Serratia marcescens suppresses host cellular immunity via the production of an adhesion-inhibitory factor against immunosurveillance cells

Abstract

Injection of a culture supernatant of Serratia marcescens into the bloodstream of the silkworm Bombyx mori increased the number of freely circulating immunosurveillance cells (hemocytes). Using a bioassay with live silkworms, serralysin metalloprotease was purified from the culture supernatant and identified as the factor responsible for this activity. Serralysin inhibited the in vitro attachment of both silkworm hemocytes and murine peritoneal macrophages. Incubation of silkworm hemocytes or murine macrophages with serralysin resulted in degradation of the cellular immune factor BmSPH-1 or calreticulin, respectively. Furthermore, serralysin suppressed in vitro phagocytosis of bacteria by hemocytes and in vivo bacterial clearance in silkworms. Disruption of the ser gene in S. marcescens attenuated its host killing ability in silkworms and mice. These findings suggest that serralysin metalloprotease secreted by S. marcescens suppresses cellular immunity by decreasing the adhesive properties of immunosurveillance cells, thereby contributing to bacterial pathogenesis.

Keywords: Adhesion; Bacterial Toxins; Cellular Immune Response; Innate Immunity; Insect Immunity; Macrophages; Metalloprotease; Protein Purification; Silkworm.

FIGURE 1.

Decrease in free hemocytes by injection stimulation and suppression of this response by S. marcescens. A, alteration of hemocyte density after wounding or liquid injection. Silkworm larvae (day 2 of 5th instar, 2 g/larva) were either injured by needles or injected with 50 μl of liquid samples (Milli-Q water, saline, LPS-free saline, IPS, 50 mm Tris-HCl (pH 8.0), 10 mg/ml bovine serum albumin, LB10 culture medium, or BHI culture medium). After 0.5 h, hemolymph was collected, and the number of hemocytes was counted under a microscope. Data represent mean ± S.D. of 4–6 larvae. Statistical analysis was performed using one-way analysis of variance (ANOVA) with Tukey's multiple comparison tests. Statistically significant differences were observed between columns with different letters (a–d; p < 0.05). B, 10 silkworms per group were injected with 100 μl of either BHI culture medium and live bacterial suspensions (10¹⁰ cells/ml) of S. aureus, E. coli, or S. marcescens. Hemolymph collected from 10 silkworms after 3 h were pooled, and hemocyte density was determined under a microscope. Data represent mean ± S.D. of 3–4 experiments. Statistical analysis was performed using a one-way ANOVA, and statistical differences compared with the control “BHI medium” group were analyzed by Dunnett's multiple comparison test (*, p < 0.001). C, silkworms were injected with 100 μl of either culture medium (open circle) or a filtered fraction of an overnight BHI culture supernatant of S. marcescens (closed circle), and the number of free hemocytes was counted at the indicated time points. Data represent mean ± S.D. of five larvae. Statistical analysis was performed using Student's t test between two groups at each time point (#, p < 0.005; *, p < 0.001). D, effect of heat treatment on hemocyte-increasing activity of the BHI culture supernatant of S. marcescens. Silkworms were injected with 100 μl of either a filtered fraction of an overnight BHI culture supernatant of S. marcescens (closed circle, Control) or the supernatant heated at 100 °C for 30 min (open circle, Heat-treated). After 0.5 h, the hemolymph was collected, and hemocyte numbers were counted. Data represent mean ± S.D. of four larvae. Statistical analysis was performed using Student's t test between two groups at injection volumes of 13, 25, 50, and 100 μl (#, p < 0.01; *, p < 0.001).

FIGURE 2.

Hemocyte number-increasing activities of eluted fractions of culture supernatant of S. marcescens from DEAE-Toyopearl column. A, silkworm hemocyte-increasing activity of S. marcescens IPS culture supernatant. Overnight-cultured S. marcescens cells were suspended in IPS (10¹¹ cells/ml) and statically incubated at 30 °C for 1 day. The suspension was then centrifuged, and the supernatant was filtered through a 0.22-μm filter. Three larvae per group (day 2 of 5th instar) were injected with 100 μl of each diluted sample, and the hemolymph was collected after 0.5 h. Hemolymph from three larvae was pooled, and hemocyte numbers were counted under a microscope. Activity with a 1.5-fold increase in the number of hemocytes relative to that obtained from IPS-injected silkworms was defined as 1 unit. B, effect of heat treatment on hemocyte-increasing activity of the IPS culture supernatant of S. marcescens. Silkworms were injected with 100 μl of either a filtered fraction of an IPS culture supernatant of S. marcescens or the supernatant heated at 100 °C for 30 min. After 0.5 h, the hemolymph was collected, and hemocyte numbers were counted. Data represent mean ± S.D. of three larvae. Statistical analysis was performed by one-way ANOVA with Tukey's multiple comparison tests (*, p < 0.01). C, hemocyte number-increasing activities (filled squares) and amounts of protein (open circles) in eluted fractions of culture supernatant of S. marcescens from the DEAE-Toyopearl column. Dashed lines indicate the NaCl concentration. D and E, effect of heat treatment on hemocyte-increasing activity of eluted fractions of S. marcescens culture supernatant from the DEAE-Toyopearl column. Peak 1 or peak 2 fractions obtained by DEAE-Toyopearl column chromatography were heated at 100 °C for 30 min. Silkworms were injected with 100 μl of heat-treated peak 1 (D) or peak 2 (E) fraction. After 0.5 h, the hemolymph was collected, and hemocyte numbers were counted. Data represent mean ± S.D. of three larvae. Statistical analysis was performed using Student's t test (D) or one-way ANOVA with Tukey's multiple comparison test (E) (*, p < 0.01; #, p < 0.001). F, fractions 12–19 of the above DEAE-Toyopearl column chromatography were subjected to SDS-PAGE, and the gel was stained with Coomassie Brilliant Blue. M, molecular weight markers.

FIGURE 3.

Hemocyte number-increasing activities of eluted fractions of culture supernatant of S. marcescens from the Superose 12 gel filtration column. A, peak 2 fraction obtained from DEAE-Toyopearl column chromatography of S. marcescens culture supernatant was further applied to a Superose 12 gel filtration column. Hemocyte number-increasing activities (filled squares) and amounts of protein (open circles) in eluted fractions are shown. B, fractions 19–30 of the above Superose 12 column chromatography were subjected to SDS-PAGE, and the gel was stained with Coomassie Brilliant Blue. M, molecular weight markers.

FIGURE 4.

Expression of recombinant serralysin protein in E. coli and its hemocyte number-increasing activity. A, expression of recombinant serralysin protein in transformed E. coli cells. E. coli cells harboring the expression plasmid were incubated in the presence of isopropyl β-d-thiogalactopyranoside (IPTG), and insoluble fractions were prepared from cell lysates. Samples were then solubilized in 8 m urea and subjected to SDS-PAGE. pET28a, E. coli cells transformed with an empty vector; pET28a/ser, E. coli cells transformed with a serralysin ORF-containing vector; P, insoluble fractions of cell lysates; S, urea-soluble fractions. B, fractionation of recombinant serralysin protein by nickel affinity column chromatography. The above urea-soluble fractions were applied to Probond resin. The column was washed and eluted as described under “Experimental Procedures.” FT, flow-through fraction; W, washed fraction; E1–5, eluted fractions. C, hemocyte-increasing activity of recombinant serralysin protein. The E1 fraction of the above nickel-affinity column chromatography was dialyzed in IPS and injected into silkworm hemolymph. Three larvae (day 2 of 5th instar) were injected with 100 μl of each sample, and after 0.5 h hemolymph were collected and pooled. Hemocyte numbers were counted under a microscope. Cell counts relative to that of IPS-injected group are shown. D, effect of heat treatment on hemocyte-increasing activity of the recombinant serralysin protein. Silkworms were injected with 100 μl of either purified recombinant serralysin (2.6 μm) or the protein sample heated at 100 °C for 30 min. After 0.5 h, the hemolymph was collected, and hemocyte numbers were counted. Data represent mean ± S.D. of three larvae. Statistical analysis was performed by one-way ANOVA with Tukey's multiple comparison tests (*, p < 0.001). E, effect of EDTA on the hemocyte-increasing activity of the recombinant serralysin protein. Recombinant serralysin was incubated in the presence of 50 mm EDTA at 4 °C for 3 h according to a previous report (26), and then the sample was injected into silkworm larvae (100 μl/larva). After 0.5 h, hemocyte numbers were counted as above. Data represent mean ± S.D. of 4–5 larvae. Statistical differences between EDTA(−) and EDTA(+) at each serralysin concentration were analyzed using Student's t test (*, p < 0.005).

FIGURE 5.

Suppression of hemocyte-dependent cellular immune responses by serralysin. A, inhibition of hemocyte attachment to solid surfaces by serralysin. Silkworm hemocytes suspended in IPS were incubated in cell culture plates with or without recombinant serralysin protein at 27 °C for 3 h. Plates were mildly shaken, and hemocytes collected in aliquots were counted as detached cells. Data represent mean ± S.D. of four experiments (*, p < 0.05). B, alterations in protein patterns of hemocyte membrane fractions by serralysin. Silkworm hemocytes were suspended in IPS and incubated in the presence of recombinant serralysin (700 nm) at 27 °C for 1 h. Membrane fractions of hemocytes were subjected to two-dimensional electrophoresis. Coomassie Brilliant Blue-stained gels of either the control or serralysin-treated group are shown in the upper or middle panels, respectively. Areas surrounding spots altered by serralysin treatment are magnified in the black frames. These pattern alterations were reproducibly observed in three experiments. Lower panels show results of MASCOT analysis of amino acid sequences obtained from protein extraction of gels indicated as protein spots 1 and 2. Gene products showing the highest identity with each sequence in the database are indicated, and sequences matching those obtained from each spot are written in red. C, effect of serralysin on phagocytosis of S. aureus by silkworm hemocytes in vitro. Hemocytes were suspended in PBS and incubated with S. aureus in the presence of recombinant serralysin at 27 °C for 1 h. Hemocytes were then lysed, and numbers of incorporated S. aureus were determined. Bacterial numbers per hemocyte relative to those of nonreacted control groups incubated without recombinant serralysin at 4 °C are indicated as the phagocytic index. Data represent mean ± S.D. of four experiments (*, p < 0.01). D, effect of serralysin on changes in live cell density of S. aureus in silkworm hemolymph in vivo. S. aureus cells suspended with or without recombinant serralysin (rSer) protein were injected into silkworm larvae, and changes in the live bacterial cell density in the hemolymph were monitored at the indicated time points. Data represent mean ± S.D. of six larvae. Statistical differences were analyzed between two groups at each time point using Student's t test (*, p < 0.01).

FIGURE 6.

Decrease in the density of silkworm hemocytes by the insect cytokine PP and suppression of this effect by serralysin. A, alterations of the hemocyte density in silkworm hemolymph induced by active PP. Silkworms were injected with 50 μl of either IPS, active PP (1 μm), a truncated form of PP lacking the N-terminal ENF residues (PPΔENF, 1 μm), or a mixture of active PP (1 μm) and recombinant serralysin (rSer) (800 nm), and after 0.5 h the density of hemocytes was determined. Data represent mean ± S.D. of 4–5 silkworms. Statistical analysis was performed using a one-way ANOVA with Tukey's multiple comparison tests. Statistically significant differences were observed between columns with different letters (p < 0.05). B, effect of serralysin on the protein level of active PP. Active PP (1 μm) was incubated in IPS for 1 h with or without recombinant serralysin (800 nm). A 5-fold serial dilution of each sample was applied to electrophoresis, and PP was detected by Western blot analysis. Ref, 10 ng of chemically synthesized active PP.

FIGURE 7.

Effect of serralysin on silkworm killing by bacteria. A, requirement of the ser gene for the increase in the cell density of hemocytes induced by S. marcescens. IPS supernatant of either S. marcescens wild-type (WT) or the disrupted mutant of the ser gene (Δser) was injected into silkworms (100 μl/larva), and after 0.5 h, the cell density of hemocytes in the hemolymph collected from three larvae per group was determined. Data represent hemocyte counts relative to that of the IPS-injected group. B, hemocyte number-increasing activities of eluted fractions of culture supernatant of the ser gene-disrupted mutant from the DEAE column. Hemocyte number-increasing activities (filled squares) and amounts of protein (open circles) in eluted fractions from the DEAE-Toyopearl column using culture supernatant of the Δser mutant as the starting material are shown. Dashed lines indicate the NaCl concentration. C, requirement of the ser gene in silkworm killing by S. marcescens. Serial diluted suspension of either S. marcescens wild-type (WT) or the disrupted mutant of the ser gene (Δser) was injected into silkworms in each group, and survival rates after 16 h were determined. Each point represents the survival rate of five larvae infected with the indicated number of S. marcescens. D, effect of co-injection of serralysin on silkworm killing by S. marcescens disruption mutant of the ser gene. Silkworms were injected with either S. marcescens wild-type cells suspended in IPS (WT, IPS), the disrupted mutant of the ser gene (Δser), or Δser mutant suspension supplemented with recombinant serralysin protein (Δser, rSer). LD₅₀ values were determined from survival rates monitored after 16 h of infection. Data represent mean ± S.D. of 4–5 experiments. One-way ANOVA with Tukey's multiple comparison test was performed, and statistically significant differences were observed between columns with different letters (a or b; p < 0.05). E, effect of serralysin on silkworm killing by S. aureus. Silkworm larvae (day 2 of 5th instar) were injected with S. aureus suspension supplemented with recombinant serralysin protein. LD₅₀ values were determined from survival rates monitored after 24 h of infection. Data represent mean ± S.D. of four experiments. Statistical analysis was performed using Student's t test (*, p < 0.05).

FIGURE 8.

Involvement of serralysin in S. marcescens virulence against mice. A, inhibition of murine peritoneal macrophage attachment to solid surfaces by serralysin. Macrophages suspended in PBS were preincubated with or without recombinant serralysin (800 nm) at 37 °C for 1 h. Cell suspensions were diluted and placed into cell culture dishes, and aliquots were removed after incubation at 37 °C for 2 h. Cells detached from the plate surfaces by cell scrapers were counted. Data represent mean ± S.D. of triplicates (*, p < 0.05). B, alterations in protein patterns of macrophage membrane fractions by serralysin. Mouse peritoneal macrophages were suspended in IPS and incubated in the presence of recombinant serralysin (700 nm) at 37 °C for 1 h. Membrane fractions of macrophages were subjected to two-dimensional electrophoresis. Coomassie Brilliant Blue-stained gels of either the control or serralysin-treated groups are shown in the upper or middle panels, respectively. The spot altered by serralysin treatment is magnified in the black frames. Lower panel shows the result of MASCOT analysis of amino acid sequences obtained from protein extraction of the gel. The gene product showing the highest identity with the obtained sequence is indicated, and the matched sequences are written in red. C, degradation of calreticulin in mouse macrophages by serralysin. Western blot analysis for calreticulin was performed using membrane fractions of mouse peritoneal macrophages treated with recombinant serralysin (rSer) as above. β-Actin was detected as a loading control. D, requirement of the ser gene of S. marcescens for mouse killing. Live bacteria suspension (200 μl; 3 × 10⁸ CFU/ml) of either wild-type (WT) or the disrupted ser in gene mutant (Δser) was intravenously injected into mice (n = 6), and survival rates were monitored. A log-rank test revealed a significant difference between the survival curves of “WT” and “Δser” (p < 0.05).