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.
Multiple strains of Campylobacter coli, C. jejuni, C. helveticus, C. lari, C. sputorum, and C. upsaliensis isolated from animal, clinical, or food samples have been analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Whole bacterial cells were harvested from colonies or confluent growth on agar and transferred directly into solvent and then to a spot of dried 3-methoxy-4-hydroxycinnamic acid (matrix). Multiple ions in the 5,000-to 15,000-Da mass range were evident in spectra for each strain; one or two ions in the 9,500-to 11,000-Da range were consistently high intensity. "Species-identifying" biomarker ions (SIBIs) were evident from analyses of multiple reference strains for each of the six species, including the genome strains C. jejuni NCTC 11168 and C. jejuni RM1221. Strains grown on nine different combinations of media and atmospheres yielded SIBI masses within ؎5 Da with external instrument calibration. The highestintensity C. jejuni SIBIs were cytosolic proteins, including GroES, HU/HCj, and RplL. Multiple intraspecies SIBIs, corresponding probably to nonsynonymous nucleotide polymorphisms, also provided some intraspecies strain differentiation. MALDI-TOF MS analysis of 75 additional Campylobacter strains isolated from humans, poultry, swine, dogs, and cats revealed (i) associations of SIBI type with source, (ii) strains previously speciated incorrectly, and (iii) "strains" composed of more than one species. MALDI-TOF MS provides an accurate, sensitive, and rapid method for identification of multiple Campylobacter species relevant to public health and food safety.
We have developed web-based software for the rapid identification of protein biomarkers of bacterial microorganisms. Proteins from bacterial cell lysates were ionized by matrix-assisted laser desorption ionization (MALDI), mass isolated, and fragmented using a tandem time of flight (TOF-TOF) mass spectrometer. The sequence-specific fragment ions generated were compared to a database of in silico fragment ions derived from bacterial protein sequences whose molecular weights are the same as the nominal molecular weights of the protein biomarkers. A simple peak-matching and scoring algorithm was developed to compare tandem mass spectrometry (MS-MS) fragment ions to in silico fragment ions. In addition, a probability-based significance-testing algorithm (P value), developed previously by other researchers, was incorporated into the software for the purpose of comparison. The speed and accuracy of the software were tested by identification of 10 protein biomarkers from three Campylobacter strains that had been identified previously by bottom-up proteomics techniques. Protein biomarkers were identified using (i) their peak-matching scores and/or P values from a comparison of MS-MS fragment ions with all possible in silico N and C terminus fragment ions (i.e., ions a, b, b-18, y, y-17, and y-18), (ii) their peak-matching scores and/or P values from a comparison of MS-MS fragment ions to residue-specific in silico fragment ions (i.e., in silico fragment ions resulting from polypeptide backbone fragmentation adjacent to specific residues [aspartic acid, glutamic acid, proline, etc.]), and (iii) fragment ion error analysis, which distinguished the systematic fragment ion error of a correct identification (caused by calibration drift of the second TOF mass analyzer) from the random fragment ion error of an incorrect identification.
Some strains of Pseudomonas aeruginosa produce R-type pyocins, which are high-molecular-weight phage tail-like protein complexes that have bactericidal activity against other Pseudomonas strains. These particles recognize and bind to bacterial surface structures via tail fibers, their primary spectrum determinant. R-type pyocins kill the cell by contracting a sheath-like structure and inserting their hollow core through the cell envelope, resulting in dissipation of the cellular membrane potential. We have retargeted an R-type pyocin to Escherichia coli O157:H7 by fusing a tail spike protein from an O157-specific phage, V10, to the pyocin tail fiber. The V10 tail spike protein recognizes and degrades the O157 lipopolysaccharide. This engineered pyocin, termed AVR2-V10, is sensitive and specific, killing 100% of diverse E. coli O157:H7 isolates but no other serotypes tested. AVR2-V10 can kill E. coli O157:H7 on beef surfaces, making it a candidate agent for the elimination of this pathogen from food products. All rare AVR2-V10-resistant mutants isolated and examined have lost the ability to produce the O157 antigen and are expected to have compromised virulence. In addition, E. coli O157:H7 exposed to and killed by AVR2-V10 do not release Shiga toxin, as is often the case with many antibiotics, suggesting potential therapeutic applications. The demonstration that a novel R-type pyocin can be created in the laboratory by fusing a catalytic tail spike from the family Podoviridae to a tail fiber of a member of the family Myoviridae is evidence that the plasticity observed among bacteriophage tail genes can, with modern molecular techniques, be exploited to produce nonnatural, targeted antimicrobial agents.
We have developed two sets of Campylobacter shuttle vectors containing either the gfp (green fluorescent protein), yfp (yellow fluorescent protein), or cfp (cyan fluorescent protein) reporter gene. In one set, the reporter gene is fused to a consensus Campylobacter promoter sequence (P c ). The other set contains a pUC18 multicloning site upstream of the reporter gene, allowing the construction of transcriptional fusions using known promoters or random genomic fragments. C. jejuni cells transformed with the P c fusion plasmids are strongly fluorescent and easily visualized on chicken skin, on plant tissue, and within infected Caco-2 cells. In each C. jejuni strain tested, these plasmids were maintained over several passages in the absence of antibiotic selection. Also, in many C. jejuni strains, >91% of the cells transformed with the P c fusion plasmids remained fluorescent after several days. Experiments with yellow fluorescent and cyan fluorescent C. jejuni transformants suggest that aggregates containing two or more strains of C. jejuni may be present in an enrichment broth culture. Colonies arising from these aggregates would be heterologous in nature; therefore, isolation of a pure culture of C. jejuni, by selecting single colonies, from an environmental sample may not always yield a single strain.
The content and heat stability of protease inhibitors of a standard cultivar (Williams 82) and an isoline (L81-4590) lacking the Kunitz trypsin inhibitor (KTI) were measured by using enzyme inhibition and enzyme-linked immunosorbent assays (ELISA). The KTI content of the isoline was less than 0.2% compared to Williams 82, with the exact content depending on the extent of cross-pollination of the soybeans. Steam heating of the isoline flour (121 °C, 20 min) resulted in a near-zero level of trypsin inhibitory activity, while 20% remained in the Williams 82 sample. The raw soy flour prepared from the isoline was nutritionally superior to the raw flour prepared from the standard variety, as measured by PER and pancreatic weights. The increased PER was likely due to the lower level of trypsin inhibitory activity in the isoline. Steam heating the flours for up to 30 min at 121 °C progressively increased the PER for both strains. Screening of several accessions from the USDA Soybean Germplasm Collection showed variation in the content of trypsin inhibitor, sulfur amino acids, and lectins, indicating that further screening studies could lead to the discovery of soybeans which yield flour that is safe and nutritious, with minimal heating.
We have identified a prominent approximately 10-kDa protein biomarker observed in the matrix-assisted laser desorption/ionization time-of-flight mass spectra (MALDI-TOF-MS) of cell lysates of five thermophilic species of Campylobacter: jejuni, coli, lari, upsaliensis, and helveticus. The biomarker was unambiguously identified by genomic and proteomic sequencing as a DNA-binding protein HU. We report the amino acid sequence of HU as determined by sequencing the hup gene of four species (12 strains): C. jejuni (2), C. coli (4), C. upsaliensis (4) and C. lari(2). Confirmation of the amino acid sequence was obtained by nanoflow high-performance liquid chromatography-tandem mass spectrometry of the tryptic peptides of the extracted/digested HU protein. Protein identification was also confirmed by comparison of the molecular weight (MW) predicted from the hup gene and the MW of HU as measured by high-resolution mass spectrometry. We found the HU protein to be particularly useful as a biomarker in that it strongly ionizes by MALDI and its MW varies between species and among strains within a species. Intra- and interspecies variation of the HU MW is due to changes in the amino acid sequence of the HU protein and not due to co- or posttranslational modifications. The strong ionization efficiency of HU by MALDI is likely due, in part, to four lysine residues clustered at the carboxyl end of the protein. We also report identification of the HU protein biomarker for a C. helveticus strain, whose hup gene was not sequenced, but whose HU amino acid sequence was partially conserved in C. upsaliensis strains. We have also tentatively assigned a approximately 10.5-kDa protein biomarker of a C. concisus strain as an HU protein.
Curli are adhesive fimbriae of Enterobacteriaceae and are involved in surface attachment, cell aggregation, and biofilm formation. Here, we report that both inter-and intrastrain variations in curli production are widespread in enterohemorrhagic Escherichia coli O157:H7. The relative proportions of curli-producing variants (C ؉ ) and curli-deficient variants (C ؊ ) in an E. coli O157:H7 cell population varied depending on the growth conditions. In variants derived from the 2006 U.S. spinach outbreak strains, the shift between the C ؉ and C ؊ subpopulations occurred mostly in response to starvation and was unidirectional from C ؊ to C ؉ ; in variants derived from the 1993 hamburger outbreak strains, the shift occurred primarily in response to oxygen depletion and was bidirectional. Furthermore, curli variants derived from the same strain displayed marked differences in survival fitness: C ؉ variants grew to higher concentrations in nutrient-limited conditions than C ؊ variants, whereas C ؊ variants were significantly more acid resistant than C ؉ variants. This difference in acid resistance does not appear to be linked to the curli fimbriae per se, since a csgA deletion mutant in either a C ؉ or a C ؊ variant exhibited an acid resistance similar to that of its parental strain. Our data suggest that natural curli variants of E. coli O157:H7 carry several distinct physiological properties that are important for their environmental survival. Maintenance of curli variants in an E. coli O157:H7 population may provide a survival strategy in which C ؉ variants are selected in a nutrient-limited environment, whereas C ؊ variants are selected in an acidic environment, such as the stomach of an animal host, including that of a human.
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