Michael R. Wessels

Streptolysin O (SLO) is a virulence determinant of group A Streptococcus ( S. pyogenes ), the agent of streptococcal sore throat and severe invasive infections. SLO is a member of a family of bacterial pore-forming toxins known as cholesterol-dependent cytolysins, which require cell membrane cholesterol for pore formation. While cholesterol is essential for cytolytic activity, accumulating data suggest that cell surface glycans may also participate in the binding of SLO and other cholesterol-dependent cytolysins to host cells. Here, we find that unbiased CRISPR screens for host susceptibility factors for SLO cytotoxicity identified genes encoding enzymes involved in the earliest steps of glycosphingolipid (GSL) biosynthesis. Targeted knockouts of these genes conferred relative resistance to SLO cytotoxicity in two independent human cell lines. Inactivation of ugcg , which codes for UDP-glucose ceramide glucosyltransferase, the enzyme catalyzing the first glycosylation step in GSL biosynthesis, reduced the clustering of SLO on the cell surface. This result suggests that binding to GSLs serves to cluster SLO molecules at lipid rafts where both GSLs and cholesterol are abundant. SLO clustering and susceptibility to SLO cytotoxicity were restored by reconstituting the GSL content of ugcg knockout cells with ganglioside GM1, but susceptibility to SLO cytotoxicity was not restored by a GM1 variant that lacks an oligosaccharide head group required for SLO binding, nor by a variant with a “kinked” acyl chain that prevents efficient packing of the ganglioside ceramide moiety with cholesterol. Thus, SLO appears to co-opt cell surface glycosphingolipids to gain access to lipid rafts for increased efficiency of pore formation and cytotoxicity. IMPORTANCE: Group A Streptococcus is a global public health concern as it causes streptococcal sore throat and less common but potentially life-threatening invasive infections. Invasive infections have been associated with bacterial strains that produce large amounts of a secreted toxin, streptolysin O (SLO), which belongs to a family of pore-forming toxins produced by a variety of bacterial species. This study reveals that SLO binds to a class of molecules known as glycosphingolipids on the surface of human cells and that this interaction promotes efficient binding of SLO to cholesterol in the cell membrane and enhances pore formation. Understanding how SLO damages human cells provides new insight into streptococcal infection and may inform new approaches to treatment and prevention.

Variability in disease severity caused by a microbial pathogen is impacted by each infection representing a unique combination of host and pathogen genomes. Here, we show that the outcome of invasive Streptococcus pyogenes infection is regulated by an interplay between human STING genotype and bacterial NADase activity. S. pyogenes-derived c-di-AMP diffuses via streptolysin O pores into macrophages where it activates STING and the ensuing type I IFN response. However, the enzymatic activity of the NADase variants expressed by invasive strains suppresses STING-mediated type I IFN production. Analysis of patients with necrotizing S. pyogenes soft tissue infection indicates that a STING genotype associated with reduced c-di-AMP-binding capacity combined with high bacterial NADase activity promotes a ‘perfect storm’ manifested in poor outcome, whereas proficient and uninhibited STING-mediated type I IFN production correlates with protection against host-detrimental inflammation. These results reveal an immune-regulating function for bacterial NADase and provide insight regarding the host-pathogen genotype interplay underlying invasive infection and interindividual disease variability.

Nicotinamide adenine dinucleotide (NAD⁺) is an essential co-factor for cellular metabolism and serves as a substrate in enzymatic processes. NAD⁺ is produced by de novo synthesis or salvage pathways in nearly all bacterial species. Haemophilus influenzae lacks the capacity for de novo synthesis, so it is dependent on import of NAD⁺ from the external environment or salvage biosynthetic pathways for recycling of NAD⁺ precursors and breakdown products. However, the actual sources of NAD⁺ utilized by H. influenzae in the respiratory tract are not well defined. In this study, we found that a variety of bacteria, including species found in the upper airway of humans, released NAD⁺ that was readily detectable in extracellular culture fluid, and which supported growth of H. influenzae in vitro. By contrast, certain strains of Streptococcus pyogenes (group A streptococcus or GAS) inhibited growth of H. influenzae in vitro by secreting NAD⁺-glycohydrolase (NADase), which degraded extracellular NAD⁺. Conversely, GAS strains that lacked enzymatically active NADase released extracellular NAD⁺, which could support H. influenzae growth. Our results suggest that many bacterial species, including normal flora of the upper airway, release NAD⁺ into the environment. GAS is distinctive in its ability to both release and degrade NAD⁺. Thus, colonization of the airway with H. influenzae may be promoted or restricted by co-colonization with GAS in a strain-specific manner that depends, respectively, on release of NAD⁺ or secretion of active NADase. We suggest that, in addition to its role as a cytotoxin for host cells, NADase may serve a separate function by restricting growth of H. influenzae in the human respiratory tract.

NADase and streptolysin O (SLO) are secreted toxins important for pathogenesis of group A Streptococcus , the agent of strep throat and severe invasive infections. The two toxins interact in solution and mutually enhance cytotoxic activity.

Group A Streptococcus (GAS) is an important public health threat as a cause of sore throat, skin infections, life-threatening invasive infections, and the postinfectious complications of acute rheumatic fever, a leading cause of acquired heart disease. This work characterizes CsrRS, a GAS system for the detection of environmental signals that enables adaptation of the bacteria for survival in the human throat by regulating the production of products that allow the bacteria to resist clearance by the human immune system.

Streptococcus pyogenes or group A Streptococcus (GAS) is a leading cause of bacterial pharyngitis, skin and soft tissue infections, life-threatening invasive infections, and the post-infectious autoimmune syndromes of acute rheumatic fever and post-streptococcal glomerulonephritis. Genetic manipulation of this important pathogen is complicated by resistance of the organism to genetic transformation. Very low transformation efficiency is attributed to recognition and degradation of introduced foreign DNA by a type I restriction-modification system encoded by the hsdRSM locus. DNA sequence analysis of this locus in ten GAS strains that had been previously transformed with an unrelated plasmid revealed that six of the ten harbored a spontaneous mutation in hsdR, S, or M. The mutations were all different, and at least five of the six were predicted to result in loss of function of the respective hsd gene product. The unexpected occurrence of such mutations in previously transformed isolates suggested that the process of transformation selects for spontaneous inactivating mutations in the Hsd system. We investigated the possibility of exploiting the increased transformability of hsd mutants by constructing a deletion mutation in hsdM in GAS strain 854, a clinical isolate representative of the globally dominant M1T1 clonal group. Mutant strain 854ΔhsdM exhibited a 5-fold increase in electrotransformation efficiency compared to the wild type parent strain and no obvious change in growth or off-target gene expression. We conclude that genetic transformation of GAS selects for spontaneous mutants in the hsdRSM restriction modification system. We propose that use of a defined hsdM mutant as a parent strain for genetic manipulation of GAS will enhance transformation efficiency and reduce the likelihood of selecting spontaneous hsd mutants with uncharacterized genotypes.

Streptococcus pyogenes or group A Streptococcus (GAS) is a leading cause of bacterial pharyngitis, skin and soft tissue infections, life-threatening invasive infections, and the post-infectious autoimmune syndromes of acute rheumatic fever and post-streptococcal glomerulonephritis. Genetic manipulation of this important pathogen is complicated by resistance of the organism to genetic transformation. Very low transformation efficiency is attributed to recognition and degradation of introduced foreign DNA by a type I restriction-modification system encoded by the hsdRSM locus. DNA sequence analysis of this locus in ten GAS strains that had been previously transformed with an unrelated plasmid revealed that six of the ten harbored a spontaneous mutation in hsdR, S , or M . The mutations were all different, and at least five of the six were predicted to result in loss of function of the respective hsd gene product. The unexpected occurrence of such mutations in previously transformed isolates suggested that the process of transformation selects for spontaneous inactivating mutations in the Hsd system. We investigated the possibility of exploiting the increased transformability of hsd mutants by constructing a deletion mutation in hsdM in GAS strain 854, a clinical isolate representative of the globally dominant M1T1 clonal group. Mutant strain 854Δ hsdM exhibited a 5-fold increase in transformation efficiency compared to the wild type parent strain and no obvious change in growth or off-target gene expression. We conclude that genetic transformation of GAS selects for spontaneous mutants the hsdRSM restriction modification system. We propose that use of a defined hsdM mutant as a parent strain for genetic manipulation of GAS will enhance transformation efficiency and reduce the likelihood of selecting spontaneous hsd mutants with uncharacterized genotypes.

Most clinical isolates ofStreptococcus pyogeneselaborate a capsular polysaccharide, which is composed of hyaluronic acid, a high-molecular-mass polymer of alternating residues ofN-acetyl glucosamine and glucuronic acid. Certain strains, particularly those of the M18 serotype, produce abundant amounts of capsule, resulting in formation of large, wet-appearing, translucent or “mucoid” colonies on solid media, whereas strains of M-types 4 and 22 produce none. Studies of acapsular mutant strains have provided evidence that the capsule enhances virulence in animal models of infection, an effect attributable, at least in part, to resistance to complement-mediated opsonophagocytic killing by leukocytes. The presence of the hyaluronic acid capsule may mask adhesins on the bacterial cell wall. However, the capsule itself can mediate bacterial attachment to host cells by binding to the hyaluronic-acid binding protein, CD44. Furthermore, binding of theS. pyogenescapsule to CD44 on host epithelial cells can trigger signaling events that disrupt cell-cell junctions and facilitate bacterial invasion into deep tissues. This article summarizes the biochemistry, genetics, regulation, and role in pathogenesis of this important virulence determinant.

The globally dominant, invasive M1T1 strain of group A Streptococcus (GAS) harbors polymorphisms in the promoter region of an operon that contains the genes encoding streptolysin O (SLO) and NAD ⁺ -glycohydrolase (NADase), resulting in high-level expression of these toxins. While both toxins have been shown experimentally to contribute to pathogenesis, many GAS isolates lack detectable NADase activity. DNA sequencing of such strains has revealed that reduced or absent enzymatic activity can be associated with a variety of point mutations in nga , the gene encoding NADase; a commonly observed polymorphism associated with near-complete abrogation of activity is a substitution of aspartic acid for glycine at position 330 (G330D). However, nga has not been observed to contain early termination codons or mutations that would result in a truncated protein, even when the gene product contains missense mutations that abrogate enzymatic activity. It has been suggested that NADase that lacks NAD-glycohydrolase activity retains an as-yet-unidentified inherent cytotoxicity to mammalian cells and thus is still a potent virulence factor. We now show that expression of NADase, either enzymatically active or inactive, augments SLO-mediated toxicity for keratinocytes. In culture supernatants, SLO and NADase are mutually interdependent for protein stability. We demonstrate that the two proteins interact in solution and that both the translocation domain and catalytic domain of NADase are required for maximal binding between the two toxins. We conclude that binding of NADase to SLO stabilizes both toxins, thereby enhancing GAS virulence. IMPORTANCE The global increase in invasive GAS infections in the 1980s was associated with the emergence of an M1T1 clone that harbors a 36-kb pathogenicity island, which codes for increased expression of toxins SLO and NADase. Polymorphisms in NADase that render it catalytically inactive can be detected in clinical isolates, including invasive strains. However, such isolates continue to produce full-length NADase. The rationale for this observation is not completely understood. This study characterizes the binding interaction between NADase and SLO and reports that the expression of each toxin is crucial for maximal expression and stability of the other. By this mechanism, the presence of both toxins increases toxicity to keratinocytes and is predicted to enhance GAS survival in the human host. These observations provide an explanation for conservation of full-length NADase expression even when it lacks enzymatic activity and suggest a critical role for binding of NADase to SLO in GAS pathogenesis.