The Journal of Allergy and Clinical Immunology
Volume 125, Issue 1 , Pages 39-49, January 2010

Secreted virulence factor comparison between methicillin-resistant and methicillin-sensitive Staphylococcus aureus, and its relevance to atopic dermatitis

  • Patrick M. Schlievert, PhD

      Affiliations

    • Department of Microbiology, University of Minnesota Medical School, Minneapolis, Minn
    • Corresponding Author InformationReprint requests: Patrick M. Schlievert, PhD, Department of Microbiology, Medical School, University of Minnesota, 420 Delaware Street SE, Minneapolis, MN 55455.
    • ,
  • Kristi L. Strandberg, PhD

      Affiliations

    • Department of Microbiology, University of Minnesota Medical School, Minneapolis, Minn
    • ,
  • Ying-Chi Lin, MS

      Affiliations

    • Department of Experimental and Clinical Pharmacology, College of Pharmacy, Minneapolis, Minn
    • ,
  • Marnie L. Peterson, PharmD, PhD

      Affiliations

    • Department of Experimental and Clinical Pharmacology, College of Pharmacy, Minneapolis, Minn
    • ,
  • Donald Y.M. Leung, MD, PhD

      Affiliations

    • Department of Pediatrics, National Jewish Health, Denver, Colo

Received 27 August 2009; received in revised form 23 October 2009; accepted 26 October 2009.

Article Outline

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) strains have emerged as serious health threats in the last 15 years. They are associated with large numbers of atopic dermatitis skin and soft tissue infections, but when they originate from skin and mucous membranes, have the capacity to produce sepsis and highly fatal pulmonary infections characterized as necrotizing pneumonia, purpura fulminans, and postviral toxic shock syndrome. This review is a discussion of the emergence of 3 major CA-MRSA organisms, designated CA-MRSA USA400, followed by USA300, and most recently USA200. CA-MRSA USA300 and USA400 isolates and their methicillin-sensitive counterparts (community-associated methicillin-sensitive S aureus) typically produce highly inflammatory cytolysins α-toxin, γ-toxin, δ-toxin (as representative of the phenol soluble modulin family of cytolysins), and Panton Valentine leukocidin. USA300 isolates produce the superantigens enterotoxin-like Q and a highly pyrogenic deletion variant of toxic shock syndrome toxin 1 (TSST-1), whereas USA400 isolates produce the superantigens staphylococcal enterotoxin B or staphylococcal enterotoxin C. USA200 CA-MRSA isolates produce small amounts of cytolysins but produce high levels of TSST-1. In contrast, their methicillin-sensitive S aureus counterparts produce various cytolysins, apparently in part dependent on the niche occupied in the host and levels of TSST-1 expressed. Significant differences seen in production of secreted virulence factors by CA-MRSA versus hospital-associated methicillin-resistant S aureus and community-associated methicillin-sensitive S aureus strains appear to be a result of the need to specialize as the result of energy drains from both virulence factor production and methicillin resistance.

Key words: Atopic dermatitis, Staphylococcus aureus, superantigens, cytolysins, methicillin resistance

Abbreviations used: AD, Atopic dermatitis, CA-MRSA, Community-associated methicillin-resistant Staphylococcus aureus, CA-MSSA, Community-associated methicillin-sensitive Staphylococcus aureus, CDC, Centers for Disease Control and Prevention, HA-MRSA, Hospital-associated methicillin-resistant Staphylococcus aureus, MRSA, Methicillin-resistant Staphylococcus aureus, MSCRAMM, Microbial surface component recognizing adhesive matrix molecules, MSSA, Methicillin-sensitive Staphylococcus aureus, PBP2a, Alternative penicillin-binding protein 2, PSM, Phenol soluble modulin, PVL, Panton Valentine leukocidin, SCC mec, Staphylococcal chromosome cassette methicillin resistance gene, SE, Staphylococcal enterotoxin, SEl, Staphylococcal enterotoxin–like, TSS, Toxic shock syndrome, TSST, Toxic shock syndrome toxin, Vβ-TCR, Variable region of the β-chain of the T-cell receptor

 

Staphylococcus aureus is a gram-positive bacterium that produces a remarkable array of cell-surface and secreted virulence factors (Fig 1) to facilitate disease causation, and rapidly develops antimicrobial resistance almost as quickly as new therapeutic agents are developed.1 The cell-surface virulence factors include microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) as receptors in the human host, other surface proteins such as iron-regulated proteins, polysaccharide intercellular adhesin, and capsular polysaccharides.2, 3, 4 The cell-surface MSCRAMMs typically are produced during exponential phase of growth. The function of these various factors is to provide nutrients (such as iron-uptake proteins) required for survival in the host, and microbial cell protection from the host immune system during lesion formation (such as protein A, fibronectin-binding proteins, and capsules). The secreted virulence factors (Fig 1), typically produced during the postexponential and stationary phases, include a large group of exoenzymes, such as proteases, glycerol ester hydrolase (lipase), and nucleases that make nutrients available to the micro-organism, but also detoxify various innate immune mechanisms (for example, protease cleavage of host cytokines). Importantly, the secreted virulence factors also include a large group of true exotoxins such as highly inflammatory cytolysins (mainly α, β, γ, and δ–toxins and Panton-Valentine leukocidin [PVL]); superantigens, including staphylococcal enterotoxins ([SEs]; SEA, SEB, SECn, SED, SEE, and SEI), SE-like proteins ([SEls]; SEl-G, SEl-H, and SEl-J to SEl-U), and toxic shock syndrome toxin (TSST)–1; and exfoliative toxins A and B.5, 6, 7

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  • Fig 1. 

    Virulence factor production by S aureus. The organism produces cell surface virulence factors (MSCRAMMs) during the exponential phase and exoproteins and exopolysaccharides during the postexponential/stationary phase.

The Centers for Disease Control and Prevention (CDC) and affiliated state health departments in 2007 reported from data obtained in 2005 that S aureus is the most significant cause of serious infections in the United States.8 This is certainly the result of the ubiquity of the organism (as many as 40% of human beings may be colonized on mucosal surfaces, such as the anterior nares or vagina, or nearly all atopic dermatitis [AD] skin by various strains of the organism), production of a myriad of virulence factors, and ease of development of antibiotic resistance.1 A great concern in clinical management of serious staphylococcal infections is the development of methicillin resistance, predictive of global resistance to β-lactam antibiotics. For many years it has been known that more than 50% of hospital-associated S aureus isolates are methicillin-resistant (HA-MRSA). Recently, even a small number of vancomycin-resistant strains have been isolated in Michigan and Pennsylvania from patients in hospital settings.9 In addition, as many as 30% of community-associated strains of S aureus today are also methicillin-resistant (CA-MRSA). Methicillin resistance is not a direct factor that increases virulence of S aureus, but rather functions indirectly to influence production of secreted virulence factors, and clearly makes clinical management of staphylococcal infections more difficult.

This review is a discussion of secreted virulence factor production by methicillin-resistant S aureus (MRSA) strains compared with methicillin-sensitive S aureus (MSSA) strains with respect to emergence of CA-MRSA isolates, noting the influence of methicillin resistance on selective production of certain secreted virulence factors. Key clinical concepts for AD, relative to CA-MRSA versus HA-MRSA and MSSA, are summarized in Table I.

Table I. Clinical implications for atopic dermatitis of CA-MRSA versus HA-MRSA and CA-MSSA and hospital-associated MSSA
One third of S aureus from patients with AD is CA-MRSA; these strains are sometimes resistant to clindamycin. HA-MRSA is resistant to additional antibiotics. MSSA produces β-lactamases and is resistant to penicillin, ampicillin, and possibly some other β-lactam antibiotics.
Production of inflammatory cytolysins and high-level SAgs by CA-MRSA increases their ability to cause extensive AD infections. SAgs (particularly TSST-1, SEB, and SEC) prevent healing of skin lesions in many patients. These patients respond to (1) inclusion of antibiotics such as vancomycin, rifampin (because of great penetrating ability), and clindamycin (potential to shut off exotoxin production); (2) in difficult cases, use of IVIG; and (3) antimicrobial scrubs.
Most patients with AD respond to topical corticosteroids. However, there are patients with steroid-resistant AD; their S aureus strains have been selected for great diversity in SAg types and likelihood of producing high-level SAgs. Patients with steroid-resistant AD respond to other T-cell immunosuppressive agents, such as calcineurin inhibitors.
Skin sites with CA-MRSA, as well as consequent nasal carriage, can be associated with fatal pulmonary infections, including necrotizing pneumonia, purpura fulminans, and postinfluenza TSS. These patients should be treated with (1) antibiotics such as vancomycin, rifampin, and clindamycin; (2) fluid and electrolyte supportive care including use of vasopressors; and (3) possibly IVIG and activated protein C (drotrecogin-α).

IVIG, Intravenous immunoglobulin; SAg, superantigen.

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Superantigens 

Superantigens were originally defined by their capacities to induce fever, to amplify the lethal effects of other toxic molecules including LPS,10, 11 and in the case of SEs to cause the emesis seen in staphylococcal food poisoning.12 Their abilities to stimulate T-cell proliferation has been known for a long time,13, 14 but in 1990, Marrack and Kappler15 described the novel mechanism by which superantigens stimulate T cells and macrophages to produce massive amounts of cytokines, leading to toxic shock syndrome (TSS). It is now well established that superantigens stimulate T-cell proliferation by forming a cross-bridge between certain variable parts of the β-chains of T-cell receptors (Vβ-TCRs) and invariant regions on either or both of the α and β–chains of MHC II molecules on antigen-presenting cells, including macrophages.7, 16, 17 Each superantigen has a unique set of Vβ-TCRs with which it interacts. However, the consequence of the interaction of superantigens with T cells and macrophages is the same: massive cytokine production that includes IL-2, TNF-β, and IFN-γ from CD4+T cells, and IL-1β and TNF-α from macrophages. Collectively, these cytokines cause most of the clinical features of TSS. It has been shown that the ability of SEs to cause food poisoning is separable from superantigen activity and appears to depend on amino acid structural elements within and adjacent to a cystine loop in the molecules.18, 19 In addition, superantigens stimulate epithelial and endothelial cells to produce IL-8 and macrophage inflammatory protein 3α (as well as other cytokines).20, 21 These 2 chemokines are attractants for PMNs and dendritic cells and likely participate early in S aureus infections through induction of inflammation, particularly on skin and mucosal surfaces. A model for superantigen stimulation of human T cells is presented in Fig 2.

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  • Fig 2. 

    Model for the activation of CD4+T cells and macrophages by the superantigen (SAg) SEB compared with antigenic peptide activation of the same cells. Ribbon diagrams of TCRs, MHC II molecules, and SEB adapted from previously published studies.95, 96, 97

Three superantigens are most associated with production of TSS: (1) menstrual TSS caused nearly exclusively by TSST-1, and (2) nonmenstrual TSS caused by TSST-1, SEB, and SEC.14, 22, 23, 24, 25 These 3 superantigens typically are produced in the highest concentrations (of the superantigens) by S aureus strains as tested in vitro, with amounts produced in broth (planktonic) cultures often in the 5 to 50 μg/mL range. Other superantigens typically are produced in vitro in amounts 104 to 106 lower than these 3 superantigens. These differences are important because it has been shown that superantigen amounts as low as 0.1 μg/human being are able to cause TSS symptoms.26 Like cytolysins, superantigens are usually redundantly produced and made by the majority of S aureus strains, with some strains producing as many as 15 superantigen types.27 Although it is not entirely clear why strains produce so many superantigens, it is known that certain host niches appear to favor disease causation by superantigens; for example, menstrual TSS is nearly exclusively caused by TSST-1.14, 22 Other superantigens do not appear to be able to penetrate mucosal barriers as effectively as TSST-1, and this may prevent their association with menstrual illness.18

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Cytolysins 

The cytolysins, as originally described, are hemolytic for red blood cells, but variability occurs in susceptibility dependent on the species source of the red cells. However, it is more likely that the major function of cytolysins is to kill immune cells that are drawn into local areas of infection. It is useful to think of these factors as the second line of defense against the host acting locally, the first line being systemic action of superantigens to interfere with the immune system, and the third line being MSCRAMMs and other bacterial surface factors that function at the individual bacterial cell level. In addition, the cytolysins are exceptionally proinflammatory at subcytolytic doses,21, 28, 29, 30 with proinflammatory activities as much as 106 more than cell surface factors, including cell wall–associated peptidoglycan and lipoteichoic acids, and as much as 103 more inflammatory than superantigens.

Since the initial description of PVL's association with CA-MRSA USA400, and later USA300 strains, investigators have undertaken studies to determine whether PVL has a causal role in skin and severe pulmonary diseases or is simply a marker for the strains. Through use of isogenic PVL+ and PVL- CA-MRSA strains, the DeLeo, Otto, and Schneewind laboratories collaboratively determined in mice, and later in rabbits, that PVL is not necessary for fatal pulmonary diseases.31, 32, 33 Both PVL+ and PVL- strains killed the mice comparably. In contrast, their studies strongly implicated α-toxin, and later also phenol soluble modulins (PSMs), such as δ-toxin, in disease causation.34, 35, 36 Vaccination against α-toxin effectively protected mice from fatal pulmonary diseases.35 At the same time, a series of contradictory studies were published by another collaborative research group from France and the United States, suggesting that PVL is critical to severe pulmonary diseases in mice as caused by these same CA-MRSA strains.37 In addition, vaccination against PVL provided protection to the mice.38 To date there has not been a complete resolution of these apparently contradictory findings. However, studies suggest that PVL has minimal toxicity to mouse PMNs, compared with high toxicity to human PMNs, bringing into question how PVL can be critical in causing mouse pulmonary diseases.39, 40 Our unpublished studies performed in rabbits, with use of the same isogenic CA-MRSA USA400 strains as the DeLeo, Otto, and Schneewind collaborative group used, suggest that PVL is not required for lethal pulmonary diseases; both PVL+ and PVL- strains were comparably lethal when administered intrabronchially.

It appears to us that the cytolysins are likely to contribute variably to serious human pulmonary infections when they are made by the causative organisms. There are likely to be 2 important pathways to fatal pulmonary diseases: (1) hemorrhagic and inflammatory pneumonias in which cytolysins, particularly α-toxin, play major roles; and (2) highly fatal superantigen-induced TSS-like pulmonary illnesses, with activity potentially amplified by cytolysins. Because the cytolysins also have exceptionally strong proinflammatory effects on epithelial and endothelial cells, they are likely to be required for or contribute to skin and soft tissue infections,5 other systemic infections,5 and menstrual TSS (through disruption of the vaginal epithelial barrier and facilitating TSST-1 penetration).20, 21, 41 Their abilities to be produced by S aureus strains, the concentrations made by causative bacteria, and host susceptibility to their various activities likely determine the relative contributions of each. Importantly, multiple cytolysins typically are produced by individual S aureus strains, making it difficult to assign a disease-causing role to any single toxin.5

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Atopic dermatitis and S aureus 

S aureus is a common commensal organism that colonizes as many as 40% of human beings mainly on mucous membranes.42 The organism often colonizes AD-damaged skin and anterior nares and from these sites may spread to infect any other body sites. Nearly all patients with AD may be colonized with S aureus.43, 44 This is likely the result of a combination of host factors including skin barrier dysfunction as well as impaired host immune responses in AD.45, 46 As well, there are many regulatory mechanisms within S aureus that lead to the organism's ability to colonize or infect almost any niche within the host. These mechanisms include both global transcriptional control systems and very recently identified posttranscriptional mechanisms. The global transcriptional regulatory systems have been extensively reviewed recently47, 48, 49 and thus are not highly discussed in this review; 1 example is given as applied to clinical disease.

Of importance to this review is that we are now coming to recognize that S aureus strains appear often to be selected for host niches based on staphylococcal nontranscriptional controls, including (1) “read-through” mechanisms for protein translation through stop codons and (2) altered protein secretion based on virulence factor selection for bacterial survival in various niches. Examples of these mechanisms are given in a subsequent section (COMPARISON OF EXOTOXIN PRODUCTION BY USA400 CA-MRSA VERSUS CA-MSSA), but briefly, menstrual TSS S aureus isolates nearly always have stop codons in the structural genes for the highly inflammatory α-toxin. However, the organisms produce small amounts of α-toxin by “reading through” the stop codons in the messenger RNAs. This allows α-toxin to facilitate TSST-1 penetration of the vaginal mucosa without completely destroying the mucosa.21 In contrast, S aureus strains require wild-type α-toxin production for skin survival.

It is also likely that multiple host factors will participate in regulation of niche selection of S aureus strains, but to date these have not been well studied. One example that has been clarified, however, is that TSS S aureus isolates do not produce the superantigens and cytolysins in blood. This is the result of α and β–globin chains of hemoglobin altering the activity of staphylococcal 2-component systems that are normally required to upregulate exotoxin production.50 Counterintuitively, this suggests that TSST-1 and cytolysins are not produced vaginally in tampon regions containing menstrual blood, but rather are produced in tampon regions that lack menstrual blood. In addition, the data suggest that, although S aureus is a major cause of bloodstream infections,51 the organism may not produce superantigens and cytolysins in blood. It has been proposed that this host action to suppress exotoxin production induced S aureus strains to adapt to host niches in which the organism is walled off from blood through the actions of coagulase and clumping factors, even when present in the bloodstream, allowing production of its critical secreted virulence factors.50 It has also been suggested that positively charged stretches of amino acid residues in the α and β–globin chains may be responsible for the exotoxin-suppressive activity through their interaction with bacterial 2-component systems.50 Chitosan is another highly positively charged molecule that has a similar capacity to interfere with exotoxin production with S aureus through its effects on 2-component systems.52 These studies collectively suggest that other positively charged peptides, such as skin and mucous membrane defensins and cathelicidins,53, 54 may inhibit S aureus exotoxin production at subbacterial growth–inhibiting concentrations. However, this remains to be determined.

Another example of S aureus selection by the host occurs in AD. AD is a T-cell–mediated skin disease that can significantly compromise quality of life because patients have sleep disturbances, social embarrassment, and emotional distress. S aureus infection contributes to the worsening of skin inflammation in AD.55, 56, 57 These organisms have been shown to produce superantigens, including SEA, SEB, SEC, and TSST-1. In addition, Leung et al58 have shown that these superantigens can serve as allergens, stimulating specific IgE responses in patients with AD. However, until recently the full spectrum of superantigens produced by S aureus infecting patients with AD had not been thoroughly examined.27 Furthermore, superantigens have been demonstrated to induce corticosteroid resistance of T cells in vitro.59 This could contribute to difficulty in management of AD because topical corticosteroids are the most common medication used for treatment of AD.

The recent large study mentioned in reference 27 was conducted to examine superantigen production by S aureus strains from patients with AD, both steroid-resistant and nonselected for steroid-resistance, compared with normal vaginal mucosal isolates.27 Several highly important findings were reported:

1.Isolates of S aureus from patients with steroid-resistant AD produced significantly more superantigen types per organism than did nonselected AD and vaginal isolates (these last 2 groups generally were not significantly different from each other in production of superantigen types).

2.Superantigen production by isolates from patients with steroid-resistant AD appeared dysregulated in many respects. For example, SEA typically is produced in only picogram amounts and is thought to be produced during exponential growth of S aureus.60 However, isolates from steroid-resistant patients with AD produced microgram amounts of SEA (>10,000-fold increase), and the toxin was postexponential phase–regulated (typical of staphylococcal exotoxins that are produced in high amounts).47 In addition, some isolates from steroid-resistant patients showed mutations in a global regulator of exotoxin production, designated staphylococcal respiratory response A-B, whose function appears to include acting transcriptionally to prevent exotoxin production when S aureus is grown under anaerobic conditions.61, 62, 63 These isolates could produce exotoxins, even under anaerobic conditions, for example as would be expected to occur vaginally in the absence of tampons. This predicts that in the future menstrual TSS could be caused by TSST-1 production by such S aureus strains in the absence of tampon use; the major role of tampons in menstrual TSS appears to be providing oxygen necessary for TSST-1 production.64

3.The major superantigens associated with nonmenstrual TSS were produced by large numbers of isolates from patients with steroid-resistant AD, and the isolates produced these 3 superantigens in unusual combinations. For example, until this study, in which 10% of S aureus isolates were shown to produce both SEB and TSST-1, these 2 superantigens were not coproduced in tests of over 5000 isolates from patients with TSS. Collectively, this study suggested that use of topical steroids to manage AD is selecting for S aureus strains that are adapting to their “new” skin surface niche by increasing their virulence capabilities. It is also likely that use of additional T-cell immunosuppressive agents, such as tacrolimus and cyclosporine A, to manage steroid-resistant AD will ultimately select for S aureus strains with even greater ability to produce diverse types and amounts of superantigens to continue causation of AD. In addition, it is likely that the skin of such patients will serve as a reservoir for S aureus isolates that can further adapt to new host niches, carrying along their newly acquired virulence capabilities.

We have recently tested a small group of AD isolates for methicillin resistance and have found that 25% are MRSA. MRSA may have either staphylococcal chromosome cassette methicillin resistance gene (SCC mec)–II or SCC mecIV DNA elements. As expected, all skin strains, whether MRSA or MSSA, from patients produced comparable amounts of α-toxin, a cytolysin that appears critical for enhanced survival on skin. We have only recently initiated studies to examine differences in superantigen production by these strains. However, evidence is presented in the next section showing that S aureus strains differ not only in production of secreted virulence factors based on host niche but also dependent on the presence or absence of SCC mec DNA.

CA-MRSA 

Although previously described by Herold and colleagues,65 the emergence of CA-MRSA primarily came to the attention of the medical and scientific communities as a result of the description by the Minnesota Department of Health, the CDC, and colleagues of 4 infant deaths in the Upper Midwest in 1999.66 These infants developed pulmonary infections characterized as necrotizing pneumonia with septicemia. The 4 isolated CA-MRSA were reported as USA400 as determined by pulsed-field gel electrophoresis analysis of isolate DNAs (pulsed-field gel electrophoresis designations as described by the CDC have been adopted for use by many investigators to categorize CA-MRSA strains and are used throughout this article). Since the original description of the disease association with these strains, additional articles also noted the association of CA-MRSA USA400 strains with a variety of severe pulmonary diseases, including necrotizing pneumonia, purpura fulminans, and postviral TSS.67, 68 At the same time, it was noted that an even larger number of the same strains were associated with highly significant skin and soft tissue infections.69 The data suggest that these strains predominantly cause skin and soft tissue infections but can gain access to the lungs to cause necrotizing pneumonia, purpura fulminans, or TSS after upper respiratory viral infections or in patients who are predisposed to lung infections as a result of asthma or other respiratory conditions.

The S aureus strains from the 4 infants were immediately analyzed for a variety of factors that possibly could explain the virulence of the strains, the first being identification of the DNA element encoding methicillin resistance. Multiple DNA elements have been described that account for methicillin resistance, the major gene (mecA) encoding an altered penicillin-binding protein referred to as PBP2a.70 PBP2a is able to mediate peptidoglycan cross-bridging in staphylococcal cell division in the presence of β-lactam antibiotics. PBP2a is encoded on a DNA element referred to as SCC mec. There are multiple SCC mec DNAs, but the major HA-MRSA DNA element is designated SCC mecII71 and is encoded on a large (>50-kb) heterologous DNA insert into the staphylococcal chromosome. SCC mecII appears uncommonly to transfer from 1 S aureus strain to another because of its large size. The isolates from the 4 infants infected with CA-MRSA USA400 did not contain SCC mecII but rather had a much smaller, previously unrecognized SCC mec, now referred to as SCC mecIV71; this SCC mec DNA is more likely to be easily transferrable from 1 S aureus strain to another because of its small size. For a time, the presence of SCC mecIV could be used to distinguish CA-MRSA from HA-MRSA (SCC mecII+), but as might be expected on the basis of the versatility of S aureus, we are now seeing some blending of SCC mec DNAs in MRSA, with both SCC mecII and mecIV (and other SCC mecs) progressively distributed between both CA-MRSA and HA-MRSA.

Additional analyses of CA-MRSA USA400 strains noted the production of a cytolysin that is uncommonly seen in human S aureus strains.67 This cytolysin, PVL, is a hetero-heptamer pore-forming toxin that belongs to the larger γ-toxin family.72 PVL is composed of 2 peptide chains, designated F (Fast) and S (Slow) for elution from a chromatographic column, that polymerize into hetero-chain heptamers and insert into host plasma membranes to exert their cytolytic activity. PVL and the other γ-toxin hetero-heptamers were originally defined by their hemolytic activity, but PVL differs from other γ-toxins in that it has greater cytotoxic activity for PMNs than other family members. PVL is produced by large numbers, but not all, of both CA-MRSA and community-associated methicillin-sensitive S aureus (CA-MSSA) USA400 strains, compared with other S aureus strains, giving rise to the hypothesis that the toxin has a causative role in serious pulmonary diseases.67 Other cytolysins are also produced by CA-MRSA (and CA-MSSA) USA400 strains and include α-toxin, γ-toxins, and δ-toxin (a member of the PSM family)73; the staphylococcal β-toxin, a sphingomyelinase,74 may also occasionally be produced, but the toxin gene is most often disrupted by bacteriophage integration and is thus nonfunctional. α-Toxin, like PVL and other members of the γ-toxin family, is a heptamer pore-forming toxin, except that α-toxin forms a homo-chain heptamer (Fig 3).75 δ-Toxin and other PSMs are small peptides that form α-helical chains and appear to disrupt host cell membranes through formation of small pores combined with membrane solubilization.5, 73 The possible role of these cytolysins in S aureus diseases is discussed in more detail.

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  • Fig 3. 

    Three-dimensional cartoon diagram of the staphylococcal α-toxin homo-chain heptamer adapted from Gouaux et al.75 γ-Toxins and PVL structures are similar except the heptamers are composed of combinations of F (Fast) and S (Slow) peptides.72 Gray is α-toxin monomer (A); multicolored is α-toxin heptamer (B).

In the initial description of the 4 CA-MRSA USA400 strains associated with infant pulmonary diseases, it was reported that 2 of the S aureus strains produce the superantigen SEB, and the remaining 2 isolates produce the superantigen SEC.66 Superantigens, importantly TSST-1, SEB, and SEC, cause staphylococcal TSS, characterized by high fever, hypotension (which may progress to shock and death), a scarlet fever–like rash, peeling of the skin if the patient recovers, and a variable multiorgan component, most often seen as vomiting and diarrhea, and confusion or combativeness.14, 22, 25, 76, 77, 78, 79 Soon after the initial description of TSS, it became clear that many patients with TSS do not exhibit all of the defining clinical features, with rash and visible skin peeling often missing. Thus, categories of probable TSS and toxin-mediated disease have been established to describe patients who do not meet all of the defining criteria.80, 81 Two major categories of TSS have been described on the basis of body sites of infection by S aureus, including menstrual TSS, which is highly associated with vaginal S aureus colonization and use of certain tampons,77, 78 and nonmenstrual TSS, which may occur in association with any other type of staphylococcal infection or colonization.81 Superantigens cause TSS through combinations of their abilities to induce massive cytokine production from both CD4+T cells and macrophages7, 15 and directly stimulate epithelial and endothelial cells (most likely also through cytokine production by these cells).20, 21, 82, 83 The superantigens SEB and SEC were later shown to be highly associated with both CA-MRSA USA400 and their CA-MSSA USA400 counterparts, whether from skin and soft tissue infections, or from serious pulmonary diseases.25, 66, 69, 71

In 2003, the CDC and colleagues reported the emergence of a new clone of CA-MRSA, referred to as CA-MRSA USA300.84, 85 These isolates were also highly associated with skin and soft tissue infections and serious pulmonary diseases similar to USA400 isolates.

Numerous studies have shown that CA-MRSA USA300 strains not only emerged but also began to replace CA-MRSA USA400 strains, such that the majority of skin and soft tissue infections (and consequently serious pulmonary infections) became associated with CA-MRSA USA300. Our laboratory tests S aureus strains submitted by physicians, primarily from Minnesota, who suspect TSS in their patients, for production of PVL and superantigens. We also noted the relative reduction in nonmenstrual TSS isolates submitted that were USA400 (both CA-MRSA and CA-MSSA), beginning in 2003, and replaced by CA-MRSA and CA-MSSA USA300 strains. However, beginning in June 2008, we noted a resurgence of USA400 strains and a significant relative reduction of submitted USA300 strains. It is probable that variations in dominant S aureus strains continue to occur as has happened many times in the past, with strains emerging and disappearing in roughly 10-year cycles.86, 87, 88

The USA300 strains have been thoroughly examined for production of factors that may account for their emergence. CA-MRSA USA300 strains most often contain the small SCC mecIV DNA element that explains their methicillin resistance.84, 85 The organisms, whether CA-MRSA or CA-MSSA, often produce PVL, and they make other cytolysins such as α-toxin, γ-toxins, and δ-toxin (and other PSMs); they occasionally produce β-toxin. Unlike USA400 strains, CA-MRSA and CA-MSSA USA300 do not produce the superantigens SEB and SEC, but instead produce SEl-Q (and other superantigens including occasionally SEA and often SEl-K) and apparently an N-terminal one-half deletion variant of TSST-1.84, 85, 89, 90 Although its activity is incompletely characterized, the deletion variant of TSST-1 has recently been associated with a newly described illness, extreme pyrexia syndrome caused by S aureus, in which patients rapidly develop fevers in excess of 108°F and quickly succumb; all described patients thus far have died.89

Finally, although not well described in the literature, CA-MRSA USA200 strains are emerging. These strains also cause skin and soft tissue infections and are associated with serious pulmonary diseases, but their CA-MSSA counterparts also are well known to cause menstrual TSS. On the basis of our tests of the organisms, regional differences exist in SCC mec DNAs associated with the strains. In Minnesota, nearly all of the isolates have SCC mecII, reminiscent of HA-MRSA, but in isolates tested from New York, both SCC mecII and IV may be present. The isolates rarely produce PVL; the strains variably produce α-toxin, β-toxin, and γ-toxin; but the strains all produce δ-toxin and presumably other PSMs. These isolates also produce the superantigen TSST-1, because these isolates are the predominant causes of both menstrual and one-half of nonmenstrual TSS.

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Comparison of exotoxin production by USA200 CA-MRSA versus CA-MSSA 

In 1982, Schlievert et al91 showed that menstrual, vaginal mucosal TSS S aureus strains produce less α-toxin and more TSST-1 than skin S aureus isolates. It was hypothesized that high levels of cytolysins are required for producing inflammation to disrupt the intact skin barrier and facilitate S aureus infection. Conversely, production of the same amounts of cytolysins on mucosal surfaces would lead to such strong host responses that the host would either quickly succumb to overwhelming infection or the microbe would quickly be eliminated by the host immune system. These studies have been confirmed by others who reported that most menstrual TSS S aureus isolates have a defective α-toxin structural gene, containing a stop codon within the gene.92

These studies suggest that S aureus strains may be selected to occupy niches in the host based on secreted virulence factor production,91 but the data highlight an unresolved issue. Our previous studies suggested that menstrual, vaginal TSS isolates made reduced amounts of α-toxin, but not zero α-toxin.91 In contrast, the presence of the stop codon within the α-toxin gene indicated there should be no α-toxin produced.92 With use of hyperimmune antibodies to α-toxin, we have recently studied production of α-toxin (Table II) in vitro by representative menstrual TSS CA-MSSA USA200 isolates (example: TSST-1+ CDC587, containing a stop codon within the α-toxin gene), nonmenstrual TSS CA-MSSA USA200 (example: TSST-1+ MNPE with wild-type α-toxin gene), and typical menstrual TSS CA-MRSA USA200 (example: TSST-1+ isolate MNWH with a stop codon in the α-toxin gene). None of these isolates produce PVL and β-toxin. (It is important to note that the USA200 strains used in the following discussion are typical of large numbers of organism analyzed.) Strain MNPE, with a wild-type α-toxin gene, as expected produced 55 μg/mL α-toxin by red cell lysis bioassay. Surprisingly, CDC587 also produced α-toxin in spite of the stop codon in the α-toxin gene, but at approximately 10-fold lower concentrations (5 μg/mL). In addition, the CA-MRSA USA200 isolate MNWH produced 3.5μg/mL α-toxin despite having the α-toxin gene stop codon. Thus, menstrual TSS S aureus strains have developed “read through” mechanisms to allow some translation of α-toxin protein, even in the presence of a stop codon. Although not previously described for S aureus, at least 2 mechanisms have been described for this phenomenon in gram-negative bacteria: (1) altered transfer RNAs to allow an amino acid to be inserted instead of recognition of the stop signal, and (2) altered ribosome structure. Both of these mechanisms can allow “read through” at approximately 1/10 wild-type level.

Table II. Production of virulence factors by TSST-1+ CA-MRSA and CA-MSSA USA200 S aureus
Strainsα-Toxin (μg/mL)TSST-1 (μg/mL)Pigment (yes/no)
Menstrual CA-MSSA TSS (CDC587)5.03Yes
Postviral respiratory TSS (MNPE)553Yes
Menstrual CA-MRSA TSS (MNWH)3.515No

Total exoprotein production by the same strains was also examined, including the production of TSST-1 (Fig 4). CDC587, with the stop codon in the α-toxin gene, produced many exoproteins, but in low amounts, and produced approximately 3 μg/mL TSST-1. Strain MNPE, with wild-type α-toxin, produced fewer exoproteins than CDC587, but also produced approximately 3 μg/mL TSST-1. Finally, strain MNWH produced almost no exoproteins except TSST-1, with TSST-1 produced in higher concentrations than in the CA-MSSA strains. These data suggest that total protein production burden as a function of host niche can posttranscriptionally control exotoxin production. Thus, MNPE is presumed to be a skin isolate of S aureus because of its wild-type α-toxin gene; the strain unfortunately was isolated from a death case of postinfluenza pulmonary TSS93 with the assumption that like USA300 and USA400 CA-MRSA, the organisms are skin isolates that occasionally are transferred into the lungs to cause highly fatal TSS-like illnesses.66, 69, 84 The production of high levels of α-toxin appears required for skin survival of S aureus, and in producing high levels of α-toxin, the organism, MNPE, reduces production of many other, less essential secreted virulence factors than seen with CDC587. When the additional burden of the large SCC mecII DNA element is added into the USA200 organism, as in MNWH, even fewer secreted virulence factors are produced, but TSST-1 amounts are increased. It appears that high-level production of TSST-1 is required, more so than other secreted factors including cytolysins, for survival of the organism.

  • View full-size image.
  • Fig 4. 

    Secreted protein profiles of CA-MSSA USA200 strains CDC587 and MNPE and CA-MRSA USA200 strain MNWH. All organisms were cultured to stationary phase, cells removed by centrifugation, sterile supernates concentrated 10-fold, SDS-PAGE performed, and gel stained with Coomassie brilliant blue R-250.

S aureus strains also produce carotenoid pigments, which in certain host niches appear to function as virulence factors.94 Fig 5 shows carotenoid pigment production by strains CDC587, MNPE, and MNWH. Both CDC587 and MNPE produce the typical gold pigment expected from S aureus strains. In contrast, the CA-MRSA USA200 strain does not produce detectable pigment. As noted, the presence of the large SCC mecII DNA may have required selection in the host of an organism that cannot produce pigment (as well as not producing other secreted virulence factors).

  • View full-size image.
  • Fig 5. 

    Carotenoid pigment production by CA-MSSA USA200 strains CDC587 and MNPE, and CA-MRSA USA200 strain MNWH. Organisms were cultured to the stationary phase, and cells were pelleted by centrifugation.

Our analysis of the CA-MRSA and CA-MSSA USA200 strains suggests that production of secreted virulence factors among these highly related strains can vary considerably on the basis of host niche and virulence factor gene load, and determine collective production of secreted virulence factors. One additional observation should be noted with respect to the typical menstrual USA200 CA-MSSA TSST-1+ organisms with the stop codon in the α-toxin gene. In many ways, the organisms resemble normal flora coagulase-negative staphylococci, except for low-level production of cytolysins and production of TSST-1. Our recent studies suggest that production of α-toxin in low amounts by these isolates facilitates penetration of TSST-1 across the mucosal barrier.21 We hypothesize that the low production of α-toxin by these strains in the presence of a stop codon in the structural gene provides a novel, nontranscriptional regulatory mechanism for the organism. It is possible that these organisms have developed mechanisms to “read through” the stop codon. If strain USA200 CA-MSSA MNPE were to be found vaginally, the strain would either cause so much inflammation that the organism would be quickly eliminated by the host immune system, or the organism would quickly kill the host through increased TSST-1 penetration or sepsis.

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Comparison of exotoxin production by USA400 CA-MRSA versus CA-MSSA 

As S aureus strains colonize human mucous membranes and skin, as observed in patients with AD, the organism is present primarily as thin biofilms of organisms growing on surfaces, rather than as planktonic (broth) cultures. As noted earlier, even in the bloodstream, it appears S aureus quickly becomes walled off from the blood into biofilms through the actions of coagulase and clumping factors.47 The data are consistent with the organism having difficulty producing secreted virulence factors when present in blood50; thus, it may be selectively advantageous for the organisms to be sequestered from the blood to facilitate production of cytolysins and superantigens, both of which may be critical to protection from the host immune system.

We have recently performed studies to assess production of TSST-1 and SEC in vitro by CA-MRSA and CA-MSSA USA200 and USA400 strains, respectively, in biofilm versus planktonic cultures, in the absence of blood. The studies confirmed the studies in Fig 5 showing that CA-MRSA USA200 strains produce more TSST-1 in planktonic cultures than their CA-MSSA counterparts, but the studies also showed that this difference is magnified in biofilm cultures. CA-MRSA USA200 strains produced approximately 4000 μg/mL TSST-1 in tampon biofilms in vitro, compared with 1000 μg/mL produced by CA-MSSA USA200 isolates under the same conditions. The data also emphasized that biofilm cultures may have 200 times more TSST-1 produced compared with planktonic cultures, presumably as a result of quorum-sensing and other global transcription regulation.47 We have also compared CA-MRSA USA400 strains with CA-MSSA USA400 strains for production of SEC in biofilm versus planktonic cultures. Results similar to those with USA200 strains were obtained, in that CA-MRSA USA400 strains made more SEC than CA-MSSA USA400 strains in planktonic cultures, that both organisms made as much as 200 times more SEC in biofilm cultures than planktonic cultures, and that CA-MRSA USA400 strains made more SEC in biofilms than CA-MSSA USA400 strains.

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Conclusions, future research directions, and clinical implications 

One purpose of this review was to emphasize that S aureus strains have become selected for their various niches within human beings as a function of secreted virulence factors produced. When they become stressed by the host, as in attempting to survive on the skin versus on mucous membranes, or when they must grow in the presence of antibiotic therapy, as in acquisition of SCC mec DNA, the organisms specialize in production of secreted virulence factors, progressively emphasizing production of “most necessary” factors, including cytolysins and superantigens. Thus, the mucosal, menstrual vaginal TSST-1+ CA-MSSA USA200 isolate is the least specialized of organisms this review discusses. It appears the organism maintains its ability to colonize mucosal surfaces and resemble normal flora coagulase-negative staphylococci by production of only low levels of secreted virulence factors. In contrast, when the same organism produces wild-type α-toxin, needed for skin survival, and combined with TSST-1 production, the organism loses ability to produce large numbers of secreted factors. The same is the case when the organism acquires the SCC mec DNA. The organism upregulates production of TSST-1 but produces almost no other secreted factors, as if gene expression from the SCC mec element places an excessive burden on the microbe.

There appear to be multiple mechanisms that regulate production of secreted virulence factors as a function of host niche. First, the niche itself regulates virulence factor production through influences of nutrient availability and the presence of the innate and adaptive immune systems. S aureus also has at least 2 major types of regulatory systems that influence secreted virulence factor production: (1) global transcriptional regulators, and (2) previously unrecognized posttranscriptional regulators, examples of which are discussed in this review. Through combinations of these mechanisms, S aureus has become the master commensal and ultimate pathogen, causing more total and more types of infections than any other organism. At the same time, the abundance of these mechanisms makes it difficult to sort out their relative contributions to virulence factor expression and may have contributed to our lack of recognition of the importance of S aureus in numerous human diseases. For example, our current studies are the first to explore the major influence of SCC mecII DNA on virulence factor secretion. It is our opinion that studies of the influence of posttranscriptional mechanisms provide cutting edge research for the next 5 or more years.

It is well recognized that S aureus strains colonize the anterior nares of human beings. Indeed, it is commonly thought that this body site most often is the source of staphylococcal diseases occurring at other body sites. This thought must now change. With the emergence of CA-MRSA USA300 and 400 isolates, we now recognize that the skin is often the source of potentially highly virulent S aureus strains, such as those causing fatal pulmonary infections. Patients with AD provide an exceptionally rich source of S aureus strains, and as shown in this review, required treatment of such patients can select for a variety of even more pathogenic strains. This makes it clear that we as a research community must develop as great an understanding of skin S aureus strains in AD, as the research community is attempting to do with understanding mucosal isolates. This is necessary not only to manage AD, but also to understand and manage the contribution of S aureus to noncutaneous infections.

Finally, what are the potential clinical implications in AD of the emergence of CA-MRSA versus HA-MRSA and MSSA strains? First, it is important to recognize that CA-MRSA generally differs from both HA-MRSA and MSSA strains in antibiotic susceptibility patterns.8, 69 CA-MRSA strains are methicillin-resistant, and some have become clindamycin-resistant, but generally these organisms are susceptible to non–β-lactam antibiotics (Table I). In contrast, HA-MRSA typically is resistant to larger numbers of antibiotics than CA-MRSA, and MSSA strains predictably produce β-lactamases but are generally susceptible to the majority of β-lactam antibiotics. This difference in antibiotic susceptibility patterns allows the clinician also to predict whether the patient is likely to be infected with strains that produce high levels of the superantigens, TSST-1, SEB, and SEC, exotoxins that are most often associated with serious staphylococcal diseases (including TSS, purpura fulminans, and necrotizing pneumonia). These illnesses often occur as the result of spread from skin sources. As indicated, CA-MRSA USA200 strains produce TSST-1; CA-MRSA USA300 strains produce a toxic, deletion variant of TSST-1 (and SEl-Q); and CA-MRSA USA400 strains produce either SEB or SEC. HA-MRSA strains typically do not produce these superantigens.69 In addition, we are preparing a manuscript to describe a new clinical presentation associated with S aureus superantigens that relate to skin infections: TSS associated with AD skin infections and impaired healing in patients. Some of these patients have had infections with CA-MRSA TSS S aureus (producing TSST-1, SEB, or SEC) for more than a year and have exhibited accompanying weight losses in excess of 50% of their original body weights. Resultant skin abscesses from such infections have been the size of footballs, and in many cases the infections simply do not heal. Our preliminary studies suggest that both IgE responses to superantigens and the presence of superantigens themselves contribute to the failure of skin lesion healing.

We have also demonstrated that CA-MRSA isolates typically produce more superantigens than their corresponding CA-MSSA counterparts. The clinical significance of this is incompletely understood. However, microbial stress induced by clinical treatment of patients, such as patients who develop steroid-resistant AD and those whose organisms have acquired SCC mec DNA, increases production of superantigens by the associated S aureus strains. Because we do not have a full understanding of all superantigen-induced staphylococcal illnesses, we likewise do not completely understand the significance of the increased superantigen production by the strains; presumably, increased amounts of superantigen improves their survival in the host but at the same time may lead to more serious illnesses.

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 Supported by National Institutes of Health grants R01s AI74283, AI73366, and AR41256; U54-AI57153 (Great Lakes Regional Center of Excellence in Biodefense and Emerging Infectious Diseases, where P.M.S. is a member); and contract N01 AI40029 to D.Y.M.L.

 Disclosure of potential conflict of interest: P. M. Schlievert receives research support from the NIH and has provided legal consultation/expert witness testimony in cases related to streptococcal toxic shock syndrome. M. L. Peterson receives research support from the NIH and 3M. D. Y. M. Leung receives research support from the NIH/NIAID, the NIH/NIAMS, and Novartis Pharmaceuticals. The rest of the authors have declared that they have no conflict of interest.

PII: S0091-6749(09)01605-4

doi:10.1016/j.jaci.2009.10.039

The Journal of Allergy and Clinical Immunology
Volume 125, Issue 1 , Pages 39-49, January 2010

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