Enhancement of streptolysin O activity and intrinsic cytotoxic effects of the group A streptococcal toxin, NAD-glycohydrolase.
Streptolysin O (SLO) is a cholesterol-dependent cytolysin produced by the important human pathogen, group A Streptococcus (Streptococcus pyogenes or GAS). In addition to its cytolytic activity, SLO mediates the translocation of GAS NAD-glycohydrolase (NADase) into human epithelial cells in vitro. Production of both NADase and SLO is associated with augmented host cell injury beyond that produced by SLO alone, but the mechanism of enhanced cytotoxicity is not known. We have now shown that expression of NADase together with SLO dramatically enhanced the lytic activity of GAS culture supernatants for erythrocytes but had no effect on SLO-mediated poration of synthetic cholesterol-rich liposomes. This result revealed a previously unknown contribution of NADase to the cytolytic activity associated with GAS production of SLO. Purified recombinant SLO bound NADase in vitro, supporting a specific, physical interaction of the two proteins. Exposure of human keratinocytes to wild-type GAS, but not to a NADase-deficient mutant strain, resulted in profound depletion of cellular NAD+ and ATP. Furthermore, expression of recombinant GAS NADase in yeast, in the absence of SLO, induced growth arrest, depletion of NAD+ and ATP, and cell death. These findings have provided evidence that the augmentation of SLO-mediated cytotoxicity by NADase is a consequence of depletion of host cell energy stores through the enzymatic action of NADase. Together, the results have provided mechanistic insight into the cytotoxic effects of a unique bipartite bacterial toxin.
Michos
Athanasios
A
Division of Infectious Diseases, Children's Hospital Boston, Massachusetts 02215, USA.
Gryllos
Ioannis
I
Håkansson
Anders
A
Srivastava
Amit
A
Kokkotou
Efi
E
Wessels
Michael R
MR
D000255:Adenosine Triphosphate / Q000737:chemistry; D001426:Bacterial Proteins / Q000378:metabolism; D001427:Bacterial Toxins / Q000737:chemistry; D015153:Blotting, Western; D049109:Cell Proliferation; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D017931:DNA Primers / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000378:metabolism; D005098:Exotoxins; D006461:Hemolysis; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry; D015603:Keratinocytes / Q000378:metabolism; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism; D008856:Microscopy, Fluorescence; D009243:NAD / Q000737:chemistry; D009244:NAD+ Nucleosidase / Q000378:metabolism*; D017830:Octoxynol / Q000494:pharmacology; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011994:Recombinant Proteins / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D013290:Streptococcal Infections / Q000378:metabolism; D013297:Streptococcus pyogenes / Q000737:chemistry*; D013301:Streptolysins / Q000378:metabolism*; D013997:Time Factors; D014774:Virulence
D001426:Bacterial Proteins; D001427:Bacterial Toxins; D017931:DNA Primers; D005098:Exotoxins; D007074:Immunoglobulin G; D008081:Liposomes; D011994:Recombinant Proteins; D013301:Streptolysins; C025964:streptolysin O; D009243:NAD; D000255:Adenosine Triphosphate; D017830:Octoxynol; D009244:NAD+ Nucleosidase