Characterization and Expression Analysis of Staphylococcus aureus Pathogenicity Island 3

We describe the complete sequence of the 15.9-kb staphylococcal pathogenicity island 3 encoding staphylococcal enterotoxin serotypes B, K, and Q. The island, which meets the generally accepted definition of pathogenicity islands, contains 24 open reading frames potentially encoding proteins of more than 50 amino acids, including an apparently functional integrase. The element is bordered by two 17-bp direct repeats identical to those found flanking staphylococcal pathogenicity island 1. The island has extensive regions of homology to previously described pathogenicity islands, particularly staphylococcal pathogenicity islands 1 and bov. The expression of 22 of the 24 open reading frames contained on staphylococcal pathogenicity island 3 was detected either in vitro during growth in a laboratory medium or serum or in vivo in a rabbit model of toxic shock syndrome using DNA microarrays. The effect of oxygen tension on staphylococcal pathogenicity island 3 gene expression was also examined. By comparison with the known staphylococcal pathogenicity islands in the context of gene expression described here, we propose a model of pathogenicity island origin and evolution involving specialized transduction events and addition, deletion, or recombination of pathogenicity island “modules.” We describe the complete sequence of the 15.9-kb staphylococcal pathogenicity island 3 encoding staphylococcal enterotoxin serotypes B, K, and Q. The island, which meets the generally accepted definition of pathogenicity islands, contains 24 open reading frames potentially encoding proteins of more than 50 amino acids, including an apparently functional integrase. The element is bordered by two 17-bp direct repeats identical to those found flanking staphylococcal pathogenicity island 1. The island has extensive regions of homology to previously described pathogenicity islands, particularly staphylococcal pathogenicity islands 1 and bov. The expression of 22 of the 24 open reading frames contained on staphylococcal pathogenicity island 3 was detected either in vitro during growth in a laboratory medium or serum or in vivo in a rabbit model of toxic shock syndrome using DNA microarrays. The effect of oxygen tension on staphylococcal pathogenicity island 3 gene expression was also examined. By comparison with the known staphylococcal pathogenicity islands in the context of gene expression described here, we propose a model of pathogenicity island origin and evolution involving specialized transduction events and addition, deletion, or recombination of pathogenicity island “modules.” toxic shock syndrome staphylococcal pathogenicity island toxic shock syndrome toxin 1 open reading frame Todd-Hewitt staphylococcal enterotoxin Staphylococcus aureus is a leading etiologic agent of both nosocomial and community-acquired infections worldwide. These infections range from fairly benign cutaneous infections, such as furuncles, to potentially fatal diseases, including endocarditis and toxic shock syndrome (TSS)1(reviewed in Ref. 1.Tenover F.C. Gaynes R.P. Fischetti V.A. Novick R.P. Ferretti J.J. Portnoy D.A. Rood J.I. Gram-positive Pathogens. ASM Press, Washington, D. C.2000: 414-421Google Scholar). Its ability to cause this range of disease is due in part to its elaboration of a vast array of both cell surface-associated and secreted virulence factors. Among the secreted factors are the pyrogenic toxin superantigens that have the ability to activate large populations (10–50%) of T lymphocytes in a manner specific to the variable region of the β-chain of the T-cell receptor (2.Marrack P. Kappler J. Science. 1990; 248: 705-711Crossref PubMed Scopus (1210) Google Scholar). The ensuing massive cytokine release results in the symptoms of TSS, including fever, hypotension, rash, vomiting, diarrhea, multiple organ failure, disseminated intravascular coagulation, and desquamation. The staphylococcal enterotoxins (SEs), members of the superantigen family, are associated with both TSS and food poisoning and have proven emetic activities that appear to be separable from their superantigenic activity (3.Hovde C.J. Marr J.C. Hoffmann M.L. Hackett S.P. Chi Y.I. Crum K.K. Stevens D.L. Stauffacher C.V. Bohach G.A. Mol. Microbiol. 1994; 13: 897-909Crossref PubMed Scopus (78) Google Scholar). Most, if not all, staphylococcal superantigens are encoded by accessory genetic elements that are either mobile or appear to have been mobile at one time (reviewed in Ref. 4.McCormick J.K. Yarwood J.M. Schlievert P.M. Annu. Rev. Microbiol. 2001; 55: 77-104Crossref PubMed Scopus (563) Google Scholar). These identified elements include plasmids, transposons, prophages, and the pathogenicity islands. The staphylococcal pathogenicity islands (SaPIs), of which five have, until recently, been described (SaPI1–4 and SaPIbov), are the first clearly defined pathogenicity islands in Gram-positive bacteria, and each encodes one or more of the staphylococcal superantigens (reviewed in Ref. 5.Novick R.P. Schlievert P. Ruzin A. Microbes Infect. 2001; 3: 585-594Crossref PubMed Scopus (115) Google Scholar). SEB, SEC, SEK, SEL, SEQ, and toxic shock syndrome toxin 1 (TSST-1) are known to be encoded by one or more of these phage-related elements (reviewed in Ref. 4.McCormick J.K. Yarwood J.M. Schlievert P.M. Annu. Rev. Microbiol. 2001; 55: 77-104Crossref PubMed Scopus (563) Google Scholar). More recently, Kuroda et al.(6.Kuroda M. Ohta T. Uchiyama I. Baba T. Yuzawa H. Kobayashi I. Cui L. Oguchi A. Aoki K. Nagai Y. Lian J. Ito T. Kanamori M. Matsumaru H. Maruyama A. Murakami H. Hosoyama A. Mizutani-Ui Y. Takahashi N.K. Sawano T. Inoue R. Kaito C. Sekimizu K. Hirakawa H. Kuhara S. Goto S. Yabuzaki J. Kanehisa M. Yamashita A. Oshima K. Furuya K. Yoshino C. Shiba T. Hattori M. Ogasawara N. Hayashi H. Hiramatsu K. Lancet. 2001; 357: 1225-1240Abstract Full Text Full Text PDF PubMed Scopus (1564) Google Scholar) identified six novel pathogenicity islands in the complete genomes of two S. aureus strains, N315 and Mu50, including three that carried tstH (encoding TSST-1), two that carried clusters of staphylococcal exotoxin-like proteins, and one, present in both strains, that carried clusters of serine proteases and enterotoxins. These genomic loci meet the generally accepted requirements of the pathogenicity island subgroup of “genomic islands” as defined previously (7.Hacker J. Kaper J.B. Annu. Rev. Microbiol. 2000; 54: 641-679Crossref PubMed Scopus (938) Google Scholar, 8.Hentschel U. Hacker J. Microbes Infect. 2001; 3: 545-548Crossref PubMed Scopus (87) Google Scholar). They are present in the genomes of many staphylococci but absent from closely related strains, they are relatively large genomic fragments (>15 they in from the of the they are by direct repeats of the elements the are associated with and they genetic including The staphylococcal pathogenicity island, was identified and by et Ruzin A. N. Novick R.P. Mol. Microbiol. PubMed Scopus Google Scholar) as the genetic element encoding the superantigen to be associated with of is in by a direct contains a functional and is the in also to a SEK, and part of a of has been in the of a such as Ruzin et A. J. Novick R.P. Mol. Microbiol. 2001; PubMed Scopus Google Scholar) have that to and of in a that is to that and the growth of from its in the with and is specialized transduction to a by the the which the is islands with appear to have M. Ohta T. Uchiyama I. Baba T. Yuzawa H. Kobayashi I. Cui L. Oguchi A. Aoki K. Nagai Y. Lian J. Ito T. Kanamori M. Matsumaru H. Maruyama A. Murakami H. Hosoyama A. Mizutani-Ui Y. Takahashi N.K. Sawano T. Inoue R. Kaito C. Sekimizu K. Hirakawa H. Kuhara S. Goto S. Yabuzaki J. Kanehisa M. Yamashita A. Oshima K. Furuya K. Yoshino C. Shiba T. Hattori M. Ogasawara N. Hayashi H. Hiramatsu K. Lancet. 2001; 357: 1225-1240Abstract Full Text Full Text PDF PubMed Scopus (1564) Google be that the carried by the island the in the of these toxin on mobile genetic elements their staphylococcal as as by these elements are not these mobile elements have and to an in the evolution of S. aureus as a and as a the that virulence are by particularly in nosocomial infections, and that the of accessory genetic elements a the and of mobile genetic elements J. Microbiol. 2001; PubMed Scopus Google Scholar). These islands also the toxin gene in of strains, laboratory has identified a that both and has been that these are encoded by pathogenicity islands that appear to each from their the of pathogenicity islands and their in the evolution of Staphylococcus as a the origin and of pathogenicity islands with of the have multiple open reading frames many of which have this the of or not these are and their be in the of superantigens or in the and of the islands have not been this we the complete sequence and of encoding and the more identified P.M. Novick R.P. Schlievert P.M. Infect. 2001; PubMed Scopus Google Scholar) and M. D. Y. M. H. L. A. and P. M. M. D. Y. M. H. L. A. and P. M. we describe the first time the expression of the contained on a staphylococcal pathogenicity island using DNA We the the evolution of the staphylococcal pathogenicity islands. is a of S. aureus that is by The is a of S. aureus from a of TSS in which laboratory has identified two novel P.M. Novick R.P. Schlievert P.M. Infect. 2001; PubMed Scopus Google Scholar) and aureus also a of S. was as a of genomic of virulence gene the DNA and was from a of TSS P.M. D.A. J. Infect. PubMed Scopus Google Scholar). sequence the S. aureus was from The was to by and sequence that the loci of in the genomic sequence using an model was with the of the of of S. aureus with at in either Todd-Hewitt or rabbit serum in each medium in and at the and of growth and with an cell of of and was using DNA microarrays. to oxygen 1 of was with S. aureus from an to an cell of in a cell in H. and with either oxygen or oxygen with and and at with the and of growth at and and expression of was using DNA microarrays. We have previously that is in growth or of the growth of in oxygen and oxygen with and J.M. Schlievert P.M. J. Microbiol. 2000; PubMed Google J.M. J.K. Schlievert P.M. J. 2001; PubMed Scopus Google Scholar). with SEB, which is in by in by three at with each of in of and in of of to was by of serum 1 the The two of and as with of in J.M. J.J. Infect. PubMed Google Scholar). of the and in the with of that contained the of staphylococcal relatively of of the of S. aureus in medium by from the of growth of was in of and the from the in expression by DNA microarrays. of S. aureus from the at the using a S. aureus was by and expression of was using DNA microarrays. of staphylococcal gene expression in vitro and in vivo using DNA was as described a of from S. aureus and was with to fragments of of each gene from genomic of to genomic DNA in the and the on and with the The in using a from of S. aureus in was using the to the DNA was from the using the to the from from S. aureus to be was with either or and with the microarrays. of the with a was each of two on the of in the and was as the of the each each gene was the from both and was using to expression from the two growth of as if the was at that of using not by and and if was detected in each of the each in of by either or was in the each of was on of expression and using the expression of in using the of the S. aureus the of large to not we that this sequence a novel pathogenicity island, which we the sequence and sequence as to complete the sequence of in the all, a and was to sequence of the was to have 24 potentially encoding proteins 50 amino in 3 of which encoded staphylococcal enterotoxin serotypes B, K, and and many of which have in and We also to the of in the S. aureus a known by and of the three regions as in not is an from a of TSS in which laboratory has described the of two novel P.M. Novick R.P. Schlievert P.M. Infect. 2001; PubMed Scopus Google Scholar) and The flanking identical to the of and the of pathogenicity island the of and in the the identified two with the of the island, and are to the The of was than the found the of S. aureus M. Ohta T. Uchiyama I. Baba T. Yuzawa H. Kobayashi I. Cui L. Oguchi A. Aoki K. Nagai Y. Lian J. Ito T. Kanamori M. Matsumaru H. Maruyama A. Murakami H. Hosoyama A. Mizutani-Ui Y. Takahashi N.K. Sawano T. Inoue R. Kaito C. Sekimizu K. Hirakawa H. Kuhara S. Goto S. Yabuzaki J. Kanehisa M. Yamashita A. Oshima K. Furuya K. Yoshino C. Shiba T. Hattori M. Ogasawara N. Hayashi H. Hiramatsu K. Lancet. 2001; 357: 1225-1240Abstract Full Text Full Text PDF PubMed Scopus (1564) Google of using the at in by the islands of the gene in detected in detected in S. of using the at A. J.C. Press, by the islands of the gene in a We have identified in to the pathogenicity the in is identified by the gene of this gene be as are identified using a is to from of the the be to that such as on the islands are identified to their are identified on the island to its those be to those than of the on the We have this in laboratory to of on islands, and of expression to comparison of and is in and the are identified in I. The of the three is is on the by also has a than and regions of homology three islands identified The first region of of and of this has an not present in in contains this as as an not present in either or The region of of and of three islands to the and contains an enterotoxin gene in the as tstH in and and appear to be more closely to the elements three islands and the identical in and these two islands are at the in and in in and in The and and are also in the to each and to the of and the of is of the gene that have an in the of S. Its and are at the amino and not has the identical sequence as and is secreted in by S. aureus to homology and the region encoding and extensive homology at the to a region in are or more to regions of origin in of to the of was identical to a sequence to the this region of apparently not region with as with is found the region and this region is a of that is identical to a sequence from the identified a S. aureus encoding the of in both and is a sequence staphylococcal and D. D. Mol. Microbiol. PubMed Scopus Google Scholar, J. 1990; PubMed Google Scholar, J. PubMed Google Scholar). sequence is the two proteins that and J. PubMed Google Scholar). to the and the regions with homology to of the contained on a mobile element of a of elements The potentially encoded by is to the of identified in the S. J.C. 1994; PubMed Scopus Google Scholar) amino and of H. K. D. J. 1994; PubMed Google Scholar) amino potentially encodes a with homology amino to the of a P.M. Rood J.I. J. Microbiol. 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DNA to each are in to each was by the of the a a The each was by the of the a a in a in growth are in staphylococcal are generally growth we first the effect of growth on the expression of all, the expression of of 24 was detected during growth of in The expression of five of these was by growth as with the expression of was by the expression of known to be a and was by in as with the of the expression of and was by growth that these are in laboratory and are to those expression was or in the as to the those expression was not detected the growth in a and are to those expression was or in the as to the those expression was not detected the growth examined. have of staphylococcal toxin associated with TSS, by and of toxin in particularly in the of J.M. Schlievert P.M. J. Microbiol. 2000; PubMed Google Scholar, J. J. Infect. PubMed Scopus Google Scholar, J.K. A. J. Infect. 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The expression of of 24 was detected in and during in vivo in the as with the and by in the The expression of and was by of in the at in which they with and of the contained on in the of or be with the enterotoxins carried by the island, the to the of their expression The expression of the secreted toxin and the in these are as virulence factors. the and expression of and are not by the of or of the described pathogenicity islands. clusters of The expression of closely with that of and not of the the expression of and was found to be to that of the and of in their expression is the of of these is that of of the on those in the island with the of expression was detected in a of growth a with variable expression was not with of the examined. the of these array not a that of the are the of to one and the of their expression are and which are by as and by and by also have expression a of or but their expression was not detected in a of to a their also a of or reading and which by in their expression The of not be if the pathogenicity island was of are in genomes such as are encoded by to at the The of that as is in 1. The of these is with those that are known or to be such as and as as with those that be such as all, we to the expression of 22 of the 24 by in vivo and in in vitro and not in growth examined. The expression of either of was more detected than that of contained in the region of the with a these islands in of staphylococcal the expression of the and was detected multiple growth the here, we have described a novel pathogenicity island, in S. aureus and We propose that the identified island meets the of a pathogenicity island (7.Hacker J. Kaper J.B. Annu. Rev. Microbiol. 2000; 54: 641-679Crossref PubMed Scopus (938) Google including the of virulence on the island and the of the island in closely related strains, the of a relatively large genomic region a than the S. the of flanking direct and the of factors of the contained on the island are to contained on described that this described is of we have detected the expression of 22 of the 24 contained on the island, that many of be in the of the island or in of the associated enterotoxins. the origin of the staphylococcal pathogenicity islands are large of pathogenicity islands the gene of with which staphylococci is in or on its and and from which staphylococci these elements this superantigen has been identified in a of that these elements are not in The be until the genomes of of staphylococci are that are carried by genetic such as and and have of recombination in one more These recombination events be by in the accessory genetic elements and the pathogenicity island, to the islands. this not the origin of superantigen on the apparently of superantigen on pathogenicity islands, and the the mobile pathogenicity islands in and evolution of which we elements of the first with the of the of a genetic element to and and the of and islands recombination of the pathogenicity islands identified a genetic element and that these islands have in part specialized transduction and recombination The of the islands, specific is with encoded on either of the islands of tstH is identical in and SaPIbov), and the on the of the islands of the the of the islands multiple of apparently the region in and and and is identical to regions in of these islands. we propose a model the origin and evolution of staphylococcal pathogenicity islands The of the on the of the islands be by a specialized transduction involving an in either Staphylococcus or a with which Staphylococcus has in gene this a element from the to a superantigen and a the enterotoxin either or a of DNA complete as as with the expression of which is known to be by the staphylococcal accessory gene in not with the expression of gene contained on the island but with the gene which is not associated with the that these an The of on the of the island, the be due to addition, deletion, or of large pathogenicity island fragments or “modules.” The of recombination events by regions of sequence homology is by both the of of homology regions of the islands and the expression described in this regions of such as that and are by regions with homology the islands. of these regions appear to be from such as and appear to have the present on and and have these elements from a addition, we found that we expression of on either of the island, particularly the more to and be of the of these have they proteins in the of the encoding and are at the of the the and D.L. Press, Scholar). these be the pathogenicity island is the regions these be or the expression of these at time and the of a The that are to and in their expression of these and the of this enterotoxin to the pathogenicity the DNA sequence is and to a is not the and of these be by recombination events that of DNA in the genomes or are more or The also be that staphylococcal pathogenicity islands have in part as a of from pyrogenic a to be more closely related to staphylococcal enterotoxins SEB, and than superantigens J.K. Yarwood J.M. Schlievert P.M. Annu. Rev. Microbiol. 2001; 55: 77-104Crossref PubMed Scopus (563) Google Scholar). 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