Identification and characterization of SpyA, a mono-ADP-ribosyltransferase from Streptococcus pyogenes.

摘要

Streptococcus pyogenes is the etiological agent of both respiratory and skin infections and releases numerous exotoxins to establish an infection. Analysis of the S. pyogenes M1 (SF370) genome revealed the presence of a putative exotoxin termed SpyA for S&barbelow;treptococcus pyogenes A&barbelow;DP-ribosylating toxin. SpyA, MW 24.9 kDa, shares amino acid sequence identity with the ADP-ribosyltransferases (ADPRTs) EDIN from Staphylococcus aureus and C3 from Clostridium botulinum. The SpyA gene is present in all sequenced S. pyogenes genomes as well as all clinical isolates tested. SpyA-encoding mRNA is expressed at mid-log phase in the S. pyogenes strains SF370 and JRS4 grown in liquid culture and is upregulated in S. pyogenes passaged through a murine subcutaneous chamber. In addition others have demonstrated that spyA transcript is in the top 6% of all transcripts by relative abundance when S. pyogenes is grown in saliva. Recombinant SpyA is a NAD-glycohydrolase, which is a trait shared by all ADPRTs, and like other biglutamate ADPRTs, SpyA is able to ADP-ribosylate poly-L-arginine. SpyA modifies numerous proteins in cell lysates as well as performs auto-ADP-ribosylation. Its enzymatic activity was determined to be dependent on the catalytic glutamate 187, serine 147, threonine 148 and arginine 100 and as predicted glutamate 185 is indispensable for the modification of the substrate. Two-dimensional gel analysis and MALDI-TOF MS analysis of modified proteins indicated that the cytoskeletal proteins vimentin, tropomyosin, alpha-tubulin, and actin are targets. SpyA modified all actin isoforms in vitro and intracellular expression of SpyA resulted in the disruption of actin filaments. SpyA fused to the first 225 amino acids of lethal factor (LFn) from Bacillus anthracis was constructed as a mechanism to deliver SpyA into eukaryotic cells. Intoxication of HeLa cells with LFn-SpyA resulted in the collapse of vimentin filaments around the nucleus. In addition SpyA modifies vimentin at multiple arginine sites. One of these sites of modification was discovered by V8 protease digestion to lie within the amino terminal region known to be essential for polymerization. MALDI-MS analysis revealed that vimentin is also modified on the col1B region. It was determined that SpyA gains entrance into the eukaryotic cell via Streptolysin O mediated mechanism where it associates with the eukaryotic membrane. Future experiments are to identify other cellular targets of SpyA and determine the role of the toxin in S. pyogenes pathogenesis.