Molecular and biological characterization of Streptococcal SpyA-mediated ADP-ribosylation of intermediate filament protein vimentin.

The Gram-positive bacterial pathogen Streptococcus pyogenes produces a C3 family ADP-ribosyltransferase designated SpyA (S. pyogenes ADP-ribosyltransferase). Our laboratory has identified a number of eukaryotic protein targets for SpyA, prominent among which are the cytoskeletal proteins actin and vimentin. Because vimentin is an unusual target for modification by bacterial ADP-ribosyltransferases, we quantitatively compared the activity of SpyA on vimentin and actin. Vimentin was the preferred substrate for SpyA (k(cat), 58.5 ± 3.4 min(-1)) relative to actin (k(cat), 10.1 ± 0.6 min(-1)), and vimentin was modified at a rate 9.48 ± 1.95-fold greater than actin. We employed tandem mass spectrometry analysis to identify sites of ADP-ribosylation on vimentin. The primary sites of modification were Arg-44 and -49 in the head domain, with several additional secondary sites identified. Because the primary sites are located in a domain of vimentin known to be important for the regulation of polymerization by phosphorylation, we investigated the effects of SpyA activity on vimentin polymerization, utilizing an in vitro NaCl-induced filamentation assay. SpyA inhibited vimentin filamentation, whereas a catalytic site mutant of SpyA had no effect. Additionally, we demonstrated that expression of SpyA in HeLa cells resulted in collapse of the vimentin cytoskeleton, whereas expression in RAW 264.7 cells impeded vimentin reorganization upon stimulation of this macrophage-like cell line with LPS. We conclude that SpyA modification of vimentin occurs in an important regulatory region of the head domain and has significant functional effects on vimentin assembly.

SpyA is a membrane-bound ADP-ribosyltransferase of Streptococcus pyogenes which modifies a streptococcal peptide, SpyB.

All sequenced genomes of Streptococcus pyogenes (Group A Streptococcus, GAS) encode a protein, SpyA, with homology to C3-like ADP-ribosyltransferase toxins. SpyA is a novel virulence factor which plays a role in pathogenesis in a mouse model of soft-tissue infection. In this study we demonstrate that SpyA is a surface-exposed membrane protein which is anchored to the streptococcal membrane by an N-terminal transmembrane sequence. We identified a small gene upstream of spyA, designated spyB, which encodes a peptide of 35 amino acids, and is co-transcribed with spyA. Expression of spyBA is strongly influenced by translational coupling: mutational inactivation of spyB translation completely abolishes translation of spyA. spyB expression increases with increasing cell density and reaches its maximum at late exponential growth phase. The SpyB N-terminus is predicted to fold into an amphipathic α-helix, a structural motif that targets a protein to the cytoplasmic membrane. Consistent with the prediction, we found that a SpyB fusion with peptide affinity tags is located in the streptococcal membrane. An ADP-ribosylation assay with recombinant SpyA demonstrated that SpyA modifies SpyB. Thus, our study suggests that ADP-ribosylation of SpyB may be an important function of SpyA.

Behavioral alterations in adolescent and adult rats caused by a brief subtoxic exposure to chlorpyrifos during neurulation.

The widely used organophosphate insecticide, chlorpyrifos (CPF), elicits neurobehavioral abnormalities after apparently subtoxic neonatal exposures. In the current study, we administered 1 or 5 mg/kg/day of CPF to pregnant rats on gestational days 9-12, the embryonic phase spanning formation and closure of the neural tube. Although there were no effects on growth or viability, offspring showed behavioral abnormalities when tested in adolescence and adulthood. In the CPF-exposed groups, locomotor hyperactivity was noted in early T-maze trials, and in the elevated plus-maze; alterations in the rate of habituation were also identified. Learning and memory were adversely affected, as assessed using the 16-arm radial maze. Although all CPF-exposed animals eventually learned the task, reference and working memory were impaired in the early training sessions. After training, rats in the CPF group did not show the characteristic amnestic effect of scopolamine, a muscarinic acetylcholine antagonist, suggesting that, unlike the situation in the control group, muscarinic pathways were not used to solve the maze. These results indicate that apparently subtoxic CPF exposure during neurulation adversely affects brain development, leading to behavioral anomalies that selectively include impairment of cholinergic circuits used in learning and memory. The resemblance of these findings to those of late gestational or neonatal CPF exposure indicates a prolonged window of vulnerability of brain development to CPF.