Clifton K. Fagerquist

Top-Down Proteomic Identification of Shiga Toxin 2 Subtypes from Shiga Toxin-Producing Escherichia coli by Matrix-Assisted Laser Desorption Ionization–Tandem Time of Flight Mass Spectrometry

ABSTRACT We have analyzed 26 Shiga toxin-producing Escherichia coli (STEC) strains for Shiga toxin 2 (Stx2) production using matrix-assisted laser desorption ionization (MALDI)–tandem time of flight (TOF-TOF) tandem mass spectrometry (MS/MS) and top-down proteomic analysis. STEC strains were induced to overexpress Stx2 by overnight culturing on solid agar supplemented with either ciprofloxacin or mitomycin C. Harvested cells were lysed by bead beating, and unfractionated bacterial cell lysates were ionized by MALDI. The A2 fragment of the A subunit and the mature B subunit of Stx2 were analyzed by MS/MS. Sequence-specific fragment ions were used to identify amino acid subtypes of Stx2 using top-down proteomic analysis using software developed in-house at the U.S. Department of Agriculture (USDA). Stx2 subtypes (a, c, d, f, and g) were identified on the basis of the mass of the A2 fragment and the B subunit as well as from their sequence-specific fragment ions by MS/MS (postsource decay). Top-down proteomic identification was in agreement with DNA sequencing of the full Stx2 operon (stx 2) for all strains. Top-down results were also compared to a bioassay using a Vero-d2EGFP cell line. Our results suggest that top-down proteomic identification is a rapid, highly specific technique for distinguishing Stx2 subtypes.

Evolutionary silence of the acid chaperone protein HdeB in enterohemorrhagic Escherichia coli O157:H7.

ABSTRACT The periplasmic chaperones HdeA and HdeB are known to be important for cell survival at low pH (pH < 3) in Escherichia coli and Shigella spp. Here we investigated the roles of HdeA and HdeB in the survival of various enterohemorrhagic E. coli (EHEC) following exposure to pH 2.0. Similar to K-12 strains, the acid protections conferred by HdeA and HdeB in EHEC O145 were significant: loss of HdeA and HdeB led to over 100- to 1,000-fold reductions in acid survival, depending on the growth condition of prechallenge cells. However, this protection was much less in E. coli O157:H7 strains. Deletion of hdeB did not affect the acid survival of cells, and deletion of hdeA led to less than a 5-fold decrease in survival. Sequence analysis of the hdeAB operon revealed a point mutation at the putative start codon of the hdeB gene in all 26 E. coli O157:H7 strains analyzed, which shifted the ATG start codon to ATA. This mutation correlated with the lack of HdeB in E. coli O157:H7; however, the plasmid-borne O157-hdeB was able to restore partially the acid resistance in an E. coli O145ΔhdeAB mutant, suggesting the potential function of O157-HdeB as an acid chaperone. We conclude that E. coli O157:H7 strains have evolved acid survival strategies independent of the HdeA/B chaperones and are more acid resistant than nonpathogenic K-12 for cells grown under nonfavorable culturing conditions such as in Luria-Bertani no-salt broth at 28°C. These results suggest a divergent evolution of acid resistance mechanisms within E. coli.

Top-Down Proteomic Identification of Furin-Cleaved α-Subunit of Shiga Toxin 2 from Escherichia coli O157:H7 Using MALDI-TOF-TOF-MS/MS

A method has been developed to identify the α-subunit of Shiga toxin 2 (α-Stx2) from Escherichia coli O157:H7 using matrix-assisted laser desorption/ionization time-of-flight-time-of-flight tandem mass spectrometry (MALDI-TOF-TOF-MS/MS) and top-down proteomics using web-based software developed in-house. Expression of Stx2 was induced by culturing E. coli O157:H7 on solid agar supplemented with an antibiotic that elicits the bacterial SOS-response. Bacterial cell lysates were incubated in the presence of furin, a human enzyme, that cleaves α-Stx2 into A1 (~28 kDa) and A2 (~5 kDa) protein fragments. A subsequent disulfide reduction step unlinked A1 from A2. MALDI-TOF-MS of the furin-digested/disulfide-reduced sample showed a peak at mass-to-charge (m/z) 5286 that corresponded to the A2 fragment. No peak was observed that corresponded to the A1 fragment although its presence was confirmed by bottom-up proteomics. The peak at m/z 5286 was definitively identified by MALDI-TOF-TOF-MS/MS and top-down proteomics as the A2 fragment of α-Stx2.

The need for agriculture phenotyping: “Moving from genotype to phenotype”

UNLABELLED Increase in the world population has called for the increased demand for agricultural productivity. Traditional methods to augment crop and animal production are facing exacerbating pressures in keeping up with population growth. This challenge has in turn led to the transformational change in the use of biotechnology tools to meet increased productivity for both plant and animal systems. Although many challenges exist, the use of proteomic techniques to understand agricultural problems is steadily increasing. This review discusses the impact of genomics, proteomics, metabolomics and phenotypes on plant, animal and bacterial systems to achieve global food security and safety and we highlight examples of intra and extra mural research work that is currently being done to increase agricultural productivity. BIOLOGICAL SIGNIFICANCE This review focuses on the global demand for increased agricultural productivity arising from population growth and how we can address this challenge using biotechnology. With a population well above seven billion humans, in a very unbalanced nutritional state (20% overweight, 20% risking starvation) drastic measures have to be taken at the political, infrastructure and scientific levels. While we cannot influence politics, it is our duty as scientists to see what can be done to feed humanity. Hence we highlight the transformational change in the use of biotechnology tools over traditional methods to increase agricultural productivity (plant and animal). Specifically, this review deals at length on how a three-pronged attack, namely combined genomics, proteomics and metabolomics, can help to ensure global food security and safety. This article is part of a Special Issue entitled: Translational Plant Proteomics.

An Antibiotic Linked to Peptides and Proteins is Released by Electron Capture Dissociation Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

Desfuroylceftiofur (DFC) is a bioactive β-lactam antibiotic metabolite that has a free thiol group. Previous experiments have shown release of DFC from plasma extracts after addition of a disulfide reducing agent, suggesting that DFC may be bound to plasma and tissue proteins through disulfide bonds. We have reacted DFC with [Arg8]-vasopressin (which has one disulfide bond) and bovine insulin (which has three disulfide bonds) and analyzed the reaction products by use of electron capture dissociation Fourier transform ion cyclotron resonance mass spectrometry (ECD FT-ICR MS), which has previously shown preferential cleavage of disulfide bonds. We observe cleavage of DFC from vasopressin and insulin during ECD, suggesting that DFC is indeed bound to peptides and proteins through disulfide bonds. Specifically, we observed dissociative loss of one, as well as two, DFC species during ECD of [vasopressin + 2(DFC-H)+2H]2+ from a single electron capture event. Loss of two DFCs could arise from either consecutive or simultaneous loss, but in any case implies a gas phase disulfide exchange step. ECD of [insulin + DFC + 4H]4+ shows preferential dissociative loss of DFC. Combined with HPLC, ECD FT-ICR-MS may be an efficient screening method for detection of drug-biomolecule binding.

Quantification and characterization of mucosa-associated and intracellular Escherichia coli in inflammatory bowel disease.

Background:Mucosa-associated Escherichia coli are abundant in inflammatory bowel disease (IBD), but whether these bacteria gain intracellular access within the mucosa is uncertain. If E. coli does gain intracellular access, the contribution of bacterial pathogenicity to this requires further elucidation. This study aimed to quantify and characterize mucosa-associated and intracellular E. coli in patients with IBD and in healthy control subjects (HC). Methods:Mucosal biopsies from 30 patients with Crohns disease (CD), 15 with ulcerative colitis (UC), and 14 HC were cultured with or without gentamicin protection to recover intracellular or mucosa-associated E. coli, respectively. Overall, 40 strains (CD: n = 24, UC: n = 9, and HC: n = 7) were characterized by phylogenetic typing, adhesion and invasion assays, detection of virulence factors, antimicrobial resistance genes, and proteomic analysis. Results:Mucosa-associated E. coli were more abundant in CD and UC than in HC (2750 versus 1350 versus 230 median colony-forming units per biopsy; P = 0.01). Intracellular E. coli were more prevalent in CD (90%) than in UC (47%) or HC mucosal biopsies (0%) (P < 0.001). Of 24 CD strains, 2 were adherent and invasive, but there were no unifying pathogenicity determinants that could distinguish most CD strains from UC or HC strains, or intracellular isolates from mucosa-associated isolates. Conclusions:Intracellular E. coli are more common in CD than in UC and not identified in HC. Most intracellular E. coli did not have characterizing pathogenic features, suggesting a significant role for defects in mucosal immunity or barrier dysfunction in their ability to gain intracellular access.

A new calibrant for matrix-assisted laser desorption/ionization time-of-flight-time-of-flight post-source decay tandem mass spectrometry of non-digested proteins for top-down proteomic analysis

RATIONALE Matrix-assisted laser desorption/ionization (MALDI) time-of-flight-time-of-flight (TOF-TOF) post-source decay (PSD) tandem mass spectrometry (MS/MS) has seen increasing use for analysis of non-digested protein ions for top-down proteomic identification. However, there is no commonly accepted calibrant for this purpose beyond the use of peptide calibrants whose fragment ions span a lower mass-to-charge (m/z) range. METHODS We have used the PSD-generated fragment ions of disulfide-reduced/alkylated thioredoxin (AlkTrx) for TOF-TOF calibration in reflectron mode for the purpose of PSD-MS/MS analysis. The average m/z values of AlkTrx fragment ions were used for calibration. The quality of the calibration was assessed from the observed fragment ion mass error of MS/MS of the YahO protein from an unfractionated bacterial cell lysate of Escherichia coli O157:H7 as well as from MS/MS of bovine ubiquitin. The fragment ion mass errors of these two analytes were also used to assess instrument calibration using the monoisotopic fragment ions of [Glu(1)]-fibrinopeptide B (GluFib). RESULTS A general improvement in fragment ion mass accuracy was observed using the AlkTrx calibration compared to the GluFib calibration which resulted in a more significant top-down proteomic identification of these analyte proteins. CONCLUSIONS Our results suggest that AlkTrx may be useful as a calibrant for MALDI-TOF-TOF-PSD-MS/MS of small and modest-sized protein ions. The uniform fragmentation efficiency of YahO across its sequence suggests that it may be useful as a post-calibration standard to assess PSD-MS/MS instrument performance as well as establishing appropriate top-down proteomic fragment ion tolerances.

Peptides Identified in Soybean Protein Increase Plasma Cholesterol in Mice on Hypercholesterolemic Diets

The in vitro micellar cholesterol displacement assay has been used to identify peptides that may potentially reduce cholesterol in vivo. Two of these peptides, LPYPR and WGAPSL, derived from soybean protein (SP) that have been reported to displace cholesterol from micelles were tested by feeding them as a part of a hypercholesterolemic diet to mice for 3 weeks. Except reduction of very low-density lipoprotein cholesterol (VLDL-C) and triglyceride contents, the peptide-containing diets increased plasma cholesterol content with the increasing dose of the peptides. Mice fed diets supplemented with the peptides also had lower fecal bile acid excretion. Negative correlations between fecal bile acid excretion and plasma total cholesterol content (r = -0.876, P = 0.062) and non-HDL-C content (r = -0.831, P = 0.084) were observed. The mRNA levels of the genes for cholesterol and bile acid metabolism, CYP51, LDLR, CYP7A1, and LPL, were up-regulated in mice fed diets supplemented with peptides except the group fed the low dose of WGAPSL. The results suggested that higher plasma total cholesterol content possibly due to lower fecal steroid excretion as well as lower VLDL-C and triglyceride contents might due to the up-regulated expression levels of the genes CYP51, LDLR, and LPL.

A Designed Experiments Approach to Optimizing MALDI-TOF MS Spectrum Processing Parameters Enhances Detection of Antibiotic Resistance in Campylobacter jejuni

MALDI-TOF MS has been utilized as a reliable and rapid tool for microbial fingerprinting at the genus and species levels. Recently, there has been keen interest in using MALDI-TOF MS beyond the genus and species levels to rapidly identify antibiotic resistant strains of bacteria. The purpose of this study was to enhance strain level resolution for Campylobacter jejuni through the optimization of spectrum processing parameters using a series of designed experiments. A collection of 172 strains of C. jejuni were collected from Luxembourg, New Zealand, North America, and South Africa, consisting of four groups of antibiotic resistant isolates. The groups included: (1) 65 strains resistant to cefoperazone (2) 26 resistant to cefoperazone and beta-lactams (3) 5 strains resistant to cefoperazone, beta-lactams, and tetracycline, and (4) 76 strains resistant to cefoperazone, teicoplanin, amphotericin, B and cephalothin. Initially, a model set of 16 strains (three biological replicates and three technical replicates per isolate, yielding a total of 144 spectra) of C. jejuni was subjected to each designed experiment to enhance detection of antibiotic resistance. The most optimal parameters were applied to the larger collection of 172 isolates (two biological replicates and three technical replicates per isolate, yielding a total of 1,031 spectra). We observed an increase in antibiotic resistance detection whenever either a curve based similarity coefficient (Pearson or ranked Pearson) was applied rather than a peak based (Dice) and/or the optimized preprocessing parameters were applied. Increases in antimicrobial resistance detection were scored using the jackknife maximum similarity technique following cluster analysis. From the first four groups of antibiotic resistant isolates, the optimized preprocessing parameters increased detection respective to the aforementioned groups by: (1) 5% (2) 9% (3) 10%, and (4) 2%. An additional second categorization was created from the collection consisting of 31 strains resistant to beta-lactams and 141 strains sensitive to beta-lactams. Applying optimal preprocessing parameters, beta-lactam resistance detection was increased by 34%. These results suggest that spectrum processing parameters, which are rarely optimized or adjusted, affect the performance of MALDI-TOF MS-based detection of antibiotic resistance and can be fine-tuned to enhance screening performance.

Unlocking the proteomic information encoded in MALDI-TOF-MS data used for microbial identification and characterization

ABSTRACT Introduction: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is increasingly utilized as a rapid technique to identify microorganisms including pathogenic bacteria. However, little attention has been paid to the significant proteomic information encoded in the MS peaks that collectively constitute the MS ‘fingerprint’. This review/perspective is intended to explore this topic in greater detail in the hopes that it may spur interest and further research in this area. Areas covered: This paper examines the recent literature on utilizing MALDI-TOF for bacterial identification. Critical works highlighting protein biomarker identification of bacteria, arguments for and against protein biomarker identification, proteomic approaches to biomarker identification, emergence of MALDI-TOF-TOF platforms and their use for top-down proteomic identification of bacterial proteins, protein denaturation and its effect on protein ion fragmentation, collision cross-sections and energy deposition during desorption/ionization are also explored. Expert commentary: MALDI-TOF and TOF-TOF mass spectrometry platforms will continue to provide chemical analyses that are rapid, cost-effective and high throughput. These instruments have proven their utility in the taxonomic identification of pathogenic bacteria at the genus and species level and are poised to more fully characterize these microorganisms to the benefit of clinical microbiology, food safety and other fields.