In order to verify if proteins other than LEE proteins were being expressed by O157 upon LY2835219 concentration growth in DMEM which could have a possible role in O157 adherence to RSE cells, we analyzed the O157 proteome as expressed in DMEM. While the proteome of O157 has been analyzed under various other growth conditions [30–33] we decided to evaluate the same following growth in DMEM for several reasons, such as (i) this was the media AZD8186 used to culture both bacteria and the RSE cells, separately, prior to the adherence assays, (ii) the media closely mimicked the nutrient-limiting conditions seen in vivo, and (iii) this media closely matched that used to develop a commercially available cattle, O157 vaccine

[15, 16; http://​www.​bioniche.​com. Our observations did not support a role for other host (RSE-cell)-derived factors in this adherence of O157 and hence, we did not evaluate RSE-cell adherence of O157 cultured in eukaryotic cell-conditioned media. This inference came from the fact that similar adherence results were obtained https://www.selleckchem.com/products/gant61.html when DMEM was supplemented with norepinephrine (NE; DMEM-NE), a host neuroendocrine hormone that is encountered by O157 in vivo during the actual process of infection (data not shown). NE is reportedly a mimic of autoinduer 3 (AI-3), which regulates O157 virulence gene expression via

quorum sensing [34]. 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 [34], and by us, and also using top down proteomics by us (data not shown). A total

of 684 proteins were MycoClean Mycoplasma Removal Kit identified as being part of the O157 DMEM-proteome (13% of the O157 sequenced proteome), and these included several characterized and hypothetical/unknown proteins besides the TTSS proteins. While 171 of these proteins were uncharacterized with hypothetical functions assigned in the O157 genome [21; Figure 5, Additional files 3 and 5–12], the remaining 513 proteins localized to various bacterial cell compartments with functions including metabolic, cell division, regulatory, transport, environmental adaptation, and previously characterized O157 virulence factors [21]; Figure 5, Additional files 4 and 5 6 7 8 9 10 11 12. Proteins associated with O157 virulence or adherence in the DMEM-proteome included Tir, Intimin, EspB, LuxS, Iha, OmpA, KatP, ChuA, EspP, Stx1A, Stx1B, and Stx2B [20]; Additional files 4 and 5 6 7 8 9 10 11 12. Interestingly, 64 of the 684 (9.4%) proteins comprising the O157 DMEM-proteome were also part of the O157 immunoproteome in cattle, defined using the innovative proteome mining tool, Proteomics- based Expression Library Screening (PELS) [23]; Additional files 3 and 4. In addition, nine members of the DMEM-proteome were also part of the O157 immunome in humans [26]. Figure 5 Bacterial cell localization of proteins comprising the O157 DMEM-proteome.