Proc. Natl. Acad. Sci. USA Vol. 90, pp. 8154-8158, September 1993 Microbiology Glyceraldehyde-3-phosphate dehydrogenase on the surface of group A streptococci is also an ADP-ribosylating enzyme VUAYKUMAR PANCHOLI* AND VINCENT A. FISCHETTI Laboratory of Bacterial Pathogenesis and Immunology, The Rockefeiler University, 1230 York Avenue, New York, NY 10021 Communicated by Emil C. Gotschlich, May 19, 1993 ABSTRACT We recently identified an enzymatically ac- of this modification in prokaryotes is limited only to a few tive glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12; bacterial toxins such as diphtheria toxin (8), pseudomonas GAPDH) as a major protein on the surface of group A exotoxin A (9), pertussis toxin, and cholera toxin (10) and the streptococci (SDH), which exhibits multiple binding activity to regulation of nitrogenase activity in the photosynthetic bac- various mammalian proteins. We now report that the SDH teria Rhodospirillum rubrum (11). molecule also functions as an ADP-ribosylating enzyme, which, The ability of NO to stimulate ADP-ribosylation of eu- in the presence of NAD, is auto-ADP-ribosylated. In a crude karyotic GAPDH independent of cyclic GMP (3, 12) clearly cel wail extract of group A streptococci, SDH is the only distinguishes it from other known and characterized ADP- protein that is ADP-ribosylated. SDH found in the streptococ- ribosyl transferases that essentially involve activation of cal cytoplasmic fraction could not be ADP-ribosylated in the guanylate cyclase (10). Although the role of NO found in presence ofNAD. Treatment of ADP-ribosylated SDH with the activated macrophages is well established for its cytocidal cytoplasmic fraction removed the ADP-ribose from SDH, and cytostasis activity against fungal, helminthic, protozoal, suggesting the presence of an ADP-ribosyl hydrolase in the and bacterial pathogens (13), the role of NO-mediated ADP- cytoplasmic compartment. The covalent linkage ofADP-ribose ribosylation in bacteria and its implications in the pathogen- to SDH was stable to neutral hydroxylamine, sensitive to esis of the disease, however, have not been studied. HgCl2, and inhibitable by free cysteine, indicating that the Because eukaryotic GAPDHs have been found, in addition modification was at a cysteine residue of SDH. In addition to to their glycolytic activity, to perform various functions (see its auto-ADP-ribosylation activity, purified SDH or strepto- ref. 1) including ADP-ribosylation (3-5), we examined coccal cell wall extracts were able to transfer the ADP-ribose whether SDH also had ADP-ribosylating activity. We here moiety of NAD specifically to free cysteine, resulting in a true report that SDH is an ADP-ribosylating enzyme with auto- thioglycosidic linkage. Treatment ofpurified SDH or the crude ADP-ribosylating activity and the ability to specifically ADP- cell wall extract with sodium nitroprusside, which spontane- ribosylate free L-cysteine. The ADP-ribosylating activities of ously generates nitric oxide, was found to stimulate the ADP- SDH were also found to be significantly enhanced in the ribosylation of SDH in a time-dependent manner. ADP- presence of NO. Since, to our knowledge, such observations ribosylation and nitric oxide treatment inhibited the GAPDH for a surface molecule on a pathogenic organism have not activity of SDH. Since ADP-ribosylation and nitric oxide are been reported previously, our findings may open new ave- involved in signal transduction events, the ADP-ribosylating nues for understanding the pathogenesis of streptococcal activity of SDH may enable communication between host and disease. parasite during infection by group A streptococci. Streptococcal surface dehydrogenase (SDH), a 35.8-kDa MATERIALS AND METHODS protein, has recently been identified as one of the major Materials and Chemicals. SDH was purified from a strep- surface proteins ofgroup A streptococci (1). Structurally and tococcal cell wall extract (14, 15) as described (1). Rabbit functionally it is a member of the glyceraldehyde-3- polyclonal antisera against SDH were raised and affinity phosphate dehydrogenase (EC 1.2.1.12; GAPDH) family of purified as described (1). [a-32P]NAD (30 Ci/mmol; 1 Ci = 37 molecules. SDH is unique in its localization on a bacterial GBq) and [adenine-2,8-3H]NAD (25.9 Ci/mmol) were ob- surface and is found in all except a few streptococcal groups tained from NEN/DuPont. All other biochemicals were and in all group A streptococcal M types tested (1). SDH also obtained unless binds various mammalian proteins such as lysozyme, fibro- from Sigma, otherwise mentioned. nectin, and the cytoskeletal proteins actin and myosin. Re- Subcellular Fractionation of Streptococci. The cell walls cently, a structurally similar molecule has been shown to bind were digested using the amidase enzyme lysin in 30% raffi- plasmin (2). nose at pH 6.1, and the cytoplasm and membranes were Nitric oxide (NO) and NO-generating agents such as so- separated from the resulting protoplasts as described (14, 15). dium nitroprusside (SNP) were reported to stimulate mono- ADP-Ribosylation of SDH. The ADP-ribosylation of puri- ADP-ribosylation of a cytosolic 36-kDa protein found in fied SDH (20 pg) (1) was carried out in a reaction mixture (0.2 various human tissues such as platelets, liver, intestine, ml) containing 100 mM Tris HCl at pH 7.4, 10 mM dithio- heart, lung (3), brain (3, 4), and erythrocytes (5). This 36-kDa threitol, 1 mM NADP, 10 mM thymidine, and 10 uM protein has recently been identified to possess GAPDH [32P]NAD (ADPR buffer) for 1 hr at 37°C followed by the activity (4-6). Mono-ADP-ribosylation is a widely used addition of50 ,ul of 100o (wt/vol) chilled trichloroacetic acid method by which eukaryotic cells modify protein structure (TCA) to stop the reaction as described (5). The resulting and function (7). It is a covalent, posttranslational protein precipitates were washed, dried, and subjected to SDS/ modification in which the ADP-ribose moiety of NAD is PAGE (12% polyacrylamide) as described (14, 15). The gel transferred to an individual substrate (7). Our understanding Abbreviations: NO, nitric oxide; GAPDH, glyceraldehyde-3- phosphate dehydrogenase; SNP, sodium nitroprusside; SDH, strep- The publication costs of this article were defrayed in part by page charge tococcal surface dehydrogenase; TCA, trichloroacetic acid; PVDF, payment. This article must therefore be hereby marked "advertisement" poly(vinylidene difluoride). in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 8154 Downloaded by guest on September 26, 2021 Microbiology: Pancholi and Fischetti Proc. Natl. Acad. Sci. USA 90 (1993) 8155 was dried and autoradiographed at -80°C. To locate specific and autoradiographed. In a similar set of experiments, ADP- ADP-ribosylating proteins, duplicate gels were electrotrans- ribosylation was performed using 200 ,ul of streptococcal cell ferred to nitrocellulose or poly(vinylidene difluoride) (PVDF) wall extract in ADPR buffer containing 10 p.M [32P]NAD membranes, Western blotted using anti-SDH antibodies, and incubated in the presence and absence of 2 mM SNP. autoradiographed. GAPDH Activity of ADP-Ribosylated SDH. GAPDH activ- In Vitro ADP-Ribosylation of SDH. To determine the spec- ity of purified SDH (5 pg) or ADP-ribosylated SDH (5 pg) ificity of the ADP-ribosylation of SDH in streptococci, two was determined in the presence of NAD and glyceraldehyde sets of experiments were performed: (i) the cell wall, cyto- 3-phosphate by measuring the absorbance at 340 nm, showing plasm, and membrane fractions, each adjusted to 1 mg of the conversion of NAD to NADH as described (1). protein per ml in 0.2 ml of ADPR buffer, were mixed with 10 To study the effect of NAD and SNP on the GAPDH ,uM [32P]NAD and (ii) to determine the ADP-ribosylating activity of SDH, (i) SDH (5 ,ug) was preincubated with activity of intact streptococci, an overnight culture of strep- different concentrations ofNAD for 1 hr prior to determining tococci (10 ml) was washed and resuspended in ADPR buffer its GAPDH activity, (ii) different concentrations ofSNP were (0.2 ml) containing 10 AM [32P]NAD as described above. added to SDH (5 ,ug) immediately prior to the addition of Bacterial cells were then thoroughly washed with 50 mM glyceraldehyde 3-phosphate, and (iii) SDH (5 p.g) was incu- sodium phosphate buffer at pH 6.1. The cells were digested bated with 2 mM SNP for different time periods prior to with lysin and fractionated as described above to locate the determining its GAPDH activity. GAPDH activity in all three ADP-ribosylated proteins in the cell wall, cytoplasm, and sets was carried out using a constant amount of NAD and membrane fractions. glyceraldehyde 3-phosphate as described above. Specificity and Inhibition of the ADP-Ribose-Protein Bond. The effect ofdifferent concentrations ofHgCl2 (0.063-2 mM) and neutral hydroxylamine (pH 7.4, 0.63-10 mM) on the RESULTS ADP-ribosylation reaction was determined by incorporating ADP-Ribosylation of SDH. To examine whether SDH has these reagents into the ADPR buffer prior to the addition of the same ADP-ribosylating activity as recently reported for [32P]NAD to carry out the reactions. After 30 min, the eukaryotic GAPDHs (3-5), purified SDH was incubated with samples were precipitated with cold TCA, dried, and sub- [32P]NAD in ADPR buffer. After SDS/PAGE and autorad- jected to electrophoresis and autoradiography. iography, SDH was found to have incorporated the radioac- Competitive Inhibition of ADP-Ribosylation of SDH with tivity from the labeled NAD (Fig. 1, lane 9). When the Free Amino Acids and ADP-Ribosyl Transferase Activity of molecules in the subcellular fractions representing cell wall- SDH. To verify the amino acid-specific ADP-ribosylation of associated cytoplasm and membrane components were sep- SDH, the ADP-ribosylation reaction was carried out in the arated by SDS/PAGE and Western blotted with affinity- presence of increasing concentrations of L-cysteine, L-histi- purified anti-SDH antibodies, SDH could be identified in all dine, L-arginine, and L-glutamic acid.