Enhancement of Streptolysin O Activity and Intrinsic Cytotoxic Effects of the Group A Streptococcal Toxin, NAD-Glycohydrolase

Abstract

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 cholesterolrich 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 Group A Streptococcus (Streptococcus pyogenes or GAS), 2 the causative agent of streptococcal pharyngitis, skin and soft tissue infections, and severe invasive infections, produces more than 40 secreted proteins, many of which have been implicated in disease pathogenesis (1-3). Among the secreted products of GAS are streptolysin O (SLO) and NAD-glycohydrolase (NADase). SLO is a member of the pore-forming cytolysin family known as cholesterol-dependent cytolysins due to their ability to bind cholesterol in eukaryotic cell membranes (4, 5). Individual toxin molecules oligomerize and insert into the cell membrane to form transmembrane pores up to 30 nm in diameter. Sufficient doses of SLO result in cell lysis (4, 6, 7). Purified SLO is a potent toxin that is lethal within minutes of intravenous injection in animal models, primarily because of cardiotoxicity (8, 9). GASNAD-glycohydrolases are enzymes that catalyze the hydrolysis of the nicotinamide-ribose bond of NAD ϩ to yield nicotinamide and adenosine diphosphoribose. These enzymes have been described in several bacterial species and in a variety of eukaryotes (10, 11). The GAS NADase differs from most other known bacterial NAD-glycohydrolases in that it is secreted instead of being cell-associated and it possesses ADP ribosyl cyclase activity (12-14). Thus, in addition to producing adenosine diphosphoribose, the GAS NADase can convert NAD ϩ to cyclic adenosine diphosphoribose as a side reaction. Cyclic adenosine diphosphoribose is an important second messenger and signaling molecule for the regulation and mobilization of intracellular calcium stores (14 -16). The GAS genes encoding SLO (slo) and NADase (nga) are expressed from an operon that is transcribed under the control of a promoter upstream of nga. Some studies have identified downstream of nga a second weak promoter for slo (17, 18). The two proteins are functionally linked in that SLO is re...

Contribution of Secreted NADase and Streptolysin O to the Pathogenesis of Epidemic Serotype M1 Streptococcus pyogenes Infections

“…12 Furthermore, the NADase activity of SPN augments the cytotoxicity and the hemolytic activity of SLO. 4 These findings suggest the virulence function of SPN is (at least partially) dependent on SLO, and vice versa. We therefore are more inclined to believe that in epidemic M1 strains, the SPN-deficient mutant and the SLO-deficient mutants are attenuated to similar levels in animal models of invasive infection.…”

“…This result is consistent with a previous report that SPN augments SLO-mediated hemolysis and cytotoxicity. 4 Unexpectedly, strain Dnga-ifs had a further decrease in SLO activity compared to Dnga.…”

Evolutionary pathway to increased virulence and epidemic group A Streptococcus disease derived from 3,615 genome sequences

“…The molecular events included phage-mediated virulence gene acquisition, single nucleotide variant accumulation, and horizontal transfer of the 36-kb region encoding the virulence factors Nga and Slo (61)(62)(63)(64)(65)(66)(67)(68)(69)(70)(71)(72)(73), likely mediated by generalized transduction (24). It is now clear from our analysis that a complex multistep (9).…”

The Signal Recognition Particle Pathway Is Required for Virulence in Streptococcus pyogenes

“…For example, many secreted proteins, such as the SpeB cysteine protease, are trafficked into the extracellular spaces. At least one, SPN (also known as Nga), is translocated across the host cell membrane into its cytosol by a process that involves a second secreted translocator protein, SLO, that is itself delivered to the host cell membrane (33,35,37). Since M protein, SpeB, SPN, and SLO have different trafficking fates, the consequence of the loss of Ffh on their secretion was assessed.…”