Yoriko Taketo

Mutational and comparative analysis of streptolysin O, an oxygen-labile streptococcal hemolysin

The structural gene of streptolysin O was cloned from Streptococcus pyogenes strain Sa and S. equisimilis H46A, and the nucleotide sequences were compared with those of strain Richards. To obtain the minimal active fragment of the toxin and to elucidate structure-function relationships in hemolytic function, streptolysin O mutants deleted in N- and C-terminal regions were constructed. Internal amino acid residues were also replaced by introduction of point mutations. Analyses of these mutants showed that considerable activity was retained even after deletion of the N-terminal 107 residues, but genetic removal of the ultimate C-terminal residue resulted in a marked decrease in hemolytic function. By removal in succession, hemolytic activity declined exponentially, and only 0.002% of the activity remained after deletion of the C-terminal four residues. Nucleotide replacement experiments indicated pivotal roles of I202, V217, D324-L325, V339, and H469 residues in hemolysis.

Effects of Trypan Blue and Related Compounds on Production and Activity of Streptolysin S

Abstract Most dyes related to trypan blue inhibited hemolytic activity of oligonucleotide-streptolysin S (SLS) complex, an exotoxin produced by Streptococcus pyogenes. Order of the inhibition was: trypan blue > benzo blue 2B > Congo red > Evans blue > benzo purpurine 4B > thiazine red > trypan red. When resting streptococcal cells were incubated with these dyes, significant amount of the hemolysin was produced. The carrier (or inducing) activity for SLS was further manifested in growing cell system and the potency of the compounds was as follows: Congo red > benzo blue 2B > trypan blue > Evans blue > Benzo purpurine 4B > thiazine red > trypan red. In this system, Congo red was more effective than oligonucleotide fraction rich in guanyl residue. Chromotrope 2B, H acid and o-tolidine were ineffective, as the carrier as well as the inhibitor. Based on these results, structure-function relationship among SLS, the carrier and the inhibitor was discussed.

Reactivation of streptolysin S by oligonucleotide.

Abstract Oligonucleotide-streptolysin S complex inactivated by alkali treatment remains nonhemolytic, even after acidification and mixing with intact carrier oligonucleotide rich in guanyl residue. Upon dehydration, how ever, the inactive streptolysin S-oligonucleotide mixture turned to be hemolytic, and this reactivation of the hem olysin was promoted by treatment with guanidine hydrochloride. After alkaline hydrolysis, streptolysin S was freed from nucleotide moiety, by gel filtration through a Sephadex G -50 column. From this nonhemolytic apotoxin as well, active streptolysin S complex was reconstructed upon dehydration with the carrier.

Activation of streptolysin S in vitro by oligonucleotides.

When washed streptococci were incubated in a phosphate buffer containing MgSO4 and maltose (Bernheimer’s basal medium) and centrifuged, no hemolytic activity was detected in the supernatant. Although incubation of the spent medium with carrier oligonucleotide did not yield active hemolysin, the mixture turned to be significantly hemolytic, upon ethanol precipitation and dehydration. Oxygen stability, sensitivity to trypan blue, absence in the spent medium from strain C203U, as well as chromatographic properties demonstrated that the hemolytic activity was due to streptolysin S-oligonucleotide complex. The latent streptolysin S was detected in the streptococcal culture supernatant as well. These results indicate that, even in the absence of exogenous carrier, streptolysin S is produced extracellularly by hemolytic streptococci but suffers rapid denaturation. and that the carrier oligonucleotide serves as an effector for the toxin peptide to assume active conformation, through noncovalent interaction. Effects of several protein denaturants were investigated on the toxin activation in vitro.

Enhanced production of cell-bound and extracellular streptolysin S by hemolytic streptococci pretreated with proteases.

The amount of streptolysin S produced by resting streptococci was considerably increased after incubation of the washed bacteria with trypsin or pronase. Production of both cell-bound and free forms of the toxin was enhanced by the protease treatment. By addition of trypsin, streptolysin S yield was considerably increased in growing culture as well. Treatment with lysozyme was ineffective, and the toxin production was only slightly promoted by preincubation with hyaluronidase or chymotrypsin. In contrast, pretreatment witn chymotrypsin caused increased production of an extracellular nuclease, whereas the yield of this enzyme was reduced after incubation of the cocci with pronase. Evidence was obtained indicating de novo synthesis of the exotoxin in the protease-treated bacteria.

Effects of norfloxacin and rifampicin on growth and streptolysin S production in hemolytic streptococci.

Abstract Norfloxacin, a nalidixic acid analogue, inhibited streptolysin S (SLS) production when added to young streptococcal culture. DNA synthesis was mainly affected, but increment of cell mass. RNA and protein was also significantly reduced in streptococci treated with norfloxacin. In stationary phase cells and in the washed resting bacteria, the toxin production was resistant to the drug. Pretreatment with norfloxacin did not abolish the cellular capacity to produce SLS. Although extracellular SLS was detectable at log phase of streptococcal growth, enhanced production of the toxin occurred upon cessation of coccal multiplication. In contrast to norfloxacin, lower concentration of rifampicin inhibited SLS production, even added at late log or early stationary phase. Roles of growth phase, medium and carrier in induction of SLS production were analyzed as well.

Effects of membrane-acting drugs and aerobiosis on production of streptolysin S and nuclease in hemolytic streptococci

Abstract Yield of streptolysin S (SLS) in streptococcal culture was considerably reduced by procaine, dibucaine, atropine or chlorpromazine at concentrations which scarecely affected production of an extracellular nuclease as well as the bacterial growth. Cerulenin was also inhibitory to SLS formation, but its effect was more pronounced on the nuclease production. By aerobiosis, amount of SLS produced into culture supernatant was increased significantly, whereas yield of the nuclease was rather unaffected.

Effect of streptococcal extracellular nuclease on the carrier activity of RNA for streptolysin S.

Upon digestion with a streptococcal extracellular nuclease, yeast RNA yielded acid-insoluble core having increased carrier activity for streptolysin S. The carrier activity was found in minor fractions of the core which were eluted from a DEAE-cellulose column at higher salt concentrations. Upon gel filtration through a Sephadex G-75 column, the effective component (Fr. I) was eluted earlier than bulk oligonucleotides (Fr. II). Nucleotide composition (in mol %) of Fr. I was AMP: 21.8; GMP: 55.1; CMP: 8.2; UMP: 14.9, whereas that of Fr. II was AMP: 38.0; GMP: 33.1; CMP: 8.0; UMP: 20.9. Chromatographic patterns of SLS complex induced by Fr. I were similar to those of the toxin formed in the presence of active fraction prepared from RNase I core. Hemolytic activity of the latter complex was, like the former, unaffected by streptococcal nuclease treatment. The carrier activity of DNA digested with the nuclease was also investigated.

Decreased production of RNA-streptolysin S in streptococci devoid of extracellular RNase activity

Abstract In cultures of certain streptococcal strains, RNA added as a carrier for streptolysin S (SLS) was hardly degraded, owing to deficiency of extracellular RNase activity. Production of RNA-SLS into culture supernatant was markedly reduced in the RNase-deficient streptococci. Even in these RNase-less strains, guanylic-acid rich oligonucleotides, polyG or trypan blue effectively induced SLS production, as in RNase-positive cells. These results demonstrate involvement of the streptococcal nuclease in manifestation of SLS-inducing effect of exogenous RNA. Additional data indicated that cellular growth was promoted by supplementation of RNA, in the nuclease-producing streptococci.

Effects of polynucleotides on production and activity of streptolysin S.

Activity of various natural and synthetic polynucleotides as a carrier for streptolysin S was tested in a resting cell system. As the carrier, intact molecules of MS2 RNA, E. coli tRNA or rat liver RNA were almost inactive, whereas RNase I core of these RNAs, especially the core-fractions eluted from DEAE-cellulose column at higher NaCl concentrations, effectively induced production of the extracellular hemolysin. The carrier activity of yeast RNA was significantly enhanced by simultaneous addition of RNase I, to the streptococcal suspension. Preincubation of yeast RNA with growing streptococci or a protein fraction from the bacterial culture supernatant increased its carrier activity. Evidences were obtained suggesting involvement of streptococcal nuclease in the enhancement of the carrier effect. Production of the streptococcal hemolysin was markedly promoted by polyguanylic acid but neither by polyadenylic acid, polycytidylic acid nor by polyuridylic acid. Like trypan blue, polyguanylic acid exerted potent inhibitory effect on hemolytic activity of streptolysin S complex. Similar but less marked effect was observed with certain RNase I core fractions of rat liver RNA. Some data concerning effect of polynucleotides on stability of the hemolysin were also presented.