Adam R. Spaulding

Staphylococcal and Streptococcal Superantigen Exotoxins

SUMMARY This review begins with a discussion of the large family of Staphylococcus aureus and beta-hemolytic streptococcal pyrogenic toxin T lymphocyte superantigens from structural and immunobiological perspectives. With this as background, the review then discusses the major known and possible human disease associations with superantigens, including associations with toxic shock syndromes, atopic dermatitis, pneumonia, infective endocarditis, and autoimmune sequelae to streptococcal illnesses. Finally, the review addresses current and possible novel strategies to prevent superantigen production and passive and active immunization strategies.

Superantigens Are Critical for Staphylococcus aureus Infective Endocarditis, Sepsis, and Acute Kidney Injury

ABSTRACT Infective endocarditis and kidney infections are serious complications of Staphylococcus aureus sepsis. We investigated the role of superantigens (SAgs) in the development of lethal sepsis, infective endocarditis, and kidney infections. SAgs cause toxic shock syndrome, but it is unclear if SAgs contribute to infective endocarditis and kidney infections secondary to sepsis. We show in the methicillin-resistant S. aureus strain MW2 that lethal sepsis, infective endocarditis, and kidney infections in rabbits are critically dependent on high-level SAgs. In contrast, the isogenic strain lacking staphylococcal enterotoxin C (SEC), the major SAg in this strain, is attenuated in virulence, while complementation restores disease production. SAgs’ role in infective endocarditis appears to be both superantigenicity and direct endothelial cell stimulation. Maintenance of elevated blood pressure by fluid therapy significantly protects from infective endocarditis, possibly through preventing bacterial accumulation on valves and increased SAg elimination. These data should facilitate better methods to manage these serious illnesses. IMPORTANCE The Centers for Disease Control and Prevention reported in 2007 that Staphylococcus aureus is the most significant cause of serious infectious diseases in the United States (R. M. Klevens, M. A. Morrison, J. Nadle, S. Petit, K. Gershman, et al., JAMA 298:1763–1771, 2007). Among these infections are sepsis, infective endocarditis, and acute kidney injury. Infective endocarditis occurs in 30 to 60% of patients with S. aureus bacteremia and carries a mortality rate of 40 to 50%. Over the past decades, infective endocarditis outcomes have not improved, and infection rates are steadily increasing (D. H. Bor, S. Woolhandler, R. Nardin, J. Brusch, D. U. Himmelstein, PLoS One 8:e60033, 2013). There is little understanding of the S. aureus virulence factors that are key for infective endocarditis development and kidney abscess formation. We demonstrate that superantigens are critical in the causation of all three infections. We show that their association results from both superantigenicity and direct toxic effects on endothelial cells, the latter likely contributing to delayed endothelium healing. Our studies contribute significantly to understanding the development of these illnesses and are expected to lead to development of important therapies to treat such illnesses. The Centers for Disease Control and Prevention reported in 2007 that Staphylococcus aureus is the most significant cause of serious infectious diseases in the United States (R. M. Klevens, M. A. Morrison, J. Nadle, S. Petit, K. Gershman, et al., JAMA 298:1763–1771, 2007). Among these infections are sepsis, infective endocarditis, and acute kidney injury. Infective endocarditis occurs in 30 to 60% of patients with S. aureus bacteremia and carries a mortality rate of 40 to 50%. Over the past decades, infective endocarditis outcomes have not improved, and infection rates are steadily increasing (D. H. Bor, S. Woolhandler, R. Nardin, J. Brusch, D. U. Himmelstein, PLoS One 8:e60033, 2013). There is little understanding of the S. aureus virulence factors that are key for infective endocarditis development and kidney abscess formation. We demonstrate that superantigens are critical in the causation of all three infections. We show that their association results from both superantigenicity and direct toxic effects on endothelial cells, the latter likely contributing to delayed endothelium healing. Our studies contribute significantly to understanding the development of these illnesses and are expected to lead to development of important therapies to treat such illnesses.

Mixed Infections and In Vivo Evolution in the Human Fungal Pathogen Cryptococcus neoformans

ABSTRACT Koch’s postulates are criteria establishing a causal relationship between a microbe and a disease that lead to the assumption that diseases are caused by a single strain or its evolved forms. Cryptococcus neoformans is a life-threatening human fungal pathogen responsible for an estimated 1 million cases of cryptococcosis/year, predominantly meningoencephalitis. To assess the molecular diversity of clinical isolates and gain knowledge of C. neoformans biology in the host, we analyzed clinical cultures collected during the prospective CryptoA/D study. Using molecular analysis of unpurified isolates, we demonstrated that mixed infections in humans are more common than previously thought, occurring in almost 20% of patients diagnosed with cryptococcosis. These mixed infections are composed of different mating types, serotypes, and/or genotypes. We also identified genetically related haploid and diploid strains in the same patients. Experimental infections and quantitative PCR show that these ploidy changes can result from endoreplication (duplication of DNA content) and that shuttling between haploid and diploid states can occur, suggesting in vivo evolution. Thus, the concept of one strain/one infection does not hold true for C. neoformans and may apply to other environmentally acquired fungal pathogens. Furthermore, the possibility of mixed and/or evolving infections should be taken into account when developing therapeutic strategies against these pathogens. IMPORTANCECryptococcus  neoformans is a life-threatening human fungal pathogen that is present in the environment and is responsible for an estimated 1 million cases of cryptococcosis/year, predominantly meningoencephalitis in HIV-infected patients. To assess the molecular diversity of clinical isolates and gain knowledge of C. neoformans biology in the host, we analyzed clinical cultures collected during a prospective study on cryptococcosis. Using molecular analysis of unpurified isolates, we uncovered an unexpectedly high frequency (almost 20%) of mixed infections. We further demonstrated that these mixed infections could result from infestation by multiple strains acquired from the environment. We also made the serendipitous discovery of in vivo evolution leading to endoreplication of the yeasts within the host. Thus, the concept of one strain causing one infection does not hold true for C. neoformans and potentially for other environmentally acquired fungal pathogens. The possibility of mixed and/or evolving infections should be taken into account when developing therapeutic strategies against these pathogens. Cryptococcus  neoformans is a life-threatening human fungal pathogen that is present in the environment and is responsible for an estimated 1 million cases of cryptococcosis/year, predominantly meningoencephalitis in HIV-infected patients. To assess the molecular diversity of clinical isolates and gain knowledge of C. neoformans biology in the host, we analyzed clinical cultures collected during a prospective study on cryptococcosis. Using molecular analysis of unpurified isolates, we uncovered an unexpectedly high frequency (almost 20%) of mixed infections. We further demonstrated that these mixed infections could result from infestation by multiple strains acquired from the environment. We also made the serendipitous discovery of in vivo evolution leading to endoreplication of the yeasts within the host. Thus, the concept of one strain causing one infection does not hold true for C. neoformans and potentially for other environmentally acquired fungal pathogens. The possibility of mixed and/or evolving infections should be taken into account when developing therapeutic strategies against these pathogens.

Immunity to Staphylococcus aureus secreted proteins protects rabbits from serious illnesses

Staphylococcus aureus causes significant illnesses throughout the world, including toxic shock syndrome (TSS), pneumonia, and infective endocarditis. Major contributors to S. aureus illnesses are secreted virulence factors it produces, including superantigens and cytolysins. This study investigates the use of superantigens and cytolysins as staphylococcal vaccine candidates. Importantly, 20% of humans and 50% of rabbits in our TSS model cannot generate antibody responses to native superantigens. We generated three TSST-1 mutants; G31S/S32P, H135A, and Q136A. All rabbits administered these TSST-1 toxoids generated strong antibody responses (titers>10,000) that neutralized native TSST-1 in TSS models, both in vitro and in vivo. These TSST-1 mutants lacked detectable residual toxicity. Additionally, the TSST-1 mutants exhibited intrinsic adjuvant activity, increasing antibody responses to a second staphylococcal antigen (β-toxin). This effect may be due to TSST-1 mutants binding to the immune co-stimulatory molecule CD40. The superantigens TSST-1 and SEC and the cytolysin α-toxin are known to contribute to staphylococcal pneumonia. Immunization of rabbits against these secreted toxins provided complete protection from highly lethal challenge with a USA200 S. aureus strain producing all three exotoxins; USA200 strains are common causes of staphylococcal infections. The same three exotoxins plus the cytolysins β-toxin and γ-toxin contribute to infective endocarditis and sepsis caused by USA200 strains. Immunization against these five exotoxins protected rabbits from infective endocarditis and lethal sepsis. These data suggest that immunization against toxoid proteins of S. aureus exotoxins protects from serious illnesses, and concurrently superantigen toxoid mutants provide endogenous adjuvant activity.

Vaccination Against Staphylococcus aureus Pneumonia

BACKGROUND Staphylococcus aureus causes serious infections in both hospital and community settings. Attempts have been made to prevent human infection through vaccination against bacterial cell-surface antigens; thus far all have failed. Here we show that superantigens and cytolysins, when used in vaccine cocktails, provide protection from S. aureus USA100-USA400 intrapulmonary challenge. METHODS Rabbits were actively vaccinated (wild-type toxins or toxoids) or passively immunized (hyperimmune serum) against combinations of superantigens (toxic shock syndrome toxin 1, enterotoxins B and C, and enterotoxin-like X) and cytolysins (α-, β-, and γ-toxins) and challenged intrapulmonarily with multiple strains of S. aureus, both methicillin-sensitive and methicillin-resistant. RESULTS Active vaccination against a cocktail containing bacterial cell-surface antigens enhanced disease severity as tested by infective endocarditis. Active vaccination against secreted superantigens and cytolysins resulted in protection of 86 of 88 rabbits when challenged intrapulmonarily with 9 different S. aureus strains, compared to only 1 of 88 nonvaccinated animals. Passive immunization studies demonstrated that production of neutralizing antibodies was an important mechanism of protection. CONCLUSIONS The data suggest that vaccination against bacterial cell-surface antigens increases disease severity, but vaccination against secreted virulence factors provides protection against S. aureus. These results advance our understanding of S. aureus pathogenesis and have important implications in disease prevention.

AhrC and Eep Are Biofilm Infection-Associated Virulence Factors in Enterococcus faecalis

ABSTRACT Enterococcus faecalis is part of the human intestinal microbiome and is a prominent cause of health care-associated infections. The pathogenesis of many E. faecalis infections, including endocarditis and catheter-associated urinary tract infection (CAUTI), is related to the ability of clinical isolates to form biofilms. To identify chromosomal genetic determinants responsible for E. faecalis biofilm-mediated infection, we used a rabbit model of endocarditis to test strains with transposon insertions or in-frame deletions in biofilm-associated loci: ahrC, argR, atlA, opuBC, pyrC, recN, and sepF. Only the ahrC mutant was significantly attenuated in endocarditis. We demonstrate that the transcriptional regulator AhrC and the protease Eep, which we showed previously to be an endocarditis virulence factor, are also required for full virulence in murine CAUTI. Therefore, AhrC and Eep can be classified as enterococcal biofilm-associated virulence factors. Loss of ahrC caused defects in early attachment and accumulation of biofilm biomass. Characterization of ahrC transcription revealed that the temporal expression of this locus observed in wild-type cells promotes initiation of early biofilm formation and the establishment of endocarditis. This is the first report of AhrC serving as a virulence factor in any bacterial species.

Comparison of Staphylococcus aureus strains for ability to cause infective endocarditis and lethal sepsis in rabbits.

Staphylococcus aureus is a major cause of infective endocarditis (IE) and sepsis. Both methicillin-resistant (MRSA) and methicillin-sensitive (MSSA) strains cause these illnesses. Common S. aureus strains include pulsed-field gel electrophoresis (PFGE) types USA200, 300, and 400 types where we hypothesize that secreted virulence factors contribute to both IE and sepsis. Rabbit cardiac physiology is considered similar to humans, and rabbits exhibit susceptibility to S. aureus superantigens (SAgs) and cytolysins. As such, rabbits are an excellent model for studying IE and sepsis, which over the course of four days develop IE vegetations and/or fatal septicemia. We examined the ability of MRSA and MSSA strains (4 USA200, 2 USA300, 2 USA400, and three additional common strains, FRI1169, Newman, and COL) to cause vegetations and lethal sepsis in rabbits. USA200, TSST-1+ strains that produce only low amounts of α-toxin, exhibited modest LD50 in sepsis (1 × 108 – 5 × 108) colony-forming units (CFUs), and 3/4 caused significant IE. USA200 strain MNPE, which produces high-levels of α-toxin, was both highly lethal (LD50 5 × 106 CFUs) and effective in causing IE. In contrast, USA300 strains were highly effective in causing lethal sepsis (LD50s 1 × 106 and 5 × 107 CFUs) but were minimally capable of causing IE. Strain Newman, which is phylogenetically related to USA300 strains, was not highly lethal (LD50 of 2 × 109 CFUs) and was effective in causing IE. USA400 strains were both highly lethal (LD50s of 1 × 107 and 5 × 107 CFUs) and highly effective causes of IE. The menstrual TSS isolate FRI1169, that is TSST-1+, produces high-levels of α-toxin, but is not USA200, was both highly lethal and effective in causing IE. Additional studies showed that phenol soluble modulins (PSMs) produced by FRI1169 were important for sepsis but did not contribute to IE. Our studies show that these clonal groups of S. aureus differ in abilities to cause IE and lethal sepsis and suggest that secreted virulence factors, including SAgs and cytolysins, account for some of these differences.

The Staphylococcus aureus ArlRS two-component system is a novel regulator of agglutination and pathogenesis.

Staphylococcus aureus is a prominent bacterial pathogen that is known to agglutinate in the presence of human plasma to form stable clumps. There is increasing evidence that agglutination aids S. aureus pathogenesis, but the mechanisms of this process remain to be fully elucidated. To better define this process, we developed both tube based and flow cytometry methods to monitor clumping in the presence of extracellular matrix proteins. We discovered that the ArlRS two-component system regulates the agglutination mechanism during exposure to human plasma or fibrinogen. Using divergent S. aureus strains, we demonstrated that arlRS mutants are unable to agglutinate, and this phenotype can be complemented. We found that the ebh gene, encoding the Giant Staphylococcal Surface Protein (GSSP), was up-regulated in an arlRS mutant. By introducing an ebh complete deletion into an arlRS mutant, agglutination was restored. To assess whether GSSP is the primary effector, a constitutive promoter was inserted upstream of the ebh gene on the chromosome in a wildtype strain, which prevented clump formation and demonstrated that GSSP has a negative impact on the agglutination mechanism. Due to the parallels of agglutination with infective endocarditis development, we assessed the phenotype of an arlRS mutant in a rabbit combined model of sepsis and endocarditis. In this model the arlRS mutant displayed a large defect in vegetation formation and pathogenesis, and this phenotype was partially restored by removing GSSP. Altogether, we have discovered that the ArlRS system controls a novel mechanism through which S. aureus regulates agglutination and pathogenesis.

Staphylococcus aureus β-toxin Production is Common in Strains with the β-toxin Gene Inactivated by Bacteriophage

BACKGROUND Staphylococcus aureus causes life-threatening infections, including infective endocarditis, sepsis, and pneumonia. β-toxin is a sphingomyelinase encoded for by virtually all S. aureus strains and exhibits human immune cell cytotoxicity. The toxin enhances S. aureus phenol-soluble modulin activity, and its activity is enhanced by superantigens. The bacteriophage φSa3 inserts into the β-toxin gene in human strains, inactivating it in the majority of S. aureus clonal groups. Hence, most strains are reported not to secrete β-toxin. METHODS This dynamic was investigated by examining β-toxin production by multiple clonal groups of S. aureus, both in vitro and in vivo during infections in rabbit models of infective endocarditis, sepsis, and pneumonia. RESULTS β-toxin phenotypic variants are common among strains containing φSa3. In vivo, φSa3 is differentially induced in heart vegetations, kidney abscesses, and ischemic liver compared to spleen and blood, and in vitro growth in liquid culture. Furthermore, in pneumonia, wild-type β-toxin production leads to development of large caseous lesions, and in infective endocarditis, increases the size of pathognomonic vegetations. CONCLUSIONS This study demonstrates the dynamic interaction between S. aureus and the infected host, where φSa3 serves as a regulator of virulence gene expression, and increased fitness and virulence in new environments.

Engineering a soluble high-affinity receptor domain that neutralizes staphylococcal enterotoxin C in rabbit models of disease

Superantigens (SAgs) are a class of immunostimulatory exotoxins that activate large numbers of T cells, leading to overproduction of cytokines and subsequent inflammatory reactions and systemic toxicity. Staphylococcal enterotoxin C (SEC), a SAg secreted by Staphylococcus aureus, has been implicated in various illnesses including non-menstrual toxic shock syndrome (TSS) and necrotizing pneumonia. SEC has been shown to cause TSS illness in rabbits and the toxin contributes to lethality associated with methicillin-resistant S.aureus (MRSA) in a rabbit model of pneumonia. With the goal of reducing morbidity and mortality associated with SEC, a high-affinity variant of the extracellular variable domain of the T-cell receptor beta-chain for SEC (~14 kDa) was generated by directed evolution using yeast display. This protein was characterized biochemically and shown to cross-react with the homologous (65% identical) SAg staphylococcal enterotoxin B (SEB). The soluble, high-affinity T-cell receptor protein neutralized SEC and SEB in vitro and also significantly reduced the bacterial burden of an SEC-positive strain of MRSA (USA400 MW2) in an infective endocarditis model. The neutralizing agent also prevented lethality due to MW2 in a necrotizing pneumonia rabbit model. These studies characterize a soluble high-affinity neutralizing agent against SEC, which is cross-reactive with SEB, and that has potential to be used intravenously with antibiotics to manage staphylococcal diseases that involve these SAgs.