Characterization and expression analysis of Staphylococcus aureus pathogenicity island 3: 1 implications for the evolution of staphylococcal pathogenicity islands

pathogenicity islands then undergo further divergent evolution in subsequent staphylococcal lineages. int , integrase gene; SE enterotoxin gene; φ , phage element.


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The prototypical staphylococcal pathogenicity island, SaPI1, was identified and characterized by Lindsay et 1 al. (9) as the genetic element encoding TSST-1, the only superantigen to be associated with nearly all cases of 2 menstrual TSS. SaPI1 is 15.2 kb in length, flanked by a 17-nt direct repeat, contains a functional integrase (int) 3 gene, and is located near the tyrB locus in strain RN4282. It also appears to encode a second superantigen, SEK, 4 and part of a third, SEQ. Mobility of SaPI1 has been demonstrated only in the presence of a helper phage, such as 5 80α. Ruzin et al. (10) have demonstrated that SaPI1 appears to parasitize excision, replication and encapsidation 6 functions of phage 80α in a relationship that is similar to that between coliphages P4 and P2. During growth of 7 phage 80α, SaPI1 excises from its unique chromosomal insertion site, att c , replicates in the linear form, interferes 8 with phage growth, and is encapsidated into specialized phage heads. Upon transduction to a recipient organism, 9 SaPI1 integrates by the classical Campbell mechanism into the att c site for which the SaPI1-coded integrase is 10 necessary. As islands with different att sites appear to have dissimilar integrases (6), it may well be that the 11 integrase carried by the island determines the integration site in the genome.

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Existence of these toxin genes on mobile genetic elements implies their transfer between staphylococcal 13 strains as well as other bacterial species by horizontal transfer. Furthermore, these elements are not uniformly 14 distributed among clinical isolates. Thus, these mobile elements likely have played, and continue to play, an 15 integral role in the evolution of S. aureus as a species and as a pathogen. Indeed, recent evidence supports the 16 hypothesis that virulence traits are spread by horizontal transfer, particularly in nosocomial infections, and that the 17 presence of accessory genetic elements with a strain may affect the acquisition and loss of other mobile genetic 18 elements (11). These islands may also form the basis for toxin gene exclusion. For instance, in testing thousands of 19 strains, our laboratory has never identified a clinical isolate that produced both TSST-1 and SEB. It has been 20 determined that these toxins are encoded by different pathogenicity islands that appear to exclude each other from 21 their respective integration sites.

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However, despite the identification of numerous pathogenicity islands and their likely importance in the 23 evolution of Staphylococcus as a pathogen, the origin and functions of pathogenicity islands remain areas with little 24 investigation. All of the SaPIs have multiple open-reading frames (ORFs), many of which have no identifiable 25 homologs. To this point, it has not been addressed whether or not these ORFs are expressed and whether their 26 function might be in the regulation of island-associated superantigens, or only in the maintenance and transfer of the 27 islands. In this study we report the complete sequence and map of SaPI3, encoding SEB and the more recently 28 by guest on March 24, 2020 http://www.jbc.org/ Downloaded from respectively, and expression of SaPI3 genes quantified using DNA microarrays. We have previously determined 1 that there is no significant difference in growth parameters (cell densities or timing of entry into the growth phases 2 of interest) between cultures grown in 1% and 21% oxygen [v/v] balanced with nitrogen and 7% carbon dioxide (15, 3 16).

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Immunization of Dutch-belted rabbits-Two Dutch-belted rabbits were immunized with SEB which is 5 made in high concentrations ( > 10 µg/ml) by MN NJ grown in vitro. Rabbits were immunized by three 6 subcutaneous injections at two week intervals with each injection containing 25µg of purified SEB resuspended in 7 0.5 ml phosphate buffer saline and emulsified in 0.5 ml incomplete Freund's adjuvant. Development of antibody to 8 SEB was determined by enzyme-linked immunosorbent assay of serum samples taken one week after the final 9 immunization. The two rabbits developed anti-SEB (IgG) titers of 1:5,120 and 1:10,240, respectively, as compared 10 to preimmune titers of <1:20.

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Subcutaneous infection model-Sterilized perforated hollow polyethylene golf balls were implanted 12 subcutaneously in four Dutch-belted rabbits (17). Implantation of the polyethylene balls and subsequent healing 13 created transudate-filled cavities in the rabbits with volumes of approximately 15 ml that contained few host cells, 14 thus enabling the preparation of staphylococcal RNA relatively free of contaminating host RNA. Six weeks after 15 implantation of the polyethylene balls, ~10 10 colony forming units (CFU) of S. aureus MN NJ grown in TH medium 16 were collected by centrifugation from the late exponential phase of growth (cell density of cultures was 6.7 x 10 8 17 CFU/ml), resuspended into two ml of phosphate buffer saline and injected into the implanted polyethylene balls.

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Samples were removed from the inoculum culture prior to centrifugation for use in expression analysis by DNA 19 microarrays. Two milliliters of transudate containing S. aureus were then removed from the infection chambers at 20 the indicated times after inoculation using a sterile syringe, S. aureus was enumerated by plating and expression of 21 SaPI3 genes quantified using DNA microarrays.  (5), as the D.

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2 nodosus vap genes appear to reside on an integrated bacteriophage (25,26). The predicted product of sapi3_18 is 3 75% similar over 58 amino acids to a putative cro-like repressor of Streptococcus thermophilus bacteriophage Sfi21 4 (27,28). Even stronger homology is seen between the predicted product of sapi3_19 and the cI-like repressor of S.

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Expression analysis of SaPI3-Studies of pathogenicity islands in other bacterial species have 10 demonstrated that genes contained on the islands act in regulation of virulence factors carried by the island 11 (reviewed in reference 7). SaPI3 contains several ORFs with no identifiable function and it was not known whether 12 or not they were expressed, and if so, whether they might act in regulation of the SaPI3 enterotoxins, seb, sek, and 13 seq, or only in the maintenance and transfer of the island itself. We thus employed DNA microarrays to examine the 14 expression of all SaPI3 ORFs potentially encoding products over 50 amino acids in size to determine whether or not 15 they were detectably expressed and if their expression profiles were similar to those of the SaPI3-associated 16 enterotoxins. DNA primers used to PCR amplify probes for each ORF are listed in Table 2.

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Expression data for SaPI3 genes in various growth conditions are summarized in Table 3. Since 18 staphylococcal exotoxins are generally growth-phase regulated, we first examined the effect of growth stage on the 19 expression of SaPI3 genes. In all, the expression of 13 of 24 ORFs was detected during growth of MN NJ in TH 20 broth. The expression of five of these genes was significantly (p < 0.05, Student's t-test) altered by post-exponential 21 growth as compared to the exponential phase of growth, while the expression of four genes was affected by 22 stationary phase growth. As expected, the expression of seb, known to be a post-exponential and stationary phase-23 produced exotoxin, was increased by 8.2-fold in stationary phase as compared to the exponential phase of growth.

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Interestingly, the expression of sek and seq was unaffected by growth phase, suggesting that these are constitutively 25 produced exotoxins in laboratory media.

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Previous studies have demonstrated repression of another staphylococcal toxin associated with TSS, TSST-of elevated carbon dioxide (15,(32)(33)(34)(35). Our laboratory has also determined that the effect of oxygen on tstH 1 expression occurs primarily at the transcriptional level and is independent of cell density and pH (15) 3 . To examine 2 the effect of oxygen concentration on the expression of SaPI3 genes, the cultures were exposed to microaerobic (1% growth conditions in 1 ml cultures of TH broth. There was no significant 4 difference in growth parameters between the microaerobic and aerobic cultures as both cultures entered their 5 respective growth phases (exponential, post-exponential and stationary) of interest at similar times. Also, 6 differences in cell densities between the microaerobic and aerobic cultures were two-fold or less in any given growth 7 phase. In all, the expression of 18 of the 24 SaPI3 ORFs was detected. The expression of four, four, and three 8 genes was significantly affected by growth in microaerobic conditions in the exponential, post-exponential and 9 stationary phases of growth, respectively. The expression of seb was somewhat repressed in the exponential and 10 post-exponential phases of growth in aerobic conditions, while being slightly upregulated during stationary phase 11 growth in aerobic conditions. The expression of no gene, however, was affected by more than approximately two-12 fold.

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To approximate in vivo conditions, and determine those genes whose expression might be artificially 14 enhanced by growth in rich lab medium, the expression of SaPI3 genes was examined during growth of MN NJ in 15 TH broth versus rabbit serum. Effect of growth in serum was examined in the exponential, post-exponential and 16 stationary phases of growth. Cells were quantified by both optical density and dry weight. genes were affected by growth in serum as compared to TH medium in the exponential, post-exponential and 1 stationary phases of growth, respectively, though no gene was affected by more than 3.3-fold. In general, 2 expression of genes on either end of the island were more consistently detected than sapi3_7-17.

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Finally, we examined the expression of SaPI3 genes during incubation of MN NJ in vivo in a rabbit model 4 of TSS using subcutaneous hollow polyethylene infection chambers (17). The expression of SaPI3 genes in vivo 5 was compared to that in the inoculum, which was harvested from cultures in the late-exponential phase of growth 6 and used to infect both nonimmune rabbits and rabbits immunized with SEB. All animals developed symptoms

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To determine whether any of the genes contained on SaPI3 might act in the regulation of, or be co-20 regulated with, the enterotoxins carried by the island, the genes were clustered according to the similarity of their 21 expression profiles (Fig. 2). The expression profiles of the secreted toxin α-hemolysin (hla) and the surface

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In all, we were able to detect the expression of 22 of the 24 SaPI3 ORFs by microarray analysis (Table 1) In the work presented here, we have described a novel pathogenicity island, SaPI3, in S. aureus strains 3 COL and MN NJ. We propose that the newly identified island meets the consensus definitions of a pathogenicity 4 island (7), including the presence of demonstrated virulence genes on the island (seb, sek and seq), the lack of the 5 island in closely related strains, the occupation of a relatively large genomic region (~16 kb), a lower G+C content 6 than the overall S. aureus genome (31.4% versus 32.8%), the presence of flanking direct repeats, and the presence of 7 mobility factors (integrase). Many of the genes contained on the island are homologous to genes contained on 8 described bacteriophages, suggesting that this SaPI, like others previously described, is of bacteriophage origin. 1 ancestral genetic element and that these islands have arisen in part through specialized transduction and 2 recombination events. The overall layout of the islands, even specific genes, is similar, with exotoxins encoded on 3 either end of the islands (the location of tstH is nearly identical in SaPIn1/SaPIm1, SaPI1 and SaPIbov), and the 4 exotoxin genes on the right end of the islands consistently located upstream of the integrase genes. Towards the 5 center of the islands lie multiple genes of apparently phage origin. Furthermore, the region in SaPI3 between 6 nucleotides 3113 and 6929 and nucleotides 9591 and 10494 is nearly identical to regions in all of these islands.

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Thus, we propose a generalized model for the origin and evolution of staphylococcal pathogenicity islands (Fig. 3). island, but rather the agr-regulated gene hla, which is not associated with SaPI3. This supports the hypothesis that 17 these exotoxins were incorporated into an existing phage.

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The presence of exotoxins on the right end of the island, inside the integrase may instead be due to 19 addition, deletion, or mutual crossover of large pathogenicity island fragments, or "modules". The hypothesis of 20 modular recombination events, perhaps mediated by key regions of sequence homology, is supported both by the 21 presence of long stretches of homology between distinct regions of the islands and the expression data described in 22 this work. Extensive regions of very high similarity (>95% identity), such as that between SaPI3 and SaPI1, are 23 abruptly interrupted by regions with very little homology between the islands. Some of these regions appear to be 24 derived from phages such as PVL-converting phage φSLT. Furthermore, SaPI1 and SaPI3 appear to have either lost 25 the sel/sek/sec3 module present on SaPIn1/SaPIm1 and SaPIbov or, alternatively, SaPIn1/SaPIm1 and SaPIbov have 26 gained these elements after diverging from a common ancestor. In addition, we found that we could consistently 27 detect expression of genes on either end of the island, particularly sapi3_19-24. However, the "core" bacteriophage genes were much more difficult to detect, and may only be strongly expressed upon mobilization of the island.
1 Though most of these central genes have no definite homolog, they may well encode proteins involved in the 2 structural components of the original phage. In phage λ, for instance, genes encoding head and tail components are 3 located together at the end of the prophage opposite the int and xis genes (37). Thus, these genes may only be 4 expressed when the pathogenicity island is mobilized. Alternatively, the promoter regions for these genes may be 5 defective or even absent, preventing the expression of these genes at any time and requiring the structural 6 components of a helper phage for transfer. The observation, then, that sapi3_19-24 are transcribed opposite to 7 sapi3_3-18 and cluster in their expression profiles, implies co-regulation of these genes and the addition of this 8 enterotoxin module to the already existing pathogenicity island. Interestingly, the DNA sequence containing 9 sapi3_3-17 is highly conserved between SaPI1 and SaPI3, and to a lesser extent, SaPIbov. It is not yet clear whether 10 the size and type of these modules might be constrained by recombination events that require conserved stretches of 11 DNA sequences in the phage genomes, or are more or less randomly generated. The possibility also cannot be 12 excluded that staphylococcal pathogenicity islands have arisen in part as a result of horizontal transfer from another 13 species. Indeed, streptococcal pyrogenic exotoxin A, a phage-encoded superantigen, appears to be more closely 14 related to staphylococcal enterotoxins SEB, SEC3 and SEG, than other streptococcal superantigens (4).

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We also propose that the promiscuity of these islands and their tendency to undergo recombination events The demonstrated mobility of SaPI1 and the presence of apparently functional integrases in all of these 5 islands (5,9,10) provides further support for the hypothesis that they remain a significant part of the evolutionary 6 scheme of S. aureus and will likely give rise to new enterotoxins and pathogenicity islands. The mobility of SaPIs 7 has led to speculation that these islands represent relatively "young", recently acquired genetic elements, as opposed 8 to islands which likely entered their host organisms millions of years ago and have become relatively immobile, 9 perhaps even part of the host core genome (7). Alternatively, the ability of staphylococci and their associated 10 pathogenicity islands to evolve may be constrained by the evolution of their human hosts. Thus, the conversion of 11 any particular SaPI into a stable element may be restricted by the need to constantly respond to the adaptive human   Table 3. Expression data for SaPI3 ORFs a . Green and red fonts are used to indicate those genes whose expression was significantly increased or decreased in the test experimental condition (blue font) as compared to the reference experimental condition (purple font). Asterisks represent significance of results as determined using the Student's t-test (* p < 0.05; ** p < 0.01; *** p < 0.001). Blank spaces indicated those ORFs whose expression was not detected under the growth conditions examined.