Generation and characterization of a defined mutant of Streptococcus suis lacking suilysin

Abstract

A defined allelic-replacement mutant of the sly gene, encoding a thiol-activated cytolysin, from a European isolate of Streptococcus suis serotype 2 was generated and characterized. Unlike the parental strain, it is nonhemolytic, noncytotoxic for cultured macrophage-like cells, avirulent in a mouse infection model, yet only slightly attenuated in a porcine model of systemic infection.

FIG. 1

Characterization of P1/7 and S7c culture supernatants. (a) Overnight culture supernatants from wild type P1/7 (wt) and S7c (S7c) were concentrated 100-fold by ammonium sulfate precipitation, and 10-μl samples, indicated by circles, were overlaid onto a 7% (vol/vol) horse blood agar plate, followed by incubation at 37°C for 60 min. Samples in track B were treated with β-mercaptoethanol to a final concentration of 1 mM. PBS, phosphate-buffered saline. (b) Western blot of culture supernatants from P1/7 and S7c using antisuilysin monoclonal antibody. Proteins secreted from overnight culture supernatants from P1/7 (track A) and S7c (track B) were concentrated 25 times, separated on a polyacrylamide gel, and Western blotted onto nitrocellulose, followed by development with a monoclonal antibody specific for suilysin. The band corresponding to suilysin in track A is completely absent from track B. Molecular weights are in thousands.

FIG. 2

Comparison of cytotoxicities of P1/7 and S7c for J774.2 cells. Monolayers of J774. 2 cells (2 × 10⁵ cells) were infected with P1/7 (P) or S7c (S). Relative levels of LDH released at 3 and 5 h were measured and plotted as percentages of the maximum lysis achieved by chemical treatment. Experiments were performed in triplicate for each datum point. Where no bars are present, the lysis was effectively zero. Spontaneous lysis (C) occurred without the addition of bacteria. It is clear that wild-type bacteria cause substantial lysis of the cell monolayer, whereas the mutant lacking suilysin is unable to lyse the cells.