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Persistence of Staphylococcus aureus on mucosal membranes: Superantigens and internalization by host cells
REVIEW ARTICLE Persistence of Staphylococcus aureus on mucosal membranes: Superantigens and internalization by host cells WITOLD A. FERENS* and GREGORY A. BOHACH MOSCOW, IDAHO
Abbreviations: IFN = interferon; IL = interleukin; MHCII = major histocompatibility complex class II; PBMC = peripheral blood mononuclear cell; PT = pyrogenic toxin; SAg = superantigen; SEC = staphylococcal enterotoxin type C; TCR = T cell receptor; Vβ = variable chain β
S
taphylococcus aureus frequently inhabits and persists in the anterior nares, female genital tract, and skin surface of human beings 1 and may also colonize certain sites such as the nostrils, rectum, and teat skin of ruminants.2 Colonization by S. aureus develops into clinically apparent infections in only a relatively small percentage of cases, usually after breaching of skin or mucosal barriers. Possible outcomes of colonization by S. aureus are grouped into at least four categories: (1) passive colonization not immediately causing clinical illness (eg, nasal carriage); (2) colonization with a potential for causing toxigenic illness (eg, menstrual toxic shock syndrome); (3) colonization with a transient inflammatory reaction, resulting in self-cure (eg, localized abscess); and (4) persistent or severe infection, with dissemination to secondary sites.
From the Department of Microbiology, Molecular Biology, and Biochemistry, University of Idaho. Supported by grants from the United States Department of Agriculture (NRI-CGP), the Public Health Service (AI28401), the United Dairymen of Idaho, and the Idaho Agricultural Experiment Station. Submitted for publication September 7, 1999; revision submitted November 29, 1999; accepted December 15, 1999. *Current address: Idaho Immunodiagnostics Inc, Business Technology Incubator, 121 Sweet Ave, Moscow, ID 83843. Reprint requests: Greg A. Bohach, PhD, Department of Microbiology, Molecular Biology, and Biochemistry, University of Idaho, College of Agriculture, Moscow, ID 83844. J Lab Clin Med 2000;135:225-30. Copyright © 2000 by Mosby, Inc. 0022-2143/2000 $12.00 + 0 5/1/105179 doi:10.1067/mlc.2000.105179
BOVINE MAMMARY MODEL OF STAPHYLOCOCCAL COLONIZATION
The mechanisms by which S. aureus persists in some hosts remain largely unknown. Two possibilities that are not mutually exclusive are (1) failure of the immune system to respond adequately to S. aureus and (2) ability of the organism to evade the immune response. Our laboratory evaluated these two possibilities by using a bovine mammary model. S. aureus is detectable in practically all dairy herds, including those not experiencing clinical infections.2 Clinical staphylococcal mastitis, or inflammation of the udder resulting from intramammary infection, is a constant threat and results in huge losses for the dairy industry. However, the presence of staphylococci in cows usually causes only mild or subclinical infections. Because the immune response involved seems to be unable to completely eliminate the organism, staphylococcal mastitis can be a valid model of persistent colonization. In several regards, this situation resembles the colonization of mucosal membranes with a potential for recurrent clinical infections in human beings. Our results suggest that two factors, (1) immunosuppression by SAg production and (2) internalization of the organism by epithelial cells, may be important in promoting the persistence of S. aureus in this model. SUPPRESSION OF THE BOVINE IMMUNE RESPONSE BY S. AUREUS SAGS Principles of SAg function. The ability of S. aureus to immunomodulate the host results from the action of multiple virulence factors expressed singly or in various combinations. These act through a variety of mech225
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Fig 1. Schematic depiction of the interaction of conventional peptide antigen and superantigen with T cell receptor and MHCII. After endocytosis and proteolytic processing via an exogenous pathway, antigenic peptides are presented within a groove of MHCII for cognate interaction with T cell receptor (left). In contrast, superantigens interact with T cell receptor and MHCII without processing, binding outside of the sites involved in the interaction of these molecules with antigenic peptides and possibly displacing these peptides from the binding groove.
anisms and include surface molecules, hemolysins, enzymes, and PTs. Staphylococcal enterotoxins and toxic shock syndrome toxin-1 belong to the PT family and are prototypic bacterial SAgs. These toxins are implicated as agents of staphylococcal food poisoning, toxic shock syndrome, and possibly other immunologic disorders in human beings.3 In contrast to immunogenic peptides that are processed within antigen-presenting cells, SAgs are not processed or expressed within the peptide-binding groove of MHCII molecules. Instead, SAgs bind to solvent-exposed external surfaces of MHCII and to conserved elements of Vβ of TCR (Fig 1). Binding to MHCII and Vβ can occur simultaneously, because the binding sites are located at different and non-overlapping regions of the SAg molecule.4 Because the SAg binding site is located outside of the site involved in antigen recognition by the TCR, SAgs bypass specific antigenic recognition mechanisms and stimulate a high proportion of T cells. The consequences of this unique interaction are not fully understood but presumably cause an abnormal signal transduction or simultaneous delivery of stimulatory signals to the T cell and antigen-presenting cell. In principle, a given SAg can interact with all T cells bearing reactive Vβ elements. Typically 10% to 30% of peripheral T cells are activated,
leading to a massive release of inflammatory cytokines systemically, particularly tumor necrosis factor-α, that are responsible for symptoms associated with toxic shock syndrome (ie, hypotension, shock, and fever). At the cellular level, SAg-induced stimulation causes a wide range of responses initiated by T cell proliferation. Although the initial oligoclonal stimulation causes an increased number of T cells bearing SAg-reactive Vβs, eventually the abnormal stimulation leads to anergy, apoptosis, and deletion of reactive T cells and other T cell populations. Effects of SAgs on bovine T cell subpopulations. Until recently, the immunosuppressive properties of SAgs have been studied nearly exclusively by using cells from primates or rodents. Because very few studies on the effects of SAgs in ruminants had been conducted previously, it was important to examine how lymphocytes from dairy animals respond to the toxins. We used SEC as a representative PT SAg for studies in the bovine system because SEC is the most common toxin produced by isolates from mastitis.5 We have shown that bovine T cells are stimulated to proliferate on exposure to SAgs and, as with primate and murine lymphocytes, they respond in a Vβ-dependent fashion. Based on the limited bovine TCR sequence information available, Deringer et al6 developed a polymerase chain
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reaction assay that could quantify the expression of six different bovine Vβs. By using this technique, it was determined that T cells expressing two bovine Vβs, designated boVβBTB13 and boVβBTB35, were selectively expanded when bovine peripheral blood mononuclear cells were exposed to SEC. Interestingly, the bovine Vβs recognized by SEC are most similar to the human Vβs 3, 12, 13, 14, 15, 17, and 20 activated by SEC. Stimulation of bovine PBMCs with SAgs in vitro can be quantified by using several parameters, and together various methods of analysis reveal an interesting change in population dynamics. When proliferation is assessed by measuring incorporation of radiolabeled thymidine into cellular DNA, SEC initially causes a delayed proliferative response. The proliferation is relatively weak during the first 4 days as compared with the response seen with continued incubation. In fact, compared with many T cell stimulants that induce peak nucleic acid synthesis within the first 4 days, stimulation by SEC reaches maximal levels at approximately 6 days. During the initial 96 hours of incubation, T cells enlarge and begin to express surface activation markers. However, despite these indications of stimulation, neither CD4+ or CD8+ T cell subpopulations divide extensively, and overall cell numbers remain relatively unchanged during this first 96 hours. In fact, with cells from most bovine donors, the number of CD4+ cells drops initially. We have attributed this effect to selective apoptosis of this subpopulation. The reduction in CD4+ cell numbers during the initial 4 days of culture is followed by a period of vigorous proliferation for approximately 3 days. Interestingly, although both populations increase in number, CD8+ cells increase more dramatically than CD4+ cells. Thus, after 7 days of culture, the CD4:CD8 ratio in PBMC cultures drops from an initial normal value of ≥1.5 to ≤0.3. The proliferating CD8+ cells are highly activated. They become enlarged and up-regulate the expression of several activation markers including MHCII and IL-2 receptor α. One other activation marker, designated ACT3, is expressed on the majority of bovine CD8+ T cells after stimulation by SEC. Despite its likely significance, the identity of ACT3 is currently unknown. Although they likely exist, its human or murine orthologs are currently unknown. This activation marker was discovered initially by reaction with monoclonal antibodies directed against T cells activated with a variety of stimulants, and it is typically observed on activated CD4+ but not CD8+ T cells. We suspect that the expression of ACT3 on CD8+ T cells is indicative of a high level, but abnormal, activation state. Cells stimulated by other mitogens such as con-
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Fig 2. Expression of cytokine mRNA by nonadherent cells in cultures of PBMCs stimulated by SEC. Agarose gel stained with ethidium bromide shows reverse transcriptase–polymerase chain reaction products resulting from primers specific for bovine cytokines. Samples were obtained from cultures with a representative donor. Collection times are indicated. Baseline RNA levels were quantitated with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a standard.
canavalin A do not express significant levels of ACT3.7 Based on the unique association of ACT3 with SAg stimulation, we are currently placing a great deal of emphasis toward its identification. The function of ACT3+CD8+ bovine T cells activated by SEC remains to be determined. In other systems, CD8+ cells induced by SAgs have been demonstrated to have several immunosuppressive properties. For example, CD8+ cells stimulated with SAgs can inhibit the proliferation of CD4+ cells8 or induce FAS-ligandmediated cell death of CD4+ cells.9 One intriguing possibility is that the SEC-induced CD8+ cells may be analogous to CD8+ cells demonstrated on exposure of mice to SAgs.10 These cells can respond vigorously to antigens but have depressed cytotoxic capacity. If SEC stimulation induces a differentiation of CD8+ cells toward a non-cytotoxic phenotype, the ability of the immune system to clear intracellular staphylococci (see below) could be compromised. SAg-induced bovine cytokines. Immune responses are orchestrated in large part by cytokines produced by T cells. The major types of immune responses are classified as type 1 or type 2, depending on whether they support cell-mediated mechanisms (type 1) or the production of humoral factors (type 2). Type 1 cytokines (eg, IL-12, IL-2, IFN-γ) are associated with responses mediated by macrophages, cytotoxic T cells, and neutrophils, whereas type 2 cytokines (eg, IL-4, IL-5, IL-
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Fig 3. Transmission electron microscopy analysis of MAC-T cells infected with S. aureus Novel. All panels represent a 3-hour coculture of MAC-T cells with S. aureus. A, Invasion of a MAC-T cell by S. aureus, illustrating contact with the MAC-T cell surface, formation of pseudopod-like structures, engulfment of bacteria, phagosome formation, and degradation of the phagosome membrane in the interior of the cell (magnification ×5000). B, Enlargement (magnification ×30,000) of the boxed area shown in A. Arrows indicate fragments of the degraded phagosome membrane. C, Enlargement (magnification ×15,000) of pseudopod-like structures and engulfment of bacteria. D, Cytoplasmic membrane contortion (magnification ×4000) associated with many of the infected MAC-T cells. (From Bayles et al. Infect Immun 1998;66:336-42. Used by permission of the American Society for Microbiology and the authors.)
6, IL-10) support B cell differentiation, proliferation, and production of antibodies in addition to suppressing macrophage function. Exposure of bovine PBMCs to SEC causes up-regulation of IL-4, a key cytokine for the induction of type 2 responses, whereas IL-12, an IL-4 antagonist, is down-regulated (Fig 2). The expression of IL-4 coincides with the lack of T cell proliferation in early cultures and with ACT3 expression on CD8+ T cells.11 Despite the demonstration that SEC causes up-regulation of IL-4, it is not presently possible to define SEC-induced bovine cytokine profiles as a type 2 response, because the cytokine signature is characteristic of both type 1 and 2 responses. For example, SEC stimulation causes an up-regulation of both IL-4 and IFN-γ. Furthermore, in experiments with other animals such as rodents, SAgs typically fail to induce expression of IL-4 but instead induce primarily type 1 cytokines.12,13 It remains to be determined whether
the effect observed with bovine PBMCs is unique to the bovine system or is a property unique for SEC. In any case, the phenotypes generated in the presence of IL-4 and the absence of IL-12 may produce an immune response that is inefficient in clearing intracellular pathogens. INTERNALIZATION OF S. AUREUS BY EPITHELIAL CELLS Molecular and cellular aspects of internalization.
Although S. aureus has not been traditionally considered an intracellular pathogen, its ability to enter and survive in phagocytic and non-phagocytic cells is now well established.14-16 Bayles et al17 initially characterized the interaction of S. aureus with an established bovine mammary epithelium cell line (MAC-T; Fig 3) as an indication of the potential for this organism to become internalized in the mammary gland. Intimate contact with the bacterial cell surface is required for host cell signal transduction, cytoskeletal rearrange-
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ments, and eventual uptake of the organism. Staphylococcal fibronectin binding protein is a required microbial ligand that mediates the uptake through a genistein-sensitive protein tyrosine kinase–mediated pathway.18 After internalization within a membranebound endosome, S. aureus cells eventually escape and multiply in the host cytoplasm. Escape from an endosome is probably mediated by one or more of the staphylococcal hemolysins. It has been shown that S. aureus actively modulates host cell physiology, leading to apoptosis. Although the identity of the apoptosis-inducing substance has not been determined, it has been shown to be regulated by the staphylococcal global regulators agr and sar.19 Significance of intracellular S. aureus. There are several potential mechanisms by which intracellular staphylococci could promote the persistence of infections. First, the existence of intracellular staphylococci could help the organism evade exposure to macrophages. It remains to be determined whether the organism could evade phagocytosis by professional phagocytic cells entirely. Alternatively, phagocytosis of organisms within apoptotic bodies could result in the use of alternate macrophage receptors and thereby hamper macrophage activation. Although there is copious evidence that S. aureus is able to enter and survive in a variety of cultured cells, further studies in this area should be aimed at determining the significance of intracellular S. aureus in vivo. Recently Hudson et al20 described the internalization of staphylococci by osteoblasts of a chicken embryo, pointing to a likely relevance of this phenomenon to S. aureus persistence. Because S. aureus pathogenesis is very complex, internalization of the organism could very well have different consequences, depending on which infection model is used. SAg production by S. aureus could further enhance the advantage of intracellular staphylococci. There is a great deal of circumstantial evidence that SAg production promotes persistence. Infections caused by SECproducing S. aureus isolates resolve poorly even when treated.21 Interestingly, in a study characterizing S. aureus strains isolated from bovine mastitis cases of varying severity, all peracute isolates produced SEC, while none of them produced protein A.22 This combination is consistent with a model in which the bacteria are protected from a largely antibody-based immune response promoted by the SAg activity of SEC. The reduced ability of macrophages to destroy bacteria internalized within apoptotic bodies would be further exacerbated by a depression of macrophage functions by SEC-induced IL-10 (Fig 2), which is known to down-regulate macrophage functions and intracellular killing.23
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CONCLUSIONS
The studies summarized in this report were conducted to explore novel mechanisms by which S. aureus could persist on mucosal surfaces. The data suggest that the organism may use a variety of mechanisms. First, SAgs can modulate the immune response in several ways, leading to an immunosuppressive state. Although the local production of SAgs may be insufficient to cause a large-scale systemic immunosuppression, a local effect at the site of colonization could promote the survival of the organism. Several effects on bovine lymphocyte subpopulations mimic those seen in other systems, including a reversal of the CD4:CD8 T cell ratio. We have attributed this effect to the apoptosis of CD4+ cells and the emergence of a highly activated CD8+ T cell subpopulation characterized by the expression of the ACT3 marker. In contrast to many other microbial pathogens, antibodies against S. aureus do not seem to fully protect against colonization or infection.24 Although this is often attributed to the anti-opsonic properties of protein A, several lines of experimental evidence including work with protein A negative isolates or mutants suggest that this traditional explanation does not adequately explain the persistence.22,25 The studies conducted here provide alternative explanations for the evasion of antibody-mediated clearance by this organism. First, the importance of intracellular S. aureus is becoming more clear. There is strong evidence that a specific molecular interaction occurs between the staphylococcal and host cell surfaces. Interestingly, there is a potential link between SAg production and intracellular S. aureus in the bovine system. Our analysis of SAg-induced cytokine profiles suggests that an immune response less capable of dealing with intracellular pathogens is generated. The evidence suggests that, at least on long-term exposure, SAgs might cause a shift in the immune response to one that is not competent to clear intracellular pathogens. Until recently this would not have been likely to be an important consideration for S. aureus, which has been traditionally described as a pyogenic extracellular pathogen. One of the most frequently asked questions related to S. aureus pathogenesis is “What advantage do superantigen toxins provide to S. aureus?” The most wellknown properties of the toxins are their effects on the cardiovascular system, leading to systemic effects such as toxic shock syndrome. However, it is unlikely that a successful pathogen would benefit from a toxin that is acutely lethal to the host. Therefore it is more likely that genes encoding SAgs are retained by this pathogen because they reduce the effectiveness of immune responses via mechanisms outlined in this review. The ability of staphylococci to induce premature production
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of type 2 cytokines, in conjunction with the invasion of epithelial cells and a non-stimulatory entry into macrophages, may enable S. aureus to survive in an immunocompetent host. Because the association of staphylococci with ruminants most likely preceded, in evolutionary terms, the association of these pathogens with human beings, the SAgs produced by human strains may constitute an evolving element of this interaction. In this review we present our view that SAgs have a potential to act as immunoregulatory factors, facilitating the survival of S. aureus. Although we suspect that SAgs promote S. aureus persistence, the organism produces numerous other toxins (eg, leukocidin, hemolysins) capable of immunomodulation by damaging eukaryotic cells. Elucidation of the role of staphylococcal toxins and internalization in the pathogenicity of this organism clearly requires further experimentation. Staphylococcal mastitis of ruminants can provide a useful model for work in this area. We thank our collaborators on the projects described in this article: Ken Bayles, Bill Davis, Kasia Dziewanowska, Larry Fox, Will Goff, Linda Liou, Yong Ho Park, Joe Patti, Bill Trumble, and Carla Wesson. REFERENCES
1. Kluytmans J, van Belkum A, Verbrugh H. Nasal carriage of Staphylococcus aureus: epidemiology, underlying mechanisms, and associated risks. Clin Microbiol Rev 1997;10: 505-20. 2. Davidson I. Observations on the pathogenic staphylococci in a dairy herd during a period of six years. Res Vet Sci 1961;2:22-40. 3. Schlievert P. Role of superantigens in human diseases. J Infect Dis 1993;167:997-1002. 4. Leder L, Llera A, Lavoie PM, Lebedeva MI, Li H, Sekaly RP, et al. A mutational analysis of the binding of staphylococcal enterotoxins B and C3 to the T cell receptor beta chain and major histocompatibility complex class II. J Exp Med 1998;187:823-33. 5. Kenny K, Reiser RF, Bastida-Corcuera FD, Norcross NL. Production of enterotoxins and toxic shock syndrome toxin by bovine mammary isolates of Staphylococcus aureus. J Clin Microbiol 1993;31:706-7. 6. Deringer JR, Ely RJ, Monday SR, Stauffacher CV, Bohach GA. Vβ dependent stimulation of bovine and human T cells by host-specific staphylococcal enterotoxins. Infect Immun 1997;65:4048-54. 7. Ferens WA, Davis WC, Hamilton MJ, Park YH, Deobald CF, Fox L, et al. Activation of bovine lymphocyte subpopulations by staphylococcal enterotoxin C. Infect Immun 1998;66:57380. 8. Noble A, Pestano GA, Cantor H. Suppression of immune responses by CD8 cells. I. Superantigen-activated CD8 cells induce unidirectional Fas-mediated apoptosis of antigen-activated CD4 cells. J Immunol 1998;159:559-65.
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9. Ettinger RD, Panka J, Wang JKM, Stanger BZ, Ju S-T, Marshak-Rothstein A. Fas ligand-mediated cytotoxicity is directly responsible for apoptosis of normal CD4+ T cells responding to a bacterial superantigen. J Immunol 1995;154:4302-8. 10. Sundstedt A, Hoiden I, Hansson J, Hedlund G, Kalland T, Dohlsten M. Superantigen-induced anergy in cytotoxic CD8+ T cells. J Immunol 1995;154:6306-13. 11. Ferens WA, Goff WL, Davis WC, Fox LK, Deobald C, Hamilton MJ, et al. Induction of type 2 cytokines by a staphylococcal enterotoxin superantigen. J Nat Toxins 1998; 7:193-213. 12. Litton MJ, Sander B, Murphy E, O’Garra A, Abrams JS. Early expression of cytokines in lymph nodes after treatment in vivo with Staphylococcus enterotoxin B. J Immunol Methods 1994;175:47-58. 13. Sriskandan S, Evans TJ, Cohen J. Bacterial superantigeninduced human lymphocyte responses are nitric oxide dependent and mediated by IL-12 and IFN-γ. J Immunol 1996;156: 2430-5. 14. Craven N, Anderson JC. Phagocytosis of Staphylococcus aureus by bovine mammary gland macrophages and intracellular protection from antibiotic action in vitro and in vivo. J Dairy Res 1984;51:513-23. 15. Hudson MC, Ramp WK, Nicholson NC, Williams AS, Nousiainen MT. Internalization of Staphylococcus aureus by cultured osteoblasts. Microbial Pathogenesis 1995;19:409-19. 16. Vann JM, Proctor RA. Ingestion of Staphylococcus aureus by bovine endothelial cells results in time- and dose-dependent damage to endothelial cell monolayers. Infect Immun 1987;55:2155-63. 17. Bayles KW, Wesson CA, Liou LE, Fox LK, Bohach GA, Trumble WR. Intracellular Staphylococcus aureus escapes the endosome and induces apoptosis in epithelial cells. Infect Immun 1998;66:336-42. 18. Dziewanowska K, Patti JM, Deobald CF, Bayles KW, Trumble WR, Bohach GA. Fibronectin binding protein and host cell tyrosine kinase are required for internalization of Staphylococcus aureus by epithelial cells. Infect Immun 1999;67: 4673-8. 19. Wesson CA, Liou LE, Todd KM, Bohach GA, Trumble WR, Bayles KW. Staphylococcus aureus Agr and Sar global regulators influence internalization and induction of apoptosis. Infect Immun 1998;66:5238-43. 20. Reilly SS, Hudson MC, Kellam JF, Ramp WK. In vivo internalization of Staphylococcus aureus by embryonic chick osteoblasts. Bone 2000;26:63-70. 21. Jones TO, Wieneke AA. Staphylococcal toxic shock syndrome. Vet Rec 1986;119:435-6. 22. Matsunaga T, Kamata S, Kakiichi N, Uchida K. Characteristics of Staphylococcus aureus isolated from peracute, acute, and chronic bovine mastitis. J Vet Med Sci 1993;55:297-300. 23. Sieling PA, Abrams JS, Yamamura M, Salgame P, Bloom BR, Rea TH, et al. Immunosuppressive roles for IL-10 and IL-4 in human infection. J Immunol 1993;150:5501-10. 24. Foster TJ. Potential for vaccination against infections caused by Staphylococcus aureus. Vaccine 1991;9:221-7. 25. Patel AH, Nowlan P, Weavers ED, Foster T. Virulence of protein A-deficient and alpha-toxin-deficient mutants of Staphylococcus aureus isolated by allele replacement. Infect Immun 1987;55:3103-10.
Abbreviations: IFN = interferon; IL = interleukin; MHCII = major histocompatibility complex class II; PBMC = peripheral blood mononuclear cell; PT = pyrogenic toxin; SAg = superantigen; SEC = staphylococcal enterotoxin type C; TCR = T cell receptor; Vβ = variable chain β
S
taphylococcus aureus frequently inhabits and persists in the anterior nares, female genital tract, and skin surface of human beings 1 and may also colonize certain sites such as the nostrils, rectum, and teat skin of ruminants.2 Colonization by S. aureus develops into clinically apparent infections in only a relatively small percentage of cases, usually after breaching of skin or mucosal barriers. Possible outcomes of colonization by S. aureus are grouped into at least four categories: (1) passive colonization not immediately causing clinical illness (eg, nasal carriage); (2) colonization with a potential for causing toxigenic illness (eg, menstrual toxic shock syndrome); (3) colonization with a transient inflammatory reaction, resulting in self-cure (eg, localized abscess); and (4) persistent or severe infection, with dissemination to secondary sites.
From the Department of Microbiology, Molecular Biology, and Biochemistry, University of Idaho. Supported by grants from the United States Department of Agriculture (NRI-CGP), the Public Health Service (AI28401), the United Dairymen of Idaho, and the Idaho Agricultural Experiment Station. Submitted for publication September 7, 1999; revision submitted November 29, 1999; accepted December 15, 1999. *Current address: Idaho Immunodiagnostics Inc, Business Technology Incubator, 121 Sweet Ave, Moscow, ID 83843. Reprint requests: Greg A. Bohach, PhD, Department of Microbiology, Molecular Biology, and Biochemistry, University of Idaho, College of Agriculture, Moscow, ID 83844. J Lab Clin Med 2000;135:225-30. Copyright © 2000 by Mosby, Inc. 0022-2143/2000 $12.00 + 0 5/1/105179 doi:10.1067/mlc.2000.105179
BOVINE MAMMARY MODEL OF STAPHYLOCOCCAL COLONIZATION
The mechanisms by which S. aureus persists in some hosts remain largely unknown. Two possibilities that are not mutually exclusive are (1) failure of the immune system to respond adequately to S. aureus and (2) ability of the organism to evade the immune response. Our laboratory evaluated these two possibilities by using a bovine mammary model. S. aureus is detectable in practically all dairy herds, including those not experiencing clinical infections.2 Clinical staphylococcal mastitis, or inflammation of the udder resulting from intramammary infection, is a constant threat and results in huge losses for the dairy industry. However, the presence of staphylococci in cows usually causes only mild or subclinical infections. Because the immune response involved seems to be unable to completely eliminate the organism, staphylococcal mastitis can be a valid model of persistent colonization. In several regards, this situation resembles the colonization of mucosal membranes with a potential for recurrent clinical infections in human beings. Our results suggest that two factors, (1) immunosuppression by SAg production and (2) internalization of the organism by epithelial cells, may be important in promoting the persistence of S. aureus in this model. SUPPRESSION OF THE BOVINE IMMUNE RESPONSE BY S. AUREUS SAGS Principles of SAg function. The ability of S. aureus to immunomodulate the host results from the action of multiple virulence factors expressed singly or in various combinations. These act through a variety of mech225
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Fig 1. Schematic depiction of the interaction of conventional peptide antigen and superantigen with T cell receptor and MHCII. After endocytosis and proteolytic processing via an exogenous pathway, antigenic peptides are presented within a groove of MHCII for cognate interaction with T cell receptor (left). In contrast, superantigens interact with T cell receptor and MHCII without processing, binding outside of the sites involved in the interaction of these molecules with antigenic peptides and possibly displacing these peptides from the binding groove.
anisms and include surface molecules, hemolysins, enzymes, and PTs. Staphylococcal enterotoxins and toxic shock syndrome toxin-1 belong to the PT family and are prototypic bacterial SAgs. These toxins are implicated as agents of staphylococcal food poisoning, toxic shock syndrome, and possibly other immunologic disorders in human beings.3 In contrast to immunogenic peptides that are processed within antigen-presenting cells, SAgs are not processed or expressed within the peptide-binding groove of MHCII molecules. Instead, SAgs bind to solvent-exposed external surfaces of MHCII and to conserved elements of Vβ of TCR (Fig 1). Binding to MHCII and Vβ can occur simultaneously, because the binding sites are located at different and non-overlapping regions of the SAg molecule.4 Because the SAg binding site is located outside of the site involved in antigen recognition by the TCR, SAgs bypass specific antigenic recognition mechanisms and stimulate a high proportion of T cells. The consequences of this unique interaction are not fully understood but presumably cause an abnormal signal transduction or simultaneous delivery of stimulatory signals to the T cell and antigen-presenting cell. In principle, a given SAg can interact with all T cells bearing reactive Vβ elements. Typically 10% to 30% of peripheral T cells are activated,
leading to a massive release of inflammatory cytokines systemically, particularly tumor necrosis factor-α, that are responsible for symptoms associated with toxic shock syndrome (ie, hypotension, shock, and fever). At the cellular level, SAg-induced stimulation causes a wide range of responses initiated by T cell proliferation. Although the initial oligoclonal stimulation causes an increased number of T cells bearing SAg-reactive Vβs, eventually the abnormal stimulation leads to anergy, apoptosis, and deletion of reactive T cells and other T cell populations. Effects of SAgs on bovine T cell subpopulations. Until recently, the immunosuppressive properties of SAgs have been studied nearly exclusively by using cells from primates or rodents. Because very few studies on the effects of SAgs in ruminants had been conducted previously, it was important to examine how lymphocytes from dairy animals respond to the toxins. We used SEC as a representative PT SAg for studies in the bovine system because SEC is the most common toxin produced by isolates from mastitis.5 We have shown that bovine T cells are stimulated to proliferate on exposure to SAgs and, as with primate and murine lymphocytes, they respond in a Vβ-dependent fashion. Based on the limited bovine TCR sequence information available, Deringer et al6 developed a polymerase chain
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reaction assay that could quantify the expression of six different bovine Vβs. By using this technique, it was determined that T cells expressing two bovine Vβs, designated boVβBTB13 and boVβBTB35, were selectively expanded when bovine peripheral blood mononuclear cells were exposed to SEC. Interestingly, the bovine Vβs recognized by SEC are most similar to the human Vβs 3, 12, 13, 14, 15, 17, and 20 activated by SEC. Stimulation of bovine PBMCs with SAgs in vitro can be quantified by using several parameters, and together various methods of analysis reveal an interesting change in population dynamics. When proliferation is assessed by measuring incorporation of radiolabeled thymidine into cellular DNA, SEC initially causes a delayed proliferative response. The proliferation is relatively weak during the first 4 days as compared with the response seen with continued incubation. In fact, compared with many T cell stimulants that induce peak nucleic acid synthesis within the first 4 days, stimulation by SEC reaches maximal levels at approximately 6 days. During the initial 96 hours of incubation, T cells enlarge and begin to express surface activation markers. However, despite these indications of stimulation, neither CD4+ or CD8+ T cell subpopulations divide extensively, and overall cell numbers remain relatively unchanged during this first 96 hours. In fact, with cells from most bovine donors, the number of CD4+ cells drops initially. We have attributed this effect to selective apoptosis of this subpopulation. The reduction in CD4+ cell numbers during the initial 4 days of culture is followed by a period of vigorous proliferation for approximately 3 days. Interestingly, although both populations increase in number, CD8+ cells increase more dramatically than CD4+ cells. Thus, after 7 days of culture, the CD4:CD8 ratio in PBMC cultures drops from an initial normal value of ≥1.5 to ≤0.3. The proliferating CD8+ cells are highly activated. They become enlarged and up-regulate the expression of several activation markers including MHCII and IL-2 receptor α. One other activation marker, designated ACT3, is expressed on the majority of bovine CD8+ T cells after stimulation by SEC. Despite its likely significance, the identity of ACT3 is currently unknown. Although they likely exist, its human or murine orthologs are currently unknown. This activation marker was discovered initially by reaction with monoclonal antibodies directed against T cells activated with a variety of stimulants, and it is typically observed on activated CD4+ but not CD8+ T cells. We suspect that the expression of ACT3 on CD8+ T cells is indicative of a high level, but abnormal, activation state. Cells stimulated by other mitogens such as con-
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Fig 2. Expression of cytokine mRNA by nonadherent cells in cultures of PBMCs stimulated by SEC. Agarose gel stained with ethidium bromide shows reverse transcriptase–polymerase chain reaction products resulting from primers specific for bovine cytokines. Samples were obtained from cultures with a representative donor. Collection times are indicated. Baseline RNA levels were quantitated with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a standard.
canavalin A do not express significant levels of ACT3.7 Based on the unique association of ACT3 with SAg stimulation, we are currently placing a great deal of emphasis toward its identification. The function of ACT3+CD8+ bovine T cells activated by SEC remains to be determined. In other systems, CD8+ cells induced by SAgs have been demonstrated to have several immunosuppressive properties. For example, CD8+ cells stimulated with SAgs can inhibit the proliferation of CD4+ cells8 or induce FAS-ligandmediated cell death of CD4+ cells.9 One intriguing possibility is that the SEC-induced CD8+ cells may be analogous to CD8+ cells demonstrated on exposure of mice to SAgs.10 These cells can respond vigorously to antigens but have depressed cytotoxic capacity. If SEC stimulation induces a differentiation of CD8+ cells toward a non-cytotoxic phenotype, the ability of the immune system to clear intracellular staphylococci (see below) could be compromised. SAg-induced bovine cytokines. Immune responses are orchestrated in large part by cytokines produced by T cells. The major types of immune responses are classified as type 1 or type 2, depending on whether they support cell-mediated mechanisms (type 1) or the production of humoral factors (type 2). Type 1 cytokines (eg, IL-12, IL-2, IFN-γ) are associated with responses mediated by macrophages, cytotoxic T cells, and neutrophils, whereas type 2 cytokines (eg, IL-4, IL-5, IL-
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Fig 3. Transmission electron microscopy analysis of MAC-T cells infected with S. aureus Novel. All panels represent a 3-hour coculture of MAC-T cells with S. aureus. A, Invasion of a MAC-T cell by S. aureus, illustrating contact with the MAC-T cell surface, formation of pseudopod-like structures, engulfment of bacteria, phagosome formation, and degradation of the phagosome membrane in the interior of the cell (magnification ×5000). B, Enlargement (magnification ×30,000) of the boxed area shown in A. Arrows indicate fragments of the degraded phagosome membrane. C, Enlargement (magnification ×15,000) of pseudopod-like structures and engulfment of bacteria. D, Cytoplasmic membrane contortion (magnification ×4000) associated with many of the infected MAC-T cells. (From Bayles et al. Infect Immun 1998;66:336-42. Used by permission of the American Society for Microbiology and the authors.)
6, IL-10) support B cell differentiation, proliferation, and production of antibodies in addition to suppressing macrophage function. Exposure of bovine PBMCs to SEC causes up-regulation of IL-4, a key cytokine for the induction of type 2 responses, whereas IL-12, an IL-4 antagonist, is down-regulated (Fig 2). The expression of IL-4 coincides with the lack of T cell proliferation in early cultures and with ACT3 expression on CD8+ T cells.11 Despite the demonstration that SEC causes up-regulation of IL-4, it is not presently possible to define SEC-induced bovine cytokine profiles as a type 2 response, because the cytokine signature is characteristic of both type 1 and 2 responses. For example, SEC stimulation causes an up-regulation of both IL-4 and IFN-γ. Furthermore, in experiments with other animals such as rodents, SAgs typically fail to induce expression of IL-4 but instead induce primarily type 1 cytokines.12,13 It remains to be determined whether
the effect observed with bovine PBMCs is unique to the bovine system or is a property unique for SEC. In any case, the phenotypes generated in the presence of IL-4 and the absence of IL-12 may produce an immune response that is inefficient in clearing intracellular pathogens. INTERNALIZATION OF S. AUREUS BY EPITHELIAL CELLS Molecular and cellular aspects of internalization.
Although S. aureus has not been traditionally considered an intracellular pathogen, its ability to enter and survive in phagocytic and non-phagocytic cells is now well established.14-16 Bayles et al17 initially characterized the interaction of S. aureus with an established bovine mammary epithelium cell line (MAC-T; Fig 3) as an indication of the potential for this organism to become internalized in the mammary gland. Intimate contact with the bacterial cell surface is required for host cell signal transduction, cytoskeletal rearrange-
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ments, and eventual uptake of the organism. Staphylococcal fibronectin binding protein is a required microbial ligand that mediates the uptake through a genistein-sensitive protein tyrosine kinase–mediated pathway.18 After internalization within a membranebound endosome, S. aureus cells eventually escape and multiply in the host cytoplasm. Escape from an endosome is probably mediated by one or more of the staphylococcal hemolysins. It has been shown that S. aureus actively modulates host cell physiology, leading to apoptosis. Although the identity of the apoptosis-inducing substance has not been determined, it has been shown to be regulated by the staphylococcal global regulators agr and sar.19 Significance of intracellular S. aureus. There are several potential mechanisms by which intracellular staphylococci could promote the persistence of infections. First, the existence of intracellular staphylococci could help the organism evade exposure to macrophages. It remains to be determined whether the organism could evade phagocytosis by professional phagocytic cells entirely. Alternatively, phagocytosis of organisms within apoptotic bodies could result in the use of alternate macrophage receptors and thereby hamper macrophage activation. Although there is copious evidence that S. aureus is able to enter and survive in a variety of cultured cells, further studies in this area should be aimed at determining the significance of intracellular S. aureus in vivo. Recently Hudson et al20 described the internalization of staphylococci by osteoblasts of a chicken embryo, pointing to a likely relevance of this phenomenon to S. aureus persistence. Because S. aureus pathogenesis is very complex, internalization of the organism could very well have different consequences, depending on which infection model is used. SAg production by S. aureus could further enhance the advantage of intracellular staphylococci. There is a great deal of circumstantial evidence that SAg production promotes persistence. Infections caused by SECproducing S. aureus isolates resolve poorly even when treated.21 Interestingly, in a study characterizing S. aureus strains isolated from bovine mastitis cases of varying severity, all peracute isolates produced SEC, while none of them produced protein A.22 This combination is consistent with a model in which the bacteria are protected from a largely antibody-based immune response promoted by the SAg activity of SEC. The reduced ability of macrophages to destroy bacteria internalized within apoptotic bodies would be further exacerbated by a depression of macrophage functions by SEC-induced IL-10 (Fig 2), which is known to down-regulate macrophage functions and intracellular killing.23
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CONCLUSIONS
The studies summarized in this report were conducted to explore novel mechanisms by which S. aureus could persist on mucosal surfaces. The data suggest that the organism may use a variety of mechanisms. First, SAgs can modulate the immune response in several ways, leading to an immunosuppressive state. Although the local production of SAgs may be insufficient to cause a large-scale systemic immunosuppression, a local effect at the site of colonization could promote the survival of the organism. Several effects on bovine lymphocyte subpopulations mimic those seen in other systems, including a reversal of the CD4:CD8 T cell ratio. We have attributed this effect to the apoptosis of CD4+ cells and the emergence of a highly activated CD8+ T cell subpopulation characterized by the expression of the ACT3 marker. In contrast to many other microbial pathogens, antibodies against S. aureus do not seem to fully protect against colonization or infection.24 Although this is often attributed to the anti-opsonic properties of protein A, several lines of experimental evidence including work with protein A negative isolates or mutants suggest that this traditional explanation does not adequately explain the persistence.22,25 The studies conducted here provide alternative explanations for the evasion of antibody-mediated clearance by this organism. First, the importance of intracellular S. aureus is becoming more clear. There is strong evidence that a specific molecular interaction occurs between the staphylococcal and host cell surfaces. Interestingly, there is a potential link between SAg production and intracellular S. aureus in the bovine system. Our analysis of SAg-induced cytokine profiles suggests that an immune response less capable of dealing with intracellular pathogens is generated. The evidence suggests that, at least on long-term exposure, SAgs might cause a shift in the immune response to one that is not competent to clear intracellular pathogens. Until recently this would not have been likely to be an important consideration for S. aureus, which has been traditionally described as a pyogenic extracellular pathogen. One of the most frequently asked questions related to S. aureus pathogenesis is “What advantage do superantigen toxins provide to S. aureus?” The most wellknown properties of the toxins are their effects on the cardiovascular system, leading to systemic effects such as toxic shock syndrome. However, it is unlikely that a successful pathogen would benefit from a toxin that is acutely lethal to the host. Therefore it is more likely that genes encoding SAgs are retained by this pathogen because they reduce the effectiveness of immune responses via mechanisms outlined in this review. The ability of staphylococci to induce premature production
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of type 2 cytokines, in conjunction with the invasion of epithelial cells and a non-stimulatory entry into macrophages, may enable S. aureus to survive in an immunocompetent host. Because the association of staphylococci with ruminants most likely preceded, in evolutionary terms, the association of these pathogens with human beings, the SAgs produced by human strains may constitute an evolving element of this interaction. In this review we present our view that SAgs have a potential to act as immunoregulatory factors, facilitating the survival of S. aureus. Although we suspect that SAgs promote S. aureus persistence, the organism produces numerous other toxins (eg, leukocidin, hemolysins) capable of immunomodulation by damaging eukaryotic cells. Elucidation of the role of staphylococcal toxins and internalization in the pathogenicity of this organism clearly requires further experimentation. Staphylococcal mastitis of ruminants can provide a useful model for work in this area. We thank our collaborators on the projects described in this article: Ken Bayles, Bill Davis, Kasia Dziewanowska, Larry Fox, Will Goff, Linda Liou, Yong Ho Park, Joe Patti, Bill Trumble, and Carla Wesson. REFERENCES
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