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Heme-containing chlorite dismutases (Clds) catalyze a highly unusual O–O bond forming reaction. The O–O cleaving reactions of hydrogen peroxide and peracetic acid (PAA) with the Cld from Dechloromonas aromatica (DaCld) were studied to better understand the Cl–O cleavage of the natural substrate and subsequent O–O bond formation. While reactions with H2O2 resulted in slow destruction of the heme, at acidic pH, heterolytic cleavage of the O–O bond of PAA cleanly yielded the ferryl porphyrin cation radical (Compound I). At alkaline pH, the reaction proceeds more rapidly and the first observed intermediate is a ferryl heme. Freezequench EPR confirmed that the latter has an uncoupled protein-based radical, indicating that Compound I is the first intermediate formed at all pH values and that radical migration is faster at alkaline pH. These results suggest by analogy that two-electron Cl–O bond cleavage to yield a ferryl-porphyrin cation radical is the most likely initial step in O–O bond formation from chlorite.

Streptococcus pyogenes, or group A Streptococcus (GAS), is a human bacterial pathogen that can manifest as a range of diseases from pharyngitis and impetigo to severe outcomes such as necrotizing fasciitis and toxic shock syndrome. GAS disease remains a global health burden with cases estimated at over 700 million annually and over half a million deaths due to severe infections(1). For over 100 years, a clinical hallmark of diagnosis has been the appearance of complete (beta) haemolysis when grown in the presence of blood. This activity is due to the production of a small peptide toxin by GAS known as streptolysin S. Although it has been widely held that streptolysin S exerts its lytic activity through membrane disruption, its exact mode of action has remained unknown. Here, we show, using high-resolution live cell imaging, that streptolysin S induces a dramatic osmotic change in red blood cells, leading to cell lysis. This osmotic change was characterized by the rapid influx of Cl(-) ions into the red blood cells through SLS-mediated disruption of the major erythrocyte anion exchange protein, band 3. Chemical inhibition of band 3 function significantly reduced the haemolytic activity of streptolysin S, and dramatically reduced the pathology in an in vivo skin model of GAS infection. These results provide key insights into the mechanism of streptolysin S-mediated haemolysis and have implications for the development of treatments against GAS.

Background: Host complement-mediated opsonophagocytosis is an innate immune response to GAS. Results: Inactivation of the covS component of the covRS system allows GAS to avoid opsonization. Conclusion: Bacterial genes that subvert opsonization of GAS are regulated by the covRS system. Significance: The covRS system regulates complement inhibitor binding to GAS via the products of the fba and enn genes.

The cluster of virulence sensor (CovS)/responder (CovR) two-component operon (CovRS) regulates ∼15% of the genes of the Group A Streptococcal pyogenes (GAS) genome. Bacterial clones containing inactivating mutations in the covS gene have been isolated from patients with virulent invasive diseases. We report herein an assessment of the nature and types of covS mutations that can occur in both virulent and nonvirulent GAS strains, and assess whether a nonvirulent GAS can attain enhanced virulence through this mechanism. A group of mice were infected with a globally-disseminated clonal M1T1 GAS (isolate 5448), containing wild-type (WT) CovRS (5448/CovR+S+), or less virulent engineered GAS strains, AP53/CovR+S+ and Manfredo M5/CovR+S+. SpeB negative GAS clones from wound sites and/or from bacteria disseminated to the spleen were isolated and the covS gene was subjected to DNA sequence analysis. Numerous examples of inactivating mutations were found in CovS in all regions of the gene. The mutations found included frame-shift insertions and deletions, and in-frame small and large deletions in the gene. Many of the mutations found resulted in early translation termination of CovS. Thus, the covS gene is a genomic mutagenic target that gives GAS enhanced virulence. In cases wherein CovS− was discovered, these clonal variants exhibited high lethality, further suggesting that randomly mutated covS genes occur during the course of infection, and lead to the development of a more invasive infection.

The first genome sequence of a group A Streptococcus pyogenes serotype M23 (emm23) strain (M23ND), isolated from an invasive human infection, has been completed. The genome of this opacity factor-negative (SOF ؊ ) strain is composed of a circular chromosome of 1,846,477 bp. Gene profiling showed that this strain contained six phage-encoded and 24 chromosomally inherited well-known virulence factors, as well as 11 pseudogenes. The bacterium has acquired four large prophage elements, ⌽M23ND.1 to ⌽M23ND.4, harboring genes encoding streptococcal superantigen (ssa), streptococcal pyrogenic exotoxins (speC, speH, and speI), and DNases (spd1 and spd3), with phage integrase genes being present at one flank of each phage insertion, suggesting that the phages were integrated by horizontal gene transfer. Comparative analyses revealed unique large-scale genomic rearrangements that result in genomic rearrangements that differ from those of previously sequenced GAS strains. These rearrangements resulted in an imbalanced genomic architecture and translocations of chromosomal virulence genes. The covS sensor in M23ND was identified as a pseudogene, resulting in the attenuation of speB function and increased expression of the genes for the chromosomal virulence factors multiple-gene activator (mga), M protein (emm23), C5a peptidase (scpA), fibronectinbinding proteins (sfbI and fbp54), streptolysin O (slo), hyaluronic acid capsule (hasA), streptokinase (ska), and DNases (spd and spd3), which were verified by PCR. These genes are responsible for facilitating host epithelial cell binding and and/or immune evasion, thus further contributing to the virulence of M23ND. In conclusion, strain M23ND has become highly pathogenic as the result of a combination of multiple genetic factors, particularly gene composition and mutations, prophage integrations, unique genomic rearrangements, and regulated expression of critical virulence factors.

The genome of an invasive skin-tropic strain (AP53) of serotype M53 group A Streptococcus pyogenes (GAS) is composed of a circular chromosome of 1,860,554 bp and carries genetic markers for infection at skin locales, viz., emm gene family pattern D and FCT type 3. Through genome-scale comparisons of AP53 with other GAS genomes, we identified 596 candidate single-nucleotide polymorphisms (SNPs) that reveal a potential genetic basis for skin tropism. The genome of AP53 differed by ϳ30 point mutations from a noninvasive pattern D serotype M53 strain (Alab49), 4 of which are located in virulence genes. One pseudogene, yielding an inactive sensor kinase (CovS ؊ ) of the two-component transcriptional regulator CovRS, a major determinant for invasiveness, severely attenuated the expression of the secreted cysteine protease SpeB and enhanced the expression of the hyaluronic acid capsule compared to the isogenic noninvasive AP53/CovS ؉ strain. The collagen-binding protein transcript sclB differed in the number of 5=-pentanucleotide repeats in the signal peptides of AP53 and Alab49 (9 versus 15), translating into different lengths of their signal peptides, which nonetheless maintained a full-length translatable coding frame. Furthermore, GAS strain AP53 acquired two phages that are absent in Alab49. One such phage (⌽AP53.2) contains the known virulence factor superantigen exotoxin gene tandem speK-slaA. Overall, we conclude that this bacterium has evolved in multiple ways, including mutational variations of regulatory genes, short-tandem-repeat polymorphisms, large-scale genomic alterations, and acquisition of phages, all of which may be involved in shaping the adaptation of GAS in specific infectious environments and contribute to its enhanced virulence. IMPORTANCEInfectious strains of S. pyogenes (GAS) are classified by their serotypes, relating to the surface M protein, the emm-like subfamily pattern, and their tropicity toward the nasopharynx and/or skin. It is generally agreed that M proteins from pattern D strains, which also directly bind human host plasminogen, are skin tropic. We have sequenced and characterized the genome of an invasive pattern D GAS strain (AP53) in comparison to a very similar strain (Alab49) that is noninvasive and developed a genomic rationale as to possible reasons for the skin tropicity of these two strains and the greater invasiveness of AP53. G roup A Streptococcus pyogenes (GAS) is a beta-hemolytic, Gram-positive, human-pathogenic bacterium that infects multiple epithelial surfaces and, in some cases, is able to invade deeper soft tissue and cause severe disease (1). An estimated 700 million cases of GAS infection occur worldwide per year (2), ranging from benign treatable clinical conditions, e.g., pharyngitis and impetigo, to more invasive and potentially lethal infections, including necrotizing fasciitis (NF), streptococcal toxic shock syndrome (STSS), acute rheumatic fever (ARF), and acute glomerulonephritis (AGN) (3, 4). The wide range of infectious niches and broad varianc...

Cluster 2b streptokinase (SK2b), secreted by invasive skin-trophic strains of Streptococcus pyogenes (GAS), is a human plasminogen (hPg) activator that optimally functions when human plasma hPg is bound, via its kringle-2 domain, to cognizant bacterial cells through the a1a2 domain of the major cellular hPg receptor, Plasminogen-binding group A streptococcal M-like protein (PAM). Another class of streptokinases (SK1), secreted primarily by GAS strains that possess affinity for pharyngeal infections, does not require PAM-bound hPg for optimal activity. We find herein that replacement of the central β-domain of SK2b with the same module from SK1 reduces the dependency of SK2b on PAM, and the converse is true when the β-domain of SK1 is replaced with this same region of SK2b. These data suggest that simple evolutionary shuttling of protein domains in GAS can be employed by GAS to rapidly generate strains that differ in tissue tropism and invasive capability and allow the bacteria to survive different challenges by the host.