Table 1.
List of oligonucleotides for PCR.
Fig 1.
Effect of salt and rat serum on cellular Sph2 production in different strains of L. interrogans.
L. interrogans serovar Lai strain 56601 (A), serovar Manilae strain L495 (B), serovar Pomona strain LC82-25 (C), and serovar Copenhageni Fiocruz L1-130 (D) were grown in EMJH (lane 1), EMJH supplemented with 120 mM sodium chloride (lane 2) or 10% rat serum (lane 3) or both sodium chloride and rat serum (lane 4) for 4 hours at 30°C. Whole-cell lysates were analyzed by immunoblotting with anti-Sph2 and anti-LipL41 antisera. Arrows identify Sph2, SphH, and LipL41. The anti-Sph2 antiserum cross-reacts with SphH. Positions of molecular mass standards are shown in kilodaltons.
Fig 2.
Cross-reaction of anti-Sph2 antibody with other sphingomyelinase-like proteins.
50 ng of recombinant Sph1, Sph2, Sph3, and Sph4 were subjected to SDS-polyacryamide gel electrophoresis in duplicate gels. One gel was stained with Coomassie blue (A), and the second was analyzed by Western blot with anti-Sph2 antiserum (B). Positions of molecular mass standards are shown in kilodaltons.
Fig 3.
Effect of salt and serum on the amount of Sph2 released by L. interrogans.
Sph2 was recovered by immunoprecipitation from spent growth medium of L. interrogans Manilae L495 (lanes 2–4) and Pomona LC82-25 (5–7) strains grown under different conditions for 4 hours at 30°C in EMJH (lanes 2 and 5), EMJH with 120 mM sodium chloride (lanes 3 and 6), and EMJH with sodium chloride and 10% rat serum (lanes 4 and 7). Spent medium was collected, and Sph2 was detected by immunoprecipitation (lanes 2–7). Cell lysates are shown for comparison (lanes 1 and 8). The Pomona cell lysate was diluted four fold before loading. Arrows indicate Sph2 and SphH observed in cell lysates. The anti-Sph2 antiserum cross-reacts with SphH. Arrowheads mark Sph2 detected in the spent medium. Positions of molecular mass standards are shown in kilodaltons.
Fig 4.
Effect of salt and serum on sph2 transcript levels in L. interrogans.
Transcript levels were determined by quantitative RT-PCR. Ct values for sph2 transcript were normalized to those for lipL41 transcript. Fold differences between growth conditions are shown in italics. Mean and standard deviation from three biological replicates are shown; p < 0.001 for all group comparisons (one-way ANOVA, Tukey multiple comparisons post-test).
Fig 5.
Hemolytic activity in spent growth medium of L. interrogans.
A. Hemolytic activity associated with spent growth medium from L. interrogans Manilae L495 and Pomona LC82-25 strains grown in EMJH, EMJH supplemented with 120 mM sodium chloride (Na) and EMJH supplemented with 120 mM sodium chloride and 10% rat serum (Na+serum) was examined on blood agar plates. 50 mU of Bacillus cereus sphingomyelinase (Bc SMase) was spotted onto the agar as a positive control. B. Hemolytic activities of spent growth medium from Manilae L495 and Pomona LC82-25 cultures incubated in EMJH and EMJH supplemented with 120 mM sodium chloride were measured by a quantitative hemolytic assay. The spent medium from the Pomona LC82-25 cultures were diluted 10 fold into EMJH plus 120 mM sodium chloride prior to conducting the assay. Measurements obtained with the diluted Pomona samples were multiplied by ten and plotted on the graph. Data are presented as mean ± standard deviation of triplicates. *, p < 0.05; **, p < 0.01; ***, p < 0.001 (one-way ANOVA, Tukey multiple comparisons post-test).
Fig 6.
Sphingomyelinase activity in spent growth medium of L. interrogans.
A sphingomyelinase assay was conducted with the spent growth medium of L. interrogans Manilae L495 and Pomona LC82-25 strains grown in EMJH (blue) and in EMJH supplemented with 120 mM NaCl (purple). The dashed line indicates the background enzymatic activity of EMJH with 120 mM NaCl alone. Data are presented as mean ± standard deviation of three biological replicates; ***, p < 0.001 (one-way ANOVA, Tukey multiple comparisons post-test).
Fig 7.
Location of tranposons in an L. interrogans sph2 mutant and in sph2 complemented isolates.
A. Genetic organization of L. interrogans sph1-sph2 locus. The location of the transposon insertion in the sph2 mutant is designated with "Tn." The ends of the sequence cloned for complementation studies are marked with dashed lines. The segment of sph2 encoding the enzymatic domain is shaded gray, and the catalytic residues demonstrated experimentally to be necessary for Bacillus subtilis sphingomyelinase activity are abbreviated by their single-letter amino acid codes. B. Genetic structure of the complementing transposon. The sph2 sequences were cloned between the KpnI and XhoI restriction sites in the transposon. The aadA gene encodes resistance to spectinomycin. C. Insertion sites of the complementing transposon. The location of the complementing transposon in four transconjugants (TC) is indicated by the vertical line. The arrow above the vertical line depicts the orientation of the sph2 gene in the transposon. The dashed open arrow indicates a pseudogene. HP, hypothetical protein.
Fig 8.
Immunoblot analysis of the L. interrogans sph2 mutant and its complemented isolates.
A. Immunoblot analysis of the sph2 mutant. The wild-type (WT) and mutant (sph2-) strains were incubated in EMJH (-) or EMJH with 120 mM sodium chloride (Na) for six hours, and cell lysates were examined by immunoblot analysis with anti-Sph2 and anti-LipL41 antisera. The anti-Sph2 antiserum cross-reacts with SphH. B. Screen for complemented isolates producing Sph2. The sph2 gene was introduced into the sph2 mutant by conjugation. 15 transconjugants were examined for production of Sph2 following incubation in EMJH with 120 mM sodium chloride for six hours. The immunoblot was probed with anti-Sph2 antiserum. C. Regulation of Sph2 production in three transconjugants. The transconjugants TC4, TC10, and TC14 were examined for Sph2 production following incubation in EMJH (odd-numbered lanes) and EMJH with 120 mM sodium chloride (even-number lanes). The immunoblot was probed with anti-Sph2 and anti-LipL41 antisera.
Fig 9.
Hemolytic and sphingomyelinase activities secreted from L. interrogans sph2 mutant and complemented isolates.
Culture supernatant fluid from wild-type strain Lai L391, sph2 mutant, and sph2 mutant complemented with wild-type sph2 (TC4, TC8, and TC14) incubated in EMJH and EMJH with 120 mM sodium chloride for 24 hours was assayed for hemolytic activity (A) and sphingomyelinase activity (B). The dashed line indicates the background activity in culture medium (EMJH plus 120 mM NaCl) alone. Mean and standard deviation from three biological replicates are shown; *, p < 0.05; ***, p < 0.001 (one-way ANOVA, Tukey multiple comparisons post-test).