Clifton Fagerquist (Presenter)
Agricultural Research Service, USDA
Bio: Dr. Clifton K. Fagerquist received his PhD in physical chemistry from UCLA in 1994 where he used fast atom bombardment and sector-field mass spectrometry to study stable and metastable gas phase clusters. After post-doctoral research at UC Berkeley and two years directing a mass spectrometry facility at University of Minnesota, Minneapolis, he joined the Agricultural Research Service (ARS) in Philadelphia. After two years in Philadelphia, he transferred to the ARS facility in Albany, California where he has been using advanced mass spectrometry and proteomic techniques to identify and characterize foodborne pathogens and their toxins.
Authorship: Clifton K. Fagerquist (1), William J. Zaragoza (2), Beatriz Quinones (3).
Agricultural Research Service, USDA
We analyzed 45 Shiga toxin-producing Escherichia coli (STEC) strains (environmental isolates collected from Northern California agricultural regions) for expression of Shiga toxin (Stx) using MALDI-TOF-TOF-MS/MS and top-down proteomic analysis. Strains were grown overnight on agar supplemented with a sub-inhibitory level of DNA-damaging antibiotics. Nineteen STEC strains produced clinical subtype Stx2a, fifteen strains produced clinical subtype Stx2c, nine strains produced clinical subtype Stx2c or Stx2d (weak inducers), three strains induced but expressed an unknown protein biomarker close in mass to Stx but not Stx. Four control strains were also correctly identified.
Shiga toxin-producing Escherichia coli (STEC) can be a potentially deadly contaminant in the agricultural food chain. As such, it represents a significant public health hazard as well as a potential liability to the food industry. Methods are needed to rapidly detect and characterize STEC strains and the Shiga toxin (Stx) they produce (not all of which are clinically relevant). Stx is an AB5 protein toxin composed of six non-covalently assembled proteins. Stx has five identical B-subunits that form a torus (donut) shape quaternary structure having five-fold symmetry. Each B-subunit has its own intramolecular disulfide bond. Part of the single A-subunit (A2) fits within the donut “hole” providing stability. The rest of the A-subunit (A1) is positioned on one side of the donut. The A-subunit is a single continuous polypeptide chain, however a highly accessible 20-residue loop in the chain delineates A1 from A2. In addition, there is an intramolecular disulfide bond at the base of this loop.
The AB5 holotoxin attaches to specific surface receptors (globotriaosylceramide or Gb3) of certain eukaryotic cells (e.g. kidney cells) after which it is enveloped by the cell and following a retrograde pathway eventually to the endoplasmic reticulum where the 20-residue loop of the A-subunit undergoes proteolytic cleavage by an endopeptidase (furin) followed by reduction of the disulfide bond at its base resulting in release of A1 from the holotoxin complex. A1 then travels to the cytoplasm where it disrupts protein synthesis leading to cell death .
Stx has two types: Stx1 and Stx2. Stx1 has three subtypes (all clinical): a, c and d. Stx2 has seven subtypes: a, b, c, d, e, f, g (and perhaps more). Only Stx2a-d are clinically relevant . The clinical subtypes of Stx2 have been linked to major foodborne pathogen outbreaks. What distinguishes subtypes of Stx2 (as well as Stx1) are amino acid substitutions (AAS) primarily in the B-subunit and the A2 of the A-subunit. AAS can play a significant role in the quaternary structure of AB5 holotoxin complex affecting its binding affinity for eukaryotic surface receptors (e.g. Gb3). In consequence, distinguishing types and subtypes of Stx provides important information about the potential virulence and pathogenicity of any putative STEC strain.
Our laboratory has developed a technique to rapidly detect and identify Stx types and subtypes produced from putative STEC strains using MALDI-TOF-TOF tandem mass spectrometry (MS/MS) and top-down proteomic analysis. Over the past few years, our method has undergone a number of improvements, primarily with respect to sample protocol that enhance the sensitivity of the technique.
The protocol has been described in detail previously [3-6]. Briefly, putative STEC strains were cultured from glycerol stocks for 4 hours in Luria-Bertani broth (LBB) prior to plating on solid LB agar that is supplemented (on separate plates) with two different concentrations of two DNA-damaging antibiotics: ciprofloxacin (10 and 20 ng/mL) and mitomycin-C (800 and 1200 ng/mL). The response of a putative STEC strain to an antibiotic is largely unknown, however it is strain dependent. The initial screen with different antibiotics at different concentrations was used to determine the optimum sub-inhibitory level of antibiotic for a particular STEC strain. After culturing overnight, cells were harvested and transferred to 300 µL of water in an Eppendorf tube, briefly vortexed and then centrifuged. No mechanical cell lysis was utilized as the antibiotic-induction triggers a bacterial response from the strain that causes lysis of its membranes releasing the toxin. The advantage of this approach is that non-induced cells (i.e. no toxin) do not lyse . Sample fractionation is not performed nor is it necessary. Supernatant is spotted onto the stainless steel target which, after drying, is overlayed with a saturated solution of sinapinic acid (67% water, 33% acetonitrile, 0.1% TFA). For analysis of the reduced B-subunit, the sample was reduced with dithiolthreitol (DTT) and heated for 10 minutes at 70 °C. For detection of the A2 fragment of the A-subunit, the extraction was performed in 3 mM CaCl2 in water to which was added 1 µL of furin (New England Biolabs) and allowed to incubate for 1 hour at 37 °C after which the sample was reduced with DTT for 10 minutes at 70 °C. Samples were then spotted onto the MALDI target as before.
The mass spectrometer used was a 4800 MALDI-TOF-TOF (Sciex). Sample spots were first analyzed in linear MS mode. Detection of the disulfide bond-intact B-subunit at 7.8 kDa (no DTT and no furin) in linear mode was then followed by MS/MS post-source decay (PSD) in reflectron mode. Fragment ion triplets confirm the characteristic lasso-looped disulfide bond-intact structure of the B-subunit. Disulfide bond-reduced and furin digested/reduced samples were also analyzed by MS and MS/MS-PSD for the reduced B-subunit and the A2 fragment (5.3 kDa) of the A-subunit.
The A2 fragment and the reduced B-subunit were analyzed by top-down proteomic analysis using software developed in-house . MS/MS spectra of top identifications were also manually inspected.
Forty-five environmental STEC strains were analyzed for antibiotic-induced expression of Stx. Nineteen strains produced the clinical subtype Stx2a, fifteen strains produced clinical subtype Stx2c, nine strains produced clinical subtype Stx2c or Stx2d (weak inducers), three strains induced but expressed an unknown protein biomarker close in mass to Stx but not Stx. Three negative control strains were negative and one positive control was positive. No Stx1 toxin was detected.
STEC strains classified as weak inducers produced Stx at low abundance such that only the disulfide bond-intact and reduced B-subunit could be identified (but not the A2 fragment). In consequence, it was not possible to determine definitively whether these strains were subtype Stx2c or Stx2d (both clinical subtypes).
Three putative STEC strains induced when exposed to antibiotic and generated a protein biomarker close in mass to known B-subunits. However, MS/MS-PSD of this non-reduced protein biomarker that appeared at approximately 7838 Da did not show any evidence of fragment ion triplets indicative of the presence of an intramolecular disulfide bond. In addition, six prominent fragment ions were observed and each was paired to its complementary fragment ion (i.e. b/y fragment ion pair ). However, assuming that these fragment ions are the result of polypeptide backbone cleavage on the C-terminal side of aspartic acid (D) or glutamic acid (E) residues, their location in the sequence was inconsistent with the location of the D and E residues in known B-subunit sequences. Top-down proteomic analysis of this protein resulted in no significant identifications. It would seem that this antibiotic-induced protein is not a Stx B-subunit (or at least not a functional B-subunit) although its exact identity is not clear. It is probable that the protein is an inducible bacteriophage protein and the inability to identify it by top-down analysis may be due to an unknown post-translational modification (PTM). PTMs of bacteriophage proteins are not well documented. Alternatively, the stx2 operon may have insertions or frame shifts leading to a non-functional B-subunit.
Further improvements to the protocol involve adjusting the CaCl2 concentration. CaCl2 is necessary for the enzymatic activity of furin. However, salts can reduce the ionization efficiency of MALDI. We have found increased MALDI ionization by reducing the CaCl2 from 3 mM to 1 mM while increasing the digestion time from 1 hour to 2 hours.
Conclusions & Discussion
MALDI-TOF-TOF-MS/MS and top-down proteomic analysis is a rapid technique for identifying clinical and non-clinical types and subtypes of Stx from putative STEC strains. The only time-consuming step is overnight culturing. However, overnight culturing is a standard microbiological technique that demonstrates (among other things) the viability of the microorganism. The protocol developed in our laboratory relies upon antibiotic-induced production of Stx with concomitant bacterial cell lysis that releases Stx into the extraction solution. The method is robust, highly specific in the determination of types and subtypes and complementary to DNA-based techniques such as PCR and WGS that can only confirm the presence/absence of stx genes but not their expression. Our results clearly demonstrate that Stx expression is highly strain dependent.
References & Acknowledgements:
1. Johannes L, Römer W. Shiga toxins--from cell biology to biomedical applications. Nat Rev Microbiol. 2010; 8:105-16. Review.
2. Scheutz F, Teel LD, Beutin L, Piérard D, Buvens G, Karch H, Mellmann A, Caprioli A, Tozzoli R, Morabito S, Strockbine NA, Melton-Celsa AR, Sanchez M, Persson S, O'Brien AD. Multicenter evaluation of a sequence-based protocol for subtyping Shiga toxins and standardizing Stx nomenclature. J Clin Microbiol. 2012; 50: 2951-2963.
3. Fagerquist CK, Sultan O. Induction and identification of disulfide-intact and disulfide-reduced β-subunit of Shiga toxin 2 from Escherichia coli O157:H7 using MALDI-TOF-TOF-MS/MS and top-down proteomics. Analyst. 2011; 136:1739-1746.
4. Fagerquist CK, Sultan O. Top-down proteomic identification of furin-cleaved α-subunit of Shiga toxin 2 from Escherichia coli O157:H7 using MALDI-TOF-TOF-MS/MS. J Biomed Biotechnol. 2010; 2010:123460.
5. Fagerquist CK, Zaragoza WJ, Sultan O, Woo N, Quiñones B, Cooley MB, Mandrell RE. 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. Appl Environ Microbiol. 2014; 80:2928-2940.
6. Fagerquist CK, Zaragoza WJ. Bacteriophage cell lysis of Shiga toxin-producing Escherichia coli for top-down proteomic identification of Shiga toxins 1 & 2 using matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry. Rapid Commun Mass Spectrom. 2016; 30:671-680.
7. Fagerquist CK, Garbus BR, Williams KE, Bates AH, Boyle S, Harden LA. Web-based software for rapid top-down proteomic identification of protein biomarkers, with implications for bacterial identification. Appl Environ Microbiol. 2009; 75: 4341-53.
8. Fagerquist CK, Zaragoza WJ. Complementary b/y fragment ion pairs from post-source decay of metastable YahO for calibration of MALDI-TOF-TOF-MS/MS. Intl J Mass Spectrometry. 415, 2017, 29-37.
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