Sub-Speciating Campylobacter jejuni by Proteomic Analysis of Its Protein Biomarkers and Their Post-Translational Modifications

Abstract

We have identified several protein biomarkers of three Campylobacter jejuni strains (RM1221, RM1859, and RM3782) by proteomic techniques. The protein biomarkers identified are prominently observed in the time-of-flight mass spectra (TOF MS) of bacterial cell lysate supernatants ionized by matrix-assisted laser desorption/ionization (MALDI). The protein biomarkers identified were: DNA-binding protein HU, translation initiation factor IF-1, cytochrome c553, a transthyretin-like periplasmic protein, chaperonin GroES, thioredoxin Trx, and ribosomal proteins: L7/L12 (50S), L24 (50S), S16 (30S), L29 (50S), and S15 (30S), and conserved proteins similar to strain NCTC 11168 proteins Cj1164 and Cj1225. The protein biomarkers identified appear to represent high copy, intact proteins. The significant findings are as follows: (1) Biomarker mass shifts between these strains were due to amino acid substitutions of the primary polypeptide sequence and not due to changes in post-translational modifications (PTMs). (2) If present, a PTM of a protein biomarker appeared consistently for all three strains, which supported that the biomarker mass shifts observed between strains were not due to PTM variability. (3) The PTMs observed included N-terminal methionine (N-Met) cleavage as well as a number of other PTMs. (4) It was discovered that protein biomarkers of C. jejuni (as well as other thermophilic Campylobacters) appear to violate the N-Met cleavage rule of bacterial proteins, which predicts N-Met cleavage if the penultimate residue is threonine. Two protein biomarkers (HU and 30S ribosomal protein S16) that have a penultimate threonine residue do not show N-Met cleavage. In all other cases, the rule correctly predicted N-Met cleavage among the biomarkers analyzed. This exception to the N-Met cleavage rule has implications for the development of bioinformatics algorithms for protein/pathogen identification. (5) There were fewer biomarker mass shifts between strains RM1221 and RM1859 compared to strain RM3782. As the mass shifts were due to the frequency of amino acid substitutions (and thus underlying genetic variations), this suggested that strains RM1221 and RM1859 were phylogenetically closer to one another than to strain RM3782 (in addition, a protein biomarker prominent in the spectra of RM1221 and RM1859 was absent from the RM3782 spectrum due to a nonsense mutation in the gene of the biomarker). These observations were confirmed by a nitrate reduction test, which showed that RM1221 and RM1859 were C. jejuni subsp. jejuni whereas RM3782 was C. jejuni subsp. doylei. This result suggests that detection/identification of protein biomarkers by pattern recognition and/or bioinformatics algorithms may easily subspeciate bacterial microorganisms. (6) Finally, the number and variation of PTMs detected in this relatively small number of protein biomarkers suggest that bioinformatics algorithms for pathogen identification may need to incorporate many more possible PTMs than suggested previously in the literature.

Microbial Typing by Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry: Do We Need Guidance for Data Interpretation?

“…The apparent m/z value is only one (simple) characteristic of an individual protein and could be considered the "shadow" of a protein. As described before, a large number of proteins with entirely different functions and sequences can produce peaks with similar m/z values (24). In addition, the m/z value for a given protein can change depending on, for instance, the degree of posttranslational modification or complexing of cofactors.…”

Web-Based Software for Rapid Top-Down Proteomic Identification of Protein Biomarkers, with Implications for Bacterial Identification

“…Bacterial proteins were extracted from Campylobacter bacterial cells using a technique that has been previously reported (9)(10)(11)(12)29). Briefly, Campylobacter upsaliensis strain RM3195, C. coli strain RM2228, and C. lari strain RM2100 were each cultured on nonselective growth media for 24 to 48 h. Bacterial cells were harvested with a 1-l loop (an amount which corresponded to 10 9 cells) and transferred to a microcentrifuge tube containing 0.5 ml of extraction solvents (67% water, 33% acetonitrile, and 0.1% trifluoroacetic acid) and 40 mg of 0.1-mm zirconia-silica beads (BioSpec Products Inc., Bartlesville, OK).…”

“…The likelihood of identification would depend on the number and location of the amino acid substitutions. The number of amino acid variations is dependent on the phylogenetic distance between the unknown and genomically sequenced strains (10). The more closely related the two strains are, the fewer the amino acid substitutions and the greater the probability that a protein from an unknown strain could be identified based on its sequence homology to a protein from a genomically sequenced strain (10).…”

Identification of Bacillus anthracis by Using Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry and Artificial Neural Networks

“…Ribosomal proteins and certain housekeeping proteins have been suggested to be responsible for many mass signals detected by ICMS (12,27,34). Since up to 21% of the cell's overall protein content is ribosomal (2) and because of the fact that ribosomal proteins, as part of the cellular translational machinery, are constitutively expressed in vegetative cells, these proteins constitute a stable ensemble of protein biomarkers suitable for use by fingerprinting techniques.…”