Mark J. Walker

DNase Sda1 provides selection pressure for a switch to invasive group A streptococcal infection.

Most invasive bacterial infections are caused by species that more commonly colonize the human host with minimal symptoms. Although phenotypic or genetic correlates underlying a bacteriums shift to enhanced virulence have been studied, the in vivo selection pressures governing such shifts are poorly understood. The globally disseminated M1T1 clone of group A Streptococcus (GAS) is linked with the rare but life-threatening syndromes of necrotizing fasciitis and toxic shock syndrome. Mutations in the GAS control of virulence regulatory sensor kinase (covRS) operon are associated with severe invasive disease, abolishing expression of a broad-spectrum cysteine protease (SpeB) and allowing the recruitment and activation of host plasminogen on the bacterial surface. Here we describe how bacteriophage-encoded GAS DNase (Sda1), which facilitates the pathogens escape from neutrophil extracellular traps, serves as a selective force for covRS mutation. The results provide a paradigm whereby natural selection exerted by the innate immune system generates hypervirulent bacterial variants with increased risk of systemic dissemination.

Molecular insight into invasive group A streptococcal disease

Streptococcus pyogenes is also known as group A Streptococcus (GAS) and is an important human pathogen that causes considerable morbidity and mortality worldwide. The GAS serotype M1T1 clone is the most frequently isolated serotype from life-threatening invasive (at a sterile site) infections, such as streptococcal toxic shock-like syndrome and necrotizing fasciitis. Here, we describe the virulence factors and newly discovered molecular events that mediate the in vivo changes from non-invasive GAS serotype M1T1 to the invasive phenotype, and review the invasive-disease trigger for non-M1 GAS. Understanding the molecular basis and mechanism of initiation for streptococcal invasive disease may expedite the discovery of novel therapeutic targets for the treatment and control of severe invasive GAS diseases.

Disease Manifestations and Pathogenic Mechanisms of Group A Streptococcus

SUMMARY Streptococcus pyogenes, also known as group A Streptococcus (GAS), causes mild human infections such as pharyngitis and impetigo and serious infections such as necrotizing fasciitis and streptococcal toxic shock syndrome. Furthermore, repeated GAS infections may trigger autoimmune diseases, including acute poststreptococcal glomerulonephritis, acute rheumatic fever, and rheumatic heart disease. Combined, these diseases account for over half a million deaths per year globally. Genomic and molecular analyses have now characterized a large number of GAS virulence determinants, many of which exhibit overlap and redundancy in the processes of adhesion and colonization, innate immune resistance, and the capacity to facilitate tissue barrier degradation and spread within the human host. This improved understanding of the contribution of individual virulence determinants to the disease process has led to the formulation of models of GAS disease progression, which may lead to better treatment and intervention strategies. While GAS remains sensitive to all penicillins and cephalosporins, rising resistance to other antibiotics used in disease treatment is an increasing worldwide concern. Several GAS vaccine formulations that elicit protective immunity in animal models have shown promise in nonhuman primate and early-stage human trials. The development of a safe and efficacious commercial human vaccine for the prophylaxis of GAS disease remains a high priority.

Trigger for group A streptococcal M1T1 invasive disease

The globally disseminated Streptococcus pyogenes M1T1 clone causes a number of highly invasive human diseases. The transition from local to systemic infection occurs by an unknown mechanism; however invasive M1T1 clinical isolates are known to express significantly less cysteine protease SpeB than M1T1 isolates from local infections. Here, we show that in comparison to the M1T1 strain 5448, the isogenic mutant ΔspeB accumulated 75‐fold more human plasmin activity on the bacterial surface following incubation in human plasma. Human plasminogen was an absolute requirement for M1T1 strain 5448 virulence following subcutaneous (s.c.) infection of humanized plasminogen transgenic mice. S. pyogenes M1T1 isolates from the blood of infected humanized plasminogen transgenic mice expressed reduced levels of SpeB in comparison with the parental 5448 used as inoculum. We propose that the human plasminogen system plays a critical role in group A streptococcal M1T1 systemic disease initiation. SpeB is required for S. pyogenes M1T1 survival at the site of local infection, however, SpeB also disrupts the interaction of S. pyogenes M1T1 with the human plasminogen activation system. Loss of SpeB activity in a subpopulation of S. pyogenes M1T1 at the site of infection results in accumulation of surface plasmin activity thus triggering systemic spread.—Cole, J. N., McArthur, J. D., McKay, F. C., Sanderson‐Smith, M. L., Cork, A. J., Ranson, M., Rohde, M., Itzek, A., Sun, H., Ginsburg, D., Kotb, M., Nizet, V., Chhatwal, G. S., Walker, M. J. Trigger for group A streptococcal M1T1 invasive disease. FASEB J. 20, E1139–E1145 (2006)

Distribution of Intimin Subtypes among Escherichia coli Isolates from Ruminant and Human Sources

ABSTRACT The intimin gene eae, located within the locus of enterocyte effacement pathogenicity island, distinguishes enteropathogenic Escherichia coli (EPEC) and some Shiga toxin-producing E. coli (STEC) strains from all other pathotypes of diarrheagenic E. coli. EPEC is a leading cause of infantile diarrhea in developing countries, and intimin-positive STEC isolates are typically associated with life-threatening diseases such as hemolytic-uremic syndrome and hemorrhagic colitis. Here we describe the development of a PCR-restriction fragment length polymorphism (RFLP) assay that reliably differentiates all 11 known intimin types (α1, α2, β, γ, κ, ε, η, ι, λ, θ, and ζ) and three new intimin genes that show less than 95% nucleotide sequence identity with existing intimin types. We designated these new intimin genes Int-μ, Int-ν, and Int-ξ. The PCR-RFLP assay was used to screen 213 eae-positive E. coli isolates derived from ovine, bovine, and human sources comprising 60 serotypes. Of these, 82 were STEC isolates, 89 were stx-negative (stx−) and ehxA-positive (ehxA+) isolates, and 42 were stx− and ehxA-negative isolates. Int-β, the most commonly identified eae subtype (82 of 213 [38.5%] isolates), was associated with 21 serotypes, followed by Int-ζ (39 of 213 [18.3%] isolates; 11 serotypes), Int-θ (25 of 213 [11.7%] isolates; 15 serotypes), Int-γ (19 of 213 [8.9%] isolates; 9 serotypes), and Int-ε (21 of 213 [9.9%] isolates; 5 serotypes). Intimin subtypes α1, α2, κ, λ, ξ, μ, ν, and ι were infrequently identified; and Int-η was not detected. Phylogenetic analyses with the Phylip package of programs clustered the intimin subtypes into nine distinct families (α, β-ξ, γ, κ, ε-η-ν, ι-μ, λ, θ, and ζ). Our data confirm that ruminants are an important source of serologically and genetically diverse intimin-containing E. coli strains.

A Systematic and Functional Classification of Streptococcus pyogenes That Serves as a New Tool for Molecular Typing and Vaccine Development

Streptococcus pyogenes ranks among the main causes of mortality from bacterial infections worldwide. Currently there is no vaccine to prevent diseases such as rheumatic heart disease and invasive streptococcal infection. The streptococcal M protein that is used as the substrate for epidemiological typing is both a virulence factor and a vaccine antigen. Over 220 variants of this protein have been described, making comparisons between proteins difficult, and hindering M protein-based vaccine development. A functional classification based on 48 emm-clusters containing closely related M proteins that share binding and structural properties is proposed. The need for a paradigm shift from type-specific immunity against S. pyogenes to emm-cluster based immunity for this bacterium should be further investigated. Implementation of this emm-cluster-based system as a standard typing scheme for S. pyogenes will facilitate the design of future studies of M protein function, streptococcal virulence, epidemiological surveillance, and vaccine development.

Surface Analyses and Immune Reactivities of Major Cell Wall-Associated Proteins of Group A Streptococcus

ABSTRACT A proteomic analysis was undertaken to identify cell wall-associated proteins of Streptococcus pyogenes. Seventy-four distinct cell wall-associated proteins were identified, 66 of which were novel. Thirty-three proteins were immunoreactive with pooled S. pyogenes-reactive human antisera. Biotinylation of the GAS cell surface identified 23 cell wall-associated proteins that are surface exposed.

stx1c Is the Most Common Shiga Toxin 1 Subtype among Shiga Toxin-Producing Escherichia coli Isolates from Sheep but Not among Isolates from Cattle

ABSTRACT Unlike Shiga toxin 2 (stx2) genes, most nucleotide sequences of Shiga toxin 1 (stx1) genes from Shiga toxin-producing Escherichia coli (STEC), Shigella dysenteriae, and several bacteriophages (H19B, 933J, and H30) are highly conserved. Consequently, there has been little incentive to investigate variants of stx1 among STEC isolates derived from human or animal sources. However stx1OX3, originally identified in an OX3:H8 isolate from a healthy sheep in Germany, differs from other stx1 subtypes by 43 nucleotides, resulting in changes to 12 amino acid residues, and has been renamed stx1c. In this study we describe the development of a PCR-restriction fragment length polymorphism (RFLP) assay that distinguishes stx1c from other stx1 subtypes. The PCR-RFLP assay was used to study 378 stx1-containing STEC isolates. Of these, 207 were isolated from sheep, 104 from cattle, 45 from humans, 11 from meat, 5 from swine, 5 from unknown sources, and 1 from a cattle water trough. Three hundred fifty-five of the 378 isolates (93.9%) also possessed at least one other associated virulence gene (ehxA, eaeA, and/or stx2); the combination stx1, stx2, and ehxA was the most common (175 of 355 [49.3%]), and 90 of 355 (25.4%) isolates possessed eaeA. One hundred thirty-six of 207 (65.7%) ovine isolates possessed stx1c alone and belonged to 41 serotypes. Seventy-one of 136 (52.2%) comprised the common ovine serotypes O5:H−, O128:H2, and O123:H−. Fifty-two of 207 isolates (25.1%) possessed an stx1 subtype; 27 (51.9%) of these belonged to serotype O91:H−. Nineteen of 207 isolates (9.2%) contained both stx1c and stx1 subtypes, and 14 belonged to serotype O75:H8. In marked contrast, 97 of 104 (93.3%) bovine isolates comprising 44 serotypes possessed an stx1 subtype, 6 isolates possessed stx1c, and the remaining isolate possessed both stx1c and stx1 subtypes. Ten of 11 (91%) isolates cultured from meat in New Zealand possessed stx1c (serotypes O5:H−, O75:H8/H40, O81:H26, O88:H25, O104:H−/H7, O123:H−/H10, and O128:H2); most of these serotypes are commonly recovered from the feces of healthy sheep. Serotypes containing stx1 recovered from cattle rarely were the same as those isolated from sheep. Although an stx1c subtype was never associated with the typical enterohemorrhagic E. coli serogroups O26, O103, O111, O113, and O157, 13 human isolates possessed stx1c. Of these, six isolates with serotype O128:H2 (from patients with diarrhea), four O5:H− isolates (from patients with hemolytic-uremic syndrome), and three isolates with serotypes O123:H− (diarrhea), OX3:H8 (hemolytic-uremic syndrome), and O81:H6 (unknown health status) represent serotypes that are commonly isolated from sheep.

M Protein and Hyaluronic Acid Capsule Are Essential for In Vivo Selection of covRS Mutations Characteristic of Invasive Serotype M1T1 Group A Streptococcus

ABSTRACT The initiation of hyperinvasive disease in group A Streptococcus (GAS) serotype M1T1 occurs by mutation within the covRS two-component regulon (named covRS for control of virulence regulatory sensor kinase), which promotes resistance to neutrophil-mediated killing through the upregulation of bacteriophage-encoded Sda1 DNase. To determine whether other virulence factors contribute to this phase-switching phenomenon, we studied a panel of 10 isogenic GAS serotype M1T1 virulence gene knockout mutants. While loss of several individual virulence factors did not prevent GAS covRS switching in vivo, we found that M1 protein and hyaluronic acid capsule are indispensable for the switching phenotype, a phenomenon previously attributed uniquely to the Sda1 DNase. We demonstrate that like M1 protein and Sda1, capsule expression enhances survival of GAS serotype M1T1 within neutrophil extracellular traps. Furthermore, capsule shares with M1 protein a role in GAS resistance to human cathelicidin antimicrobial peptide LL-37. We conclude that a quorum of GAS serotype M1T1 virulence genes with cooperative roles in resistance to neutrophil extracellular killing is essential for the switch to a hyperinvasive phenotype in vivo. IMPORTANCE The pathogen group A Streptococcus (GAS) causes a wide range of human infections ranging from the superficial “strep throat” to potentially life-threatening conditions, such as necrotizing fasciitis, also known as “flesh-eating disease.” A marked increase in the number of cases of severe invasive GAS infection during the last 30 years has been traced to the emergence and spread of a single clone of the M1T1 serotype. Recent studies have shown that GAS serotype M1T1 bacteria undergo a genetic “switch” in vivo to a hypervirulent state that allows dissemination into the bloodstream. The present study was undertaken to identify specific GAS serotype M1T1 virulence factors required for this switch to hypervirulence. The surface-anchored GAS M1 protein and hyaluronic acid capsule are found to be essential for the switching phenotype, and a novel role for capsule in GAS resistance to host defense peptides and neutrophil extracellular killing is revealed. The pathogen group A Streptococcus (GAS) causes a wide range of human infections ranging from the superficial “strep throat” to potentially life-threatening conditions, such as necrotizing fasciitis, also known as “flesh-eating disease.” A marked increase in the number of cases of severe invasive GAS infection during the last 30 years has been traced to the emergence and spread of a single clone of the M1T1 serotype. Recent studies have shown that GAS serotype M1T1 bacteria undergo a genetic “switch” in vivo to a hypervirulent state that allows dissemination into the bloodstream. The present study was undertaken to identify specific GAS serotype M1T1 virulence factors required for this switch to hypervirulence. The surface-anchored GAS M1 protein and hyaluronic acid capsule are found to be essential for the switching phenotype, and a novel role for capsule in GAS resistance to host defense peptides and neutrophil extracellular killing is revealed.

Streptococcus agalactiae clones infecting humans were selected and fixed through the extensive use of tetracycline

Streptococcus agalactiae (Group B Streptococcus, GBS) is a commensal of the digestive and genitourinary tracts of humans that emerged as the leading cause of bacterial neonatal infections in Europe and North America during the 1960s. Due to the lack of epidemiological and genomic data, the reasons for this emergence are unknown. Here we show by comparative genome analysis and phylogenetic reconstruction of 229 isolates that the rise of human GBS infections corresponds to the selection and worldwide dissemination of only a few clones. The parallel expansion of the clones is preceded by the insertion of integrative and conjugative elements conferring tetracycline resistance (TcR). Thus, we propose that the use of tetracycline from 1948 onwards led in humans to the complete replacement of a diverse GBS population by only few TcR clones particularly well adapted to their host, causing the observed emergence of GBS diseases in neonates.