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Mobile genetic elements and bacterial toxinoses: the superantigen-encoding pathogenicity islands of Staphylococcus aureus
Plasmid 49 (2003) 93–105 www.elsevier.com/locate/yplas
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Mobile genetic elements and bacterial toxinoses: the superantigen-encoding pathogenicity islands of Staphylococcus aureus Richard P. Novick* Molecular Pathogenesis Program, Departments of Microbiology and Medicine, Skirball Institute, New York University School of Medicine, New York, NY 10016, USA Received 17 September 2002, revised 4 October 2002
Abstract It is a remarkable observation that virtually all bacterial toxins associated with specific clinical conditions (toxinoses) are encoded by mobile (and therefore variable) genetic elements. Remarkably, these rarely, if ever, carry determinants of antibiotic resistance. Examples are the toxins responsible for diphtheria, anthrax, tetanus, botulism, cholera, toxic shock, scarlet fever, exfoliative dermatitis, food poisoning, travelersÕ diarrhea, shigella dysentery, necrotizing pneumonia, and others. A recently discovered example of this phenomenon is the family of related staphylococcal pathogenicity islands encoding superantigens (SAgs). These are 15–20 kb elements that occupy constant positions in the chromosomes of toxigenic strains, and are characterized by certain phage-related features, namely genes encoding integrases, helicases, and terminases, and the presence of flanking direct repeats. The prototype, SaPI1 of Staphylococcus aureus, encodes TSST-1 plus two newly described SAgs, SEK and SEL. Other members of the family encode enterotoxins B (SaPI3) and C (SaPI4), plus at least two other SAgs each. SaPI1 and SaPI2, also encoding TSST-1, are excised and induced to replicate by certain staphylococcal phages, and are then encapsidated at high efficiency into phage-like infectious particles with heads about 1/3 the size of the helper phage heads, commensurate with the sizes of the respective genomes. This results in transfer frequencies of the order of 108 /ml, and is presumably responsible for the spread of these elements as well as for their acquisition in the first place. In the absence of a helper phage, these two islands are highly stable; neither excision, loss, or transfer occurs at detectable frequency. Several general implications of this phenomenon will be discussed. One is that the determinants of these toxins have been imported from other species and therefore are not components of the basic genome of the extant producing organisms. This raises the question of the biological (adaptive?) roles of these toxins. Another is that the toxin-carrying units can spread among different (though probably related) species. An interesting question is that of the biological basis for the separation of toxin and resistance determinants. Ó 2003 Published by Elsevier Science (USA).
1. Introduction
*
Fax: 1-212-263-5711. E-mail address: [email protected].
A wide variety of bacterial accessory functions, including inducible catabolic pathways, restriction-modification systems, antibiotic resistance,
0147-619X/03/$ - see front matter Ó 2003 Published by Elsevier Science (USA). doi:10.1016/S0147-619X(02)00157-9
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and pathogenesis are determined by variable genes—genes that belong to larger units that are present in some strains of a given species and entirely absent in others. Some of these, such as plasmids, prophages, and functional transposons are clearly mobile; others, such as integrated plasmid-like elements, defective prophages and most chromosomal islands, are probably remnants of formerly mobile units. Among the variable genes involved in pathogenesis are those encoding toxins, of which a major subset is responsible for toxinoses—diseases of which a single toxin is both a necessary and sufficient cause. A remarkable feature of these toxins is that, with one known exception, Clostridium perfringens a-toxin, which is a standard chromosomal gene and, on its own, causes gangrene (J. Rood, personal communication), all are encoded by highly mobile genetic elements. As listed in Table 1, these elements include plasmids, prophages, transposons, and mobile pathogenicity islands. It is very likely that all such elements have been acquired from other species, though no suspects have yet been identified. Nevertheless, the toxin genes are all controlled by chromosomally encoded regulatory factors, raising the important question of how such regulation
may have evolved, and suggesting, incidentally, that the putative donor species must be fairly closely related to the extant toxin-producers. Mobile genetic elements are arguably selfish in that their evolution is driven by selective forces that operate on the elements themselves, independently of the host organisms within which they must of necessity reside. Nevertheless, these elements often encode products that have obvious selective value for the host. Among these is pathogenesis, which can be viewed as a consequence of the organismÕs attempt to thrive in the hostile environment of the metazoan body. The selectivity of toxinosis-causing toxins however, is not always obvious. Many of the enterotoxins, for example, cause disease in the absence of the producing organism. And even when the organism is present, it derives no discernable advantage from production of the toxin. Among these organisms are bacilli (Bacillus cereus), staphylococci (Staphylococcus aureus), and clostridia (C. perfringens, Clostridium botulinum), which produce enterotoxins causing food poisoning but are not enteric pathogens per se. Three possible explanations for this curious feature are (i) the toxin is a protein with some unknown function that is accidentally toxic; (ii) the toxicity
Table 1 Toxinoses and variable genetic elements Disease
Toxin
Organism
Element
Features
Diphtheria Anthrax Cholera Dysentery Botulism Enterocolitis Gangrene Tetanus Enterocolitis Diarrhea Diarrhea Toxic shock syndrome Food poisoning, TSS Food poisoning, TSS Food poisoning, TSS Scalded skin syndrome Scalded skin syndrome Necrotizing pneumonia Scarlet fever
Diphtheria toxin Anthrax toxin Cholera toxin Shiga toxin Botulin toxin Cpe toxin a-toxin Tetanus toxin Enterotoxin Labile toxin (LT) Stable toxin (ST) TSST-1 Enterotoxin A Enterotoxin B, C Enterotoxin D Exfoliatin A Exfoliatin B Leukocidin SPEA, C
Corynebacterium diphtheriae Bacillus anthracis Vibrio cholerae Shigella dysenteriae Clostridium botulinum Clostridium perfringens Clostridium perfringens Clostridiun tetani Clostridium difficile Escherichia coli Escherichia coli Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Streptococcus pyogenes
b-Prophage Plasmid Prophage Prophage Prophage Transposon Chromosomea Plasmid Path islet Plasmid Transposon Path island Prophage Path island Plasmid Prophage Plasmid Prophage Prophage
35 kb 110 kb Filamentous Lambdoid 110–165 kb IS14702 ::cpe 6 kb
a
‘‘Large’’ 3 kb 90 kb F-like IS12 ::cat 3 kb 15 kb /-related 45 kb 15 kb /-related 30 kb 45 kb 30 kb 45 kb 45 kb
C. perfringens a-toxin is the single exception to the rule that toxinosis-causing toxins are encoded by mobile genetic elements.
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of the protein does, in fact, provide the organism with a selective advantage, for example, toxicity for some organism that is either predatory or is a source of nutrition for the toxin-producer; (iii) the toxin gene is selected by evolutionary forces that are specific for the toxin-encoding element and have nothing to do with the biology of the host bacterium. Other toxins provide a clear selective advantage. These include cholera, diphtheria, anthrax, and C. perfringens a toxins, and the superantigens (SAgs) of S. aureus and streptococci. This presentation is focused on the staphylococcal SAgs, many of which are encoded by novel pathogenicity islands, the first elements of this type for which mobility has definitively been demonstrated (Lindsay et al., 1998). Different S. aureus strains produce different combinations of superantigen toxins. Strains lacking a given toxin also lack the gene that encodes it plus flanking chromosomal sequences. The first observations of this involved enterotoxins A (SEA) and D (SED) which are encoded by prophages (Betley and Mekalanos, 1985) and plasmids (Bayles and Iandolo, 1989), respectively. More recently, we have identified a series of discrete 15–20 kb chromosomal elements that encode SAgs, reside at specific locations and are mobilized at high frequencies by certain staphylococcal phages. These are referred to as staphylococcal pathogenicity islands (SaPIs). The genes for toxic shock toxin and for enterotoxins B, C, K, and L are all carried by elements of this type. Several of these have been sequenced and their genetic maps are shown in Fig. 1. The prototype of the family is SaPI1, whose genetic analysis was greatly aided by the construction of a derivative with tetM inserted into tst, the gene for toxic shock syndrome toxin-1 (TSST-1) (Sloane et al., 1991). Two properties of SaPI1 led to realization that it was a novel type of mobile element: first, the transduction frequency of SaPI1::tetM was extremely high—about 101 — with phage 80a, whereas it was about 107 with the closely related generalized transducing phage, /11. Second, extrachromosomal SaPI1::tetM DNA could be detected in 80a lysogens but not in /11 lysogens (Lindsay et al., 1998). Following UV induction of an 80a lysogens carrying the element, a
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SaPI1-specific band with mobility corresponding to a linear form of the element appeared in agarose gel separations of whole cell DNA and was amplified during the phage lytic cycle. A similar band was seen upon infection of a non-lysogen with an 80a-SaPI1 mixed lysate (Fig. 2A). This band was shown by Southern blotting to represent SaPI1 (Fig. 2C). No such band was seen with /11 or with several other staphylococcal transducing phages, none of which transduced SaPI1 at elevated frequencies. Superinfection with /13 caused the appearance of SaPI1-specific circular supercoiled material that did not detectably replicate (Ruzin, 1999), and was not incorporated into transducing particles by this phage, which is not a generalized transducing phage. However, a similar band was seen following superinfection of several menstrual toxic shock strains, in which tst is at a different location than that of SaPI1 (Chu et al., 1988), with the related phage 80 (Fig. 3). This species had a number of different mobility variants but always migrated considerably more rapidly than the SaPI1 and has been designated SaPI2. It is not presently known whether this band represents linear or circular DNA. Thus far, phage-induced excision/replication has yet to be demonstrated for the other SaPIs illustrated in Fig. 1. SaPI1 transducing particles could be readily separated from 80a plaque-forming particles on the basis of slower sedimentation in a sucrose gradient but could not be separated on the basis of buoyant density, suggesting that the transducing particles were smaller. Electron microscopy (Fig. 4) showed that the SaPI1 particles were morphologically similar to the 80a particles but had smaller heads, with a volume about 1/3 that of the plaque-forming particles, commensurate with the difference in genome size (18 kb for SaPI1::tetM, 45 kb for 80a (Lindsay et al., 1998)). Thus, SaPI1 is evidently encapsidated in the monomeric form by SaPI1induced small-headed 80a-like phage particles. The monomeric form was confirmed by infecting a standard recipient with purified SaPI1 particles and then analyzing the infected cells for extrachromosomal dna. Again, a DNA species corresponding to SaPI1 linears was seen immediately following infection; however, in this case, no amplification was observed and the SaPI1 band gradually diminished
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Fig. 1. Comparative linear maps for seven SaPIs. Numbered arrows represent open reading frames >65 codons. Arrows in red represent reading frames similar to known or suspected phage genes; arrows in aqua represent reading frames conserved among the SaPIs of known sequence; those in blue represent non-conserved reading frames of unknown function; those in green represent genes involved in pathogenesis. Flanking chromosomal sequences are indicated as gray boxes. TetM and erm are selective marker insertions (Fitzgerald et al., 2001; Sloane et al., 1991). Ear is an apparently incomplete ORF potentially encoding part of a b-lactamase-like protein (P. Orwin and P. Schlievert, personal communication).
in intensity during an 800 time course (Fig. 2D). It is suggested that the high frequency transfer of SaPI1 by 80a, which differs considerably from conventional transduction, represents a mechanism of horizontal dissemination of the pathogenicity island. The identity of the capsid protein(s) of the small particles and the mechanism by which they are produced, however, remains to be determined.
the absence of SaPI1. This is reflected in commensurately low titers of phage lysates and in a dramatic reduction in phage sensitivity in spot tests. The latter has provided a means of scoring for the presence of SaPI1 and has enabled transduction of the native SaPI1 element without the need for a selective marker (Ruzin, 1999). Precisely which phage functions are parasitized by SaPI1 has not been determined, though it is likely that replication and encapsidation functions are involved.
2. Interference When propagated on a SaPI1-containing strain, either by UV induction of an 80a lysogen or by exogenous infection of a non-lysogen, the single burst size of the phage is about 1% of that seen in
3. Integration Following transfer, by either transformation or transduction, SaPI1 integrates, presumably by the
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Fig. 2. The fate of incoming SaPI1 DNA. (A) Co-infection with 80a and SaPI1. Bacteria (RN450) were infected with a mixed 80a lysate at a multiplicity of 1:1. Infected bacteria were incubated at 32 °C in CY broth + phage buffer and sampled at the indicated times for preparation of SDS minilysates, which were analyzed by agarose gel electrophoresis. Control (c)—minilysate of SaPI1 strain infected with 80a and sampled at 45 min post-infection. (B) Southern blot hybridization pattern of the gel in A. The gel was transferred to a nylon membrane and hybridized overnight with peroxidase-labelled (ECL, Amersham) SaPI1-specific probe (PCR product using primers p95 and p96 (Ruzin et al., 2001)). (C) Infection of RN27 (80a lysogen) with the same mixed lysate. Bacteria (RN27) were infected with the same lysate as in A, and analyzed as above. (D) Infection of non-lysogenic recipient with purified SaPI1 transducing particles. Bacteria (RN450) were infected with purified SaPI1 transducing particles, separated by sucrose gradient sedimentation from plaque-forming particles (Ruzin et al., 2001), and analyzed as above (adapted from Ruzin et al., 2001). (E) Plot of the relative change in SaPI1 DNA with time derived from the data in panels A, C, and D, respectively.
classical Campbell mechanism, into a unique chromosomal recognition site, attC , which is present in many strains and contains a copy of the 17 nt repeat sequence found at the ends of the integrated element (Figs. 1 and 4). A SaPI1 subclone containing only int and attS integrated efficiently, whereas a derivative of this clone with a deletion affecting int did not (Ruzin, 1999), indicating that the SaPI1-encoded integrase is necessary and is the only SaPI1 function required for integration. No
phage function is required. The efficiency of integration is probably close to 100%, as judged by enumeration of the two types of particles seen in the electron microscope in comparison with transducing and plaque-forming titers determined for the corresponding lysate. Following infection with a mixed lysate of an 80a lysogen or of a nonlysogen (Figs. 2A and B), the incoming linear SaPI1 DNA gives rise to the circular form, which is first detectable about 40 min after infection. This
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Fig. 3. Excision of SaPI-like elements in naturally occurring TSST-1-producing strains by phage 80. Mid-exponential phase cultures were superinfected with phage 80 at a multiplicity of 3. Samples were removed 45 min after phage infection and used to prepare standard mini-lysates which were separated on 0.7% agarose in TBE, stained with ethidium bromide and photographed. RN3984 is a prototypical menstrual TSS isolate (‘‘Harrisburg’’). Numbers 1–7 represent naturally occurring menstrual TSS isolates sensitive to phage 80, all kindly provided by P. Schlievert (Ruzin, 1999).
Fig. 4. Electron microscopy. Phage particles from the lysate used in Fig. 2A were purified, mounted on carbon grids, stained with 2% PTA and photographed at 31,500 magnification. Note that there are particles of two different sizes, the smaller ones (arrow) corresponding to SaPI1 transducing particles, the larger to 80a particles (reproduced from (Ruzin et al., 2001), with kind permission of the publisher).
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is followed by the appearance of a SaPI1 signal in the chromosomal gel band (Fig. 2C), which is assumed to represent integration. This process seems to be relatively slow, since a strong linear SaPI1 DNA band persists for over 2 h. In a recipient containing SaPI1, an incoming SaPI1::tetM precisely replaces the resident SaPI1, even in a recA recipient (Lindsay et al., 1998). The replaced copy disappears since the transductants no longer produce TSST-1. This is different from what is usually seen with a superinfecting temperate phage, such as k, which integrates either at an unlinked secondary site or at one end of the resident prophage genome, with the latter remaining in place (Hendrix, 1983). It is likely that the SaPI1 integrase is responsible for this, since the element does not encode any detectable xis function (see below), and since it occurs with a SaPI1 subclone containing only int and attS (Ruzin, 1999; Ruzin et al., 2001).
4. Excision The SaPI1 sequence does not contain any ORF resembling phage xis determinants, and spontaneous SaPI1 excision has not been detected in the absence of vegetative phage, even by (single-stage) PCR analysis (Ruzin, 1999). Nor do subclones containing int induce the excision of a resident SaPI1 (Ruzin et al., 2001). It thus seems very likely that SaPI1 does not encode any xis function, and that SaPI1 integrase cannot mediate excision of the element. Clearly, 80a encodes the xis function that is used for SaPI1 excision, and it is assumed that this is the same function used by the phage for its own excision, following induction of a lysogen. If this is correct, it means that excision is not sequence-specific, since the phage and SaPI1 att sites are unrelated. Among several other staphylococcal phages tested, only one, /13, also catalyzed SaPI1 excision. However, the consequences of excision by the two phages were very different; whereas the product of 80a excision was immediately converted to the linear replicating form, the product of /13 excision persisted as the circular duplex monomer and did not replicate to a significant extent (Ruzin et al., 2001).
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5. Replication Following excision by 80a, SaPI1 DNA replicates autonomously to a level of at least 120 copies per cell. The only topological form that can be detected during the early stages of this amplification appears to correspond to linear monomers (Fig. 2). Thus, the SaPI1-specific band that appears following UV induction of an 80a lysogen has the same mobility as a linear SaPI1 monomer and is the same as that seen following the introduction of SaPI1 by phage infection. Though it is possible to detect SaPI1 DNA with joined ends by PCR immediately after excision, circular monomers are not seen in agarose gels or by Southern blotting until late in the infective cycle (Fig. 2); thus, the circular form must be linearized very rapidly after 80a-induced excision. Further, the incoming linear monomers, unlike the classical temperate phages, seem to enter the replication pathway directly, without going through a circular intermediate. It is suggested on the basis of these results that SaPI1 replicates as a linear molecule. Of several staphylococcal phages tested, only 80a induced SaPI1 replication, indicating an intimate relation between the two genetic elements. Attempts to define a phage-responsive replication origin for SaPI1 have thus far been unsuccessful, though the entire element has been subcloned as overlapping fragments to a plasmid vector with a thermosensitive replicon. Although a subclone containing int and attS was transduced at a much higher frequency than any of the other subclones (Ruzin et al., 2001), it is not clear whether this represents phage-induced replication. No other subclone supported growth at the restrictive temperature, or showed 80a-induced amplification or any elevation in transducing frequency. However, the possibility has not been ruled out that the circular subclones, which lack attC , could not be converted by 80a to a substrate (linear?) suitable for phage-induced replication.
6. Encapsidation Phage 80a dna is terminally redundant and circularly permuted (Stewart et al., 1985) and the
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same is true for SaPI1. As shown in Fig. 5 (middle), SaPI1::tetM DNA from transducing particles contains all of the restriction fragments expected from circular DNA; however, those with one end in the shaded region are always present in submolar amounts—for an example, in the HindIII digest shown inside of the circular map, the band representing the 6 kb fragment (8174–14,314) has about 1/4 of the expected intensity. Digestion with singly cutting enzymes always generates one or two smeared bands, representing linear fragments with variable ends that lie within the shaded region. The BglI digest at lower left shows one of the two expected smeared bands, with fragments between 8 and 15 kb in length. The other expected smear, with fragments from 0 to 8 kb is not seen, probably because the intensities are too low. Thus,
there is a limited number of permutations, and the element must be encapsidated from concatemers composed of the same average number of repeats as the number of permutations. For a more detailed description of these results, readers are referred to Ruzin et al. (2001), and to earlier papers describing the same phenomena in coliphage p22 (Spanova, 1992; Tye et al., 1974). At least one SaPI1 gene potentially involved in encapsidation (ter in Fig. 1) has been identified as an open reading frame whose predicted product is highly similar to the small terminase subunit encoded by several phages from Gram-positive bacteria (Chai et al., 1994). This reading frame is also present on SaPI3 (Novick et al., 2001), and SaPIbov (Fitzgerald et al., 2001), and is absolutely conserved.
Fig. 5. Demonstration of circular permutations in SaPI1 DNA. At upper right is an agarose gel electropherogram of SaPI1::tetM DNA isolated from the circular duplex band shown in Fig. 2A (left-hand lane), and the same DNA digested with the singly-cutting enzyme, NheI (right-hand lane). The upper band in the left lane represents contaminating linear SaPI1 DNA. This material is responsible for the smeared fragment pattern seen below the linear 18 kb band in the right-hand lane. At lower left is an electropherogram of SaPI1::tetM DNA isolated from transducing particles and digested with either BglI (right lane) or undigested (left lane). Note that the undigested dna is about 90% SaPI1 (18 kb) linears and about 10% (45 kb) 80a linears, and that the smeared fragments in the BglI digest range from 8–15 kb, represented by the innermost segment (arrow). Inside the SaPI1 map is an electropherogram of SaPI1 DNA from transducing particles digested with HindIII alongside the standard 1 kb ladder. The 6 kb marker indicates the fragment between the HindIII sites at 8174 and 15301, which is present in a submolar amount. This fragment is delineated by the first inner segment (arrow). The shaded area represents the apparent locations of the ends of the permuted SaPI1 molecules.
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7. Phage specificity 80a is not a naturally occurring phage, having been isolated as a host-range variant by plating typing phage 80 on strain NCTC8325. Subsequent studies have shown that 80a is not a restrictionmodification variant but is a recombinant with segments from at least two other phages, /11 and /13, that are present as prophages in NCTC8325 (Novick, 1967). Since neither phage 80 nor /11 can catalyze the excision of SaPI1, whereas /13 can, it is assumed that the SaPI1-specific xis function has been derived from /13. Since neither /11 nor /13 can support SaPI1 replication, it is assumed that the replication capability is derived from phage 80 or is a consequence of the recombination. Since 80 does not grow on SaPI1 strains, it has not been possible to determine whether it can support the replication of autonomous SaPI1.
8. The SaPI family With the advent of genome sequencing and micro-array technology, inserted chromosomal gene blocs are being increasingly recognized. A prototypical class of these is the SaPIs, phagerelated chromosomal islands that have the following features in common: size 15–20 kb, specific att sites and integrases, several phage related genes including ter, 3 or more SAg genes, and specific interactions with certain phages leading to excision, amplification, and encapsidation. In our hands, distinct SaPIs have been given different numbers; others have appended specific notations to the generic term SaPI—such as -bov (Fitzgerald et al., 2001) and -bap (Cucarella et al., 2001), referring to bovine origin and to the carriage of an adhesin gene, bap, respectively. SaPI2, SaPI4, and SaPIbov. Two other SaPIs encoding TSST-1 have been identified (SaPI2 and SaPIbov), and there are clearly others, on the basis of apparently different chromosomal locations (Lindsay et al., 1997). And TSST-O, produced by ovine strains of S. aureus (Lee et al., 1992) is likely to be encoded by a further SaPI variant. SaPI2, located within the tryptophan locus in the prototypical menstrual TSS strain, RN3984, is found in many other menstrual
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TSS strains, which appear to comprise a rather coherent biotype and probably represent a tight clone (Musser et al., 1990). SaPI2 is induced to excise and replicate by typing phages 29, 52, and 80, of which the latter transduces SaPI2::tetM at frequencies similar to the frequency of transduction of SaPI1 by 80a (Ruzin et al., 2001). As noted above, all of seven menstrual TSS strains sensitive to phage 80 showed excision and amplification of SaPI2-like bands, with some differences in mobility, following infection with this phage. Although there is only a very preliminary map of SaPI2, based on blot hybridization analysis with SaPI1specific probes (Lindsay et al., 1997), it appears on the basis of gel mobility to be considerably smaller than SaPI1. Comparison of the conserved regions of the two genomes is expected to provide important information on the SaPI-phage interaction. SaPIbov, recently identified in a bovine S. aureus strain (Fitzgerald et al., 2001) is similar in size to SaPI1. However, in contrast to SaPI1 and SaPI3, it is flanked by a 74 nt direct repeat, of which there is a single copy in a TSS-negative bovine strain. Some human strains contain part of this, but none has been found so far that contains the entire sequence (Fitzgerald et al., 2001). None of three phages tested, 80a, /11, or 85, were able to excise or transduce SaPIbov, including a derivative containing tetM in the tst gene. SaPI4, for which sequencing and analysis in progress (P. Schlievert, personal communication), is quite similar to SaPI1 but has sec in place of tst, contains the genes encoding three additional SAgs, sek, sel, and sem, and integrates at a different site (P. Orwin and P. Schlievert, personal communication). No inducing phage has been identified to date.
9. SaPI3 SaPI3, which encodes enterotoxin B (SEB), is carried by strain COL, which has recently been sequenced by Tigr (www.tigr.org), so that its sequence has been determined automatically (Novick et al., 2001). It has the same 17 nt flanking direct repeat and is located at the same site as SaPI1—which may account in part for the wellknown fact that TSST-1 and SEB are rarely if ever
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co-produced (De Boer and Chow, 1994). It is likely that SaPI3 is responsible for the problematic genetic results observed previously with SEB (Shafer and Iandolo, 1979). It is predicted, but has yet to be demonstrated, that SaPI3 will be mobilized and encapsidated, similarly to SaPI1 and SaPI2, by phages such as typing phage 29, which was observed to co-transduce seb with various other genetic elements including plasmids and the mecA element. If so this would simply mean that seb would be transferred at such a high frequency that co-transduction by phage 29 of seb with the other elements would occur for purely statistical reasons rather than indicating any specific association.
10. SaPIbap The most recent addition to the SaPI family is SaPIbap—which is an exception to the rule of SAg carriage as it encodes a novel adhesion, Bap, specific for the bovine mammary mucosa, instead of any SAg. Bap is a 240 kDa protein, encoded by a 12 kb ORF, the largest known staphylococcal gene, which results in a overall size of about 27 kb. SaPIbap encodes a putative transposase as well as the usually conserved phage-like products, and has been reported to excise spontaneously, in the absence of any vegetative phage (J. Penades, personal communication). Although it is clearly a member of the SaPI family, it significantly broadens the genotypic diversity and functional range of these elements.
11. Global repression of exoprotein genes by superantigens (Vojtov et al., 2002). It has been known for some time that strains producing TSST-1 produce other exoproteins at rather low levels (Schlievert et al., 1982) and, in the case of a-hemolysin, this has been attributable, in some strains, to mutations in the hla gene (OÕReilly et al., 1990). We have found that many TSST-1 strains do, in fact, produce active a-hemolysin, but at very low levels, consistent with a regulatory rather than a mutational explanation. As shown in Fig. 6, they also produce other exoproteins at very low levels, the
Fig. 6. Effect of tst on exoprotein patterns. Bacteria were grown to early stationary phase in CYGP in the absence of glucose and supernatants were TCA precipitated and analyzed by SDS–PAGE. Gels were stained with Coomassie brilliant blue and scanned. Following samples are shown: RN4282 with SaPI1 tst::tetM insertional inactivation (RN6938) in lane 1, RN4282 tst::tetM containing pRN5543::tst (pRN7119), expressing full-length tst (lane 2), RN4282 with SaPI1 carrying intact tst gene (lane 3), RN6734 (lane 4), RN6734 (SaPI1) strain (RN9131), expressing tst (lane 5), RN6734 containing pRN5543::tst (pRN7119) and expressing tst (lane 6). Location of TSST-1 protein is indicated by the arrow (reproduced from (Vojtov et al., 2002) with kind permission of the publisher).
total being about 10% of what is seen with isogenic strains with a tst knockout, whereas they produce TSST-1 at much higher levels. Additionally, the observed levels of TSST-1 are independent of gene dosage, suggesting that TSST-1 is autorepressed. Deletion analysis has revealed that the determinants of exoprotein synthesis inhibition and tst autoinhibition are localized to the 50 half of the gene, and gene expression analysis with b-lactamase as a reporter has shown clearly that both of
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Fig. 7. Inhibition of inflammation by a TSST-1 producing strain. Mice were injected subcutaneously with either the TSST-1 producing strain, RN4282, or the isogenic, tst::tetM strain, RN6938. Results shown are representative of each group of three mice that were tested. At left and right are reproduced the PAGE patterns for the two respective strains, as shown in Fig. 6. The black circle in the upper left panel demarcates the site of injection (adapted from Vojtov et al., 2002).
effects occur at the level of transcription for two standard exoprotein genes—sspA (encoding V8 protease) and lukS (encoding leukocidin S)—as well as for the tst gene. A frame-shift mutation within the 50 half of the gene eliminated the inhibitory effect but did not affect the synthesis of a downstream reporter protein, indicating that the TSST-1 protein itself, rather than the mRNA or DNA, is responsible. Addition of mature TSST-1 protein to cultures, however, had no effect, indicating that intracellular TSST-1, presumably the precursor, is the inhibitor. It has been observed repeatedly that post-surgical toxic shock is often very difficult to recognize, because, unlike the typical S. aureus lesion, the wound is apurulent (Fast et al., 1988; Kreiswirth et al., 1986). We have confirmed this effect in a murine model (see Fig. 7). The lack of purulence in such infections has been attributed to the induction by TSST-1 of TNFa, which has been reported to suppress the mobility of polymorphonuclear leukocytes (PMNs) (Fast
et al., 1988); It has also been reported that staphylococcal lipase is chemotactic for PMNs (Tyski et al., 1987) and we suggest that suppression of exoprotein synthesis might be of equal or greater importance, in the suppression of inflammation on that basis. We have also observed that SEB behaves similarly to TSST-1 with respect to inhibition of exoprotein synthesis and that the inhibitory region is localized to the 50 half of the structural gene (Vojtov et al., 2002). Finally, in streptococcal necrotizing fasciitis, associated with erythrogenic toxin SPEA, a SAg, the lesion is teeming with streptococci but is essentially devoid of PMNs (P. Schlievert, personal communication) (it is not yet known whether SPEA affects exoprotein synthesis).
12. Conclusions Like other pathogens, staphylococci contain accessory genetic elements that add greatly to their
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pathogenicity. Thus far, there are two well-defined classes, the SAg-bearing SaPIs and the resistancedetermining SCCmecs, plus other apparently inserted units that do not belong to any known class of accessory genetic elements. The SaPIs are the first clearly defined pathogenicity islands in Grampositive bacteria and they represent a remarkable example of a cooperative interaction with other accessory genetic elements, namely bacteriophages. This type of interaction is precedented by the well-studied interaction between two coliphages, P2 and P4 (Ghisotti et al., 1995; Lindqvist et al., 1993), and current studies are directed toward a detailed comparison of the two systems. An important unanswered question is that of how commonly SaPI strains contain prophages that can mobilize them—for example, most menstrual TSS strains appear to represent a single clone, yet they all carry the highly mobile SaPI2. Why, then, is SaPI2 not much more widely disseminated? A third system of this type, involving a cooperative interaction between a plasmid and a phage, has recently been discovered in the archaeon, Sulfolobus islandicus (Arnold et al., 1999). The three systems do not seem to be evolutionarily related, suggesting that this type of interaction must have a significant selective advantage. Finally, SAgs, encoded by accessory genetic elements, suppress local inflammation, probably at least in part by suppressing the production of chemotactic exoproteins by the infecting organism. This provides the bacteria with a field of operation that is relatively free from interference by the host innate immune response. Is this a manifestation of the selfishness of the SAg-encoding elements or does it mean that the SAgs are, themselves, ‘‘selfish’’ proteins?
References Arnold, H.P., She, Q., Phan, H., Stedman, K., Prangishvili, D., Holz, I., Kristjansson, J.K., Garrett, R., Zillig, W., 1999. The genetic element pSSVx of the extremely thermophilic crenarchaeon Sulfolobus is a hybrid between a plasmid and a virus. Mol. Microbiol. 34, 217–226. Bayles, K.W., Iandolo, J.J., 1989. Genetic and molecular analyses of the gene encoding staphylococcal enterotoxin D. J. Bacteriol. 171, 4799–4806.
Betley, M.J., Mekalanos, J.J., 1985. Staphylococcal enterotoxin A is encoded by phage. Science 229, 185–187. Chai, S., Kruft, V., Alonso, J.C., 1994. Analysis of the Bacillus subtilis bacteriophages SPP1 and SF6 gene 1 product: a protein involved in the initiation of headful packaging. Virology 202, 930–939. Chu, M.C., Kreiswirth, B.N., Pattee, P.A., Novick, R.P., Melish, M.E., James, J.J., 1988. Association of toxic shock toxin-1 determinant with a heterologous insertion at multiple loci in the Staphylococcus aureus chromosome. Infect. Immun. 56, 2702–2708. Cucarella, C., Solano, C., Valle, J., Amorena, B., Lasa, I., Penades, J.R., 2001. Bap, a Staphylococcus aureus surface protein involved in biofilm formation. J. Bacteriol. 183, 2888–2896. De Boer, M., Chow, A., 1994. Toxic shock syndrome toxin 1-producing Staphylococcus aureus isolates contain the staphylococcal enterotoxin B genetic element but do not express Staphylococcal aureus enterotoxin B. J. Infect. Dis. 170, 818–827. Fast, D.J., Schlievert, P.M., Nelson, R.D., 1988. Nonpurulent response to toxic shock syndrome toxin-1 producing Staphylococcus aureus: relationship to toxin-stimulated production of tumor necrosis factor. J. Immunol. 140, 949–953. Fitzgerald, J.R., Monday, S.R., Foster, T.J., Bohach, G.A., Hartigan, P.J., Meaney, W.J., Smyth, C.J., 2001. Characterization of a putative pathogenicity island from bovine Staphylococcus aureus encoding multiple superantigens. J. Bacteriol. 183, 63–70. Ghisotti, D., Briani, F., Forti, F., Piazza, F., Polo, S., Sabbattini, P., Sturniolo, T., Terzano, S., Zangrossi, S., Zappone, M., et al., 1995. Multiple regulatory mechanisms controlling phage-plasmid P4 propagation. FEMS Microbiol. Rev. 17, 127–134. Hendrix, R.W., 1983. Lambda II. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Kreiswirth, B.N., Kravitz, G.R., Schlievert, P.M., Novick, R.P., 1986. Nosocomial transmission of a strain of Staphylococcus aureus causing toxic shock syndrome. Ann. Intern. Med. 105, 704–707. Lee, P.K., Kreiswirth, B.N., Deringer, J.R., Projan, S.J., Eisner, W., Smith, B.L., Carlson, E., Novick, R.P., Schlievert, P.M., 1992. Nucleotide sequences and biological properties of toxic shock syndrome toxin-1 from ovine and bovineassociated Staphylococcus aureus. J. Infect. Dis. 165, 1056–1063. Lindqvist, B.H., Deho, G., Calendar, R., 1993. Mechanisms of genome propagation and helper exploitation by satellite phage P4. Microbiol. Rev. 57, 683–702. Lindsay, J.A., Kurepina, N., Novick, R.P., 1997. Clinical isolates of Staphylococcus aureus encode TSST-1 on genetic elements related to S. aureus pathogenicity island-1 (SaPI1). In: European Conference on Toxic Shock Syndrome. Royal Society of Medicine, London, UK. Lindsay, J.A., Ruzin, A., Ross, H.F., Kurepina, N., Novick, R.P., 1998. The gene for toxic shock toxin is carried by a
R.P. Novick / Plasmid 49 (2003) 93–105 family of mobile pathogenicity islands in Staphylococcus aureus. Mol. Microbiol. 29, 527–543. Musser, J.M., Schlievert, P.M., Chow, A.W., Ewan, P., Kreiswirth, B.N., Rosdahl, V.T., Naidu, A.S., Witte, W., Selander, R.K., 1990. A single clone of Staphylococcus aureus causes the majority of cases of toxic shock syndrome. Proc. Natl. Acad. Sci. USA 87, 225–229. Novick, R., 1967. Properties of a cryptic high-frequency transducing phage in Staphylococcus aureus. Virology 33, 155–166. Novick, R.P., Schlievert, P., Ruzin, A., 2001. Pathogenicity and resistance islands of staphylococci. Microbes Infect. 3, 585–594. OÕReilly, M., Kreiswirth, B., Foster, T., 1990. Molecular analysis of a nonexpressed a-toxin gene (hla) of clinical strains of Staphylococcus aureus. In: Novick, R.P. (Ed.), Molecular Biology of the Staphylococci. VCH Publishers Inc., New York, pp. 439–444. Ruzin, A., 1999. Molecular genetics of a mobile pathogenicity island encoding TSST-1 in Staphylococcus aureus. In: Biology. New York University, New York, p. 144. Ruzin, A., Lindsay, J., Novick, R.P., 2001. Molecular genetics of SaPI1—a mobile pathogenicity island in Staphylococcus aureus. Mol. Microbiol. 41, 365–377. Schlievert, P., Osterholm, M., Kelly, J., Nishimura, R., 1982. Toxin and enzyme characterization of Staphylococcus aureus isolates from patients with and without toxic-shock syndrome. Ann. Intern. Med. 96, 937–940.
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Shafer, W.M., Iandolo, J.J., 1979. Genetics of staphylococcal enterotoxin B in methicillin-resistant isolates of Staphylococcus aureus. Infect. Immun. 25, 902–911. Sloane, R., deAzavedo, J.C.S., Arbuthnott, J.P., Hartigan, P.J., Kreiswirth, B., Novick, R., Foster, T.J., 1991. A toxic shock syndrome toxin mutant of Staphylococcus aureus isolated by allelic replacement lacks virulence in a rabbit uterine model. FEMS Microbiol. Lett. 78, 239–244. Spanova, A., 1992. Comparison of permuted region lengths in the genomes of related Salmonella typhimurium phages P22 and L. Folia Microbiol. 37 (3), 188–192. Stewart, P.R., Waldron, H.G., Lee, J.S., Matthews, P.S., 1985. Molecular relationships among serogroup B bacteriophages of Staphylococcus aureus. J. Virol., 111–116. Tye, B.K., Huberman, J.A., Botstein, D., 1974. Non-random circular permutation of phage P22 DNA. J. Mol. Biol. 85 (4), 501–528. Tyski, S., Tylewska, S., Hryniewicz, W., Jeljaszewicz, J., 1987. Induction of human neutrophils chemotaxis by staphylococcal lipase. Zentralbl. Bakteriol. Mikrobiol. Hyg [A] 265, 360–368. Vojtov, N., Ross, H.F., Novick, R.P., 2002. Global repression of exotoxin synthesis by staphylococcal superantigens. Proc. Natl. Acad. Sci. USA 99 (15), 10102–10107. Communicated by R.P. Novick
Minireview
Mobile genetic elements and bacterial toxinoses: the superantigen-encoding pathogenicity islands of Staphylococcus aureus Richard P. Novick* Molecular Pathogenesis Program, Departments of Microbiology and Medicine, Skirball Institute, New York University School of Medicine, New York, NY 10016, USA Received 17 September 2002, revised 4 October 2002
Abstract It is a remarkable observation that virtually all bacterial toxins associated with specific clinical conditions (toxinoses) are encoded by mobile (and therefore variable) genetic elements. Remarkably, these rarely, if ever, carry determinants of antibiotic resistance. Examples are the toxins responsible for diphtheria, anthrax, tetanus, botulism, cholera, toxic shock, scarlet fever, exfoliative dermatitis, food poisoning, travelersÕ diarrhea, shigella dysentery, necrotizing pneumonia, and others. A recently discovered example of this phenomenon is the family of related staphylococcal pathogenicity islands encoding superantigens (SAgs). These are 15–20 kb elements that occupy constant positions in the chromosomes of toxigenic strains, and are characterized by certain phage-related features, namely genes encoding integrases, helicases, and terminases, and the presence of flanking direct repeats. The prototype, SaPI1 of Staphylococcus aureus, encodes TSST-1 plus two newly described SAgs, SEK and SEL. Other members of the family encode enterotoxins B (SaPI3) and C (SaPI4), plus at least two other SAgs each. SaPI1 and SaPI2, also encoding TSST-1, are excised and induced to replicate by certain staphylococcal phages, and are then encapsidated at high efficiency into phage-like infectious particles with heads about 1/3 the size of the helper phage heads, commensurate with the sizes of the respective genomes. This results in transfer frequencies of the order of 108 /ml, and is presumably responsible for the spread of these elements as well as for their acquisition in the first place. In the absence of a helper phage, these two islands are highly stable; neither excision, loss, or transfer occurs at detectable frequency. Several general implications of this phenomenon will be discussed. One is that the determinants of these toxins have been imported from other species and therefore are not components of the basic genome of the extant producing organisms. This raises the question of the biological (adaptive?) roles of these toxins. Another is that the toxin-carrying units can spread among different (though probably related) species. An interesting question is that of the biological basis for the separation of toxin and resistance determinants. Ó 2003 Published by Elsevier Science (USA).
1. Introduction
*
Fax: 1-212-263-5711. E-mail address: [email protected].
A wide variety of bacterial accessory functions, including inducible catabolic pathways, restriction-modification systems, antibiotic resistance,
0147-619X/03/$ - see front matter Ó 2003 Published by Elsevier Science (USA). doi:10.1016/S0147-619X(02)00157-9
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and pathogenesis are determined by variable genes—genes that belong to larger units that are present in some strains of a given species and entirely absent in others. Some of these, such as plasmids, prophages, and functional transposons are clearly mobile; others, such as integrated plasmid-like elements, defective prophages and most chromosomal islands, are probably remnants of formerly mobile units. Among the variable genes involved in pathogenesis are those encoding toxins, of which a major subset is responsible for toxinoses—diseases of which a single toxin is both a necessary and sufficient cause. A remarkable feature of these toxins is that, with one known exception, Clostridium perfringens a-toxin, which is a standard chromosomal gene and, on its own, causes gangrene (J. Rood, personal communication), all are encoded by highly mobile genetic elements. As listed in Table 1, these elements include plasmids, prophages, transposons, and mobile pathogenicity islands. It is very likely that all such elements have been acquired from other species, though no suspects have yet been identified. Nevertheless, the toxin genes are all controlled by chromosomally encoded regulatory factors, raising the important question of how such regulation
may have evolved, and suggesting, incidentally, that the putative donor species must be fairly closely related to the extant toxin-producers. Mobile genetic elements are arguably selfish in that their evolution is driven by selective forces that operate on the elements themselves, independently of the host organisms within which they must of necessity reside. Nevertheless, these elements often encode products that have obvious selective value for the host. Among these is pathogenesis, which can be viewed as a consequence of the organismÕs attempt to thrive in the hostile environment of the metazoan body. The selectivity of toxinosis-causing toxins however, is not always obvious. Many of the enterotoxins, for example, cause disease in the absence of the producing organism. And even when the organism is present, it derives no discernable advantage from production of the toxin. Among these organisms are bacilli (Bacillus cereus), staphylococci (Staphylococcus aureus), and clostridia (C. perfringens, Clostridium botulinum), which produce enterotoxins causing food poisoning but are not enteric pathogens per se. Three possible explanations for this curious feature are (i) the toxin is a protein with some unknown function that is accidentally toxic; (ii) the toxicity
Table 1 Toxinoses and variable genetic elements Disease
Toxin
Organism
Element
Features
Diphtheria Anthrax Cholera Dysentery Botulism Enterocolitis Gangrene Tetanus Enterocolitis Diarrhea Diarrhea Toxic shock syndrome Food poisoning, TSS Food poisoning, TSS Food poisoning, TSS Scalded skin syndrome Scalded skin syndrome Necrotizing pneumonia Scarlet fever
Diphtheria toxin Anthrax toxin Cholera toxin Shiga toxin Botulin toxin Cpe toxin a-toxin Tetanus toxin Enterotoxin Labile toxin (LT) Stable toxin (ST) TSST-1 Enterotoxin A Enterotoxin B, C Enterotoxin D Exfoliatin A Exfoliatin B Leukocidin SPEA, C
Corynebacterium diphtheriae Bacillus anthracis Vibrio cholerae Shigella dysenteriae Clostridium botulinum Clostridium perfringens Clostridium perfringens Clostridiun tetani Clostridium difficile Escherichia coli Escherichia coli Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Streptococcus pyogenes
b-Prophage Plasmid Prophage Prophage Prophage Transposon Chromosomea Plasmid Path islet Plasmid Transposon Path island Prophage Path island Plasmid Prophage Plasmid Prophage Prophage
35 kb 110 kb Filamentous Lambdoid 110–165 kb IS14702 ::cpe 6 kb
a
‘‘Large’’ 3 kb 90 kb F-like IS12 ::cat 3 kb 15 kb /-related 45 kb 15 kb /-related 30 kb 45 kb 30 kb 45 kb 45 kb
C. perfringens a-toxin is the single exception to the rule that toxinosis-causing toxins are encoded by mobile genetic elements.
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of the protein does, in fact, provide the organism with a selective advantage, for example, toxicity for some organism that is either predatory or is a source of nutrition for the toxin-producer; (iii) the toxin gene is selected by evolutionary forces that are specific for the toxin-encoding element and have nothing to do with the biology of the host bacterium. Other toxins provide a clear selective advantage. These include cholera, diphtheria, anthrax, and C. perfringens a toxins, and the superantigens (SAgs) of S. aureus and streptococci. This presentation is focused on the staphylococcal SAgs, many of which are encoded by novel pathogenicity islands, the first elements of this type for which mobility has definitively been demonstrated (Lindsay et al., 1998). Different S. aureus strains produce different combinations of superantigen toxins. Strains lacking a given toxin also lack the gene that encodes it plus flanking chromosomal sequences. The first observations of this involved enterotoxins A (SEA) and D (SED) which are encoded by prophages (Betley and Mekalanos, 1985) and plasmids (Bayles and Iandolo, 1989), respectively. More recently, we have identified a series of discrete 15–20 kb chromosomal elements that encode SAgs, reside at specific locations and are mobilized at high frequencies by certain staphylococcal phages. These are referred to as staphylococcal pathogenicity islands (SaPIs). The genes for toxic shock toxin and for enterotoxins B, C, K, and L are all carried by elements of this type. Several of these have been sequenced and their genetic maps are shown in Fig. 1. The prototype of the family is SaPI1, whose genetic analysis was greatly aided by the construction of a derivative with tetM inserted into tst, the gene for toxic shock syndrome toxin-1 (TSST-1) (Sloane et al., 1991). Two properties of SaPI1 led to realization that it was a novel type of mobile element: first, the transduction frequency of SaPI1::tetM was extremely high—about 101 — with phage 80a, whereas it was about 107 with the closely related generalized transducing phage, /11. Second, extrachromosomal SaPI1::tetM DNA could be detected in 80a lysogens but not in /11 lysogens (Lindsay et al., 1998). Following UV induction of an 80a lysogens carrying the element, a
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SaPI1-specific band with mobility corresponding to a linear form of the element appeared in agarose gel separations of whole cell DNA and was amplified during the phage lytic cycle. A similar band was seen upon infection of a non-lysogen with an 80a-SaPI1 mixed lysate (Fig. 2A). This band was shown by Southern blotting to represent SaPI1 (Fig. 2C). No such band was seen with /11 or with several other staphylococcal transducing phages, none of which transduced SaPI1 at elevated frequencies. Superinfection with /13 caused the appearance of SaPI1-specific circular supercoiled material that did not detectably replicate (Ruzin, 1999), and was not incorporated into transducing particles by this phage, which is not a generalized transducing phage. However, a similar band was seen following superinfection of several menstrual toxic shock strains, in which tst is at a different location than that of SaPI1 (Chu et al., 1988), with the related phage 80 (Fig. 3). This species had a number of different mobility variants but always migrated considerably more rapidly than the SaPI1 and has been designated SaPI2. It is not presently known whether this band represents linear or circular DNA. Thus far, phage-induced excision/replication has yet to be demonstrated for the other SaPIs illustrated in Fig. 1. SaPI1 transducing particles could be readily separated from 80a plaque-forming particles on the basis of slower sedimentation in a sucrose gradient but could not be separated on the basis of buoyant density, suggesting that the transducing particles were smaller. Electron microscopy (Fig. 4) showed that the SaPI1 particles were morphologically similar to the 80a particles but had smaller heads, with a volume about 1/3 that of the plaque-forming particles, commensurate with the difference in genome size (18 kb for SaPI1::tetM, 45 kb for 80a (Lindsay et al., 1998)). Thus, SaPI1 is evidently encapsidated in the monomeric form by SaPI1induced small-headed 80a-like phage particles. The monomeric form was confirmed by infecting a standard recipient with purified SaPI1 particles and then analyzing the infected cells for extrachromosomal dna. Again, a DNA species corresponding to SaPI1 linears was seen immediately following infection; however, in this case, no amplification was observed and the SaPI1 band gradually diminished
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Fig. 1. Comparative linear maps for seven SaPIs. Numbered arrows represent open reading frames >65 codons. Arrows in red represent reading frames similar to known or suspected phage genes; arrows in aqua represent reading frames conserved among the SaPIs of known sequence; those in blue represent non-conserved reading frames of unknown function; those in green represent genes involved in pathogenesis. Flanking chromosomal sequences are indicated as gray boxes. TetM and erm are selective marker insertions (Fitzgerald et al., 2001; Sloane et al., 1991). Ear is an apparently incomplete ORF potentially encoding part of a b-lactamase-like protein (P. Orwin and P. Schlievert, personal communication).
in intensity during an 800 time course (Fig. 2D). It is suggested that the high frequency transfer of SaPI1 by 80a, which differs considerably from conventional transduction, represents a mechanism of horizontal dissemination of the pathogenicity island. The identity of the capsid protein(s) of the small particles and the mechanism by which they are produced, however, remains to be determined.
the absence of SaPI1. This is reflected in commensurately low titers of phage lysates and in a dramatic reduction in phage sensitivity in spot tests. The latter has provided a means of scoring for the presence of SaPI1 and has enabled transduction of the native SaPI1 element without the need for a selective marker (Ruzin, 1999). Precisely which phage functions are parasitized by SaPI1 has not been determined, though it is likely that replication and encapsidation functions are involved.
2. Interference When propagated on a SaPI1-containing strain, either by UV induction of an 80a lysogen or by exogenous infection of a non-lysogen, the single burst size of the phage is about 1% of that seen in
3. Integration Following transfer, by either transformation or transduction, SaPI1 integrates, presumably by the
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Fig. 2. The fate of incoming SaPI1 DNA. (A) Co-infection with 80a and SaPI1. Bacteria (RN450) were infected with a mixed 80a lysate at a multiplicity of 1:1. Infected bacteria were incubated at 32 °C in CY broth + phage buffer and sampled at the indicated times for preparation of SDS minilysates, which were analyzed by agarose gel electrophoresis. Control (c)—minilysate of SaPI1 strain infected with 80a and sampled at 45 min post-infection. (B) Southern blot hybridization pattern of the gel in A. The gel was transferred to a nylon membrane and hybridized overnight with peroxidase-labelled (ECL, Amersham) SaPI1-specific probe (PCR product using primers p95 and p96 (Ruzin et al., 2001)). (C) Infection of RN27 (80a lysogen) with the same mixed lysate. Bacteria (RN27) were infected with the same lysate as in A, and analyzed as above. (D) Infection of non-lysogenic recipient with purified SaPI1 transducing particles. Bacteria (RN450) were infected with purified SaPI1 transducing particles, separated by sucrose gradient sedimentation from plaque-forming particles (Ruzin et al., 2001), and analyzed as above (adapted from Ruzin et al., 2001). (E) Plot of the relative change in SaPI1 DNA with time derived from the data in panels A, C, and D, respectively.
classical Campbell mechanism, into a unique chromosomal recognition site, attC , which is present in many strains and contains a copy of the 17 nt repeat sequence found at the ends of the integrated element (Figs. 1 and 4). A SaPI1 subclone containing only int and attS integrated efficiently, whereas a derivative of this clone with a deletion affecting int did not (Ruzin, 1999), indicating that the SaPI1-encoded integrase is necessary and is the only SaPI1 function required for integration. No
phage function is required. The efficiency of integration is probably close to 100%, as judged by enumeration of the two types of particles seen in the electron microscope in comparison with transducing and plaque-forming titers determined for the corresponding lysate. Following infection with a mixed lysate of an 80a lysogen or of a nonlysogen (Figs. 2A and B), the incoming linear SaPI1 DNA gives rise to the circular form, which is first detectable about 40 min after infection. This
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Fig. 3. Excision of SaPI-like elements in naturally occurring TSST-1-producing strains by phage 80. Mid-exponential phase cultures were superinfected with phage 80 at a multiplicity of 3. Samples were removed 45 min after phage infection and used to prepare standard mini-lysates which were separated on 0.7% agarose in TBE, stained with ethidium bromide and photographed. RN3984 is a prototypical menstrual TSS isolate (‘‘Harrisburg’’). Numbers 1–7 represent naturally occurring menstrual TSS isolates sensitive to phage 80, all kindly provided by P. Schlievert (Ruzin, 1999).
Fig. 4. Electron microscopy. Phage particles from the lysate used in Fig. 2A were purified, mounted on carbon grids, stained with 2% PTA and photographed at 31,500 magnification. Note that there are particles of two different sizes, the smaller ones (arrow) corresponding to SaPI1 transducing particles, the larger to 80a particles (reproduced from (Ruzin et al., 2001), with kind permission of the publisher).
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is followed by the appearance of a SaPI1 signal in the chromosomal gel band (Fig. 2C), which is assumed to represent integration. This process seems to be relatively slow, since a strong linear SaPI1 DNA band persists for over 2 h. In a recipient containing SaPI1, an incoming SaPI1::tetM precisely replaces the resident SaPI1, even in a recA recipient (Lindsay et al., 1998). The replaced copy disappears since the transductants no longer produce TSST-1. This is different from what is usually seen with a superinfecting temperate phage, such as k, which integrates either at an unlinked secondary site or at one end of the resident prophage genome, with the latter remaining in place (Hendrix, 1983). It is likely that the SaPI1 integrase is responsible for this, since the element does not encode any detectable xis function (see below), and since it occurs with a SaPI1 subclone containing only int and attS (Ruzin, 1999; Ruzin et al., 2001).
4. Excision The SaPI1 sequence does not contain any ORF resembling phage xis determinants, and spontaneous SaPI1 excision has not been detected in the absence of vegetative phage, even by (single-stage) PCR analysis (Ruzin, 1999). Nor do subclones containing int induce the excision of a resident SaPI1 (Ruzin et al., 2001). It thus seems very likely that SaPI1 does not encode any xis function, and that SaPI1 integrase cannot mediate excision of the element. Clearly, 80a encodes the xis function that is used for SaPI1 excision, and it is assumed that this is the same function used by the phage for its own excision, following induction of a lysogen. If this is correct, it means that excision is not sequence-specific, since the phage and SaPI1 att sites are unrelated. Among several other staphylococcal phages tested, only one, /13, also catalyzed SaPI1 excision. However, the consequences of excision by the two phages were very different; whereas the product of 80a excision was immediately converted to the linear replicating form, the product of /13 excision persisted as the circular duplex monomer and did not replicate to a significant extent (Ruzin et al., 2001).
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5. Replication Following excision by 80a, SaPI1 DNA replicates autonomously to a level of at least 120 copies per cell. The only topological form that can be detected during the early stages of this amplification appears to correspond to linear monomers (Fig. 2). Thus, the SaPI1-specific band that appears following UV induction of an 80a lysogen has the same mobility as a linear SaPI1 monomer and is the same as that seen following the introduction of SaPI1 by phage infection. Though it is possible to detect SaPI1 DNA with joined ends by PCR immediately after excision, circular monomers are not seen in agarose gels or by Southern blotting until late in the infective cycle (Fig. 2); thus, the circular form must be linearized very rapidly after 80a-induced excision. Further, the incoming linear monomers, unlike the classical temperate phages, seem to enter the replication pathway directly, without going through a circular intermediate. It is suggested on the basis of these results that SaPI1 replicates as a linear molecule. Of several staphylococcal phages tested, only 80a induced SaPI1 replication, indicating an intimate relation between the two genetic elements. Attempts to define a phage-responsive replication origin for SaPI1 have thus far been unsuccessful, though the entire element has been subcloned as overlapping fragments to a plasmid vector with a thermosensitive replicon. Although a subclone containing int and attS was transduced at a much higher frequency than any of the other subclones (Ruzin et al., 2001), it is not clear whether this represents phage-induced replication. No other subclone supported growth at the restrictive temperature, or showed 80a-induced amplification or any elevation in transducing frequency. However, the possibility has not been ruled out that the circular subclones, which lack attC , could not be converted by 80a to a substrate (linear?) suitable for phage-induced replication.
6. Encapsidation Phage 80a dna is terminally redundant and circularly permuted (Stewart et al., 1985) and the
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same is true for SaPI1. As shown in Fig. 5 (middle), SaPI1::tetM DNA from transducing particles contains all of the restriction fragments expected from circular DNA; however, those with one end in the shaded region are always present in submolar amounts—for an example, in the HindIII digest shown inside of the circular map, the band representing the 6 kb fragment (8174–14,314) has about 1/4 of the expected intensity. Digestion with singly cutting enzymes always generates one or two smeared bands, representing linear fragments with variable ends that lie within the shaded region. The BglI digest at lower left shows one of the two expected smeared bands, with fragments between 8 and 15 kb in length. The other expected smear, with fragments from 0 to 8 kb is not seen, probably because the intensities are too low. Thus,
there is a limited number of permutations, and the element must be encapsidated from concatemers composed of the same average number of repeats as the number of permutations. For a more detailed description of these results, readers are referred to Ruzin et al. (2001), and to earlier papers describing the same phenomena in coliphage p22 (Spanova, 1992; Tye et al., 1974). At least one SaPI1 gene potentially involved in encapsidation (ter in Fig. 1) has been identified as an open reading frame whose predicted product is highly similar to the small terminase subunit encoded by several phages from Gram-positive bacteria (Chai et al., 1994). This reading frame is also present on SaPI3 (Novick et al., 2001), and SaPIbov (Fitzgerald et al., 2001), and is absolutely conserved.
Fig. 5. Demonstration of circular permutations in SaPI1 DNA. At upper right is an agarose gel electropherogram of SaPI1::tetM DNA isolated from the circular duplex band shown in Fig. 2A (left-hand lane), and the same DNA digested with the singly-cutting enzyme, NheI (right-hand lane). The upper band in the left lane represents contaminating linear SaPI1 DNA. This material is responsible for the smeared fragment pattern seen below the linear 18 kb band in the right-hand lane. At lower left is an electropherogram of SaPI1::tetM DNA isolated from transducing particles and digested with either BglI (right lane) or undigested (left lane). Note that the undigested dna is about 90% SaPI1 (18 kb) linears and about 10% (45 kb) 80a linears, and that the smeared fragments in the BglI digest range from 8–15 kb, represented by the innermost segment (arrow). Inside the SaPI1 map is an electropherogram of SaPI1 DNA from transducing particles digested with HindIII alongside the standard 1 kb ladder. The 6 kb marker indicates the fragment between the HindIII sites at 8174 and 15301, which is present in a submolar amount. This fragment is delineated by the first inner segment (arrow). The shaded area represents the apparent locations of the ends of the permuted SaPI1 molecules.
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7. Phage specificity 80a is not a naturally occurring phage, having been isolated as a host-range variant by plating typing phage 80 on strain NCTC8325. Subsequent studies have shown that 80a is not a restrictionmodification variant but is a recombinant with segments from at least two other phages, /11 and /13, that are present as prophages in NCTC8325 (Novick, 1967). Since neither phage 80 nor /11 can catalyze the excision of SaPI1, whereas /13 can, it is assumed that the SaPI1-specific xis function has been derived from /13. Since neither /11 nor /13 can support SaPI1 replication, it is assumed that the replication capability is derived from phage 80 or is a consequence of the recombination. Since 80 does not grow on SaPI1 strains, it has not been possible to determine whether it can support the replication of autonomous SaPI1.
8. The SaPI family With the advent of genome sequencing and micro-array technology, inserted chromosomal gene blocs are being increasingly recognized. A prototypical class of these is the SaPIs, phagerelated chromosomal islands that have the following features in common: size 15–20 kb, specific att sites and integrases, several phage related genes including ter, 3 or more SAg genes, and specific interactions with certain phages leading to excision, amplification, and encapsidation. In our hands, distinct SaPIs have been given different numbers; others have appended specific notations to the generic term SaPI—such as -bov (Fitzgerald et al., 2001) and -bap (Cucarella et al., 2001), referring to bovine origin and to the carriage of an adhesin gene, bap, respectively. SaPI2, SaPI4, and SaPIbov. Two other SaPIs encoding TSST-1 have been identified (SaPI2 and SaPIbov), and there are clearly others, on the basis of apparently different chromosomal locations (Lindsay et al., 1997). And TSST-O, produced by ovine strains of S. aureus (Lee et al., 1992) is likely to be encoded by a further SaPI variant. SaPI2, located within the tryptophan locus in the prototypical menstrual TSS strain, RN3984, is found in many other menstrual
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TSS strains, which appear to comprise a rather coherent biotype and probably represent a tight clone (Musser et al., 1990). SaPI2 is induced to excise and replicate by typing phages 29, 52, and 80, of which the latter transduces SaPI2::tetM at frequencies similar to the frequency of transduction of SaPI1 by 80a (Ruzin et al., 2001). As noted above, all of seven menstrual TSS strains sensitive to phage 80 showed excision and amplification of SaPI2-like bands, with some differences in mobility, following infection with this phage. Although there is only a very preliminary map of SaPI2, based on blot hybridization analysis with SaPI1specific probes (Lindsay et al., 1997), it appears on the basis of gel mobility to be considerably smaller than SaPI1. Comparison of the conserved regions of the two genomes is expected to provide important information on the SaPI-phage interaction. SaPIbov, recently identified in a bovine S. aureus strain (Fitzgerald et al., 2001) is similar in size to SaPI1. However, in contrast to SaPI1 and SaPI3, it is flanked by a 74 nt direct repeat, of which there is a single copy in a TSS-negative bovine strain. Some human strains contain part of this, but none has been found so far that contains the entire sequence (Fitzgerald et al., 2001). None of three phages tested, 80a, /11, or 85, were able to excise or transduce SaPIbov, including a derivative containing tetM in the tst gene. SaPI4, for which sequencing and analysis in progress (P. Schlievert, personal communication), is quite similar to SaPI1 but has sec in place of tst, contains the genes encoding three additional SAgs, sek, sel, and sem, and integrates at a different site (P. Orwin and P. Schlievert, personal communication). No inducing phage has been identified to date.
9. SaPI3 SaPI3, which encodes enterotoxin B (SEB), is carried by strain COL, which has recently been sequenced by Tigr (www.tigr.org), so that its sequence has been determined automatically (Novick et al., 2001). It has the same 17 nt flanking direct repeat and is located at the same site as SaPI1—which may account in part for the wellknown fact that TSST-1 and SEB are rarely if ever
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co-produced (De Boer and Chow, 1994). It is likely that SaPI3 is responsible for the problematic genetic results observed previously with SEB (Shafer and Iandolo, 1979). It is predicted, but has yet to be demonstrated, that SaPI3 will be mobilized and encapsidated, similarly to SaPI1 and SaPI2, by phages such as typing phage 29, which was observed to co-transduce seb with various other genetic elements including plasmids and the mecA element. If so this would simply mean that seb would be transferred at such a high frequency that co-transduction by phage 29 of seb with the other elements would occur for purely statistical reasons rather than indicating any specific association.
10. SaPIbap The most recent addition to the SaPI family is SaPIbap—which is an exception to the rule of SAg carriage as it encodes a novel adhesion, Bap, specific for the bovine mammary mucosa, instead of any SAg. Bap is a 240 kDa protein, encoded by a 12 kb ORF, the largest known staphylococcal gene, which results in a overall size of about 27 kb. SaPIbap encodes a putative transposase as well as the usually conserved phage-like products, and has been reported to excise spontaneously, in the absence of any vegetative phage (J. Penades, personal communication). Although it is clearly a member of the SaPI family, it significantly broadens the genotypic diversity and functional range of these elements.
11. Global repression of exoprotein genes by superantigens (Vojtov et al., 2002). It has been known for some time that strains producing TSST-1 produce other exoproteins at rather low levels (Schlievert et al., 1982) and, in the case of a-hemolysin, this has been attributable, in some strains, to mutations in the hla gene (OÕReilly et al., 1990). We have found that many TSST-1 strains do, in fact, produce active a-hemolysin, but at very low levels, consistent with a regulatory rather than a mutational explanation. As shown in Fig. 6, they also produce other exoproteins at very low levels, the
Fig. 6. Effect of tst on exoprotein patterns. Bacteria were grown to early stationary phase in CYGP in the absence of glucose and supernatants were TCA precipitated and analyzed by SDS–PAGE. Gels were stained with Coomassie brilliant blue and scanned. Following samples are shown: RN4282 with SaPI1 tst::tetM insertional inactivation (RN6938) in lane 1, RN4282 tst::tetM containing pRN5543::tst (pRN7119), expressing full-length tst (lane 2), RN4282 with SaPI1 carrying intact tst gene (lane 3), RN6734 (lane 4), RN6734 (SaPI1) strain (RN9131), expressing tst (lane 5), RN6734 containing pRN5543::tst (pRN7119) and expressing tst (lane 6). Location of TSST-1 protein is indicated by the arrow (reproduced from (Vojtov et al., 2002) with kind permission of the publisher).
total being about 10% of what is seen with isogenic strains with a tst knockout, whereas they produce TSST-1 at much higher levels. Additionally, the observed levels of TSST-1 are independent of gene dosage, suggesting that TSST-1 is autorepressed. Deletion analysis has revealed that the determinants of exoprotein synthesis inhibition and tst autoinhibition are localized to the 50 half of the gene, and gene expression analysis with b-lactamase as a reporter has shown clearly that both of
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Fig. 7. Inhibition of inflammation by a TSST-1 producing strain. Mice were injected subcutaneously with either the TSST-1 producing strain, RN4282, or the isogenic, tst::tetM strain, RN6938. Results shown are representative of each group of three mice that were tested. At left and right are reproduced the PAGE patterns for the two respective strains, as shown in Fig. 6. The black circle in the upper left panel demarcates the site of injection (adapted from Vojtov et al., 2002).
effects occur at the level of transcription for two standard exoprotein genes—sspA (encoding V8 protease) and lukS (encoding leukocidin S)—as well as for the tst gene. A frame-shift mutation within the 50 half of the gene eliminated the inhibitory effect but did not affect the synthesis of a downstream reporter protein, indicating that the TSST-1 protein itself, rather than the mRNA or DNA, is responsible. Addition of mature TSST-1 protein to cultures, however, had no effect, indicating that intracellular TSST-1, presumably the precursor, is the inhibitor. It has been observed repeatedly that post-surgical toxic shock is often very difficult to recognize, because, unlike the typical S. aureus lesion, the wound is apurulent (Fast et al., 1988; Kreiswirth et al., 1986). We have confirmed this effect in a murine model (see Fig. 7). The lack of purulence in such infections has been attributed to the induction by TSST-1 of TNFa, which has been reported to suppress the mobility of polymorphonuclear leukocytes (PMNs) (Fast
et al., 1988); It has also been reported that staphylococcal lipase is chemotactic for PMNs (Tyski et al., 1987) and we suggest that suppression of exoprotein synthesis might be of equal or greater importance, in the suppression of inflammation on that basis. We have also observed that SEB behaves similarly to TSST-1 with respect to inhibition of exoprotein synthesis and that the inhibitory region is localized to the 50 half of the structural gene (Vojtov et al., 2002). Finally, in streptococcal necrotizing fasciitis, associated with erythrogenic toxin SPEA, a SAg, the lesion is teeming with streptococci but is essentially devoid of PMNs (P. Schlievert, personal communication) (it is not yet known whether SPEA affects exoprotein synthesis).
12. Conclusions Like other pathogens, staphylococci contain accessory genetic elements that add greatly to their
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pathogenicity. Thus far, there are two well-defined classes, the SAg-bearing SaPIs and the resistancedetermining SCCmecs, plus other apparently inserted units that do not belong to any known class of accessory genetic elements. The SaPIs are the first clearly defined pathogenicity islands in Grampositive bacteria and they represent a remarkable example of a cooperative interaction with other accessory genetic elements, namely bacteriophages. This type of interaction is precedented by the well-studied interaction between two coliphages, P2 and P4 (Ghisotti et al., 1995; Lindqvist et al., 1993), and current studies are directed toward a detailed comparison of the two systems. An important unanswered question is that of how commonly SaPI strains contain prophages that can mobilize them—for example, most menstrual TSS strains appear to represent a single clone, yet they all carry the highly mobile SaPI2. Why, then, is SaPI2 not much more widely disseminated? A third system of this type, involving a cooperative interaction between a plasmid and a phage, has recently been discovered in the archaeon, Sulfolobus islandicus (Arnold et al., 1999). The three systems do not seem to be evolutionarily related, suggesting that this type of interaction must have a significant selective advantage. Finally, SAgs, encoded by accessory genetic elements, suppress local inflammation, probably at least in part by suppressing the production of chemotactic exoproteins by the infecting organism. This provides the bacteria with a field of operation that is relatively free from interference by the host innate immune response. Is this a manifestation of the selfishness of the SAg-encoding elements or does it mean that the SAgs are, themselves, ‘‘selfish’’ proteins?
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