa metalloprotease are considerably less cytotoxic to mammalian cells. It was further revealed that the culture filtrates of wild-type bacteria pre-treated with EDTA or 1,10-phenanthroline (inhibitor of metalloproteases) exhibited significantly reduced cytotoxicity.…”

Molecular Characterization of Protease Activity in Serratia sp. Strain SCBI and Its Importance in Cytotoxicity and Virulence

“…Incubation of fibroblast cells with purified S. marcescens PrtA results in the destruction of more than 50% of cells within 1 h (15). Mutant strains lacking the 56-kDa metalloprotease are no longer cytotoxic toward HeLa cells (27). Proteases found in Serratia proteamaculans and Serratia grimesii also have cytotoxic properties.…”

“…Since protease activity has been identified as a cytotoxic factor for S. marcescens (27), the six Serratia sp. SCBI protease mutants were tested for their cytotoxic effects against BGMK cells.…”

Antibiotic Susceptibility and Heavy Metal Tolerance Pattern of Serratia Marcescens Isolated From Soil and Water

“…It functions as an opportunistic organism in immunocompromised patients because of its invasive ability to adhere to hospital instrumentation such as catheters, endoscopes and intravenous tubing [8]. Serratia marcescens is one of the major nosocomial pathogen found associated with urinary and respiratory tract infections, endocarditis, osteomyelitis, septicemia, wound infections, eye infections (conjunctivitis) and meningitis [9].…”