Six protein biomarkers from two strains of Escherichia coli O157:H7 and one non-O157:H7, nonpathogenic strain of E. coli have been identified by matrix-assisted laser desorption ionization time-of-flight-time-of-flight tandem mass spectrometry (MALDI-TOF-TOF-MS/MS) and top-down proteomics. Proteins were extracted from bacterial cell lysates, ionized by MALDI, and analyzed by MS/MS. Protein biomarker ions were identified from their sequence-specific fragment ions by comparison to a database of in silico fragment ions derived from bacterial protein sequences. Web-based software, developed in-house, was used to rapidly compare the mass-to-charge (m/z) of MS/MS fragment ions to the m/z of in silico fragment ions derived from hundreds of bacterial protein sequences. A peak matching algorithm and a p-value algorithm were used to independently score and rank identifications on the basis of the number of MS/MS-in silico matches. The six proteins identified were the acid stress chaperone-like proteins, HdeA and HdeB; the cold shock protein, CspC; the YbgS (or homeobox protein); the putative stress-response protein YjbJ (or CsbD family protein); and a protein of unknown function, YahO. HdeA, HdeB, YbgS, and YahO proteins were found to be modified post-translationally with removal of an N-terminal signal peptide. Gene sequencing of hdeA, hdeB, cspC, ybgS, yahO, and yjbJ for 11 strains of E. coli O157:H7 and 7 strains of the "near-neighbor" serotype O55:H7 revealed a high degree sequence homology between these two serotypes. Although it was not possible to distinguish O157:H7 from O55:H7 from these six biomarkers, it was possible to distinguish E. coli O157:H7 from a nonpathogenic E. coli by top-down proteomics of the YahO and YbgS. In the case of the YahO protein, a single amino acid residue substitution in its sequence (resulting in a molecular weight difference of only 1 Da) was sufficient to distinguish E. coli O157:H7 from a non-O157:H7, nonpathogenic E. coli by MALDI-TOF-TOF-MS/MS, whereas this would be difficult to distinguish by MALDI-TOF-MS. Finally, a protein biomarker ion at m/z approximately 9060 observed in the MS spectra of non-O157:H7 E. coli strains but absent from MS spectra of E. coli O157:H7 strains was identified by top-down analysis to be the HdeB acid stress chaperone-like protein consistent with previous identifications by gene sequencing and bottom-up proteomics.
"In order to verify if proteins other than LEE proteins were being expressed by O157 upon 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 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. "
[Show abstract][Hide abstract] ABSTRACT: In this study, we present evidence that proteins encoded by the Locus of Enterocyte Effacement (LEE), considered critical for Escherichia coli O157 (O157) adherence to follicle-associated epithelial (FAE) cells at the bovine recto-anal junction (RAJ), do not appear to contribute to O157 adherence to squamous epithelial (RSE) cells also constituting this primary site of O157 colonization in cattle.
Antisera targeting intimin-γ, the primary O157 adhesin, and other essential LEE proteins failed to block O157 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-O157 mutant lacking the intimin protein was comparable to that seen with its wild-type parent O157 strain grown in the same media. These adherence patterns were in complete contrast to that observed with HEp-2 cells (the adherence to which is mediated by intimin-γ), 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-O157 (DMEM-proteome). GeLC-MS/MS revealed that the O157 DMEM-proteome comprised 684 proteins including several components of the cattle and human O157 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 O157 DMEM proteome revealed several new O157-specific proteins with adhesin potential.
Proteins other than LEE and intimin-γ proteins are involved in O157 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 O157 vaccines.
"In another study, the membranes of the S. typhimurium and Klebsiella pneumoniae Enterobacteria were isolated, and the proteins were extracted with subsequent 2-D electrophoresis (Fagerquist, 2010). The excised protein spots were digested with trypsin and analyzed by MALDI-TOF-MS and peptide mass fingerprinting. "
"The top-down proteomics approach can distinguish E. coli O157:H7 from E. coli K-12. Reprinted and modified with permission from Fagerquist et al. (2010), copyright 2010 American Chemical Society. differentiation of closely related species (Hathout et al., 2003; Warscheid & Fenselau, 2003; Warscheid et al., 2003). "
[Show abstract][Hide abstract] ABSTRACT: Mass spectrometry (MS) has become an important technique to identify microbial biomarkers. The rapid and accurate MS identification of microorganisms without any extensive pretreatment of samples is now possible. This review summarizes MS methods that are currently utilized in microbial analyses. Affinity methods are effective to clean, enrich, and investigate microorganisms from complex matrices. Functionalized magnetic nanoparticles might concentrate traces of target microorganisms from sample solutions. Therefore, nanoparticle-based techniques have a favorable detection limit. MS coupled with various chromatographic techniques, such as liquid chromatography and capillary electrophoresis, reduces the complexity of microbial biomarkers and yields reliable results. The direct analysis of whole pathogenic microbial cells with matrix-assisted laser desorption/ionization MS without sample separation reveals specific biomarkers for taxonomy, and has the advantages of simplicity, rapidity, and high-throughput measurements. The MS detection of polymerase chain reaction (PCR)-amplified microbial nucleic acids provides an alternative to biomarker analysis. This review will conclude with some current applications of MS in the identification of pathogens.
Mass Spectrometry Reviews 11/2011; 30(6):1203-24. DOI:10.1002/mas.20320 · 7.71 Impact Factor
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