Specificity of streptolysin O in cytolysin-mediated translocation

Cytolysin-mediated translocation (CMT) is a recently described process in the Gram-positive pathogen Streptococcus pyogenes that translocates an effector protein of streptococcal origin into the cytoplasm of a host cell. At least two proteins participate in CMT, the pore-forming molecule streptolysin O (SLO) and an effector protein with the characteristics of a signal transduction protein, the Streptococcus pyogenes NAD-glycohydrolase (SPN). In order to begin to elucidate the molecular details of the translocation process, we examined whether perfringolysin O (PFO), a pore-forming protein related to SLO, could substitute for SLO in the translocation of SPN. When expressed by S. pyogenes, PFO, like SLO, had the ability to form functional pores in keratinocyte membranes. However, unlike SLO, PFO was not competent for translocation of SPN across the host cell membrane. Thus, pore formation by itself was not sufficient to promote CMT, suggesting that an additional feature of SLO was required. This conclusion was supported by the construction of a series of mutations in SLO that uncoupled pore formation and competence for CMT. These mutations defined a domain in SLO that was dispensable for pore formation, but was essential for CMT. However, introduction of this domain into PFO did not render PFO competent for CMT, implying that an additional domain of SLO is also critical for translocation. Taken together, these data indicate that SLO plays an active role in the translocation process that extends beyond that of a passive pore.     

Structural basis of Streptococcus pyogenes immunity to its NAD+ glycohydrolase toxin

The virulence of Gram-positive bacteria is enhanced by toxins like the Streptococcus pyogenes β-NAD(+) glycohydrolase known as SPN. SPN-producing strains of S. pyogenes additionally express the protein immunity factor for SPN (IFS), which forms an inhibitory complex with SPN. We have determined crystal structures of the SPN-IFS complex and IFS alone, revealing that SPN is structurally related to ADP-ribosyl transferases but lacks the canonical binding site for protein substrates. SPN is instead a highly efficient glycohydrolase with the potential to deplete cellular levels of β-NAD(+). The protective effect of IFS involves an extensive interaction with the SPN active site that blocks access to β-NAD(+). The conformation of IFS changes upon binding to SPN, with repacking of an extended C-terminal α helix into a compact shape. IFS is an attractive target for the development of novel bacteriocidal compounds functioning by blocking the bacterium's self-immunity to the SPN toxin.     

Variation in Streptococcus pyogenes NAD+ Glycohydrolase Is Associated with Tissue Tropism

Streptococcus pyogenes is an important pathogen that causes a variety of diseases. The most common infections involve the throat (pharyngitis) or skin (impetigo); however, the factors that determine tissue tropism and severity are incompletely understood. The S. pyogenes NAD⁺ glycohydrolase (SPN) is a virulence factor that has been implicated in contributing to the pathogenesis of severe infections. However, the role of SPN in determining the bacterium''s tissue tropism has not been evaluated. In this report, we examine the sequences of spn and its endogenous inhibitor ifs from a worldwide collection of S. pyogenes strains. Analysis of average pairwise nucleotide diversity, average number of nucleotide differences, and ratio of nonsynonymous to synonymous substitutions revealed significant diversity in spn and ifs. Application of established models of molecular evolution shows that SPN is evolving under positive selection and diverging into NAD⁺ glycohydrolase (NADase)-active and -inactive subtypes. Additionally, the NADase-inactive SPN subtypes maintain the characteristics of a functional gene while ifs becomes a pseudogene. Thus, NADase-inactive SPN continues to evolve under functional constraint. Furthermore, NADase activity did not correlate with invasive disease in our collection but was associated with tissue tropism. The ability to cause infection at both the pharynx and the skin (“generalist” strains) is correlated with NADase-active SPN, while the preference for causing infection at either the throat or the skin (“specialist” strains) is associated with NADase-inactive SPN. These findings suggest that SPN has a NADase-independent function and prompt a reevaluation of the role of SPN in streptococcal pathogenesis.Many bacterial pathogens that are capable of causing infection at multiple tissue sites have considerable underlying genetic diversity that is reflected by the presence or absence of different subsets of virulence genes or by the presence of alternative alleles of specific virulence genes (37, 44, 48). For the latter genes, variation in sequence may arise under pressure to avoid the immune response or reflect proteins whose functions are diverging. Horizontal gene transfer (HGT) events can initially increase diversity through the reassortment of these variant virulence genes and may result in altered pathogenicity or the ability to more efficiently exploit a given ecological niche (37). Continued selection of fitter variants adapted for infection of a specific niche can then lead to a subsequent purging of genetic diversity and a reduction in the types of clinical syndromes a particular lineage can cause (8). As a consequence, genetically discrete subpopulations with strong tropisms for different tissues emerge within the existing species, and this process may represent a key step in the formation of new species (6). Understanding the changes that occur during niche specialization can provide important insights into pathogenic mechanisms required for infection of a specific tissue.Analysis of tissue-specific adaptation is emerging as an important approach for understanding the pathogenesis of the numerous diseases caused by Streptococcus pyogenes (group A streptococcus [GAS]). This Gram-positive bacterium has a worldwide distribution and is a pathogen of humans exclusively, causing important diseases, which include those that are destructive of tissue and life-threatening (cellulitis, necrotizing fasciitis) and those associated with deregulation of immunity (glomerulonephritis, rheumatic fever) (6, 12). However, most cases of S. pyogenes disease are more superficial and self-limiting and occur at either the throat (pharyngitis) or the skin (impetigo). These two tissue sites also represent the primary reservoirs responsible for dissemination of the organism to new hosts. A large body of epidemiological evidence that suggests that there are distinct subpopulations of strains more adapted for infection of either the throat or the skin has accumulated, suggesting that specific adaptations to these two tissues are driving the evolution of its pan-genome (6). However, the specific adaptations responsible for niche specialization are not well understood.A frequently used approach for uncovering a common molecular basis behind bacterial phenotype has been to group strains based on sequence variation in housekeeping genes (18). In the case of niche specialization, continued selection for variants more highly adapted to a particular tissue will purge neutral gene diversity in the adapted population relative to the population as a whole. However, a complication in deciphering trends associated with tissue adaptation in S. pyogenes has been that despite some niche separation, there are high rates of recombination relative to mutation within the species as a whole, on par with that of Streptococcus pneumoniae, a species considered to be highly recombinogenic (6, 22, 57). Frequent recombination has resulted in a random segregation of neutral housekeeping haplotypes between S. pyogenes strains from ecologically distinct subpopulations (6). Thus, standard approaches to establishing relationships between strains have been of only limited utility for understanding niche adaptation for S. pyogenes.A more productive approach for S. pyogenes has been to look for genetic variation outside neutral housekeeping genes that is strongly associated with ecological niche. In this regard, genotypes based on the gene encoding the M protein (emm) provide a significant correlation with tissue tropism (6). The M protein is a fibrillar surface molecule that plays multiple roles in promoting virulence, and serological typing based on M protein diversity has been the traditional method for classifying S. pyogenes strains (35). It is well established that strains with certain M types have a strong preference for infection at either the throat or the skin (9, 40). There are more than 200 known M types (50), which can be divided into 4 major subfamilies based on the sequence of the peptidoglycan-spanning domain at the 3′ end of emm (25). Furthermore, the emm locus can encode one gene or a combination of subfamily genes in a tandem arrangement (7). Analyses of large strain collections have revealed that in ∼99% of strains, the organization of emm genes in the locus can be assigned to one of five patterns (designated A to E) (6). Although strains with each emm pattern may colonize the same tissue types, there is a strong correlation between emm pattern and the ability of the organism to cause disease at specific tissue sites. Strains with emm patterns A to C generally cause pharyngitis; emm pattern D strains are typically the cause of skin diseases, such as impetigo; and emm pattern E strains are “generalists,” which can cause symptomatic infection at either tissue site at approximately equal fractions of the total (6). Since emm pattern is strongly associated with tissue tropism, it is likely that characteristics consistently coinherited with the emm pattern also play a role in determining the tissue tropism of the organism (6, 29).The S. pyogenes NAD⁺ glycohydrolase (SPN, also known as Nga) is a virulence factor with characteristics that merit evaluation for a possible role in tissue tropism. This secreted toxin has an enzymatic activity (NADase) that cleaves the glycosidic bond of β-NAD⁺ to produce nicotinamide and ADP-ribose. All S. pyogenes strains examined to date possess the gene that encodes SPN (spn), but some strains produce a SPN that lacks detectable NADase activity (1, 30, 36, 42). Since there is evidence that SPN′s robust NADase activity contributes to virulence (4, 43, 52, 56), the existence of NADase-deficient SPN has yet to be explained. Epidemiological studies conducted on several limited strain collections have not been informative, as these studies have both found (1, 52) and failed to find (15) an association between NADase activity and whether a lineage has the capacity to cause invasive disease. Whether or not SPN is associated with tissue tropism is not known.SPN also has multiple complex interactions with other proteins that suggest it has an important, yet incompletely understood role in disease pathogenesis. These interactions also imply that SPN is under considerable coevolutionary pressure with its partners (47). For example, the ability of S. pyogenes to produce NADase-active SPN is absolutely dependent on the presence of an endogenous inhibitor protein, immunity factor for SPN (IFS) (31, 42). IFS is a competitive inhibitor of SPN′s β-NAD⁺ substrate and apparently acts to inhibit self-toxicity resulting from any presecretory SPN molecules that adventitiously fold prior to their export from the streptococcal cell. In the absence of IFS, SPN is lethal for S. pyogenes. Interestingly, strains that produce NADase-inactive SPN also have a truncated form of IFS (42). Once secreted, both NADase-active SPN and NADase-inactive SPN are injected into the host cell cytoplasm by a process known as cytolysin-mediated translocation (CMT), which requires interaction between multiple domains of SPN and the pore-forming cytolysin streptolysin O (SLO) (11, 20, 39, 41). When in the cytoplasmic compartment, NADase-active SPN can trigger rapid cell death, which is associated with depletion of β-NAD⁺ pools (10, 11, 39). The genes for SPN (spn), IFS (ifs), and SLO (slo) are encoded in the same operon (31, 42), as is typical of coevolving virulence factor/inhibitor pairs (47). Thus, SPN has multiple complex interactions and is suspected of being important in pathogenesis; however, there is a considerable amount of genetic and functional variation that has yet to be fully defined.In the present study, we sought to clarify the role of SPN in the infectious process through analysis of the genetic diversity in spn and ifs and the relationship this diversity has with disease severity and ecologic niche. By examining a diverse, worldwide collection of S. pyogenes strains, we identify the SPN domains evolving under positive (diversifying) and negative (purifying) selection, correlate these sites with NADase activity, and demonstrate that NADase activity is associated with tissue tropism but not invasiveness of disease.     

Self-inactivation of an erythrocyte NAD glycohydrolase

Summary NAD glycohydrolase activity was studied using bovine erythrocytes, erythrocyte ghosts and partially purified enzyme preparations. During catalysis the enzyme becomes irreversibly inactivated in a process related to substrate turnover. Self-inactivation was observed with intact cells, ghosts and solubilized enzyme and could be demonstrated with NAD, NADP and nicotinamide 1,N⁶ ethenoadenine dinucleotide as substrates. Thionicotinamide adenine dinucleotide and NADH, which are not substrates for the enzyme, do not inactivate but are reversible substrate-competitive inhibitors. Added thiols had no effect on enzyme self-inactivation. Of the reaction products, added nicotinamide partially protected the enzyme while added ADPR had no effect. Thermodynamic parameters calculated from Arrhenius plots for rate constants of self-inactivation indicate a large negative S for transition state formation suggesting a process other than extensive denaturation. Erythrocyte ghost NADases from several other mammalian sources have been demonstrated to undergo a self-inactivation similar to that observed with the bovine enzyme.This work was supported by Research Grant PCM 76-05839 from the National Science Foundation.     

NAD glycohydrolase activity in dog cardiac tissue.

1. Dog heart tissue suspension hydrolyzes NAD, NADP and NMN, and releases nicotinamide stoichiometrically. 2. Maximum activity was observed at 50 degrees C and the activation energy was 10 kcal/mol. 3. Optimum pH range was 6.2-7.6. 4. Compounds with adenine-ribose moiety increased the enzymatic activity. 5. Nicotinamide released during incubation produced reaction nonlinearity. 6. Km for NAD and NADP were about the same; Vmax was higher for NAD. Similar findings have been reported for rabbit heart. 7. Dog enzyme appears to be more sensitive than the rabbit enzyme to noncompetitive inhibitors. 8. Pyrophosphatase activity was not detected in dog heart in contrast to rabbit and rat heart preparations.     

The Streptococcus pyogenes NAD+ glycohydrolase modulates epithelial cell PARylation and HMGB1 release

Streptococcus pyogenes uses the cytolysin streptolysin O (SLO) to translocate an enzyme, the S. pyogenes NAD⁺ glycohydrolase (SPN), into the host cell cytosol. However, the function of SPN in this compartment is not known. As a complication, many S. pyogenes strains express a SPN variant lacking NAD⁺ glycohydrolase (NADase) activity. Here, we show that SPN modifies several SLO‐ and NAD⁺‐dependent host cell responses in patterns that correlate with NADase activity. SLO pore formation results in hyperactivation of the cellular enzyme poly‐ADP‐ribose polymerase‐1 (PARP‐1) and production of polymers of poly‐ADP‐ribose (PAR). However, while SPN NADase activity moderates PARP‐1 activation and blocks accumulation of PAR, these processes continued unabated in the presence of NADase‐inactive SPN. Temporal analyses revealed that while PAR production is initially independent of NADase activity, PAR rapidly disappears in the presence of NADase‐active SPN, host cell ATP is depleted and the pro‐inflammatory mediator high‐mobility group box‐1 (HMGB1) protein is released from the nucleus by a PARP‐1‐dependent mechanism. In contrast, HMGB1 is not released in response to NADase‐inactive SPN and instead the cells release elevated levels of interleukin‐8 and tumour necrosis factor‐α. Thus, SPN and SLO combine to induce cellular responses subsequently influenced by the presence or absence of NADase activity.     

Kinetic properties and physiological regulation of NAD glycohydrolase

Kinetic studies on horse spleen NAD-glycohydrolase demonstrate that the hydrolysis of NAD+ is extensively inhibited by physiological concentrations of nicotinamide and NADP+. These compounds act as reversibly released product and competitive inhibitor respectively.     

Purification of NAD glycohydrolase from Agkistrodon acutus venom

NAD glycohydrolase (NADase) from Agkistrodon acutus venom was purified to electrophoretic homogeneity by a fast, reproducible 3-step procedure including Q Sepharose Fast Flow, Superdex 75, and Mono S column chromatography. This new procedure gave a 15.6-fold purification with a recovery yield of 7.9% and a specific activity of 12.8 units/mg.     

Studies of self-inactivation of bovine seminal fluid NAD glycohydrolase

Bovine seminal fluid NAD glycohydrolase (NADase) was observed to be rapidly inactivated during catalytic hydrolysis of the substrate NAD. The first-order rate constant for the self-inactivation process was independent of enzyme concentration. The enzyme self-inactivation was a turnover-related process and the number of moles of NAD hydrolyzed required for inactivation was proportional to the enzyme concentration. A number of dinucleotides serving as substrates for the enzyme also promoted self-inactivation. The self-inactivation was an irreversible process having a different rate-limiting step from NAD hydrolysis and was not related to the reversible binding of products and substrate-competitive inhibitors. Modification of arginine residues of the enzyme resulted in the loss of NAD hydrolase activity with no differential effect on the self-inactivation process.     

Properties and applications of immobilized snake venom NAD glycohydrolase

The NAD glycohydrolase (NADase) from Bungarus fasciatus snake venom was adsorbed on concanavalin A-Sepharose, and demonstrated to retain both hydrolase and transglycosidase activities in the bound form. The matrix-bound enzyme was stable to repeated washing with buffer and storage at 4°C. The bound enzyme exhibited the same K_m value for hydrolysis of nicotinamide-1,N⁶-ethenoadenine dinucleotide as previously measured with the soluble, purified form of the enzyme. The bound NADase was used repeatedly for a preparative-scale synthesis of 3-acetylpyridine adenine dinucleotide. It was further demonstrated that the immobilized enzyme could be prepared directly from crude snake venom, thus avoiding the time required for purification. The application of the immobilized snake venom NADase for the preparation of pyridine nucleotide coenzyme analogs has many advantages over procedures used previously for analog synthesis.     

Immunohistochemical localization of NAD glycohydrolase in human and rabbit tissues

NAD glycohydrolase (NADase) is present in many organisms from bacteria to mammals. In any given organism, this enzyme is ubiquitous in many tissues. However, its precise localization and its physiological significance have not been defined. We have determined the distribution of NADase in normal human and rabbit tissues by immunoblotting and immunohistochemistry, using a polyclonal antibody raised in goats. Immunoblot analyses revealed that NADase was highly expressed in the heart, lung, stomach, and liver tissues of the rabbit. From immunohistochemical studies of NADase, high concentrations in both human and rabbit tissues were found in hepatocytes and sinusoidal lining cells, sinus histiocytes of the lymph node, spleen and thymus, glomerular capillary endothelial cells of the kidney, cardiac muscle, endothelium of blood vessles, and erythrocytes.     

Surface interactome in Streptococcus pyogenes

Very few studies have so far been dedicated to the systematic analysis of protein interactions occurring between surface and/or secreted proteins in bacteria. Such interactions are expected to play pivotal biological roles that deserve investigation. Taking advantage of the availability of a detailed map of surface and secreted proteins in Streptococcus pyogenes (group A Streptococcus (GAS)), we used protein array technology to define the "surface interactome" in this important human pathogen. Eighty-three proteins were spotted on glass slides in high density format, and each of the spotted proteins was probed for its capacity to interact with any of the immobilized proteins. A total of 146 interactions were identified, 25 of which classified as "reciprocal," namely, interactions that occur irrespective of which of the two partners was immobilized on the chip or in solution. Several of these interactions were validated by surface plasmon resonance and supported by confocal microscopy analysis of whole bacterial cells. By this approach, a number of interesting interactions have been discovered, including those occurring between OppA, DppA, PrsA, and TlpA, proteins known to be involved in protein folding and transport. These proteins, all localizing at the septum, might be part, together with HtrA, of the recently described ExPortal complex of GAS. Furthermore, SpeI was found to strongly interact with the metal transporters AdcA and Lmb. Because SpeI strictly requires zinc to exert its function, this finding provides evidence on how this superantigen, a major player in GAS pathogenesis, can acquire the metal in the host environment, where it is largely sequestered by carrier proteins. We believe that the approach proposed herein can lead to a deeper knowledge of the mechanisms underlying bacterial invasion, colonization, and pathogenesis.