BACKGROUND
escherichia coli  o <dig>  was first identified as a human enteric pathogen in  <dig> and has since been implicated in several outbreaks and sporadic infections  <cit> .
currently, this human pathogen ranks fourth after campylobacter, salmonella, and shigella among the etiologic agents causing diarrhea in north america  <cit> .
cattle are the primary reservoirs for o <dig>  with the bovine recto-anal junction  serving as the primary colonization site for o <dig>  humans acquire infection by consumption of undercooked beef products such as ground meat or foods contaminated with manure  <cit> .
the bovine raj comprises of two cell types, the follicle associated epithelium  towards the distal colon and the stratified squamous epithelium  closer to the anal canal  <cit> .
thus far, studies analyzing o <dig> persistence at the raj have focused primarily on its interactions with the fae cells  <cit> .
proteins encoded on the o <dig> pathogenicity island, locus of enterocyte effacement , have been shown to play a critical role in o <dig> adherence to fae cells.
these include the e. coli secreted proteins espa and espb, the adhesin intimin, and the translocated receptor for intimin, tir which is secreted via the lee-encoded type iii secretion system   <cit> .
hence, several pre-harvest control measures being evaluated in cattle to control or eliminate o <dig> from entering the food chain  <cit> , include vaccines targeting these lee-encoded proteins.
for instance, potter et al.
developed a vaccine comprising wild-type o <dig> culture supernatants that contain the ttss proteins, tir and esps  <cit> ; however, similar protection was noted in animals inoculated with the culture supernatant from a mutant strain of o <dig> lacking the tir gene.
in addition, the immune response of the vaccinated animals was not merely to the ttss proteins but also against a number of other proteins that were present in the supernatant.
interestingly, although the vaccine decreased both the number of e. coli o <dig> shed in the feces of vaccinated animals, and those colonizing the terminal rectum, it did not reduce the duration of shedding despite the subcutaneous administration of three doses of the vaccine  <cit> ; http://www.bioniche.com.
similar results were also observed with another vaccine that targets the o <dig> siderophore receptor and porin  proteins  <cit> ; https://animalhealth.pfizer.com.
this clearly suggests that unidentified proteins other than those constituting the ttss or srp may play a crucial role in bovine colonization, and that the identification and inclusion of such proteins is likely to increase the efficacy of vaccines for elimination of o <dig> from the gastrointestinal tracts of cattle.
further supporting this inference are reported observations that the ttss proteins do not significantly contribute to the persistence of o <dig> in feedlot cattle  <cit> , or to o <dig> invasion of crypt cells at the raj  <cit> .
in a previous study, o <dig> was observed to adhere to rse cells in vivo and in vitro, besides the fae cells  <cit>  and this observation was used to develop a unique in vitro adherence assay for o <dig> with rse cells  <cit> .
in this study, we decided to  evaluate if the lee-encoded proteins would also be critical for o <dig> adherence to rse cells, as for fae cells, and  in the event that these proteins would not play a significant role in rse cell adherence, define the proteome of o <dig> as expressed when grown in the adherence assay media, dmem, to assemble targets for future evaluation in rse adherence.
experimental and bioinformatic evaluation of such targets could in fact help identify a subset of novel adhesins that may have excellent potential to increase the efficacy of the anti-adhesion, cattle o <dig> vaccines, by eliminating o <dig> from both fae and rse cells at the raj.
methods
bacterial strains and culture conditions
the wild-type o <dig> strain edl <dig> , a sequenced isolate implicated in human disease  <cit> , was used in this study.
we cultured o <dig> in dulbecco modified eagle medium-low glucose , for the cell adherence assays described below.
the rationale for the use of this culture medium was  to reflect the growth conditions used in the eukaryotic cell adherence assays; and  to closely parallel the in vivo nutrient-limiting conditions, and conditions used to prepare the cattle-use approved, lee protein based, anti-adhesion o <dig> vaccine.
in addition, another wild-type o <dig> strain 86– <dig> , its isogenic mutant  negative for intimin, and this mutant complemented with the plasmid peb <dig> ) expressing intimin, were also tested in the adherence assay  <cit> .
the 86– <dig> strain and its derivatives were obtained from dr. a. d. o’brien, uniformed services university of the health sciences, bethesda, md.
we also cultured o <dig> in dmem for proteomic analysis.
specifically, an overnight culture of the wild-type o <dig> strain in luria-bertani  broth was pelleted and washed with sterile phosphate buffered saline , and subcultured to an initial od <dig> of  <dig>  in fresh dmem.
after incubation at 37 °c with shaking at 250 rpm to an od <dig> of  <dig>  to  <dig> , cells were harvested by centrifugation at  <dig>  rpm, 15 min at 4 °c.
cells were washed three times with an equal volume of sterile pbs , and processed to obtain cell lysate and pellet fractions for proteomic analysis as previously described  <cit> .
o157-rse cell adherence inhibition assay:  in the presence of pooled anti-lee proteins, anti-intimin and anti-h <dig> antisera
adherence of o <dig> to the rse cells was previously demonstrated and developed into an adherence assay in our laboratory  <cit> .
in this study, the ability of pooled, rabbit polyclonal antisera to interfere with and inhibit o <dig> adherence to rse cells was evaluated.
specifically, antisera generated against the recombinant lee-encoded proteins, tir, espa and espb, and intimin, in rabbits , was pooled.
rabbit antisera targeting the o <dig> flagellar antigen h <dig>  was also mixed into the pooled antisera, which was then tested at 1: <dig> and 1: <dig> dilutions.
specificity was confirmed by reacting each antiserum against both o <dig> cell lysates and the cognate protein in western blotting experiments .
rabbit sera  from healthy animals , at a 1: <dig> dilution, was used as a control.
in the presence of anti-intimin antisera alone
to specifically evaluate the role of intimin, the rabbit anti-intimin antisera was evaluated separately for its ability to prevent o <dig> adherence to rse cells at 1: <dig> and 1: <dig> dilutions.
each of the rse adherence assays was conducted in  <dig> technical and  <dig> biological replicates as described previously  <cit> , with minor modifications, as follows.
rse cells were washed and resuspended in 1 ml dulbecco modified eagle medium – no glucose  ±  <dig> % d + mannose, in  <dig> x 100 mm glass tubes, to a final concentration of  <dig> cells/ml.
although type  <dig> fimbriae are not expressed by o <dig>  we included d + mannose in parallel assays to cover any hitherto unknown transient expression especially in mutant strains.
bacterial pellets from overnight cultures in dmem, incubated at 37°c without aeration, were resuspended in sterile saline with or without antisera , and incubated at 37°c for 30 min.
the bacteria-antibody mix was then added to the rse cells suspension to final bacteria:cell ratio of 10: <dig>  and the mixture incubated with aeration .
at the end of 4 h, the mixture was pelleted and washed thoroughly, once with 14 ml dmem-ng, and twice with  <dig> mls of sterile, distilled water  before reconstituting in 100 μl dh2o.
eight 2 μl drops of this suspension were placed on polysine  slides and dried overnight under direct light to quench non-specific fluorescence, before fixing in cold 95% ethanol for 10 min.
the slides were then stained with 1% toluidine blue, or with fluorescence-tagged antibodies that specifically target o <dig> and the rse cell cytokeratins as described previously  <cit> .
each experiment was then done in duplicate.
o <dig> adherence patterns on rse cells were recorded as diffuse, or aggregative  for all positive interactions that involved direct association with the cells  <cit> .
scattered bacteria and bacterial micro-colonies not adhering to cell membranes were considered to be negative for adherence to the epithelial cells  <cit> .
a total of 100– <dig> well dispersed rse cells  were analyzed per slide as described previously  <cit> .
the percent rse cells with and without bacteria adhering to them were determined  <cit> .
if more than 50% of rse cells had > <dig> bacteria attached, the adherence was recorded as strongly positive.
for >50% rse cells with 1– <dig> adherent bacteria, the adherence was recorded as moderately positive.
for less than 50% rse cells with 1– <dig> adherent bacteria, the result was recorded as non-adherent.
o157-hep- <dig> cell adherence inhibition assay
the role played by lee-encoded proteins and intimin in the adherence of o <dig> to hep- <dig> cells, has already been defined previously  <cit>  and hence, this assay was used for comparative reasons.
the assay was conducted as described previously  <cit>  except that, the washed bacterial pellets were incubated with or without antisera , at 37°c for 30 min, prior to addition to the hep- <dig> cells.
both the pooled antisera and anti-intimin antisera, as described above, were used at dilutions ranging from 1: <dig> to 1: <dig> in these assays.
each assay was conducted in duplicate, and in 3– <dig> chambers of the chamber slides per run.
slides were stained with 1% toluidine blue, or with fluorescence-tagged antibodies that specifically target o <dig> and the hep- <dig> cell actin filaments as described previously  <cit>  and adherence patterns recorded as for rse cells .
adherence of 86– <dig>  86-24eae Δ <dig>  and 86-24eae Δ <dig>  to rse and hep- <dig> cells
wild-type 86– <dig> and its mutant derivatives were used to verify the role of intimin directly and compare the results with that of the o <dig> adherence inhibition assays.
this assay was conducted, recorded, as previously described and done in the absence of any antisera  <cit> .
onedimensional  sds-page liquid chromatography tandem mass spectrometry
top down proteomic analysis was done at the harvard partners center for genetics and genomics, cambridge, massachusetts.
o <dig> cell pellet and lysate fractions were concentrated using spin filters , fractionated on 1d sds-page, and digested in-gel with trypsin prior to tandem mass spectrometry  as described previously  <cit> .
the rationale for incorporating a 1d sds-page fractionation step is that this modification reduces complexity of protein mixtures, permits a larger dynamic range of protein identification, and allows for significantly better reproducibility  <cit> .
for mass spectrometry , samples were subjected to three different runs on an lcq deca xp plus proteome x workstation  from thermo finnigan as described earlier  <cit> .
for each run,  <dig> μl of each reconstituted sample was injected with a famos autosampler, and the separation was done on a  <dig> μm  x 20 cm column packed with c <dig> media running at a flow rate of  <dig>  μl/min provided from a surveyor ms pump with a flow splitter with a gradient of water,  <dig> % formic acid and then 5% acetonitrile,  <dig> % formic acid  over the course of 480 min .
between each set of samples, a standard of a  <dig> angiotensin mix of peptides  was run to ascertain column performance, and to observe any potential carryover that might have occurred.
the lcq was run in a top five configuration, with one ms scan and five ms/ms scans.
dynamic exclusion was set to  <dig> with a limit of  <dig> seconds.
peptide identifications were made using sequest  through the bioworks browser  <dig> , as described previously  <cit> .
sequential database searches were performed using the o <dig> strains edl <dig> and sakai fasta database from european bioinformatics institute http://www.ebi.ac.uk/newt/display using static carbamidomethyl-modified cysteines and differential oxidized methionines.
these protein databases  – tax id:  <dig> and escherichia coli  – tax id: 155864) have a total of  <dig> entries.
a reverse o <dig> strain edl <dig> fasta database was spiked in to provide noise and determine validity of the peptide hits, so that known and theoretical protein hits can be determined without compromising the statistical relevance of all the data  <cit> .
the ms data was searched with a 2-dalton window on the ms precursor with a  <dig>  dalton on the fragment ions.
peptide score cutoff values were chosen at cross-correlation values  of  <dig>  for singly charged ions,  <dig>  for doubly charged ions, and  <dig>  for triply charged ions, along with delta rank scoring preliminary cutoff  values of  <dig> , and cross-correlation normalized values  of  <dig>  the cross-correlation values chosen for each peptide assured a high confidence match for the different charge states, while the deltacn values ensured the uniqueness of the peptide hit.
the rsp value of  <dig> ensured that the peptide matched the top hit in the preliminary scoring.
at these peptide filter values, very few reverse database hits were observed, which permitted a higher confidence in the few single peptide protein identifications.
furthermore, single hit proteins were manually validated to ensure relevance.
bioinformatics
cellular location of proteins was determined using amino acid sequences of cognate proteins in the o <dig> sequence databases at http://www.ncbi.nlm.nih.gov/protein.
in addition, extracytoplasmic proteins were verified for the presence of signal sequences using the program signalp  <dig>  at http://www.cbs.dtu.dk/services/signalp, and subcellular localization of other proteins confirmed using the psort/psort-b program .
putative functions were determined by querying the conserved domain database  at http://www.ncbi.nlm.nih.gov/structure/cdd/wrpsb.cgi protein components of the o <dig> dmem-proteome with adhesion potential were shortlisted using vaxign, a reverse vaccinology based vaccine target prediction and analysis system at http://www.violinet.org that utilizes the spaan algorithm  <cit> .
RESULTS
lee- encoded proteins considered critical for o <dig> adherence to fae cells at the raj did not appear to have a role in o <dig> adherence to rse cells at this same site.
both o <dig> strains grown in dmem and pre-incubated with pooled, polyclonal antisera generated against the lee  and flagellar h <dig> proteins, or the anti-intimin antisera alone, at 1: <dig> and 1: <dig> dilution, continued to adhere to the rse cells, irrespective of the presence/absence of d + mannose.
data is shown for one of the o <dig> strains in the presence of d + mannose .
these results were consistent between all trials, irrespective of toluidine blue or immunofluorescent staining, and did not show any differences in the adherence patterns compared to the controls.
the same o157-rse cell-adherence pattern was observed in the controls with normal rabbit sera added at 1: <dig> dilution , and in the absence of any sera   <cit> , irrespective of the presence/absence of d + mannose.
the continued adherence of o <dig> to the rse cells in the presence of antibodies to the lee proteins may have been due to the masking of these antigens and the unmasking of other o <dig> adhesins targeting the receptors on the rse cells.
to that effect an increase in the total number of rse cells with adherent bacteria and decrease in the total number of rse cells with no adherent bacteria was observed, in the presence of pooled and anti-intimin antisera .
we intentionally included antisera targeting the flagellar antigen h <dig> as flagella have been demonstrated to play a role in initial adherence to plant cells and the fae  <cit> .
these results suggest that additional mechanisms of adherence, distinct from those attributable to lee, intimin and flagellar h <dig> proteins, are involved in o <dig> attachment to the raj squamous epithelial cells.
on the other hand, the lee-encoded proteins were critical to o <dig> adherence to hep- <dig> cells as demonstrated previously  <cit> , with or without d + mannose.
as shown in additional file  <dig> and figure  <dig>  panel a and figure  <dig>  preincubation with the pooled polyclonal antisera and the anti-intimin antisera significantly interfered with and prevented o <dig> adherence to hep- <dig> cells, at all dilutions tested.
an increase in number of hep- <dig> cells without any adhering bacteria was observed in the presence of either antiserum, accordingly .
however, pre-incubation with normal rabbit sera at 1: <dig> dilution  showed the same diffuse, moderate adherence as in the absence of any antisera .
the results observed with the adherence inhibition assays were further verified by the adherence patterns of o <dig> strain 86– <dig>  and its mutant derivatives on hep- <dig> and rse cells .
the intimin-negative mutant 86-24eae Δ <dig> did not adhere well to the hep- <dig> cells compared to the intimin-positive, wild-type 86– <dig> or complemented mutant, 86-24eae Δ <dig> that demonstrated diffuse, moderate adherence .
actin accumulation observed in the majority of hep- <dig> cells with 100x magnification only in the presence of 86– <dig> and 86-24eae Δ <dig>  along with an increase in the number of hep- <dig> cells without adhering bacteria in the presence of 86-24eae Δ <dig>  further verified these observations .
this confirmed the role of intimin in o <dig> adherence to hep- <dig> cells.
on the otherhand, 86– <dig> and all its mutant derivatives demonstrated diffuse, strong adherence to rse cells, irrespective of intimin expression .
infact with 86-24eae Δ <dig>  the number of rse cells with adhering bacteria actually increased, which suggested that intimin did not have a role in the adherence of o <dig> to rse cells.
in order to verify if proteins other than lee proteins were being expressed by o <dig> upon growth in dmem which could have a possible role in o <dig> adherence to rse cells, we analyzed the o <dig> proteome as expressed in dmem.
while the proteome of o <dig> has been analyzed under various other growth conditions  <cit>  we decided to evaluate the same following growth in dmem for several reasons, such as  this was the media used to culture both bacteria and the rse cells, separately, prior to the adherence assays,  the media closely mimicked the nutrient-limiting conditions seen in vivo, and  this media closely matched that used to develop a commercially available cattle, o <dig> vaccine [ <dig>  16; http://www.bioniche.com.
our observations did not support a role for other host -derived factors in this adherence of o <dig> and hence, we did not evaluate rse-cell adherence of o <dig> cultured in eukaryotic cell-conditioned media.
this inference came from the fact that similar adherence results were obtained when dmem was supplemented with norepinephrine , a host neuroendocrine hormone that is encountered by o <dig> in vivo during the actual process of infection .
ne is reportedly a mimic of autoinduer  <dig> , which regulates o <dig> virulence gene expression via quorum sensing  <cit> .
further, intimin, its receptor, tir, as well as espb were expressed in equivalent amounts in both dmem and dmem-ne, as observed using western blotting by others  <cit> , and by us, and also using top down proteomics by us .
a total of  <dig> proteins were identified as being part of the o <dig> dmem-proteome , and these included several characterized and hypothetical/unknown proteins besides the ttss proteins.
while  <dig> of these proteins were uncharacterized with hypothetical functions assigned in the o <dig> genome , the remaining  <dig> proteins localized to various bacterial cell compartments with functions including metabolic, cell division, regulatory, transport, environmental adaptation, and previously characterized o <dig> virulence factors  <cit> ; figure  <dig>  additional files  <dig> and  <dig>  proteins associated with o <dig> virulence or adherence in the dmem-proteome included tir, intimin, espb, luxs, iha, ompa, katp, chua, espp, stx1a, stx1b, and stx2b  <cit> ; additional files  <dig> and  <dig>  interestingly,  <dig> of the  <dig>  proteins comprising the o <dig> dmem-proteome were also part of the o <dig> immunoproteome in cattle, defined using the innovative proteome mining tool, proteomics- based expression library screening   <cit> ; additional files  <dig> and  <dig>  in addition, nine members of the dmem-proteome were also part of the o <dig> immunome in humans  <cit> .
given that a subset of pathogen proteins targeted by the host immune system help pathogens overcome hostile in vivo environments and rapidly adapt to host niches, counter host defenses, survive, propagate, and establish infection, it is very likely that proteins other than lee proteins in the o <dig> dmem-proteome play a role in o <dig> adherence to the rse cells at the raj.
the identification and inclusion of such proteins in anti-adhesion vaccine preparations, and their optimal administration to the host immune system, may enhance the efficacy of such vaccines in reducing or eliminating o <dig> not only from the fae cells but also from the rse cells of the bovine raj.
our strategy for selecting proteins constituting the o <dig> dmem proteome with potential to function as adhesins for further evaluation was to employ the spaan algorithm  <cit> , which is part of the web-based vaccine target prediction and analysis system at http://www.violinet.org.
in particular, we shortlisted proteins that were shared by four sequenced o <dig> strains, namely, strain edl <dig>  strain ec <dig>  strain sakai, and strain tw <dig>  our analysis identified 36/ <dig> components of the o <dig> dmem-proteome to have adhesin potential, including the extensively reported primary o <dig> adhesin, intimin-γ, and its protein receptor, tir .
three o157-specific proteins, namely chua, tere, and a putative outermembrane porin protein on o-island # <dig> , have been prioritized and are under experimental evaluation for a role in o <dig> adherence to rse cells.
also, towards studies of common stec adherence mechanisms, we have determined homologs of several of these putative adhesins including those encoded on o-islands such as, tir, intimin, chua, tere, espp, bioinformatically , in sequenced non-o <dig> shiga-toxin-producing escherichia coli  of clinical importance.
experimental evaluation of the contribution of these homologs to adherence of these non-o <dig> stec, e. coli o26:h <dig>  o103:h <dig> and o111:nm, are underway.
1pels: proteomics- based expression library screening
2iviat:in vivo-induced antigen technology
CONCLUSIONS
proteins other than the lee-encoded proteins are involved in o <dig> adherence to rse cells at the bovine raj.
such proteins, with adhesin potential, are expressed by this human pathogen during growth in vitro, in dmem.
three such putative adhesins, namely, chua, tere, and a putative outermembrane porin protein on o-island # <dig>  are currently under evaluation for their adherence potential.
such studies should provide both valuable insights into the o157-rse interactions and new targets for more efficacious anti-adhesion o <dig> cattle vaccines.
competing interests
the authors declare no competing financial interests.
authors’ contributions
itk was the project leader and designed, coordinated, obtained funding, conducted experiments, analyzed data and drafted the manuscript.
rwg conducted experiments and tabulated data.
bk and das performed proteomic analysis.
sbc assisted in design and participated in helpful discussions.
mj was the co-project leader, and designed, coordinated, analyzed results and performed bioinformatic analysis.
all authors read and approved the final manuscript.
supplementary material
additional file  <dig>
http://www.biomedcentral.com/imedia/9899042126754199/supp <dig> pdf.
table a quantitation of rse cells with adherent bacteria in the presence of d + mannose.
click here for file
additional file  <dig>
http://www.biomedcentral.com/imedia/6766700936754199/supp <dig> pdf.
table b quantitation of hep- <dig> cells with adherent bacteria in the presence of d + mannose.
click here for file
additional file  <dig>
http://www.biomedcentral.com/imedia/1105071156754199/supp <dig> pdf.
table c uncharacterized hypothetical proteins of the o <dig> dmem-proteome.
click here for file
additional file  <dig>
http://www.biomedcentral.com/imedia/1751063870675419/supp <dig> pdf.
table d previously characterized proteins of the o <dig> dmem-proteome.
click here for file
additional file  <dig>
http://www.biomedcentral.com/imedia/1777785157675419/supp <dig> pdf.
data sheets: o157-dmem ms/ms data sheet  <dig>
click here for file
additional file  <dig>
http://www.biomedcentral.com/imedia/1707955235675419/supp <dig> pdf.
data sheets: o157-dmem ms/ms data sheet  <dig>
click here for file
additional file  <dig>
http://www.biomedcentral.com/imedia/1451425738675419/supp <dig> pdf.
data sheets: o157-dmem ms/ms data sheet  <dig>
click here for file
additional file  <dig>
http://www.biomedcentral.com/imedia/3116488396754199/supp <dig> pdf.
data sheets: o157-dmem ms/ms data sheet  <dig>
click here for file
additional file  <dig>
http://www.biomedcentral.com/imedia/1233524502675419/supp <dig> pdf.
data sheets: o157-dmem ms/ms data sheet  <dig>
click here for file
additional file  <dig>
http://www.biomedcentral.com/imedia/1610501146675419/supp <dig> pdf.
data sheets: o157-dmem ms/ms data sheet  <dig>
click here for file
additional file  <dig>
http://www.biomedcentral.com/imedia/1326109329675419/supp <dig> pdf.
data sheets: o157-dmem ms/ms data sheet  <dig>
click here for file
additional file  <dig>
http://www.biomedcentral.com/imedia/1285024576754199/supp <dig> pdf.
data sheets: o157-dmem ms/ms data sheet  <dig>
click here for file
acknowledgements
this work was supported by a us national institutes of health grant r <dig> ai <dig> to i.t.k.
intellectual property rights for the o <dig> proteome identified in this study are held by massachusetts general hospital, boston, ma.
excellent technical assistance provided by bryan wheeler at the national animal disease center, ames, ia, with the eukaryotic cell adherence/adherence-inhibition assays is acknowledged.
disclaimer
mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the u.s. department of agriculture.
usda is an equal opportunity provider and employer.

 escherichia coli o was first identified as a human enteric pathogen in and has since been implicated in several outbreaks and sporadic infections. currently, this human pathogen ranks fourth after campylobacter, salmonella, and shigella among the etiologic agents causing diarrhea in north america. cattle are the primary reservoirs for o with the bovine recto-anal junction serving as the primary colonization site for o humans acquire infection by consumption of undercooked beef products such as ground meat or foods contaminated with manure. the bovine raj comprises of two cell types, the follicle associated epithelium towards the distal colon and the stratified squamous epithelium closer to the anal canal. thus far, studies analyzing o persistence at the raj have focused primarily on its interactions with the fae cells. proteins encoded on the o pathogenicity island, locus of enterocyte effacement, have been shown to play a critical role in o adherence to fae cells. these include the e. coli secreted proteins espa and espb, the adhesin intimin, and the translocated receptor for intimin, tir which is secreted via the lee-encoded type iii secretion system. hence, several pre-harvest control measures being evaluated in cattle to control or eliminate o from entering the food chain, include vaccines targeting these lee-encoded proteins. for instance, potter et al. developed a vaccine comprising wild-type o culture supernatants that contain the ttss proteins, tir and esps; however, similar protection was noted in animals inoculated with the culture supernatant from a mutant strain of o lacking the tir gene. in addition, the immune response of the vaccinated animals was not merely to the ttss proteins but also against a number of other proteins that were present in the supernatant. interestingly, although the vaccine decreased both the number of e. coli o shed in the feces of vaccinated animals, and those colonizing the terminal rectum, it did not reduce the duration of shedding despite the subcutaneous administration of three doses of the vaccine; http://www.bioniche.com. similar results were also observed with another vaccine that targets the o siderophore receptor and porin proteins; https://animalhealth.pfizer.com. this clearly suggests that unidentified proteins other than those constituting the ttss or srp may play a crucial role in bovine colonization, and that the identification and inclusion of such proteins is likely to increase the efficacy of vaccines for elimination of o from the gastrointestinal tracts of cattle. further supporting this inference are reported observations that the ttss proteins do not significantly contribute to the persistence of o in feedlot cattle, or to o invasion of crypt cells at the raj. in a previous study, o was observed to adhere to rse cells in vivo and in vitro, besides the fae cells and this observation was used to develop a unique in vitro adherence assay for o with rse cells. in this study, we decided to evaluate if the lee-encoded proteins would also be critical for o adherence to rse cells, as for fae cells, and in the event that these proteins would not play a significant role in rse cell adherence, define the proteome of o as expressed when grown in the adherence assay media, dmem, to assemble targets for future evaluation in rse adherence. experimental and bioinformatic evaluation of such targets could in fact help identify a subset of novel adhesins that may have excellent potential to increase the efficacy of the anti-adhesion, cattle o vaccines, by eliminating o from both fae and rse cells at the raj. methods bacterial strains and culture conditions the wild-type o strain edl, a sequenced isolate implicated in human disease, was used in this study. we cultured o in dulbecco modified eagle medium-low glucose, for the cell adherence assays described below. the rationale for the use of this culture medium was to reflect the growth conditions used in the eukaryotic cell adherence assays; and to closely parallel the in vivo nutrient-limiting conditions, and conditions used to prepare the cattle-use approved, lee protein based, anti-adhesion o vaccine. in addition, another wild-type o strain 86–, its isogenic mutant negative for intimin, and this mutant complemented with the plasmid peb ) expressing intimin, were also tested in the adherence assay. the 86– strain and its derivatives were obtained from dr. a. d. o’brien, uniformed services university of the health sciences, bethesda, md. we also cultured o in dmem for proteomic analysis. specifically, an overnight culture of the wild-type o strain in luria-bertani broth was pelleted and washed with sterile phosphate buffered saline, and subcultured to an initial od of in fresh dmem. after incubation at 37 °c with shaking at 250 rpm to an od of to, cells were harvested by centrifugation at  rpm, 15 min at 4 °c. cells were washed three times with an equal volume of sterile pbs, and processed to obtain cell lysate and pellet fractions for proteomic analysis as previously described. o157-rse cell adherence inhibition assay: in the presence of pooled anti-lee proteins, anti-intimin and anti-h antisera adherence of o to the rse cells was previously demonstrated and developed into an adherence assay in our laboratory. in this study, the ability of pooled, rabbit polyclonal antisera to interfere with and inhibit o adherence to rse cells was evaluated. specifically, antisera generated against the recombinant lee-encoded proteins, tir, espa and espb, and intimin, in rabbits, was pooled. rabbit antisera targeting the o flagellar antigen h was also mixed into the pooled antisera, which was then tested at 1: and 1: dilutions. specificity was confirmed by reacting each antiserum against both o cell lysates and the cognate protein in western blotting experiments. rabbit sera from healthy animals, at a 1: dilution, was used as a control. in the presence of anti-intimin antisera alone to specifically evaluate the role of intimin, the rabbit anti-intimin antisera was evaluated separately for its ability to prevent o adherence to rse cells at 1: and 1: dilutions. each of the rse adherence assays was conducted in technical and biological replicates as described previously, with minor modifications, as follows. rse cells were washed and resuspended in 1 ml dulbecco modified eagle medium – no glucose  ±  % d + mannose, in x 100 mm glass tubes, to a final concentration of cells/ml. although type fimbriae are not expressed by o we included d + mannose in parallel assays to cover any hitherto unknown transient expression especially in mutant strains. bacterial pellets from overnight cultures in dmem, incubated at 37°c without aeration, were resuspended in sterile saline with or without antisera, and incubated at 37°c for 30 min. the bacteria-antibody mix was then added to the rse cells suspension to final bacteria:cell ratio of 10: and the mixture incubated with aeration. at the end of 4 h, the mixture was pelleted and washed thoroughly, once with 14 ml dmem-ng, and twice with mls of sterile, distilled water before reconstituting in 100 μl dh2o. eight 2 μl drops of this suspension were placed on polysine slides and dried overnight under direct light to quench non-specific fluorescence, before fixing in cold 95% ethanol for 10 min. the slides were then stained with 1% toluidine blue, or with fluorescence-tagged antibodies that specifically target o and the rse cell cytokeratins as described previously. each experiment was then done in duplicate. o adherence patterns on rse cells were recorded as diffuse, or aggregative for all positive interactions that involved direct association with the cells. scattered bacteria and bacterial micro-colonies not adhering to cell membranes were considered to be negative for adherence to the epithelial cells. a total of 100– well dispersed rse cells were analyzed per slide as described previously. the percent rse cells with and without bacteria adhering to them were determined. if more than 50% of rse cells had > bacteria attached, the adherence was recorded as strongly positive. for >50% rse cells with 1– adherent bacteria, the adherence was recorded as moderately positive. for less than 50% rse cells with 1– adherent bacteria, the result was recorded as non-adherent. o157-hep- cell adherence inhibition assay the role played by lee-encoded proteins and intimin in the adherence of o to hep- cells, has already been defined previously and hence, this assay was used for comparative reasons. the assay was conducted as described previously except that, the washed bacterial pellets were incubated with or without antisera, at 37°c for 30 min, prior to addition to the hep- cells. both the pooled antisera and anti-intimin antisera, as described above, were used at dilutions ranging from 1: to 1: in these assays. each assay was conducted in duplicate, and in 3– chambers of the chamber slides per run. slides were stained with 1% toluidine blue, or with fluorescence-tagged antibodies that specifically target o and the hep- cell actin filaments as described previously and adherence patterns recorded as for rse cells. adherence of 86– 86-24eae Δ and 86-24eae Δ to rse and hep- cells wild-type 86– and its mutant derivatives were used to verify the role of intimin directly and compare the results with that of the o adherence inhibition assays. this assay was conducted, recorded, as previously described and done in the absence of any antisera. onedimensional sds-page liquid chromatography tandem mass spectrometry top down proteomic analysis was done at the harvard partners center for genetics and genomics, cambridge, massachusetts. o cell pellet and lysate fractions were concentrated using spin filters, fractionated on 1d sds-page, and digested in-gel with trypsin prior to tandem mass spectrometry as described previously. the rationale for incorporating a 1d sds-page fractionation step is that this modification reduces complexity of protein mixtures, permits a larger dynamic range of protein identification, and allows for significantly better reproducibility. for mass spectrometry, samples were subjected to three different runs on an lcq deca xp plus proteome x workstation from thermo finnigan as described earlier. for each run, μl of each reconstituted sample was injected with a famos autosampler, and the separation was done on a μm x 20 cm column packed with c media running at a flow rate of μl/min provided from a surveyor ms pump with a flow splitter with a gradient of water, % formic acid and then 5% acetonitrile, % formic acid over the course of 480 min. between each set of samples, a standard of a angiotensin mix of peptides was run to ascertain column performance, and to observe any potential carryover that might have occurred. the lcq was run in a top five configuration, with one ms scan and five ms/ms scans. dynamic exclusion was set to with a limit of seconds. peptide identifications were made using sequest through the bioworks browser, as described previously. sequential database searches were performed using the o strains edl and sakai fasta database from european bioinformatics institute http://www.ebi.ac.uk/newt/display using static carbamidomethyl-modified cysteines and differential oxidized methionines. these protein databases – tax id: and escherichia coli – tax id: 155864) have a total of entries. a reverse o strain edl fasta database was spiked in to provide noise and determine validity of the peptide hits, so that known and theoretical protein hits can be determined without compromising the statistical relevance of all the data. the ms data was searched with a 2-dalton window on the ms precursor with a dalton on the fragment ions. peptide score cutoff values were chosen at cross-correlation values of for singly charged ions, for doubly charged ions, and for triply charged ions, along with delta rank scoring preliminary cutoff values of, and cross-correlation normalized values of the cross-correlation values chosen for each peptide assured a high confidence match for the different charge states, while the deltacn values ensured the uniqueness of the peptide hit. the rsp value of ensured that the peptide matched the top hit in the preliminary scoring. at these peptide filter values, very few reverse database hits were observed, which permitted a higher confidence in the few single peptide protein identifications. furthermore, single hit proteins were manually validated to ensure relevance. bioinformatics cellular location of proteins was determined using amino acid sequences of cognate proteins in the o sequence databases at http://www.ncbi.nlm.nih.gov/protein. in addition, extracytoplasmic proteins were verified for the presence of signal sequences using the program signalp at http://www.cbs.dtu.dk/services/signalp, and subcellular localization of other proteins confirmed using the psort/psort-b program. putative functions were determined by querying the conserved domain database at http://www.ncbi.nlm.nih.gov/structure/cdd/wrpsb.cgi protein components of the o dmem-proteome with adhesion potential were shortlisted using vaxign, a reverse vaccinology based vaccine target prediction and analysis system at http://www.violinet.org that utilizes the spaan algorithm.  lee- encoded proteins considered critical for o adherence to fae cells at the raj did not appear to have a role in o adherence to rse cells at this same site. both o strains grown in dmem and pre-incubated with pooled, polyclonal antisera generated against the lee and flagellar h proteins, or the anti-intimin antisera alone, at 1: and 1: dilution, continued to adhere to the rse cells, irrespective of the presence/absence of d + mannose. data is shown for one of the o strains in the presence of d + mannose. these results were consistent between all trials, irrespective of toluidine blue or immunofluorescent staining, and did not show any differences in the adherence patterns compared to the controls. the same o157-rse cell-adherence pattern was observed in the controls with normal rabbit sera added at 1: dilution, and in the absence of any sera, irrespective of the presence/absence of d + mannose. the continued adherence of o to the rse cells in the presence of antibodies to the lee proteins may have been due to the masking of these antigens and the unmasking of other o adhesins targeting the receptors on the rse cells. to that effect an increase in the total number of rse cells with adherent bacteria and decrease in the total number of rse cells with no adherent bacteria was observed, in the presence of pooled and anti-intimin antisera. we intentionally included antisera targeting the flagellar antigen h as flagella have been demonstrated to play a role in initial adherence to plant cells and the fae. these results suggest that additional mechanisms of adherence, distinct from those attributable to lee, intimin and flagellar h proteins, are involved in o attachment to the raj squamous epithelial cells. on the other hand, the lee-encoded proteins were critical to o adherence to hep- cells as demonstrated previously, with or without d + mannose. as shown in additional file and figure panel a and figure preincubation with the pooled polyclonal antisera and the anti-intimin antisera significantly interfered with and prevented o adherence to hep- cells, at all dilutions tested. an increase in number of hep- cells without any adhering bacteria was observed in the presence of either antiserum, accordingly. however, pre-incubation with normal rabbit sera at 1: dilution showed the same diffuse, moderate adherence as in the absence of any antisera. the results observed with the adherence inhibition assays were further verified by the adherence patterns of o strain 86– and its mutant derivatives on hep- and rse cells. the intimin-negative mutant 86-24eae Δ did not adhere well to the hep- cells compared to the intimin-positive, wild-type 86– or complemented mutant, 86-24eae Δ that demonstrated diffuse, moderate adherence. actin accumulation observed in the majority of hep- cells with 100x magnification only in the presence of 86– and 86-24eae Δ along with an increase in the number of hep- cells without adhering bacteria in the presence of 86-24eae Δ further verified these observations. this confirmed the role of intimin in o adherence to hep- cells. on the otherhand, 86– and all its mutant derivatives demonstrated diffuse, strong adherence to rse cells, irrespective of intimin expression. infact with 86-24eae Δ the number of rse cells with adhering bacteria actually increased, which suggested that intimin did not have a role in the adherence of o to rse cells. in order to verify if proteins other than lee proteins were being expressed by o upon growth in dmem which could have a possible role in o adherence to rse cells, we analyzed the o proteome as expressed in dmem. while the proteome of o has been analyzed under various other growth conditions we decided to evaluate the same following growth in dmem for several reasons, such as this was the media used to culture both bacteria and the rse cells, separately, prior to the adherence assays, the media closely mimicked the nutrient-limiting conditions seen in vivo, and this media closely matched that used to develop a commercially available cattle, o vaccine [ 16; http://www.bioniche.com. our observations did not support a role for other host -derived factors in this adherence of o and hence, we did not evaluate rse-cell adherence of o cultured in eukaryotic cell-conditioned media. this inference came from the fact that similar adherence results were obtained when dmem was supplemented with norepinephrine, a host neuroendocrine hormone that is encountered by o in vivo during the actual process of infection. ne is reportedly a mimic of autoinduer, which regulates o virulence gene expression via quorum sensing. further, intimin, its receptor, tir, as well as espb were expressed in equivalent amounts in both dmem and dmem-ne, as observed using western blotting by others, and by us, and also using top down proteomics by us. a total of proteins were identified as being part of the o dmem-proteome, and these included several characterized and hypothetical/unknown proteins besides the ttss proteins. while of these proteins were uncharacterized with hypothetical functions assigned in the o genome, the remaining proteins localized to various bacterial cell compartments with functions including metabolic, cell division, regulatory, transport, environmental adaptation, and previously characterized o virulence factors; figure additional files and proteins associated with o virulence or adherence in the dmem-proteome included tir, intimin, espb, luxs, iha, ompa, katp, chua, espp, stx1a, stx1b, and stx2b; additional files and interestingly, of the proteins comprising the o dmem-proteome were also part of the o immunoproteome in cattle, defined using the innovative proteome mining tool, proteomics- based expression library screening; additional files and in addition, nine members of the dmem-proteome were also part of the o immunome in humans. given that a subset of pathogen proteins targeted by the host immune system help pathogens overcome hostile in vivo environments and rapidly adapt to host niches, counter host defenses, survive, propagate, and establish infection, it is very likely that proteins other than lee proteins in the o dmem-proteome play a role in o adherence to the rse cells at the raj. the identification and inclusion of such proteins in anti-adhesion vaccine preparations, and their optimal administration to the host immune system, may enhance the efficacy of such vaccines in reducing or eliminating o not only from the fae cells but also from the rse cells of the bovine raj. our strategy for selecting proteins constituting the o dmem proteome with potential to function as adhesins for further evaluation was to employ the spaan algorithm, which is part of the web-based vaccine target prediction and analysis system at http://www.violinet.org. in particular, we shortlisted proteins that were shared by four sequenced o strains, namely, strain edl strain ec strain sakai, and strain tw our analysis identified 36/ components of the o dmem-proteome to have adhesin potential, including the extensively reported primary o adhesin, intimin-γ, and its protein receptor, tir. three o157-specific proteins, namely chua, tere, and a putative outermembrane porin protein on o-island #, have been prioritized and are under experimental evaluation for a role in o adherence to rse cells. also, towards studies of common stec adherence mechanisms, we have determined homologs of several of these putative adhesins including those encoded on o-islands such as, tir, intimin, chua, tere, espp, bioinformatically, in sequenced non-o shiga-toxin-producing escherichia coli of clinical importance. experimental evaluation of the contribution of these homologs to adherence of these non-o stec, e. coli o26:h o103:h and o111:nm, are underway. 1pels: proteomics- based expression library screening 2iviat:in vivo-induced antigen technology  proteins other than the lee-encoded proteins are involved in o adherence to rse cells at the bovine raj. such proteins, with adhesin potential, are expressed by this human pathogen during growth in vitro, in dmem. three such putative adhesins, namely, chua, tere, and a putative outermembrane porin protein on o-island # are currently under evaluation for their adherence potential. such studies should provide both valuable insights into the o157-rse interactions and new targets for more efficacious anti-adhesion o cattle vaccines. competing interests the authors declare no competing financial interests. authors’ contributions itk was the project leader and designed, coordinated, obtained funding, conducted experiments, analyzed data and drafted the manuscript. rwg conducted experiments and tabulated data. bk and das performed proteomic analysis. sbc assisted in design and participated in helpful discussions. mj was the co-project leader, and designed, coordinated, analyzed results and performed bioinformatic analysis. all authors read and approved the final manuscript. supplementary material additional file http://www.biomedcentral.com/imedia/9899042126754199/supp pdf. table a quantitation of rse cells with adherent bacteria in the presence of d + mannose. click here for file additional file http://www.biomedcentral.com/imedia/6766700936754199/supp pdf. table b quantitation of hep- cells with adherent bacteria in the presence of d + mannose. click here for file additional file http://www.biomedcentral.com/imedia/1105071156754199/supp pdf. table c uncharacterized hypothetical proteins of the o dmem-proteome. click here for file additional file http://www.biomedcentral.com/imedia/1751063870675419/supp pdf. table d previously characterized proteins of the o dmem-proteome. click here for file additional file http://www.biomedcentral.com/imedia/1777785157675419/supp pdf. data sheets: o157-dmem ms/ms data sheet click here for file additional file http://www.biomedcentral.com/imedia/1707955235675419/supp pdf. data sheets: o157-dmem ms/ms data sheet click here for file additional file http://www.biomedcentral.com/imedia/1451425738675419/supp pdf. data sheets: o157-dmem ms/ms data sheet click here for file additional file http://www.biomedcentral.com/imedia/3116488396754199/supp pdf. data sheets: o157-dmem ms/ms data sheet click here for file additional file http://www.biomedcentral.com/imedia/1233524502675419/supp pdf. data sheets: o157-dmem ms/ms data sheet click here for file additional file http://www.biomedcentral.com/imedia/1610501146675419/supp pdf. data sheets: o157-dmem ms/ms data sheet click here for file additional file http://www.biomedcentral.com/imedia/1326109329675419/supp pdf. data sheets: o157-dmem ms/ms data sheet click here for file additional file http://www.biomedcentral.com/imedia/1285024576754199/supp pdf. data sheets: o157-dmem ms/ms data sheet click here for file acknowledgements this work was supported by a us national institutes of health grant r ai to i.t.k. intellectual property rights for the o proteome identified in this study are held by massachusetts general hospital, boston, ma. excellent technical assistance provided by bryan wheeler at the national animal disease center, ames, ia, with the eukaryotic cell adherence/adherence-inhibition assays is acknowledged. disclaimer mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the u.s. department of agriculture. usda is an equal opportunity provider and employer.
these adherence patterns were in complete contrast to that observed with hep- cells, assayed under same conditions. ongoing experiments to evaluate their role in rse adherence should provide both valuable insights into the o157-rse interactions and new targets for more efficacious anti-adhesion o vaccines. bioinformatics-based analysis of the components of the o dmem proteome revealed several new o157-specific proteins with adhesin potential. such proteins, with adhesin potential, are expressed by this human pathogen during growth in dmem. in this study, we present evidence that proteins encoded by the locus of enterocyte effacement, considered critical for escherichia coli o adherence to follicle-associated epithelial cells at the bovine recto-anal junction, do not appear to contribute to o adherence to squamous epithelial cells also constituting this primary site of o colonization in cattle. antisera targeting intimin-γ, the primary o adhesin, and other essential lee proteins failed to block o adherence to rse cells, when this pathogen was grown in dmem, a culture medium that enhances expression of lee proteins. this suggested that proteins other than intimin-γ that contribute to adherence to rse cells are expressed by this pathogen during growth in dmem. proteins other than lee and intimin-γ proteins are involved in o adherence to rse cells at the bovine raj. to identify such proteins, we defined the proteome of dmem-grown-o. in addition, rse adherence of a dmem-grown-o mutant lacking the intimin protein was comparable to that seen with its wild-type parent o strain grown in the same media. gelc-ms/ms revealed that the o dmem-proteome comprised proteins including several components of the cattle and human o immunome, orthologs of adhesins, hypothetical secreted and outer membrane proteins, in addition to the known virulence and lee proteins.
 in this study, we present evidence that proteins encoded by the locus of enterocyte effacement, considered critical for escherichia coli o adherence to follicle-associated epithelial cells at the bovine recto-anal junction, do not appear to contribute to o adherence to squamous epithelial cells also constituting this primary site of o colonization in cattle.  antisera targeting intimin-γ, the primary o adhesin, and other essential lee proteins failed to block o adherence to rse cells, when this pathogen was grown in dmem, a culture medium that enhances expression of lee proteins. in addition, rse adherence of a dmem-grown-o mutant lacking the intimin protein was comparable to that seen with its wild-type parent o strain grown in the same media. these adherence patterns were in complete contrast to that observed with hep- cells, assayed under same conditions. this suggested that proteins other than intimin-γ that contribute to adherence to rse cells are expressed by this pathogen during growth in dmem. to identify such proteins, we defined the proteome of dmem-grown-o. gelc-ms/ms revealed that the o dmem-proteome comprised proteins including several components of the cattle and human o immunome, orthologs of adhesins, hypothetical secreted and outer membrane proteins, in addition to the known virulence and lee proteins. bioinformatics-based analysis of the components of the o dmem proteome revealed several new o157-specific proteins with adhesin potential.  proteins other than lee and intimin-γ proteins are involved in o adherence to rse cells at the bovine raj. such proteins, with adhesin potential, are expressed by this human pathogen during growth in dmem. ongoing experiments to evaluate their role in rse adherence should provide both valuable insights into the o157-rse interactions and new targets for more efficacious anti-adhesion o vaccines. o157rectoanal junctionleeadherencedmemgelc-ms/ms 