SUB-SPECIATING CAMPYLOBACTER JEJUNI BY PROTEOMIC ANALYSIS OF ITS PROTEIN BIOMARKERS AND THEIR POST-TRANSLATIONAL MODIFICATIONS

Authors: Fagerquist, Clifton - Keith; Bates, Anne; Heath, Sekou; King, Bryan; Garbus, Brandon; Harden, Leslie - Les; Miller, William - Bill

Submitted to: Journal of Proteome Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/17/2006
Publication Date: 10/6/2006
Citation: Fagerquist, C.K., Bates, A.H., Heath, S., King, B.C., Garbus, B.R., Harden, L.A., Miller, W.G. Sub-speciating campylobacter jejuni by proteomic analysis of its protein biomarkers and their post-translational modifications. Journal of Proteome Research. 5:10:2527-2538

Interpretive Summary: We have identified several protein biomarkers of three strains of Campylobacter jejuni (RM1221, RM1859 and RM3782) by proteomic techniques. The protein biomarkers identified are prominently observed in the time-of-flight mass spectrum (TOF-MS) of bacterial cell lysate supernatent ionized by matrix-assisted laser desorption/ionization (MALDI). The protein biomarkers identified were DNA-binding protein HU, translation initiation factor IF-1, conserved hypothetical protein (cje1298), cytochrome c553, conserved hypothetical protein (cjb1372), transthyretin-like periplasmic protein, chaperonin (groES), thioredoxin and ribosomal proteins: 50S L7/L12, 50S L24, 30S S16, 50S L29 and 30S S15. The protein biomarkers identified appear to represent biologically functional (processed) proteins. The significant findings are as follows. 1. Biomarker mass shifts between strains were due to an amino acid substitutions of the primary polypeptide sequence and not due to variations in post-translational modifications (PTMs). 2. If present, a PTM for a protein biomarker appeared consistently for all three strains. 3. PTMs identified were N-terminal methionine (N-Met) cleavage, which is common among bacterial proteins, as well as a number of other PTMs not previously reported. 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 ribosomal protein 30S S16) whose penultimate residue is threonine 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 suggests that their may be a specific aminopeptidase for N-Met cleavage when the penultimate residue is threonine but that this enzyme may be absent from thermophilic Campylobacters. This exception to the N-Met cleavage rule has implications for the development of bioinformatics algorithms of protein/pathogen identification. 5. There were significantly fewer number of biomarker mass shifts between strains RM1221 and RM1859 than between the other strain RM3782. As the mass shifts were due to the frequency of amino acid substitutions (as a result of genetic variations), it suggested that strains RM1221 and RM1859 were phylogenetically closer to one another than to strain RM3782. These relationships were further confirmed by the nitrite test which showed that RM1221 and RM1859 were C. jejuni subspecies jejuni and RM3782 was C. jejuni subspecies doylei. This result suggests that detection/identification of protein biomarkers by pattern recognition and/or bioinformatics algorithms can easily sub-speciate bacterial microorganisms. 6. Finally, the number of different PTMs detected in this relatively small number of protein biomarkers suggest that bioinformatics algorithms for pathogen identification may need to incorporate many more PTMs than previously suggested in the literature.

Technical Abstract: We have identified several protein biomarkers of three strains of Campylobacter jejuni (RM1221, RM1859 and RM3782) by proteomic techniques. The protein biomarkers identified are prominently observed in the time-of-flight mass spectrum (TOF-MS) of bacterial cell lysate supernatent ionized by matrix-assisted laser desorption/ionization (MALDI). The protein biomarkers identified were DNA-binding protein HU, translation initiation factor IF-1, conserved hypothetical protein (cje1298), cytochrome c553, conserved hypothetical protein (cjb1372), transthyretin-like periplasmic protein, chaperonin (groES), thioredoxin and ribosomal proteins: 50S L7/L12, 50S L24, 30S S16, 50S L29 and 30S S15. The protein biomarkers identified appear to represent biologically functional (processed) proteins. The significant findings are as follows. 1. Biomarker mass shifts between strains were due to an amino acid substitutions of the primary polypeptide sequence and not due to variations in post-translational modifications (PTMs). 2. If present, a PTM for a protein biomarker appeared consistently for all three strains. 3. PTMs identified were N-terminal methionine (N-Met) cleavage, which is common among bacterial proteins, as well as a number of other PTMs not previously reported. 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 ribosomal protein 30S S16) whose penultimate residue is threonine 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 suggests that their may be a specific aminopeptidase for N-Met cleavage when the penultimate residue is threonine but that this enzyme may be absent from thermophilic Campylobacters. This exception to the N-Met cleavage rule has implications for the development of bioinformatics algorithms of protein/pathogen identification. 5. There were significantly fewer number of biomarker mass shifts between strains RM1221 and RM1859 than between the other strain RM3782. As the mass shifts were due to the frequency of amino acid substitutions (as a result of genetic variations), it suggested that strains RM1221 and RM1859 were phylogenetically closer to one another than to strain RM3782. These relationships were further confirmed by the nitrite test which showed that RM1221 and RM1859 were C. jejuni subspecies jejuni and RM3782 was C. jejuni subspecies doylei. This result suggests that detection/identification of protein biomarkers by pattern recognition and/or bioinformatics algorithms can easily sub-speciate bacterial microorganisms. 6. Finally, the number of different PTMs detected in this relatively small number of protein biomarkers suggest that bioinformatics algorithms for pathogen identification may need to incorporate many more PTMs than previously suggested in the literature.