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J. Biol. Chem., Vol. 277, Issue 15, 13138-13147, April 12, 2002
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From the
Received for publication, December 6, 2001, and in revised form, January 3, 2002
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."
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). 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 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). 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). 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). More recently, Kuroda et al. (6) 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, 8). They are present in the genomes of many staphylococci but absent from closely related strains, they are relatively large genomic fragments (>15 kb), they differ in GC content
from the rest of the chromosome, they are flanked by direct repeats
likely generated upon insertion of the elements into the genome, some
are associated with tRNA loci, and they possess genes coding for
genetic mobility, including conserved integrases.
The prototypical staphylococcal pathogenicity island, SaPI1, was
identified and characterized by Lindsay et al. (9) as the
genetic element encoding TSST-1, the only superantigen to be associated
with nearly all cases of menstrual TSS. SaPI1 is 15.2 kb in length,
flanked by a 17-nucleotide direct repeat, contains a functional
integrase (int) gene, and is located near the
tyrB locus in strain RN4282. It also appears to encode a
second superantigen, SEK, and part of a third superantigen, SEQ.
Mobility of SaPI1 has been demonstrated only in the presence of a
helper phage, such as 80 Existence of these toxin genes on mobile genetic elements implies their
transfer between staphylococcal strains as well as other bacterial
species by horizontal transfer. Furthermore, these elements are not
uniformly distributed among clinical isolates. Thus, these mobile
elements likely have played and continue to play an integral role in
the evolution of S. aureus as a species and as a pathogen.
Indeed, recent evidence supports the hypothesis that virulence traits
are spread by horizontal transfer, particularly in nosocomial
infections, and that the presence of accessory genetic elements within
a strain may affect the acquisition and loss of other mobile genetic
elements (11). These islands may also form the basis for toxin gene
exclusion. For instance, in testing thousands of strains, our
laboratory has never identified a clinical isolate that produced both
TSST-1 and SEB. It has been determined that these toxins are encoded by
different pathogenicity islands that appear to exclude each other from
their respective integration sites.
However, despite the identification of numerous pathogenicity islands
and their likely importance in the evolution of
Staphylococcus as a pathogen, the origin and functions of
pathogenicity islands remain areas with little investigation. All
of the SaPIs have multiple open reading frames (ORFs), many
of which have no identifiable homologs. To this point, the issues
of whether or not these ORFs are expressed and whether their
function might be in the regulation of island-associated superantigens
or only in the maintenance and transfer of the islands have not been
addressed. In this study, we report the complete sequence and map
of SaPI3 encoding SEB and the more recently identified enterotoxins,
SEK (12) and SEQ.2
Furthermore, we describe for the first time the expression of the
numerous genes contained on a staphylococcal pathogenicity island using
DNA microarray technology. We then discuss the implications for the
evolution of the staphylococcal pathogenicity islands.
Strains--
COL is a prototypical methicillin-resistant isolate
of S. aureus that is currently being sequenced by The
Institute for Genomic Research. MN NJ is a methicillin-sensitive
isolate of S. aureus from a case of nonmenstrual TSS in
which our laboratory has identified two novel superantigens, SEK (12)
and SEQ.2 S. aureus MN8, also a
methicillin-sensitive isolate of S. aureus, was used as a
source of genomic template for amplification of virulence gene probes
for the DNA microarrays and was isolated before 1980 from a case of
menstrual TSS (13).
Sequencing--
Preliminary sequence data for the S. aureus strain COL was obtained from The Institute for Genomic
Research website (www.tigr.org). A primer walking-based approach was
used to amplify by PCR and sequence ~3.4 kb that spanned the
unsequenced loci of SaPI3 in the COL genomic sequence data base.
Automated sequencing using an ABI model 377 was performed with the
assistance of the Advanced Genetic Analysis Center (University of
Minnesota, St. Paul, MN).
Growth of S. aureus in vitro--
50-ml cultures of S. aureus MN NJ were grown aerobically with shaking at 37 °C in
either Todd-Hewitt (TH) broth (BC PharMingen) or rabbit serum
(Invitrogen). Two independent cultures in each medium were grown in
parallel, and samples were removed at the exponential, postexponential,
and stationary phases of growth (2, 3, and 8 h after inoculation
with an initial cell density of A600 nm = 0.1). Expression of SaPI3 ORFs and other virulence-associated genes was quantified using DNA microarrays.
For cultures exposed to altered oxygen levels, 1 ml of TH broth was
inoculated with S. aureus MN NJ from an overnight culture to
an initial cell density of A600 nm = 0.1 in a
35 × 12-mm-diameter polystyrene Petri dish (Nunc, Roskilde,
Denmark). All cultures were placed into sealed, humidified Plexiglass
cell culture chambers (20 × 26 × 7.5 cm, internal
dimensions; Mishell-Dutton) (14), flushed with gas mixtures containing
either 1% oxygen (v/v) or 21% oxygen (v/v) balanced with nitrogen and
7% carbon dioxide (Praxair, St. Louis, MO), and sealed. Chambers were
then incubated at 37 °C with orbital shaking (~125 rpm). Samples
for the exponential, postexponential, and stationary phases of growth
were removed at ~2, 3, and 8 h after inoculation, respectively,
and expression of SaPI3 genes was quantified using DNA microarrays. We
have previously determined that there is no significant difference in
growth parameters (cell densities or timing of entry into the growth
phases of interest) between cultures grown in 1% oxygen and 21%
oxygen (v/v) balanced with nitrogen and 7% carbon dioxide (15,
16).
Immunization of Dutch-belted Rabbits--
Two Dutch-belted
rabbits were immunized with SEB, which is made in high concentrations
(>10 µg/ml) by MN NJ grown in vitro. Rabbits were
immunized by three subcutaneous injections at 2-week intervals, with
each injection containing 25 µg of purified SEB resuspended in
0.5 ml of phosphate-buffered saline and emulsified in 0.5 ml of
incomplete Freund's adjuvant. Development of antibody to SEB was
determined by enzyme-linked immunosorbent assay of serum samples taken
1 week after the final immunization. The two rabbits developed anti-SEB
(IgG) titers of 1:5,120 and 1:10,240, respectively, as compared with
preimmune titers of <1:20.
Subcutaneous Infection Model--
Sterilized perforated hollow
polyethylene golf balls were implanted subcutaneously in four
Dutch-belted rabbits (17). Implantation of the polyethylene balls and
subsequent healing created transudate-filled cavities in the rabbits
with volumes of ~15 ml that contained few host cells, thus enabling
the preparation of staphylococcal RNA relatively free of contaminating
host RNA. 6 weeks after implantation of the polyethylene balls,
~1010 colony-forming units of S. aureus MN NJ
grown in TH medium were collected by centrifugation from the late
exponential phase of growth (cell density of cultures was 6.7 × 108 colony-forming units/ml), resuspended in 2 ml of
phosphate-buffered saline, and injected into the implanted polyethylene
balls. Samples were removed from the inoculum culture before
centrifugation for use in expression analysis by DNA microarrays. 2 ml
of transudate containing S. aureus were then removed from
the infection chambers at the indicated times after inoculation using a
sterile syringe, S. aureus was enumerated by plating, and
expression of SaPI3 genes was quantified using DNA microarrays.
RNA Preparation and DNA Microarrays--
Analysis of
staphylococcal gene expression in vitro and in
vivo using DNA microarrays was performed as described elsewhere (www.agac.umn.edu/microarray/protocols/protocols.htm). In brief, a
library of targets representing 68 genes from S. aureus MN
NJ and MN8 was constructed with primers designed to amplify fragments of ~300 bp of each gene from genomic DNA. Two successive rounds of
PCR were performed to minimize genomic DNA contamination in the
amplification products, and the final 100-µl reactions were checked
for quality on agarose gels and purified with the QIAquick PCR
Purification Kit (Qiagen, Valencia, CA). The purified products were
printed in triplicate using a Total Array System robot (BioRobotics, Boston, MA). Cell pellets from centrifuged samples of S. aureus cultures were flash-frozen in liquid nitrogen. Total RNA
was prepared using the RNeasy Mini Kit (Qiagen) according to the
manufacturer's directions. DNA was removed from the RNA preparations
using the RNase-free DNase Set (Qiagen) according to the
manufacturer's directions. cDNA prepared from RNA from S. aureus cultures to be compared was labeled with either Cy3 or Cy5
fluorescent dye (Amersham Biosciences) and competitively hybridized
with the printed microarrays. Images of the hybridized arrays were
obtained with a Scanarray 5000 microarray scanner (GSI
Lumonics, Watertown, MA). One independent hybridization (on triplicate
arrays) was conducted for each of two independent experiments.
Fluorescence intensities for individual spots were normalized based on
the total intensity of fluorescence in the Cy3 and Cy5 channels.
Fluorescence intensity was determined as the average intensity of the
triplicate spots for each gene. Total fluorescence for each gene was
normalized between arrays for independent experiments, the data were
combined from both experiments, and statistical significance was
determined using Student's t test to compare expression
data from the two growth conditions of interest. SaPI3 ORFs were
determined as being expressed if the fluorescence intensity was at
least twice that of background levels established using negative
controls (probes for genes not expressed by strains MN NJ and COL) and
if fluorescence was detected in each of the triplicate arrays
for each independent experiment. To account for possible bias in
labeling of cDNA by either Cy3 or Cy5, dye labeling was reversed in
the second independent experiment for each of several experimental
conditions. No dye bias was detected. Clustering based on similarity of
expression profiles and visualization were performed using the software
program Spotfire DecisionSite 6.1 (www.spotfire.com). Similarities
between expression profiles of individual genes in all 11 experimental conditions were calculated using the "Euclidean distance" method.
Identification and Characterization of SaPI3--
Data base
searches of the unfinished S. aureus COL genome
(www.tigr.org) revealed the presence of large segments homologous to SaPI1. Because COL does not produce TSST-1, we hypothesized that
this sequence comprised a novel pathogenicity island, which we termed
SaPI3. Using the SaPI1 sequence and SaPI3 partial sequence as guides,
primers were designed to complete the sequence of SaPI3 in the COL
strain. In all, a PCR and primer walking-based approach was used to
sequence ~3.4 kb of the 15,936-bp SaPI3. SaPI3 was determined to have
24 ORFs potentially encoding proteins over 50 amino acids in length, 3 of which encoded staphylococcal enterotoxin serotypes B, K, and Q, and
many of which have homologs in SaPI1 and SaPIbov (Fig.
1; Table
I). We were also able to identify the
presence of SaPI3 in the clinical isolate S. aureus MN NJ, a
known SEB producer, by PCR analysis and sequencing of the same three
regions as in strain COL (data not shown). MN NJ is an isolate from a
case of nonmenstrual TSS in which our laboratory has described the
presence of two novel enterotoxins, SEK (12) and SEQ.2 The
repeated 17-nucleotide sequences flanking SaPI3 were identical to the
att sites of SaPI1 (5'-TTATTTAGCAGGATAA-3') and thus might form the basis of pathogenicity island exclusion (i.e. the
lack of TSST-1 and SEB in the same clinical isolates). In general, the
identified SaPI3 genes form two apparent transcriptional blocks with
ORFs 2-18 (including SEB) oriented toward the left of the island,
whereas ORFs 19-24 (including SEK and SEQ) are oriented to the right
(Fig. 1). The overall GC content of SaPI3 was 31.4%, somewhat lower
than the 32.8-32.9% found for the whole genome of S. aureus (6).
We have identified genes in SaPI3 according to the
following nomenclature: pathogenicity island_ORF number
(variant). Thus, the ninth ORF in SaPI3 is identified by the gene
name sapi3_9. A mutant of this gene might be designated as
sapi3_9(1). Sequential genes are identified using a hyphen
(e.g. sapi3_10-15 is used to identify all SaPI3 ORFs from 10 through 15). Upon determination of the gene's function, the name will
be altered to reflect that function, such as sapi3_int.
(Exotoxins on the islands are identified according to their own
nomenclature system.) If additional genes are identified on the island
subsequent to its initial sequencing, those genes will be numbered
sequential to those already identified, rather than renumbering all of
the genes on the island. We have implemented this nomenclature system
in our laboratory to promote systematic identification of SaPI genes,
preclude confusion regarding identically numbered ORFs on different
islands, and allow unambiguous assignation of expression data to SaPI genes.
A comparison of SaPI3, SaPI1, and SaPIbov is shown in Fig. 1, and the
corresponding ORFs are identified in Table I. The overall length of the
three SaPIs is similar, although SaPIbov is larger on the 5' end by
1915 bp. SaPIbov also has a different att site than SaPI1
and SaPI3. Two core regions of high (>92% identity) homology between
all three islands were identified (Fig. 1). The first core region
includes nucleotides 2974-6709 of SaPI1, 3113-6929 of SaPI3, and
5100-8745 of SaPIbov. Within this core region, SaPI1 has an additional
100 bp not present in SaPIbov (nucleotides 5909-6009 in SaPI1),
whereas SaPI3 contains this 100-bp stretch as well as an additional 67 bp not present in either SaPI1 or SaPIbov (nucleotides 6063-6230). The
second core region includes nucleotides 9380-10,284 of SaPI1,
9591-10,494 of SaPI3, and 11,419-12,429 of SaPIbov. All three islands
contain int genes adjacent to the attR sites, and
SaPI3 contains an enterotoxin gene (seb) in the same
position as tstH in SaPI1 and SaPIbov. SaPI1 and SaPI3
appear to be even more closely related. In addition to the shared
elements among all three islands and the identical attachment sites in SaPI1 and SaPI3, these two islands are highly homologous (>93% identity) at the right end (nucleotides 10,285-11,046 in SaPI1 and
10,494-11,254 in SaPI3; nucleotides 12,248-15,250 in SaPI1 and
12,891-15,953 in SaPI3) (Fig. 1). The ear
(sapi1_1 and sapi3_1) and sek genes
are also in the same relative positions to each other and to the
att sites of SaPI1 and SaPI3. Although the function of
ear is unknown, several properties of the gene suggest that it may have an important function in the life cycle of S. aureus. Its position and predicted product are conserved (~75%
identity at the amino acid level) among SaPI1, SaPI3, and SaPI4 (data
not shown), it has the identical signal sequence as TSST-1, and it is
secreted in abundant quantities by S. aureus RN4282. In
addition to homology between SaPI1, SaPI3, and SaPIbov, the region
encoding sapi3_3-9 and sapi3_15 shares extensive
homology (>95% at the nucleotide level) to a matching region in
SaPIn1/SaPIm1, whereas sapi3_10-14 are 87% or more similar
to regions presumably of phage origin in strain Mu50.
A locus of ~900 bp adjacent to the attL site of SaPI3 was
95% identical to a phage 80
In addition to the integrase and the noncoding regions with homology to
phage DNA, several of the genes contained on SaPI3 suggest a mobile
element of phage origin, perhaps a conglomeration of several phage
elements (Table I). The terminase potentially encoded by
sapi3_3 is homologous to the small subunit of identified terminases in the bacteriophages Expression Analysis of SaPI3--
Studies of pathogenicity islands
in other bacterial species have demonstrated that genes contained on
the islands act in regulation of virulence factors carried by the
island (reviewed in Ref. 7). SaPI3 contains several ORFs with no
identifiable function, and it was not known whether or not they were
expressed, and, if so, whether they might act in regulation of the
SaPI3 enterotoxins, seb, sek, and seq,
or only in the maintenance and transfer of the island itself. We thus
used DNA microarrays to examine the expression of all SaPI3 ORFs
potentially encoding products over 50 amino acids in size to determine
whether or not they were detectably expressed and whether their
expression profiles were similar to those of the SaPI3-associated
enterotoxins. DNA primers used to PCR-amplify probes for each ORF are
listed in Table II.
Expression data for SaPI3 genes in various growth conditions are
summarized in Table III. Because
staphylococcal exotoxins are generally growth phase-regulated, we first
examined the effect of growth stage on the expression of SaPI3 genes.
In all, the expression of 13 of 24 ORFs was detected during growth of
MN NJ in TH broth. The expression of five of these genes was
significantly (p < 0.05, Student's t test)
altered by postexponential growth as compared with exponential phase
growth, whereas the expression of four genes was affected by stationary
phase growth. As expected, the expression of seb, known to
be a postexponential and stationary phase-produced exotoxin, was
increased by 8.2-fold in stationary phase as compared with the
exponential phase of growth. Interestingly, the expression of
sek and seq was unaffected by growth phase, suggesting that these are constitutively produced exotoxins in laboratory media.
Previous studies have demonstrated repression of another staphylococcal
toxin associated with TSS, TSST-1, by anaerobic conditions and
enhancement of toxin production in aerobic conditions, particularly in
the presence of elevated carbon dioxide (15, 32-35). Our laboratory has also determined that the effect of oxygen on tstH
expression occurs primarily at the transcriptional level and is
independent of cell density and pH
(15).3 To examine the effect
of oxygen concentration on the expression of SaPI3 genes, the cultures
were exposed to microaerobic (1% O2 (v/v)) and aerobic
(21% O2 (v/v)) growth conditions in 1-ml cultures of TH
broth. There was no significant difference in growth parameters between
the microaerobic and aerobic cultures because both cultures entered
their respective growth phases (exponential, postexponential, and
stationary) of interest at similar times. Also, differences in cell
densities between the microaerobic and aerobic cultures were 2-fold or
less in any given growth phase. In all, the expression of 18 of the 24 SaPI3 ORFs was detected. The expression of four, four, and three genes
was significantly affected by growth in microaerobic conditions in the
exponential, postexponential, and stationary phases of growth,
respectively. The expression of seb was somewhat repressed
in the exponential and postexponential phases of growth in aerobic
conditions and was slightly up-regulated during stationary phase growth
in aerobic conditions. The expression of no gene, however, was affected
by more than approximately 2-fold.
To approximate in vivo conditions and determine those genes
whose expression might be artificially enhanced by growth in rich laboratory medium, the expression of SaPI3 genes was examined during
growth of MN NJ in TH broth versus rabbit serum. Effect of
growth in serum was examined in the exponential, postexponential, and
stationary phases of growth. Cells were quantified by both absorbance and dry weight. No difference in the time at which TH
or serum cultures entered their respective growth phases of interest
was observed. A600 nm values for S. aureus grown in TH medium were 0.83, 1.69, and 2.16 for the
exponential, postexponential, and stationary phases of growth,
respectively. A600 nm values for S. aureus grown in rabbit serum were 0.33, 0.97, and 1.27 for the
same growth phases. As measured by dry weight, cell mass at 8 h
was 8.0 mg/ml in TH medium and 8.3 mg/ml in rabbit serum.
Quantification of S. aureus growth in serum in
vitro is problematic because cells clump tightly and are resistant
to dispersal, even by ultrasonication, thus preventing accurate counts
by plating or absorbance. Alternatively, dry weight analysis is
hampered by the tendency of S. aureus to bind serum
components and thus add "artificial" cell mass. Thus, an effect of
cell density on SaPI3 gene expression cannot be ruled out in these
serum cultures. Taken as a whole, however, the data reflect the fact
that growth in standard rich laboratory media may well enhance or
repress expression of virulence-associated genes in a manner
inconsistent with what occurs in vivo. In all, the
expression of 19 of 24 SaPI3 ORFs was detected in these experiments.
The expression of five, six, and three genes were affected by growth in
serum as compared with TH medium in the exponential, postexponential,
and stationary phases of growth, respectively, although no gene was
affected by more than 3.3-fold. In general, expression of genes on
either end of the island was more consistently detected than
sapi3_7-17.
Finally, we examined the expression of SaPI3 genes during incubation of
MN NJ in vivo in a rabbit model of TSS using subcutaneous hollow polyethylene infection chambers (17, 36). The expression of
SaPI3 genes in vivo was compared with that in the inoculum, which was harvested from cultures in the late exponential phase of
growth and used to infect both nonimmune rabbits and rabbits immunized
with SEB. All animals developed symptoms consistent with TSS, including
hypotension, respiratory distress, and obvious discomfort in the
vicinity of the infection chamber. The SEB-immunized rabbits
experienced a delayed onset of symptoms and were euthanized 22 h
after inoculation, whereas the nonimmune rabbits died several hours
after inoculation. Cell densities recovered from the infection chambers
did not vary more than 0.22 log units from the cell density of the
inoculum before concentration (6.7 × 108
colony-forming units/ml), thus we were able to effectively eliminate the potential effects of cell density and growth phase on gene expression in vivo. Using this subcutaneous infection model,
we were previously able to demonstrate up to 18-fold changes in
virulence-associated gene expression between cells in the inoculum and
cells in vivo (36). The expression of 13 of 24 SaPI3 ORFs
was detected in vivo. Sapi3_1 (ear)
and seb were significantly up-regulated during incubation
in vivo in the nonimmune rabbits as compared with the inoculum and were affected very little by incubation in the immune animals. The expression of sek and seq was only
significantly affected by incubation of MN NJ in the immune animals at
8 h, in which case they were repressed, together with
sapi3_20, sapi3_21, and
sapi3_24.
To determine whether any of the genes contained on SaPI3 might act in
the regulation of or be co-regulated with the enterotoxins carried by
the island, the genes were clustered according to the similarity of
their expression profiles (Fig. 2). The
expression profiles of the secreted toxin
Although the resolution of these array experiments did not allow a
conclusive determination that any of the SaPI3 genes are co-transcribed, the proximity of several ORFs to one another and the
clustering of their expression profiles are suggestive. For instance,
sapi3_4 and sapi3_5, which are separated by only
2 nucleotides, cluster together, as do sapi3_19 and
sapi3_20, separated by 11 nucleotides. Interestingly,
sapi3_21 and seq, separated by 11 nucleotides,
also have similar expression profiles. Genes sapi3_9-11 form
a set of overlapping or nearly overlapping ORFs, but their expression
was not detected in a sufficient number of conditions to make a
conclusion regarding their possible co-transcription. Genes
sapi3_14-18 also form a set of overlapping or nearly
overlapping reading frames; however, only sapi3_15 and
sapi3_16, which overlap by 13 nucleotides, cluster in their
expression profiles. The presence of co-transcribed genes would not be
unexpected if the pathogenicity island was indeed of phage origin.
Co-transcribed genes are common in phage genomes because numerous
products, such as structural elements, are encoded by polycistronic
transcripts to achieve coordinate temporal regulation at critical
points within the phage lytic cycle. The presence of several stem-loop
structures that may serve as rho-independent transcription terminators
is indicated in Fig. 1. The placement of these structures is consistent
with those genes that are known or likely to be independently
transcribed, such as seb, sapi3_1
(ear), sek, and int, as well as with
those genes that may well be co-transcribed, such as
sapi3_14-18.
In all, we were able to detect the expression of 22 of the 24 SaPI3
ORFs by microarray analysis (Table I). 13 ORFs were detectably expressed in vivo and in vitro, 9 ORFs were
detectably expressed in vitro only, and 2 ORFs were not
detectably expressed in any growth condition examined. The expression
of genes toward either end of SaPI3 was more consistently detected than
that of genes contained in the central region of the island. Consistent
with a role for these islands in dissemination of staphylococcal
enterotoxins, the expression of the toxins seb,
sek, and seq was consistently detected under
multiple growth conditions.
In the work presented here, we have described a novel
pathogenicity island, SaPI3, in S. aureus strains COL and MN
NJ. We propose that the newly identified island meets the consensus
definitions of a pathogenicity island (7), including the presence of
demonstrated virulence genes on the island (seb,
sek, and seq), the lack of the island in closely
related strains, the occupation of a relatively large genomic region
(~16 kb), a lower GC content than the overall S. aureus
genome (31.4% versus 32.8%), the presence of flanking direct repeats, and the presence of mobility factors (integrase). Many
of the genes contained on the island are homologous to genes contained
on described bacteriophages, suggesting that this SaPI, like others
described previously, is of bacteriophage origin. Furthermore, we have
detected the expression of 22 of the 24 genes contained on the island,
suggesting that many of them may be active in the maintenance of the
island or in regulation of the associated enterotoxins.
Several explanations for the origin of the staphylococcal pathogenicity
islands are possible. A large number of pathogenicity islands may
circulate through the combined gene pool of numerous bacterial species
with which staphylococci is transiently associated, in or on its human
and animal hosts, and from which staphylococci acquires these elements
through horizontal transfer. However, this hypothesis seems unlikely
because superantigen production has been identified in a very limited
number of bacterial species, suggesting that these elements are not in
general circulation. The possibility cannot be eliminated completely,
however, until the genomes of currently unidentified bacterial
associates of staphylococci are characterized. A second hypothesis
suggests that exotoxins are carried primarily by other genetic
elements, such as plasmids and phage, and have integrated into sites of high recombination frequency in one more ancestral strains. These recombination events might be mediated by homologous genes in the
accessory genetic elements and the pathogenicity island, giving rise to
the exotoxin-carrying islands. However, this hypothesis does not
account for the origin of superantigen genes on transmissible elements,
the apparently exclusive presence of certain superantigen genes on only
pathogenicity islands, and the likely role for the demonstrably mobile
pathogenicity islands in dissemination and evolution of superantigens.
A third hypothesis, which we favor, combines certain elements of the
first two, with the addition of the assumption of a common ancestral
genetic element to SaPI1-4, SaPIbov, and SaPIm1/SaPIn1 and the
continual generation of unique toxins and islands through modular
recombination events.
Several features of the enterotoxin-encoding pathogenicity islands
identified thus far suggest a common ancestral genetic element and that
these islands have arisen in part through specialized transduction and
recombination events. The overall layout of the islands, even specific
genes, is similar, with exotoxins encoded on either end of the islands
(the location of tstH is nearly identical in SaPIn1/SaPIm1,
SaPI1, and SaPIbov), and the exotoxin genes on the right end of the
islands consistently located upstream of the integrase genes. Toward
the center of the islands lie multiple genes of apparently phage
origin. Furthermore, the region in SaPI3 between nucleotides 3113 and
6929 and nucleotides 9591 and 10,494 is nearly identical to regions in
all of these islands. Thus, we propose a generalized model for the
origin and evolution of staphylococcal pathogenicity islands (Fig.
3). The presence of the exotoxin genes on
the left ends of the islands can be explained by a specialized
transduction event involving an ancestral, exotoxin-free bacteriophage
in either Staphylococcus or a bacterial species with which
Staphylococcus has subsequently engaged in horizontal gene
transfer. In this model, a bacteriophage element excised from the
chromosome adjacent to a superantigen locus and through a
mis-recombination event gained the enterotoxin while either simultaneously or subsequently losing a segment of phage DNA necessary for complete phage function (such as xis). It thus became
dependent upon wild-type helper phage for excision, packaging, and/or
mobilization, as observed with SaPI1. Indeed, the expression of
seb, which is known to be regulated by the staphylococcal
accessory gene regulator (agr) in vitro, did not
correlate with the expression of any other gene contained on the island
but rather correlated with the agr-regulated gene
hla, which is not associated with SaPI3. This
supports the hypothesis that these exotoxins were incorporated into an
existing phage.
The presence of exotoxins on the right end of the island, inside the
integrase, may instead be due to addition, deletion, or mutual
crossover of large pathogenicity island fragments or "modules." The
hypothesis of modular recombination events perhaps mediated by key
regions of sequence homology is supported by both the presence of long
stretches of homology between distinct regions of the islands and the
expression data described in this work. Extensive regions of very high
similarity (>95% identity), such as that between SaPI3 and SaPI1, are
abruptly interrupted by regions with very little homology between the
islands. Some of these regions appear to be derived from phages such as
Panton-Valentine leukocidin-converting phage We also propose that the promiscuity of these islands and their
tendency to undergo recombination events underlie the evolutionary divergence of staphylococcal superantigens. The presence of an ancestral island in multiple staphylococcal lineages undergoing separate genetic events (e.g. point mutations, genetic
rearrangements, and insertion of other mobile genetic elements) and
evolutionary pressures in various communities and hosts would lead to a
great diversity of exotoxin genes as well as pathogenicity islands
(Fig. 3). Further recombination events upon mixing of these various staphylococcal lineages would lead to even greater diversity among the
pathogenicity islands and perhaps other mobile genetic elements as
well. However, the evolutionary fitness that superantigenic toxins
confer to the recipient strains is not yet completely understood. It is
clear that these superantigens have significant immunodeletory effects,
including inducing anergy and deletion of a large population of T cells
(38-40) as well as preventing the development of antibody to the toxin
itself (41-44). There is also evidence that superantigens destroy
endothelial cells (45) and that they may exclude neutrophils from
infection foci (46-48). Thus, superantigens likely confer immunological protection to their host strains and perhaps prevent clearance of those strains from the human host.
The demonstrated mobility of SaPI1 and the presence of apparently
functional integrases in all of these islands (5, 9, 10) provide
further support for the hypothesis that they remain a significant part
of the evolutionary scheme of S. aureus and will likely give
rise to new enterotoxins and pathogenicity islands. The mobility of
SaPIs has led to speculation that these islands represent relatively
"young," recently acquired genetic elements, as opposed to islands
that likely entered their host organisms millions of years ago and have
become relatively immobile, perhaps even part of the host core genome
(7). Alternatively, the ability of staphylococci and their associated
pathogenicity islands to evolve may be constrained by the evolution of
their human hosts. Thus, the conversion of any particular SaPI into a
stable element may be restricted by the need to constantly respond to
the adaptive human immune response. Constant generation of new
superantigens and pathogenicity islands might enable S. aureus to colonize and infect human populations that may already
have acquired immunity to ancestral exotoxins. Indeed, lack of antibody
to exotoxin is a key risk factor for the development of staphylococcal
TSS (41, 49, 50).
As additional S. aureus genome sequences and SaPIs become
available and are associated with various staphylococcal lineages and
infection types, it may become easier to identify a common ancestor of
staphylococcal pathogenicity islands and superantigens. Regardless,
continued research into the mechanisms of superantigen and SaPI
evolution and acquisition will allow more accurate epidemiological investigations as well as a greater understanding of which potential therapeutic interventions might be viable, particularly in the development of toxoid vaccines.
We thank Barbara May for assistance with
reverse transcription-PCR and Peter Southern for helpful discussions
regarding SaPI evolutionary models. Preliminary sequence data were
obtained from The Institute for Genomic Research website
(www.tigr.org) with support provided by the National Institute of
Allergy and Infectious Diseases and Merck Genome Research Institute.
*
This work was supported in part by National Institutes of
Health Grant AI22159 (to P. M. S.).The costs of publication of this article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF410775.
§
Supported by a Howard Hughes Predoctoral Fellowship in the
Biological Sciences.
**
To whom correspondence should be addressed: Dept. of Microbiology,
University of Minnesota Medical School, MMC 196, 420 Delaware St. SE,
Minneapolis, MN 55455. Tel.: 612-624-9471; Fax: 612-626-0623; E-mail:
pats@lenti.med.umn.edu.
Published, JBC Papers in Press, January 30, 2002, DOI 10.1074/jbc.M111661200
2
P. M. Orwin, D. Y. M. Leung,
H. L. Donahue, G. A. Bohach, and P. M. Schlievert,
submitted for publication.
3
J. M. Yarwood and P. M. Schlievert,
unpublished observations.
The abbreviations used are:
TSS, toxic shock
syndrome;
SaPI, staphylococcal pathogenicity island;
TSST-1, toxic
shock syndrome toxin 1;
ORF, open reading frame;
TH, Todd-Hewitt;
SE, staphylococcal enterotoxin.
Characterization and Expression Analysis of
Staphylococcus aureus Pathogenicity Island 3
IMPLICATIONS FOR THE EVOLUTION OF STAPHYLOCOCCAL PATHOGENICITY
ISLANDS*
§,
,
,**
Department of Microbiology, University of
Minnesota Medical School, Minneapolis, Minnesota 55455, and
¶ Department of Veterinary Pathobiology and Biomedical Genomics
Center, University of Minnesota, St. Paul, Minnesota 55108
ABSTRACT
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-chain of the T-cell receptor (2). 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).
. Ruzin et al. (10) have
demonstrated that SaPI1 appears to parasitize excision, replication,
and encapsidation functions of phage 80 in a relationship that is
similar to that between coliphages P4 and P2. During the growth of
phage 80, SaPI1 excises from its unique chromosomal insertion site,
attc, replicates in the linear form, interferes with
phage growth, and is encapsidated into specialized phage heads. Upon
transduction to a recipient organism, SaPI1 integrates by the classical
Campbell mechanism into the attc site for which the
SaPI1-coded integrase is necessary. Because islands with different
att sites appear to have dissimilar integrases (6), it may
well be that the integrase carried by the island determines the
integration site in the genome.
MATERIALS AND METHODS
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MATERIALS AND METHODS
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RESULTS
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View larger version (23K):
[in a new window]
Fig. 1.
Comparative map of pathogenicity islands 3 (SaPI3), 1 (SaPI1), and bov
(SaPIbov). Arrows represent the
location and orientation of open reading frames or previously described
genes greater than 50 amino acids in length. Regions of sequence
homology greater than 92% between the islands are indicated by
shading. Genes encoding enterotoxins B (seb), K
(sek), Q (seq), and the bovine variant of C
(sec-bov) are indicated. Seq' potentially encodes
a truncated enterotoxin L. sapi3_1 (ear) encodes
a putative -lactamase-like protein. Also shown are the likely
integrase genes (int) as well as the attachment
(att) sequences. Stem-loop structures that may serve as
rho-independent transcription terminators are indicated for SaPI3
(predicted using DNA Strider version 1.2).
Description of SaPI3 ORFs
sequence adjacent to the putative phage amidase gene. However, this region of SaPI3 apparently does not encode
for any protein. A ~1.6-kb region SaPI3 with significant variance as
compared with SaPI1 is found spanning the region between sapi3_17 and seq. Within this region is a stretch
of ~500 nucleotides that is nearly identical to a sequence from the
recently identified 
SLT, a temperate S. aureus phage
encoding the Panton-Valentine leukocidin. Immediately upstream of
int in both SaPI1 and SaPI3 is a 46-bp sequence conserved
among staphylococcal phages 11, 13, 42, and L54a (18-20).
This sequence is the binding site for two phage 11 proteins that
regulate int expression, RinA and RinB (21).
15 (22) (52% similarity over 162 amino acids) and PBSX of Bacillus subtilis (23) (50%
similarity over 102 amino acids). Sapi3_12 potentially
encodes a product with high homology (54% similarity over 333 amino
acids) to the virulence-associated protein (VapE) of
Dichelobacter nodosus, a sheep pathogen (24).
(Sapi1_11 is also a VapE homolog 9.) This has led to the
supposition that this gene was acquired by S. aureus through
horizontal transfer from D. nodosus during co-colonization or infection of sheep (5) because the D. nodosus vap genes appear to reside on an integrated bacteriophage (25, 26). The predicted
product of sapi3_18 is 75% similar over 58 amino acids to a
putative cro-like repressor of Streptococcus
thermophilus bacteriophage Sfi21 (27, 28). Even stronger homology
is seen between the predicted product of sapi3_19 and the
cI-like repressor of S. thermophilus phage Sfi21
(27, 28) (84% similarity over 67 amino acids) and Lactobacillus
casei phage A2 repressor (29) (80% similarity over 76 amino
acids). Sapi3_20 potentially encodes a glycoprotein similar
to one found in bacteriophage A118 (30) (54% similarity over 123 amino
acids) and is strongly similar to ORF 135 from the recently described
S. aureus temperate phage SLT carrying Panton-Valentine
leukocidin (31) (85% similarity over 153 amino acids).
Primers used to PCR-amplify SaPI3 microarray
probesa
Expression data for SaPI3 ORFsa
-hemolysin
(hla) and the surface molecule protein A (spa) in
these experimental conditions are provided as representative virulence
factors. To our knowledge, the presence and expression of
hla and spa are not affected by the presence of
SaPI3 or any of the other described pathogenicity islands. Three
primary clusters of SaPI3 genes were observed. The expression profile
of seb matched most closely with that of hla and
not any of the other SaPI3 genes. In contrast, the expression of
sek and seq was found to be most similar to that
of the surrounding genes (sapi3_19-21 and
sapi3_24). This clustering of sapi3_19-24 in
their expression profiles is suggestive because the direction of
transcription of these genes is opposite that of nearly all of the
remaining genes (sapi3_2-18) on SaPI3. Finally, those genes centrally located in the island (sapi3_3-18, with the
exception of sapi3_6), whose expression was detected only in
a limited number of growth conditions, form a third group with variable
expression patterns. Although it was consistently detected,
sapi3_1 (ear) did not group with any of the other
genes examined.
View larger version (36K):
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Fig. 2.
Expression profiles and hierarchical
clustering of the genes for -hemolysin
(hla) and protein A (spa) and the 24 SaPI3 ORFs potentially encoding products of more than 50 amino
acids. Red and green represent fold
decrease and increase, respectively, in gene expression in response to
later growth phase (lanes 1 and 2), growth in
microaerobic versus aerobic conditions (lanes
3-5), growth in rabbit serum versus laboratory medium
(lanes 6-8), and incubation in vivo as compared
with the inoculum (lanes 9-11). Gray shading
indicates those ORFs whose expression was not detected in the
corresponding growth condition. Clustering based on similarity of
expression profiles and visualization were performed using the software
program Spotfire DecisionSite 6.1 (www.spotfire.com). Similarities
between expression profiles of individual genes in all 11 experimental
conditions were determined using the Euclidean distance method.
exp, exponential; post-exp, postexponential;
stat, stationary.
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View larger version (27K):
[in a new window]
Fig. 3.
A generalized model for the origination and
evolution of staphylococcal pathogenicity islands. A rare excision
error by a fully functional bacteriophage leads to the addition of an
ancestral exotoxin (SEn) gene to the phage genome
and loss of critical phage functions. The defective phage, now
dependent on helper phage for transfer into recipient strains,
undergoes divergent evolution in multiple staphylococcal lineages.
Distinct enterotoxins arise, and integration sites may be altered
through evolution of the integrase genes. As distinct pathogenicity
islands then insert into identical host strains, addition, deletion,
and recombination of large segments or modules of the pathogenicity
islands occur. The cycle is repeated in various permutations as the
resulting pathogenicity islands then undergo further divergent
evolution in subsequent staphylococcal lineages. int,
integrase gene; SE, enterotoxin gene; , phage
element.
SLT. Furthermore, SaPI1
and SaPI3 appear to have lost the sel/sek/sec3 module present on
SaPIn1/SaPIm1 and SaPIbov, or, alternatively, SaPIn1/SaPIm1 and SaPIbov
have gained these elements after diverging from a common ancestor. In
addition, we found that we could consistently 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. Although most of these central genes have no definite homolog,
they may well encode proteins involved in the structural components of the original phage. In phage 
, for instance, genes encoding head and
tail components are located together at the end of the prophage opposite the int and xis genes (37). Thus, these
genes may only be expressed when the pathogenicity island is mobilized.
Alternatively, the promoter regions for these genes may be defective or
even absent, preventing the expression of these genes at any time and requiring the structural components of a helper phage for transfer. The
observation, then, that sapi3_19-24 are transcribed opposite to sapi3_3-18 and cluster in their expression profiles
implies co-regulation of these genes and the addition of this
enterotoxin module to the already existing pathogenicity island.
Interestingly, the DNA sequence containing sapi3_3-17 is
highly conserved between SaPI1 and SaPI3 and, to a lesser extent,
SaPIbov. It is not yet clear whether the size and type of these modules
might be constrained by recombination events that require conserved
stretches of DNA sequences in the phage genomes or are more or less
randomly generated. The possibility also cannot be excluded that
staphylococcal pathogenicity islands have arisen in part as a result of
horizontal transfer from another species. Indeed, streptococcal
pyrogenic exotoxin A, a phage-encoded superantigen, appears to be more
closely related to staphylococcal enterotoxins SEB, SEC3, and SEG, than
other streptococcal superantigens (4).
ACKNOWLEDGEMENTS
FOOTNOTES
Present address: Dept. of Environmental Science and
Engineering, California Institute of Technology, 209 Keck Laboratories, M/C 138-78, Pasadena, CA 91125-7800.
Present address: The Lawson Health Research Inst., The
University of Western Ontario, Grosvenor Campus, 268 Grovenor Street, Rm. H-323, London, Ontario, Canada N6A4V2.
ABBREVIATIONS
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Williams, K. L.,
Appel, R. D.,
and Hochstrasser, D. F.
(1998)
in
2-D Proteome Analysis Protocols
(Link, A. J., ed)
, pp. 531-552, Humana Press, Totowa, NJ
Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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