Mutational and Comparative Analysis of Streptolysin O, an Oxygen-labile Streptococcal Hemolysin

Yamamoto, Ikuo; Kimoto, Hisashi; Taketo, Yoriko; Taketo, Akira

doi:10.1271/bbb.65.2682

Cited by 10 publications

(12 citation statements)

References 18 publications

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“…Although overproduction of active SLO was attained in E. coli (22), clones with the NADase gene insert were not obtained at all (16). Taking possible host toxicity of this enzyme into consideration, the NADase gene, nga, was subcloned (Fig.…”

Section: Expression Of Streptococcal Nadase In Ementioning

confidence: 99%

Genetic and Biochemical Properties of Streptococcal NAD-glycohydrolase Inhibitor

Kimoto

Fujii

Hirano³

et al. 2006

Journal of Biological Chemistry

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The gene encoding streptolysin O (slo), a cytolysin of hemolytic streptococci, is transcribed polycistronically from the promoter of the preceding NAD-glycohydrolase (NADase) gene (nga). Between nga and slo, a putative open reading frame (orf1) is located whose function has been totally unknown. Present investigation demonstrated that the orf1 encodes a protein designated as streptococcal NADase inhibitor (SNI). From its nucleotide sequence, SNI was inferred to comprise 161 amino acid residues and the deduced molecular weight was 18,800. This protein was detectable only within cells. Coexpression of SNI was essential for production of streptococcal NADase, and NADase precursor existed as an inactive complex with SNI, in recombinant Escherichia coli. Monomeric NADase and SNI rapidly formed in vitro a stable heterodimer complex in the ratio 1:1, resulting in complete suppression of the hydrolase activity. Unlike other bacterial NADase inhibitors, SNI was thermostable. This protein, coexpressed and complexed with NADase, may protect the producer cocci from exhaustion of NAD.Besides the Lancefield group A (Streptococcus pyogenes, GAS), 3 human isolates of group C (GCS, including Streptococcus dysgalactiae subsp. equisimilis) and G (GGS) streptococci have been implicated as causative agents in outbreaks of purulent pharyngitis, wound infections, and streptococcal toxic shock-like syndrome (1-3). Streptolysin O (SLO), an oxygen-labile cholesterol-dependent cytolysin, is an extracellular protein produced by most strains of GAS and many strains of GCS and GGS (4). SLO belongs to a single, highly homologous family of cytolysins found in species of five genera: Streptococcus, Bacillus, Clostridium, Listeria, and Arcanobacterium (5-10). Upon binding to cholesterol-containing membrane as monomers, these proteins oligomerize to form large pores, comprising ϳ50 monomer subunits with internal diameter of up to 30 nm (9, 10). These large pores permit permeation of not only ions and small metabolites but also macromolecules. Madden et al. (11) proposed, for the first time from Gram-positive bacteria, a cytolysin-mediated translocation system that is a functional equivalent to the type III secretion system in Gram-negative bacteria (12)(13)(14)(15). Their data support a model in which the effector NAD-glycohydrolase (NADase), encoded by nga (spn) located upstream of the slo gene, is transported through the SLO pore into the host cell (11). Thus SLO is of considerable biochemical and clinical interest, and elucidation of expression mechanism of slo gene is important for understanding streptococcal pathogenesis as well.Recently, we demonstrated that slo is a member of an operon covering the upper-stream nusG and nga genes (nusG-nga-orf1-slo), from which transcription of slo proceeds polycistronically, and major transcript is produced by readthrough from nga promoter (16). The arrangement commonly conserved in streptococci was limited to the region from nusG to slo (17-21). These results suggest intimate relationship among NADase, ...

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Section: Expression Of Streptococcal Nadase In Ementioning

confidence: 99%

Genetic and Biochemical Properties of Streptococcal NAD-glycohydrolase Inhibitor

Kimoto

Fujii

Hirano³

et al. 2006

Journal of Biological Chemistry

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“…For construction of pSLO::EM, plasmid clone 3, 24) containing streptolysin O (SLO) gene was digested with EcoRV. Plasmid pE194 was partially digested with AlwI, and a resultant 2.1 kb Em r gene was blunt-ended with T4 DNA polymerase and ligated with the clone 3 predigested with EcoRV, to yield the plasmid P1.…”

Section: Polymerase Chain Reaction (Pcr)mentioning

confidence: 99%

“…Targeted mutagenesis of slo 24) and sagB 25) The established electroporation system was used for the insertional inactivation of chromosomal genes slo and sagB, using composite plasmids pSLO::Em and pSAGB::Sp. These pUC19-derived plasmids contain a thermosensitive replication origin from pIC333 and a drug-resistance gene (Em-or Spresistant) in each target locus (Fig.…”

Section: Electrotransformation Of Various Streptococcal Strains With mentioning

confidence: 99%

Efficient Electrotransformation System and Gene Targeting in Pyogenic Streptococci

Kimoto

Taketo

2003

Bioscience, Biotechnology, and Biochemistry

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“…Only a single amino acid deletion in this region disabled the cholesterol-binding property of streptolysin O (38). In contrast, the NH 2 -terminal region of streptolysin O is more tolerant and deletions of >100 amino acids retained streptolysin O function (38). These structural properties were repeated by streptolysin O protein synthesized within eukaryotic cells (Fig.…”

Section: Discussionmentioning

confidence: 97%

“…The COOH-terminal region of streptolysin O is the cholesterol-binding region, and the structure of this region has been suggested to be very compact. Only a single amino acid deletion in this region disabled the cholesterol-binding property of streptolysin O (38). In contrast, the NH 2 -terminal region of streptolysin O is more tolerant and deletions of >100 amino acids retained streptolysin O function (38).…”

Section: Discussionmentioning

confidence: 97%

Suicide cancer gene therapy using pore-forming toxin, streptolysin O

Yang

Park

Yoon

et al. 2006

Molecular Cancer Therapeutics

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We cloned the streptolysin O gene from the Streptococcus pyogenes genome and tested the possibility of using it as an anticancer reagent. Transient transfection of the streptolysin O gene efficiently killed 293T cells after 12 hours of transfection as determined by lactate dehydrogenase release and propidium iodide uptake. No caspase activity was observed and necrosis was prominent during streptolysin O-induced cell death. Biochemical analysis of streptolysin O protein revealed that the deletion of only 5 amino acids from the COOH-terminal region of streptolysin O, which is essential for cholesterol binding activity, abolished its cell-killing activity, whereas the NH 2 -terminal region was more resilient, i.e., up to 115 amino acids could be deleted without changing its cell-killing activity. We generated a streptolysin O-expressing adenovirus and injected it into human cervical cancer cell -derived tumors grown in a nude mouse model. Twenty-one days postinjection, the average size of tumors in the streptolysin O adenovirus -injected group was 29.3% of that of the control PBS-treated group. Our results show that the genes of pore-forming toxins, like streptolysin O protein, have the potential to establish a novel class of suicide gene therapeutic reagents. [Mol Cancer Ther 2006;5(6):1610 -9]

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Mutational and Comparative Analysis of Streptolysin O, an Oxygen-labile Streptococcal Hemolysin

Cited by 10 publications

References 18 publications

Genetic and Biochemical Properties of Streptococcal NAD-glycohydrolase Inhibitor

Genetic and Biochemical Properties of Streptococcal NAD-glycohydrolase Inhibitor

Efficient Electrotransformation System and Gene Targeting in Pyogenic Streptococci

Suicide cancer gene therapy using pore-forming toxin, streptolysin O

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