is a gram-negative enteric bacterium that can function as an opportunistic pathogen within immunocompromised hosts (7
). 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
), and is relatively resistant to standard sterilization and disinfection protocols (3
). Resistances to β-lactams, cephalosporins, and aminoglycosides have been reported, thereby complicating treatment of S. marcescens
nosocomial infections (5
Upon introduction into the host, S. marcescens
can infect numerous sites, including the urinary (42
) and respiratory (2
) epithelia, muscle and subcutaneous tissues (17
), the kidneys (25
), the lungs (13
), and also the heart and pericardium (58
). In addition, S. marcescens
eye infections are common and are a frequent cause of keratitis (4
). In general, S. marcescens
infections induce inflammation and fever, but fatal bacteremia can develop in patients weakened by previous infection, surgery, or immunosuppression (24
). Despite numerous reported S. marcescens
infections and the emergence of antibiotic-resistant strains (7
), 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
). 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 identified in these studies. S. marcescens
secretes many known extracellular proteins, including chitinases, a lecithinase, a hemolysin, siderophores, lipases, proteases, and a nuclease (5
). Because a number of these extracellular factors are hydrolytic in nature, it is reasonable to hypothesize that one or more of the factors may directly contribute to cellular cytotoxicity by exerting their damaging effects upon host cells. Alternatively, an unidentified factor may be responsible for inducing cellular cytotoxicity.
MATERIALS AND METHODS
Preparation of culture filtrates.
The S. marcescens
and Escherichia coli
strains used in this study are listed in Table 1
.All bacterial strains were cultured into stationary phase at 37°C in liquid Luria-Bertani medium (Difco, Detroit, Mich.) while being shaken on a rotary platform shaker at 250 rpm. The cultures were harvested by centrifugation at 16,000 × g
. The supernatants were immediately filter sterilized by passage through Millipore 0.2-μm-pore-size syringe filters. The culture filtrates were divided into aliquots and stored at 4°C, under which conditions cytotoxic activity did not detectably diminish for at least 2 weeks. The supernatants were assayed for total protein content using the Coomassie protein assay (Pierce, Rockford, Ill.).
HeLa cells were cultured at 37°C under 5% CO2 and 90% humidity in 25-cm2 tissue culture flasks (Corning Costar Corp, Cambridge, Mass.) using minimal essential medium supplemented with 10% (vol/vol) fetal bovine serum, 1% (vol/vol) l-glutamine, 5,000 U of penicillin/ml, and 0.85% streptomycin (Invitrogen, Carlsbad, Calif.). Twenty-four hours prior to each experiment, 96-well tissue culture plates (Corning Costar Corp, Cambridge, Mass.) were seeded with 4.0 × 104 cells/well.
Bacterial culture filtrates were applied to monolayers of HeLa cells as indicated for each experiment and were incubated for 24 h at 37°C under 5% CO2. The monolayers were washed with phosphate-buffered saline (PBS) and incubated with 5 mg of 3-(4,5-dimethyl thiazolyl-2)-2,5-diphenyl tetrazolium bromide (MTT) (Amersham Life Sciences, Arlington Heights, Ill.)/ml of RPMI 1240 medium (Sigma, Detroit, Mich.) for 3 to 4 h at 37°C. The HeLa cells were washed with PBS, and the colored formazan product was solubilized by treating the cells with a lysis solution composed of 90% isopropanol containing 40.6 mM HCl and 0.5% sodium dodecyl sulfate (SDS). Conversion of MTT to formazan was quantified by measuring the optical density at 570 nm with subtraction of background absorbance at 690 nm using a Dynatech MR 5000 plate reader. The relative cytotoxicity was calculated by subtracting from a value of 1.0 the fraction of total metabolic activity detected in a monolayer of cells treated with bacterial culture filtrates relative to control monolayers treated with PBS.
Heat treatment of culture filtrates.
Culture filtrates were heated at 100°C for 15 min. The filtrates were then immediately placed on ice until they were applied to monolayers of HeLa cells.
Pronase treatments of culture filtrates.
E. coli or S. marcescens culture filtrates (10 mg of total protein/ml of filtrate) were treated with 1% (wt/wt) pronase (CalBiochem Inc., San Diego, Calif.) in ammonium bicarbonate buffer (pH 8) for 24 h at 37°C. The treated filtrates were immediately added to monolayers of HeLa cells.
Biochemical evaluation of the role of zinc in the cytotoxic activity of S. marcescens culture filtrates.
S. marcescens culture filtrates were treated with the metalloprotease inhibitors 50 mM EDTA and 50 mM 1,10-phenanthroline (Sigma, St. Louis, Mo.) for 24 h at 4°C and then for 1 h at 37°C. As controls, the filtrates were treated with PBS (pH 7.2) for 24 h at 4°C and then for 1 h at 37°C. Each treated filtrate was dialyzed into PBS (pH 7.2) at 4°C, with three changes of buffer (100× volume). Culture filtrates were treated in an identical fashion with the highest manufacturer (Calbiochem, La Jolla, Calif.)-recommended concentrations of Nα-p-tosyl-l-lysine chloromethyl ketone (TLCK) (100 μM), 4-(2-aminoethyl) benzenesulfonylfluoride-HCl (AEBSF) (1 mM), E-64 (10 μM), and (2S, 3S)-trans-epoxysuccinyl-l-leucylamido-3-methylbutane ethyl ester (EST) (100 μM). For restoration of cytotoxic activity, culture filtrates previously treated with metalloprotease inhibitors were incubated with 2.1 mM ZnSO4 at 22°C for 1 h.
Aliquots of culture filtrates were denatured in SDS-polyacrylamide gel electrophoresis (PAGE) loading buffer and heated for 5 min at 90°C. The denatured proteins were fractionated by SDS-12% PAGE and electrotransferred to a polyvinylidene difluoride (PVDF) membrane (Osmonics, Westborough, Mass.). The membrane was probed with antiserum raised against the S. marcescens
56-kDa metalloprotease (28
) and then with alkaline phosphatase-conjugated secondary antibodies (Sigma). The Western blots were visualized by chemiluminescence (Genor Technology, St. Louis, Mo.).
Data analyses were conducted using a Student's paired t test. A P value of less than 0.05 was considered statistically significant.
Because S. marcescens
infections occur at many target tissues within the host, it is likely that the bacterium exhibits virulence strategies common to bacterial pathogens known as “generalists” (56
). These pathogens, which include Staphylococcus aureus
, Pseudomonas aeruginosa
, and many Streptococcus
species, elaborate virulence factors that facilitate colonization of multiple niches within the host and encompass different cell types and overall environments. A common strategy used by bacterial pathogens is to secrete toxins and other factors that modulate the properties of host cell tissues (20
). The identification of bacterial factors that are important for host cell interactions will be critical to our understanding of S. marcescens
Carbonell and coworkers (11
) demonstrated that culture filtrates prepared from strains of S. marcescens
caused cytotoxic effects on both HeLa and Vero cells. Importantly, cytotoxicity was found in the culture filtrates of all S. marcescens
strains that were screened. Based on the cytotoxicity observed in these studies, S. marcescens
strains were proposed to secrete one or more cytotoxic factors that induced morphological changes in cultured mammalian cells and also reduced the viability of cellular monolayers. S. marcescens
secretes a broad array of factors, including a hemolysin, a nuclease, chitinases, a metalloprotease, serine proteases, siderophores, and lipases (5
). Each of these factors by itself has the potential to exert a cytotoxic effect on mammalian cells. Our primary objective focused on identifying which, if any, of these secreted factors within culture filtrates contribute to the previously established cytotoxic activity.
In this investigation, we used both genetic and biochemical approaches to identify the previously described 56-kDa metalloprotease as a significant, and perhaps the dominant, source of in vitro cytotoxicity within S. marcescens
culture filtrates. S. marcescens
mutant strains that were deficient in metalloprotease production demonstrated decreased cytotoxicity to HeLa cells. Importantly, the 56-kDa metalloprotease has been reported to be secreted from essentially every S. marcescens
strain and is, in fact, a marker for identifying S. marcescens
). This is consistent with our finding, and those previously published (11
), that cytotoxic activity is present in all culture filtrates that have been screened.
The S. marcescens
metalloprotease is known to contain a bound Zn2+
that is essential for enzymatic activity (44
). It was previously shown that when the active-site Zn2+
was extracted with strong divalent-cation chelating agents, the enzymatic activity of the 56-kDa metalloprotease was inhibited (44
). Significantly, we eliminated the cytotoxic activity by treating S. marcescens
culture filtrates with either EDTA or 1,10-phenanthroline. Moreover, we restored the cytotoxic activity to previously detoxified culture filtrates by reintroducing Zn2+
, which reactivates the catalytic activity of the apoenzyme (44
). Because we could modulate cytotoxic activity by treating S. marcescens
culture filtrates in a manner that directly affects metalloprotease enzymatic activity, it is likely that the catalytic activity of the 56-kDa metalloprotease is required for cytotoxicity. These data, however, do not by themselves rule out the possibility that EDTA or 1,10-phenanthroline inactivates another unknown factor produced by S. marcescens
that exerts cytotoxic activity towards HeLa cells.
Perhaps the strongest evidence implicating the S. marcescens 56-kDa metalloprotease as the primary cytotoxic factor is the dramatic elevation in cytotoxicity demonstrated by culture filtrates prepared from a nonpathogenic E. coli strain transformed with a plasmid harboring the gene encoding the 56-kDa metalloprotease. This is the first evidence that when expressed in a different genetic background, the 56-kDa metalloprotease is sufficient to confer a cytotoxic phenotype on culture filtrates.
Our results indicate, for the first time, that among the broad array of potentially hydrolytic and cytotoxic factors secreted by S. marcescens
, the 56-kDa metalloprotease is a dominant source of observed cytotoxicity toward mammalian cells. Interestingly, the 56-kDa metalloprotease has previously been proposed to be involved in pathogenesis (37
). The purified enzyme has been used in a model system to study keratitis (33
), and its enzymatic activity has been characterized and shown to rapidly degrade a wide range of structural and serum proteins (48
). Moreover, purified 56-kDa metalloprotease demonstrated a marked cytotoxic effect when applied to human fibroblast cells (48
). Thus, our identification of the 56-kDa metalloprotease as a dominant source of cytotoxicity within culture filtrates strongly supports the hypothesis that this secreted factor could play a role in pathogenesis.
Zinc-dependent metalloprotease activity is exhibited by some of the most potent toxins produced by bacterial pathogens (26
), including the lethal botulinum, tetanus, and anthrax toxins. Interestingly, each of these toxins functions from an intracellular site of action, thus requiring entry into host cells (46
). These intracellularly acting toxins generally possess an A-B domain structure, with the B fragment binding the toxin to sensitive cells and facilitating translocation of a catalytic A fragment into the cytosol. It is not yet clear whether the S. marcescens
metalloprotease also possesses a B fragment for transporting the catalytic fragment into the cell. Importantly, the purified 56-kDa metalloprotease was previously shown to be internalized in fibroblasts but required the formation of a complex with α-macroglobulin for successful entry (39
). These results suggest that the 56-kDa metalloprotease may possess a binding site for specific host proteins that are internalized by an endocytic mechanism, which represents an interesting mechanism for active transport of a cytotoxic factor into host cells. In the previous study, it was not established that upon entry into sensitive mammalian cells, the 56-kDa metalloprotease acts upon a specific intracellular target, as in the case of anthrax, botulinum, and tetanus toxins (50
). However, the possibility exists that the 56-kDa metalloprotease could specifically target a host cellular protein, thereby altering its function to result in the modulation of cellular properties during S. marcescens
In summary, we have confirmed previous reports that culture filtrates prepared from S. marcescens strains are cytotoxic to mammalian cells. Significantly, we employed genetic and biochemical approaches to identify the secreted 56-kDa metalloprotease, common to all S. marcescens strains, as a dominant contributor to in vitro cytotoxicity. The loss of cytotoxicity in S. marcescens strains deficient in metalloprotease production, as well as the gain of a cytotoxic phenotype in E. coli strains expressing and secreting the recombinant 56-kDa metalloprotease, strongly suggests that this extracellular factor could be important for S. marcescens pathogenesis within immunocompromised hosts. Additional investigations will not only reveal the cytotoxic mechanism of the 56-kDa metalloprotease but will be necessary to assess the role of this secreted factor in S. marcescens pathogenesis within the host.