The autolytic enzyme LytA of Streptococcus pneumoniae is not responsible for releasing pneumolysin

Abstract

It was previously proposed that autolysin's primary role in the virulence of pneumococci was to release pneumolysin to an extracellular location. This interpretation came into question when pneumolysin was observed to be released in significant amounts from some pneumococci during log-phase growth, because autolysis was not believed to occur at this time. We have reexamined this phenomenon in detail for one such strain, WU2. This study found that the extracellular release of pneumolysin from WU2 was not dependent on autolysin action. A mutant lacking autolysin showed the same pattern of pneumolysin release as the wild-type strain. Addition of mitomycin C to a growing WU2 culture did not induce lysis, indicating the absence of resident bacteriophages that could potentially harbor lytA-like genes. Furthermore, release of pneumolysin was unaltered by growth in 2% choline, a condition which is reported to inactivate autolysin, as well as most known pneumococcal phage lysins. Profiles of total proteins in the cytoplasm and in the supernatant media supported the hypothesis that release of pneumolysin is independent of pneumococcal lysis. Finally, under some infection conditions, mutations in pneumolysin and autolysin had different effects on virulence.

FIG. 1

Southern blot analysis of transformants. Chromosomal DNAs were extracted from the various pneumococcal strains and digested with HindIII (a) or ClaI (b). The samples were electrophoresed on a 1% agarose gel and subjected to Southern blot analyses, using a digoxigenin-labeled PCR-generated probe specific for the autolysin gene (a) or the pneumolysin gene (b). (a) Lane 1, D39 (wild type); lane 2, WU2 (wild type); lane 3, AL-2 (D39 lytA mutant); lane 4, SM321.3 (WU2 lytA mutant); lane 5, SM121.10 (WU2 lytA mutant); lane 6, SM122.2 (WU2 lytA mutant). (b) Lane 1, UB2271 (WU2 ply mutant); lane 2, UB1122 (WU2 ply mutant); lane 3, UB1129 (WU2 ply mutant); lane 4, PLN-A (D39 ply mutant); lane 5, WU2 (wild type).

FIG. 2

Pneumolysin expression and release by wild-type and autolysin-deficient pneumococci. WU2 and SM121.10 (WU2 lytA mutant) were grown at 37°C in THY. Samples were withdrawn at different times, and the OD were determined (right y axis). The pneumolysin titers for cell pellets and supernatants were determined (left y axis). The pneumolysin titers for the first two points were below the detection limits of the assay (titer = 3) and were not plotted.

FIG. 3

Western blot analyses of pneumolysin expression during growth. Samples were withdrawn at different times during growth. Pellets were lysed to release the cytoplasmic proteins. Supernatant proteins were concentrated by precipitation with 10% TCA. Pneumolysin was detected using an antipneumolysin rabbit polyclonal antibody.

FIG. 4

Effect of mitomycin C on growth of WU2. WU2 was grown in THY at 37°C until the OD₆₀₀ was approximately 0.3. Then 0.1 μg of mitomycin C or penicillin or nothing was added. The OD of the cultures were monitored over the next 5 h.

FIG. 5

Pneumolysin expression and release by WU2 in the presence and absence of choline. WU2 was grown in THY (a) or THY plus 2% choline chloride (b). Samples were withdrawn at different times, and the OD were determined (right y axis). Pneumolysin titers of the pellets and the supernatants were estimated (left y axis). The pneumolysin titers for the first two points were below the detection limits of the assay (titer = 3) and were not plotted.

FIG. 6

Analyses of total proteins in the cytoplasm and supernatant during growth. WU2 was grown in THY at 37°C. Samples were withdrawn at various times. The cells were pelleted and lysed. The proteins present in the lysed pellets (cytoplasmic) and the culture supernatant (secreted) were precipitated with 10% TCA. The pellet (a) and supernatant (b) proteins were separated on a 10% polyacrylamide gel and stained with Coomassie brilliant blue R-250. THY alone was run as a control for the supernatant samples (b).

FIG. 7

Effect of inactivation of lytA on the virulence of WU2. CBA/N mice (a and c) or BALB/cByJ mice (b and d) were challenged i.v. (a and b) or i.p. (c and d). The hours to death were determined in each case. Control mice were given Ringer's solution. Test mice were given 300 CFU (a), 2.5 × 10⁷ CFU (b), 200 CFU (c), 3 × 10⁶ CFU (d).