BACKGROUND
s. aureus is one of the leading causes of nosocomial infections and is re-emerging as a major threat among hospitals due to the spread of methicillin resistant strains  <cit> .
furthermore, the occurrence of community acquired mrsa  is on the rise in this country and many others  <cit> .
s. aureus has a multitude of virulence factors that allow for host immune evasion, adherence to host tissues, biofilm formation, toxin production, and dissemination during infection  <cit> .
as the biological functions of cellular components continue to be elucidated,  <cit>  more and more virulence factors are added to this extensive list.
in a study designed to elucidate potential vaccine targets in s. aureus, lorenz et al identified a protein, which they designated the immunodominant surface antigen b , that elicited an immune response during mrsa septicemia.
isab is a  <dig>  kda s. aureus protein with no significant homology to other proteins with known function  <cit> .
another study demonstrated a mutation in the gene encoding isab in a hyper-virulent musculoskeletal isolate, leading the authors to suggest that mutation or loss of isab may increase immune evasion in the s. aureus isolate under investigation  <cit> .
other labs have reported microarray data showing that isab expression is increased in response to neutrophil exposure, in biofilms, under anaerobic conditions, and following internalization into human epithelial cells  <cit> .
all of these phenomena suggest that in spite of its role in eliciting an immune response, isab expression is induced during infection.
currently, isab is annotated as a putative virulence factor, however its function has yet to be determined.
biofilms have been shown to be a critical component of certain s. aureus infections, as these structures confer increased survival of the bacteria under many stressful conditions such as low nutrient availability, antibiotic challenge, oxidative stress, and host immune defenses  <cit> .
the major intercellular adhesin in s. aureus biofilms is the polysaccharide poly-n-acetylglucosamine , which is encoded by the intercellular adhesin locus   <cit> .
we and others have previously studied the regulation of pnag production and ica expression at the transcriptional level  <cit> .
with the aim of characterizing post-transcriptional regulation of ica expression, we designed an rna affinity chromatography assay to isolate proteins with high affinity for the 5'-untranslated region of the ica transcript.
we isolated a single protein, isab.
subsequently, we found that isab did not play a role in regulation of ica expression, was not localized to the cytoplasm where it could potentially play a regulatory role but rather was secreted and partially associated with the bacterial cell surface, and bound to rna, ssdna, and dsdna with no apparent sequence specificity.
because a number of studies have shown a role for extracellular dna in biofilm formation, we hypothesized that the extracellular dna-binding protein isab could play a role in this process  <cit> .
however, we found that isab did not contribute to biofilm formation under a variety of conditions.
this study is the first to assign a function to the putative virulence factor isab.
the physiologic role of binding extracellular nucleic acids is still unclear.
RESULTS
isolation of isab by rna affinity chromatography
we hypothesized that an rna-binding protein could regulate ica expression at the post-transcriptional level through binding to the 5'-untranslated region .
to isolate factors that bound to the 5'-utr, we designed an rna affinity chromatography assay using a biotinylated chimeric oligonucleotide  based on the sequence upstream from the ica locus as shown in table  <dig>  the 3-nt at the beginning and end were synthesized as deoxyribonucleotides to protect the oligo from exoribonuleases, and the remaining 40-nt were ribonucleotides.
the chimeric oligo was immobilized on streptavidin-coated magnetic particles, which were used to isolate proteins from whole cell lysates of s. aureus strain mn <dig>  a single  <dig>  kda protein was detectable by coomassie staining , and was identified by mass spectral analysis as the immunodominant surface antigen b .
deoxyribonucleotides are shown in capital letters and ribonucleotides are shown in lower case.
we extensively analyzed the effect of deleting and over-expressing the isab gene on ica expression but found that it played no role in this capacity, suggesting that our hypothesis that it was a post-transcriptional regulator of ica expression was invalid .
localization of isab
in order to characterize the rna binding activity of isab we cloned the gene into the expression vector pykb <dig> and purified untagged protein using a chitin affinity column .
polyclonal antiserum against the purified protein was used to localize isab within s. aureus .
because the antiserum cross-reacted with other staphylococcal proteins, cellular fractions from an isogenic isab deletion mutant were included for the definitive identification of isab bands.
isab was found in both the spent medium and cell surface extracts of s. aureus, while it was absent in both the cell membrane and cytoplasmic fractions.
gel shift analysis revealed a lack of sequence specificity by isab
to confirm the rna-binding activity of purified isab, electrophoretic mobility shift assays  were performed.
as shown in figure 3a, isab binds rna and produces an observable shift.
as is commonly noted for nucleic acid binding proteins, in the absence of carrier dna, much of the probe rna remained trapped in the well.
addition of sonicated salmon sperm dna abolished not only retention of the probe within the wells, but the shift as well, indicating that isab readily interacted with the carrier dna.
when the ratio of labeled rna to unlabeled dna was 2: <dig>  the salmon sperm prevented the shift observed with our labeled rna oligo , which suggested a greater affinity of isab for the carrier dna than for the rna.
in order to test the sequence specificity of isab, we used a panel of divergent dna and rna oligonucleotide probes and found that the nucleic acid-binding activity of isab was not specific with regard to sequence .
gel shift analysis revealed affinity for polymeric rna and dna but not nucleotides
in order to further characterize the nucleic acid binding activity of isab, emsas were performed using unlabeled double-stranded dna , yeast trna, and deoxyribonucleotides  as competitors .
as figure  <dig> shows, both yeast trna and dna completely inhibited the isab-rna shift.
however, the equivalent concentration of dntps was unable to inhibit the shift, indicating that isab specifically bound to polymeric nucleic acids and not to free dntps.
biacore analysis of isab
the affinity of isab for nucleic acids was characterized by biacore surface plasmon resonance.
using biotinylated dna, rna, or double-stranded dna bait oligonucleotides, we obtained affinities of isab to each of these ligands .
these data, in agreement with the emsas, suggest that isab binds with the highest affinity to double stranded dna.
deletion of isab reduced the accumulation of extracellular dna on the bacterial cell surface
to determine whether native, cell surface-associated isab was capable of binding extracellular dna, wildtype strains  <dig> and sa <dig> and mutants 10833Δisab::erm and sa113Δisab::erm were combined with fluorescently labeled salmon sperm dna.
relative fluorescence that bound to the bacteria was measured with a fluorimeter.
as shown in figure  <dig> more fluorescent dna bound to the wildtype strains.
specifically, there was a  <dig> -fold increase in fluorescence of  <dig> versus 10833Δisab::erm and a  <dig> -fold increase in sa <dig> versus sa113Δisab::erm.
deletion of isab did not affect biofilm formation
isogenic isab deletion mutants exhibited no apparent growth defects under any conditions tested .
microtiter assays for biofilm formation in a variety of media did not reveal any contribution of isab to biofilm formation and there was no significant difference between 10833Δisab::erm and sa113Δisab::erm and their respective wildtype parental strains in tsb, tsbg, bhi, bhig, or lb .
surprisingly, although there was no obvious visible difference, there was a statistically significant increase in the od <dig> nm in the isab deletion mutants of both strains in lbg.
this was consistent between technical and biologic replicates.
as extracellular dna has been shown to affect biofilm development in flow cells  <cit> , we also tested the wildtype and mutant strains under flow conditions.
however, there were no observable differences in biofilm formation or maintenance between the isab deletion mutants and their respective wildtype strains .
discussion
immunodominant antigen b  was first described by lorenz et al for its immunogenicity in patients recovering from septicemia  <cit> .
while isab has been referred to as a virulence factor  <cit> , the amino acid sequence does not display significant homology to other proteins of known function, and to date its function remains unknown.
in this study we serendipitously discovered the nucleic acid-binding activity of isab in a rna affinity chromatography assay designed to identify factors that regulate ica expression post-transcriptionally.
however, further experiments indicated that while isab binds the transcript, it does not affect ica expression, and does not play a significant role in the post-transcriptional regulation of ica.
furthermore, isab was present in the spent medium and associated with the bacterial cell surface but was not found in the cytoplasm where it could function as a post-transcriptional regulatory factor.
to further characterize the rna-binding activity of isab we used emsas and found that, while isab did bind rna, the interaction was not sequence-specific and it was also capable of binding to single-stranded and double-stranded dna.
however, we did find that isab only binds to polymeric nucleic acids and not to deoxyribonucleotides, suggesting that the nucleic acid binding activity is not a side-effect of a nucleotide-binding site.
isab contains an amino-terminal signal peptide and is predicted by psortb to be secreted  <cit> .
we found that indeed, isab is secreted into the spent medium, but a significant fraction was associated with the cell wall.
according to analysis with psortb, isab lacks an lpxtg motif, so it is not immediately clear how it is retained on the cell surface.
in a recent study rice et al found that extracellular dna  can contribute to the structural stability of biofilms in s. aureus, and that dnase-induced degradation of the edna leads to dissolution of the biofilm  <cit> .
furthermore, isab expression was found to be upregulated within biofilms  <cit> , which lead us to hypothesize that binding of edna by isab could play a role in the establishment or maturation of biofilms, which are a critical component of disease establishment and progression of s. aureus.
we found, using fluorescently-labeled dna, that isab does play a role in accumulation of extracellular dna on the bacterial cell surface, however, under our experimental growth conditions, isab did not contribute to biofilm-forming capacity.
surprisingly, deletion of isab actually increased biofilm formation slightly, but significantly, in lb containing 1% glucose.
this suggests that the role of isab may differ depending upon the growth conditions.
we are therefore currently exploring the possibility that isab may play a more significant role in biofilm formation under more physiologic conditions, and whether or not it contributes to virulence in an animal model of bacteremia.
isab elicits an immune response during sepsis, suggesting that it is expressed during infection  <cit> .
its expression is also induced by neutrophils and following internalization in human epithelial cells, again suggesting expression during infection and a role in virulence  <cit> .
however, it is not immediately clear how an extracellular dna-binding protein could play a role in virulence.
edna present at the site of an infection may come from a variety of sources including lysed neutrophils or neutrophils actively releasing nets  or from lysed bacterial cells  <cit> .
if isab does not play a role in biofilm formation, then binding of extracellular dna to the cell surface could be a mechanism of immune evasion by mimickry or it could result in repulsive forces between the dna-coated bacteria and the dna in nets.
we are currently investigating these potential functions of isab.
CONCLUSIONS
isab is a putative virulence factor and its expression is induced as a result of a variety of conditions related to the in vivo disease state.
in the data presented here we show that isab is an extracellular nucleic acid binding protein with a greater affinity for dsdna than for ssdna or rna.
using isogenic deletion mutants we were unable to demonstrate a role for isab on biofilm formation.
further studies are necessary to determine what role isab and its nucleic acid-binding activity play in establishment and/or progression of s. aureus infection.

 s. aureus is one of the leading causes of nosocomial infections and is re-emerging as a major threat among hospitals due to the spread of methicillin resistant strains. furthermore, the occurrence of community acquired mrsa is on the rise in this country and many others. s. aureus has a multitude of virulence factors that allow for host immune evasion, adherence to host tissues, biofilm formation, toxin production, and dissemination during infection. as the biological functions of cellular components continue to be elucidated, more and more virulence factors are added to this extensive list. in a study designed to elucidate potential vaccine targets in s. aureus, lorenz et al identified a protein, which they designated the immunodominant surface antigen b, that elicited an immune response during mrsa septicemia. isab is a kda s. aureus protein with no significant homology to other proteins with known function. another study demonstrated a mutation in the gene encoding isab in a hyper-virulent musculoskeletal isolate, leading the authors to suggest that mutation or loss of isab may increase immune evasion in the s. aureus isolate under investigation. other labs have reported microarray data showing that isab expression is increased in response to neutrophil exposure, in biofilms, under anaerobic conditions, and following internalization into human epithelial cells. all of these phenomena suggest that in spite of its role in eliciting an immune response, isab expression is induced during infection. currently, isab is annotated as a putative virulence factor, however its function has yet to be determined. biofilms have been shown to be a critical component of certain s. aureus infections, as these structures confer increased survival of the bacteria under many stressful conditions such as low nutrient availability, antibiotic challenge, oxidative stress, and host immune defenses. the major intercellular adhesin in s. aureus biofilms is the polysaccharide poly-n-acetylglucosamine, which is encoded by the intercellular adhesin locus. we and others have previously studied the regulation of pnag production and ica expression at the transcriptional level. with the aim of characterizing post-transcriptional regulation of ica expression, we designed an rna affinity chromatography assay to isolate proteins with high affinity for the 5'-untranslated region of the ica transcript. we isolated a single protein, isab. subsequently, we found that isab did not play a role in regulation of ica expression, was not localized to the cytoplasm where it could potentially play a regulatory role but rather was secreted and partially associated with the bacterial cell surface, and bound to rna, ssdna, and dsdna with no apparent sequence specificity. because a number of studies have shown a role for extracellular dna in biofilm formation, we hypothesized that the extracellular dna-binding protein isab could play a role in this process. however, we found that isab did not contribute to biofilm formation under a variety of conditions. this study is the first to assign a function to the putative virulence factor isab. the physiologic role of binding extracellular nucleic acids is still unclear.  isolation of isab by rna affinity chromatography we hypothesized that an rna-binding protein could regulate ica expression at the post-transcriptional level through binding to the 5'-untranslated region. to isolate factors that bound to the 5'-utr, we designed an rna affinity chromatography assay using a biotinylated chimeric oligonucleotide based on the sequence upstream from the ica locus as shown in table the 3-nt at the beginning and end were synthesized as deoxyribonucleotides to protect the oligo from exoribonuleases, and the remaining 40-nt were ribonucleotides. the chimeric oligo was immobilized on streptavidin-coated magnetic particles, which were used to isolate proteins from whole cell lysates of s. aureus strain mn a single kda protein was detectable by coomassie staining, and was identified by mass spectral analysis as the immunodominant surface antigen b. deoxyribonucleotides are shown in capital letters and ribonucleotides are shown in lower case. we extensively analyzed the effect of deleting and over-expressing the isab gene on ica expression but found that it played no role in this capacity, suggesting that our hypothesis that it was a post-transcriptional regulator of ica expression was invalid. localization of isab in order to characterize the rna binding activity of isab we cloned the gene into the expression vector pykb and purified untagged protein using a chitin affinity column. polyclonal antiserum against the purified protein was used to localize isab within s. aureus. because the antiserum cross-reacted with other staphylococcal proteins, cellular fractions from an isogenic isab deletion mutant were included for the definitive identification of isab bands. isab was found in both the spent medium and cell surface extracts of s. aureus, while it was absent in both the cell membrane and cytoplasmic fractions. gel shift analysis revealed a lack of sequence specificity by isab to confirm the rna-binding activity of purified isab, electrophoretic mobility shift assays were performed. as shown in figure 3a, isab binds rna and produces an observable shift. as is commonly noted for nucleic acid binding proteins, in the absence of carrier dna, much of the probe rna remained trapped in the well. addition of sonicated salmon sperm dna abolished not only retention of the probe within the wells, but the shift as well, indicating that isab readily interacted with the carrier dna. when the ratio of labeled rna to unlabeled dna was 2: the salmon sperm prevented the shift observed with our labeled rna oligo, which suggested a greater affinity of isab for the carrier dna than for the rna. in order to test the sequence specificity of isab, we used a panel of divergent dna and rna oligonucleotide probes and found that the nucleic acid-binding activity of isab was not specific with regard to sequence. gel shift analysis revealed affinity for polymeric rna and dna but not nucleotides in order to further characterize the nucleic acid binding activity of isab, emsas were performed using unlabeled double-stranded dna, yeast trna, and deoxyribonucleotides as competitors. as figure shows, both yeast trna and dna completely inhibited the isab-rna shift. however, the equivalent concentration of dntps was unable to inhibit the shift, indicating that isab specifically bound to polymeric nucleic acids and not to free dntps. biacore analysis of isab the affinity of isab for nucleic acids was characterized by biacore surface plasmon resonance. using biotinylated dna, rna, or double-stranded dna bait oligonucleotides, we obtained affinities of isab to each of these ligands. these data, in agreement with the emsas, suggest that isab binds with the highest affinity to double stranded dna. deletion of isab reduced the accumulation of extracellular dna on the bacterial cell surface to determine whether native, cell surface-associated isab was capable of binding extracellular dna, wildtype strains and sa and mutants 10833Δisab::erm and sa113Δisab::erm were combined with fluorescently labeled salmon sperm dna. relative fluorescence that bound to the bacteria was measured with a fluorimeter. as shown in figure more fluorescent dna bound to the wildtype strains. specifically, there was a -fold increase in fluorescence of versus 10833Δisab::erm and a -fold increase in sa versus sa113Δisab::erm. deletion of isab did not affect biofilm formation isogenic isab deletion mutants exhibited no apparent growth defects under any conditions tested. microtiter assays for biofilm formation in a variety of media did not reveal any contribution of isab to biofilm formation and there was no significant difference between 10833Δisab::erm and sa113Δisab::erm and their respective wildtype parental strains in tsb, tsbg, bhi, bhig, or lb. surprisingly, although there was no obvious visible difference, there was a statistically significant increase in the od nm in the isab deletion mutants of both strains in lbg. this was consistent between technical and biologic replicates. as extracellular dna has been shown to affect biofilm development in flow cells, we also tested the wildtype and mutant strains under flow conditions. however, there were no observable differences in biofilm formation or maintenance between the isab deletion mutants and their respective wildtype strains. discussion immunodominant antigen b was first described by lorenz et al for its immunogenicity in patients recovering from septicemia. while isab has been referred to as a virulence factor, the amino acid sequence does not display significant homology to other proteins of known function, and to date its function remains unknown. in this study we serendipitously discovered the nucleic acid-binding activity of isab in a rna affinity chromatography assay designed to identify factors that regulate ica expression post-transcriptionally. however, further experiments indicated that while isab binds the transcript, it does not affect ica expression, and does not play a significant role in the post-transcriptional regulation of ica. furthermore, isab was present in the spent medium and associated with the bacterial cell surface but was not found in the cytoplasm where it could function as a post-transcriptional regulatory factor. to further characterize the rna-binding activity of isab we used emsas and found that, while isab did bind rna, the interaction was not sequence-specific and it was also capable of binding to single-stranded and double-stranded dna. however, we did find that isab only binds to polymeric nucleic acids and not to deoxyribonucleotides, suggesting that the nucleic acid binding activity is not a side-effect of a nucleotide-binding site. isab contains an amino-terminal signal peptide and is predicted by psortb to be secreted. we found that indeed, isab is secreted into the spent medium, but a significant fraction was associated with the cell wall. according to analysis with psortb, isab lacks an lpxtg motif, so it is not immediately clear how it is retained on the cell surface. in a recent study rice et al found that extracellular dna can contribute to the structural stability of biofilms in s. aureus, and that dnase-induced degradation of the edna leads to dissolution of the biofilm. furthermore, isab expression was found to be upregulated within biofilms, which lead us to hypothesize that binding of edna by isab could play a role in the establishment or maturation of biofilms, which are a critical component of disease establishment and progression of s. aureus. we found, using fluorescently-labeled dna, that isab does play a role in accumulation of extracellular dna on the bacterial cell surface, however, under our experimental growth conditions, isab did not contribute to biofilm-forming capacity. surprisingly, deletion of isab actually increased biofilm formation slightly, but significantly, in lb containing 1% glucose. this suggests that the role of isab may differ depending upon the growth conditions. we are therefore currently exploring the possibility that isab may play a more significant role in biofilm formation under more physiologic conditions, and whether or not it contributes to virulence in an animal model of bacteremia. isab elicits an immune response during sepsis, suggesting that it is expressed during infection. its expression is also induced by neutrophils and following internalization in human epithelial cells, again suggesting expression during infection and a role in virulence. however, it is not immediately clear how an extracellular dna-binding protein could play a role in virulence. edna present at the site of an infection may come from a variety of sources including lysed neutrophils or neutrophils actively releasing nets or from lysed bacterial cells. if isab does not play a role in biofilm formation, then binding of extracellular dna to the cell surface could be a mechanism of immune evasion by mimickry or it could result in repulsive forces between the dna-coated bacteria and the dna in nets. we are currently investigating these potential functions of isab.  isab is a putative virulence factor and its expression is induced as a result of a variety of conditions related to the in vivo disease state. in the data presented here we show that isab is an extracellular nucleic acid binding protein with a greater affinity for dsdna than for ssdna or rna. using isogenic deletion mutants we were unable to demonstrate a role for isab on biofilm formation. further studies are necessary to determine what role isab and its nucleic acid-binding activity play in establishment and/or progression of s. aureus infection. 
staphylococcus aureus immunodominant surface antigen b elicits an immune response during septicemia and is generally classified as a virulence factor, but its biological function remains completely undefined. because extracellular dna has been shown to play a role in biofilm formation, we investigated the biofilm-forming capacity of an isogenic isab deletion mutant but we found that isab did not contribute to biofilm formation under any conditions tested. in an attempt to identify staphylococcal rna-binding proteins, we designed an rna affinity chromatography assay and subsequently isolated isab. isab is an extracellular nucleic acid binding protein, with little to no sequence specificity, but its role in virulence remains unclear. gel shift analysis confirmed the rna binding activity but revealed that isab bound to any nucleic acid without sequence specificity.
 staphylococcus aureus immunodominant surface antigen b elicits an immune response during septicemia and is generally classified as a virulence factor, but its biological function remains completely undefined. in an attempt to identify staphylococcal rna-binding proteins, we designed an rna affinity chromatography assay and subsequently isolated isab.  western analysis indicated that isab was both secreted and cell-surface associated. gel shift analysis confirmed the rna binding activity but revealed that isab bound to any nucleic acid without sequence specificity. isab exhibited the highest affinity for double-stranded dna followed by single-stranded dna and rna. because extracellular dna has been shown to play a role in biofilm formation, we investigated the biofilm-forming capacity of an isogenic isab deletion mutant but we found that isab did not contribute to biofilm formation under any conditions tested.  isab is an extracellular nucleic acid binding protein, with little to no sequence specificity, but its role in virulence remains unclear. 