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The development of a vaccine against Staphylococcus aureus infection has been the subject of an intensive research effort, but none of the vaccine trials has been successful so far. In this Opinion article, the authors suggest that an over-reliance on mouse models and a focus on targeting cell surface components have been major contributing factors to this failure. Staphylococcus aureus is a highly successful bacterial pathogen owing to its abundance of cell surface and secreted virulence factors. It is estimated that 30% of the population is colonized with S. aureus , usually on mucosal surfaces, and methicillin-resistant S. aureus is a major public health concern. There have been multiple attempts to develop an S. aureus vaccine using one or more cell surface virulence factors as antigens; all of these vaccine trials have failed. In this Opinion article, we suggest that an over-reliance on rodent models and a focus on targeting cell surface components have been major contributing factors to this failure.

Staphylococcus aureus is a human commensal and opportunistic pathogen that causes devastating infections in a wide range of locations within the body. One of the defining characteristics of S. aureus is its ability to form clumps in the presence of soluble fibrinogen, which likely has a protective benefit and facilitates adhesion to host tissue. We have previously shown that the ArlRS two-component regulatory system controls clumping, in part by repressing production of the large surface protein Ebh. In this work we show that ArlRS does not directly regulate Ebh, but instead ArlRS activates expression of the global regulator MgrA. Strains lacking mgrA fail to clump in the presence of fibrinogen, and clumping can be restored to an arlRS mutant by overexpressing either arlRS or mgrA, indicating that ArlRS and MgrA constitute a regulatory pathway. We used RNA-seq to show that MgrA represses ebh, as well as seven cell wall-associated proteins (SraP, Spa, FnbB, SasG, SasC, FmtB, and SdrD). EMSA analysis showed that MgrA directly represses expression of ebh and sraP. Clumping can be restored to an mgrA mutant by deleting the genes for Ebh, SraP and SasG, suggesting that increased expression of these proteins blocks clumping by steric hindrance. We show that mgrA mutants are less virulent in a rabbit model of endocarditis, and virulence can be partially restored by deleting the genes for the surface proteins ebh, sraP, and sasG. While mgrA mutants are unable to clump, they are known to have enhanced biofilm capacity. We demonstrate that this increase in biofilm formation is partially due to up-regulation of SasG, a surface protein known to promote intercellular interactions. These results confirm that ArlRS and MgrA constitute a regulatory cascade, and that they control expression of a number of genes important for virulence, including those for eight large surface proteins.

The human pathogen Neisseria gonorrhoeae ascends into the upper female reproductive tract to cause damaging inflammation within the Fallopian tubes and pelvic inflammatory disease (PID), increasing the risk of infertility and ectopic pregnancy. The loss of ciliated cells from the epithelium is thought to be both a consequence of inflammation and a cause of adverse sequelae. However, the links between infection, inflammation, and ciliated cell extrusion remain unresolved. With the use of ex vivo cultures of human Fallopian tube paired with RNA sequencing we defined the tissue response to gonococcal challenge, identifying cytokine, chemokine, cell adhesion, and apoptosis related transcripts not previously recognized as potentiators of gonococcal PID. Unexpectedly, IL-17C was one of the most highly induced genes. Yet, this cytokine has no previous association with gonococcal infection nor pelvic inflammatory disease and thus it was selected for further characterization. We show that human Fallopian tubes express the IL-17C receptor on the epithelial surface and that treatment with purified IL-17C induces pro-inflammatory cytokine secretion in addition to sloughing of the epithelium and generalized tissue damage. These results demonstrate a previously unrecognized but critical role of IL-17C in the damaging inflammation induced by gonococci in a human explant model of PID. In pelvic inflammatory disease, host immune responses to Neisseria gonorrhoeae damage Fallopian tube tissue and cause infertility. Here, Garcia et al. show that the cytokine IL-17C induces inflammatory responses, and peptidoglycan fragments reduce transcripts related to tissue integrity.

Infectious diseases caused by Staphylococcus aureus present a significant clinical and public health problem. S. aureus causes some of the most severe hospital-associated and community-acquired illnesses. Specifically, it is the leading cause of infective endocarditis and osteomyelitis, and the second leading cause of sepsis in the USA. While pathogenesis of S. aureus infections is at the center of current research, many questions remain about the mechanisms underlying staphylococcal toxic shock syndrome (TSS) and associated adaptive immune suppression. Both conditions are mediated by staphylococcal superantigens (SAgs)—secreted staphylococcal toxins that are major S. aureus virulence factors. Toxic shock syndrome toxin-1 (TSST-1) is the SAg responsible for almost all menstrual TSS cases in the USA. TSST-1, staphylococcal enterotoxin B and C are also responsible for most cases of non-menstrual TSS. While SAgs mediate all of the hallmark features of TSS, such as fever, rash, hypotension, and multi-organ dysfunction, they are also capable of enhancing the toxic effects of endogenous endotoxin. This interaction appears to be critical in mediating the severity of TSS and related mortality. In addition, interaction between SAgs and the host immune system has been recognized to result in a unique form of adaptive immune suppression, contributing to poor outcomes of S. aureus infections. Utilizing rabbit models of S. aureus infective endocarditis, pneumonia and sepsis, and molecular genetics techniques, we aim to elucidate the mechanisms of SAg and endotoxin synergism in the pathogenesis of TSS, and examine the cellular and molecular mechanisms underlying SAg-mediated immune dysfunction.

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.

S. aureus is the leading cause of infective endocarditis in the developed world, affecting ~40,000 individuals each year in the United States, and the second leading cause of bacteremia (D. R. Murdoch et al., Arch Intern Med 169:463–473, 2009, http://dx.doi.org/10.1001/archinternmed.2008.603 , and H. Wisplinghoff et al., Clin Infect Dis 39:309–317, 2004, http://dx.doi.org/10.1086/421946 ). Even with current medical advances, S. aureus bloodstream infections and infective endocarditis carry mortality rates of 20 to 66% (S. Y. Tong et al., Clin Microbiol Rev 28:603–661, 2015, http://dx.doi.org/10.1128/CMR.00134-14 ). S. aureus lineages associated with human disease worldwide include clonal complex 5 (CC5)/USA100, CC30/USA200, CC8/USA300, CC1/USA400, and CC45/USA600. The CC5/USA100, CC30/USA200, and CC45/USA600 lineages cause invasive disease yet remain poorly characterized. USA300 and cytotoxins are central to most S. aureus virulence studies, and yet, we find evidence that clonal groups are quite heterogeneous in parameters canonically used to measure virulence, including cytotoxicity, biofilm formation, and blood survival, and that the superantigen profile is an important parameter to consider when defining the virulence of S. aureus strains. Staphylococcus aureus diseases affect ~500,000 individuals per year in the United States. Worldwide, the USA100, USA200, USA400, and USA600 lineages cause many of the life-threatening S. aureus infections, such as bacteremia, infective endocarditis, pneumonia, toxic shock syndrome, and surgical site infections. However, the virulence mechanisms associated with these clonal lineages, in particular the USA100 and USA600 isolates, have been severely understudied. We investigated the virulence of these strains, in addition to strains in the USA200, USA300, and USA400 types, in well-established in vitro assays and in vivo in the rabbit model of infective endocarditis and sepsis. We show in the infective endocarditis and sepsis model that strains in the USA100 and USA600 lineages cause high lethality and are proficient in causing native valve infective endocarditis. Strains with high cytolytic activity or producing toxic shock syndrome toxin 1 (TSST-1) or staphylococcal enterotoxin C (SEC) caused lethal sepsis, even with low cytolytic activity. Strains in the USA100, USA200, USA400, and USA600 lineages consistently contained genes that encode for the enterotoxin gene cluster proteins, SEC, or TSST-1 and were proficient at causing infective endocarditis, while the USA300 strains lacked these toxins and were deficient in promoting vegetation growth. The USA100, USA200, and USA400 strains in our collection formed strong biofilms in vitro , whereas the USA200 and USA600 strains exhibited increased blood survival. Hence, infective endocarditis and lethal sepsis are multifactorial and not intrinsic to any one individual clonal group, further highlighting the importance of expanding our knowledge of S. aureus pathogenesis to clonal lineages causative of invasive disease. IMPORTANCE S. aureus is the leading cause of infective endocarditis in the developed world, affecting ~40,000 individuals each year in the United States, and the second leading cause of bacteremia (D. R. Murdoch et al., Arch Intern Med 169:463–473, 2009, http://dx.doi.org/10.1001/archinternmed.2008.603 , and H. Wisplinghoff et al., Clin Infect Dis 39:309–317, 2004, http://dx.doi.org/10.1086/421946 ). Even with current medical advances, S. aureus bloodstream infections and infective endocarditis carry mortality rates of 20 to 66% (S. Y. Tong et al., Clin Microbiol Rev 28:603–661, 2015, http://dx.doi.org/10.1128/CMR.00134-14 ). S. aureus lineages associated with human disease worldwide include clonal complex 5 (CC5)/USA100, CC30/USA200, CC8/USA300, CC1/USA400, and CC45/USA600. The CC5/USA100, CC30/USA200, and CC45/USA600 lineages cause invasive disease yet remain poorly characterized. USA300 and cytotoxins are central to most S. aureus virulence studies, and yet, we find evidence that clonal groups are quite heterogeneous in parameters canonically used to measure virulence, including cytotoxicity, biofilm formation, and blood survival, and that the superantigen profile is an important parameter to consider when defining the virulence of S. aureus strains.

Superantigens are indispensable virulence factors for Staphylococcus aureus in disease causation. Superantigens stimulate massive immune cell activation, leading to toxic shock syndrome (TSS) and contributing to other illnesses. However, superantigens differ in their capacities to induce body-wide effects. For many, their production, at least as tested in vitro, is not high enough to reach the circulation, or the proteins are not efficient in crossing epithelial and endothelial barriers, thus remaining within tissues or localized on mucosal surfaces where they exert only local effects. In this study, we address the role of TSS toxin-1 (TSST-1) and most importantly the enterotoxin gene cluster (egc) in infective endocarditis and sepsis, gaining insights into the body-wide versus local effects of superantigens. We examined S. aureus TSST-1 gene (tstH) and egc deletion strains in the rabbit model of infective endocarditis and sepsis. Importantly, we also assessed the ability of commercial human intravenous immunoglobulin (IVIG) plus vancomycin to alter the course of infective endocarditis and sepsis. TSST-1 contributed to infective endocarditis vegetations and lethal sepsis, while superantigens of the egc, a cluster with uncharacterized functions in S. aureus infections, promoted vegetation formation in infective endocarditis. IVIG plus vancomycin prevented lethality and stroke development in infective endocarditis and sepsis. Our studies support the local tissue effects of egc superantigens for establishment and progression of infective endocarditis providing evidence for their role in life-threatening illnesses. In contrast, TSST-1 contributes to both infective endocarditis and lethal sepsis. IVIG may be a useful adjunct therapy for infective endocarditis and sepsis.

Infective endocarditis (IE) is one of the most feared and lethal diseases caused by Staphylococcus aureus. Once established, the infection is fast-progressing and tissue destructive. S. aureus of the clonal complex 5 (CC5) commonly cause IE yet are severely understudied. IE results from bacterial colonization and formation of tissue biofilms (known as vegetations) on injured or inflamed cardiac endothelium. S. aureus IE is promoted by adhesins, coagulases, and superantigens, with the exotoxins and exoenzymes likely contributing to tissue destruction and dissemination. Expression of the large repertoire of virulence factors required for IE and sequelae is controlled by complex regulatory networks. We investigated the temporal expression of the global regulators agr ( RNAIII ), rot , sarS , sarA , sigB , and mgrA in 8 invasive CC5 isolates and established intrinsic expression patterns associated with IE outcomes. We show that vegetation formation, as tested in the rabbit model of IE, inversely correlates with RNAIII and sarA expression during growth in Todd-Hewitt broth (TH). Large vegetations with severe sequelae arise from strains with high-level expression of colonization factors but slower transition towards expression of the exotoxins. Overall, strains proficient in vegetation formation, a hallmark of IE, exhibit lower expression of RNAIII and sarA . Simultaneous high expression of RNAIII , sarA , sigB , and mgrA is the one phenotype assessed in this study that fails to promote IE. Thus, RNAIII and sarA expression that provides for rheostat control of colonization and virulence genes, rather than an on and off switch, promote both vegetation formation and lethal sepsis.

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.

Excessive weight and obesity are associated with the development of diabetes mellitus type 2 (DMII) in humans. They also pose high risks of Staphylococcus aureus colonization and overt infections. S. aureus causes a wide range of severe illnesses in both healthy and immunocompromised individuals. Among S. aureus virulence factors, superantigens are essential for pathogenicity. In this study, we show that rabbits that are chronically exposed to S. aureus superantigen toxic shock syndrome toxin-1 (TSST-1) experience impaired glucose tolerance, systemic inflammation, and elevated endotoxin levels in the bloodstream, all of which are common findings in DMII. Additionally, such DMII-associated findings are also seen through effects of TSST-1 on isolated adipocytes. Collectively, our findings suggest that chronic exposure to S. aureus superantigens facilitates the development of DMII, which may lead to therapeutic targeting of S. aureus and its superantigens. IMPORTANCE Obesity has a strong correlation with type 2 diabetes, in which fatty tissue, containing adipocytes, contributes to the development of the illness through altered metabolism and chronic inflammation. The human microbiome changes in persons with obesity and type 2 diabetes, including increases in Staphylococcus aureus colonization and overt infections. While the microbiome is essential for human wellness, there is little understanding of the role of microbes in obesity or the development of diabetes. Here, we demonstrate that the S. aureus superantigen toxic shock syndrome toxin-1 (TSST-1), an essential exotoxin in pathogenesis, induces inflammation, lipolysis, and insulin resistance in adipocytes both in vitro and in vivo . Chronic stimulation of rabbits with TSST-1 results in impaired systemic glucose tolerance, the hallmark finding in type 2 diabetes in humans, suggesting a role of S. aureus and its superantigens in the progression to type 2 diabetes. Obesity has a strong correlation with type 2 diabetes, in which fatty tissue, containing adipocytes, contributes to the development of the illness through altered metabolism and chronic inflammation. The human microbiome changes in persons with obesity and type 2 diabetes, including increases in Staphylococcus aureus colonization and overt infections. While the microbiome is essential for human wellness, there is little understanding of the role of microbes in obesity or the development of diabetes. Here, we demonstrate that the S. aureus superantigen toxic shock syndrome toxin-1 (TSST-1), an essential exotoxin in pathogenesis, induces inflammation, lipolysis, and insulin resistance in adipocytes both in vitro and in vivo . Chronic stimulation of rabbits with TSST-1 results in impaired systemic glucose tolerance, the hallmark finding in type 2 diabetes in humans, suggesting a role of S. aureus and its superantigens in the progression to type 2 diabetes.