Mass spectrometry-based phyloproteomics (MSPP) was used to type a collection of Campylobacter jejuni ssp. doylei isolates at the strain level in comparison to multilocus sequence typing (MLST).
MALDI-TOF MS offers the possibility to differentiate some bacteria not only at the species and subspecies level but even below, at the strain level. Allelic isoforms of the detectable biomarker ions result in isolate-specific mass shifts. Mass spectrometry-based phyloproteomics (MSPP) is a novel technique that combines the mass spectrometric detectable biomarker masses in a scheme that allows deduction of phyloproteomic relations from isolate specific mass shifts compared to a genome sequenced reference strain. The deduced amino acid sequences are then used to calculate MSPP-based dendrograms.
Here we describe the workflow of MSPP by typing a Campylobacter jejuni ssp. doylei isolate collection of seven strains. All seven strains were of human origin and multilocus sequence typing (MLST) demonstrated their genetic diversity. MSPP-typing resulted in seven different MSPP sequence types, sufficiently reflecting their phylogenetic relations.
The C. jejuni ssp. doylei MSPP scheme includes 14 different biomarker ions, mostly ribosomal proteins in the mass range of 2 to 11 kDa. MSPP can in principle, be adapted to other mass spectrometric platforms with an extended mass range. Therefore, this technique has the potential to become a useful tool for strain level microbial typing.
During the last decade, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has advanced to be a highly valued standard method for microbial genus and species identification in clinical microbiology1,2. Species identification is based on the recording of small protein fingerprints of intact cells or cell lysates. The typical mass range for a mass spectrometer used in routine clinical microbiology is 2-20 kDa. Additionally, the resulting spectra can be used to discriminate strains at the below-species and below-subspecies level3. Early pioneering studies have identified specific biomarker ions for a particular subgroup of strains in Campylobacter jejuni4, Clostridium difficile5, Salmonella enterica ssp. enterica serovar Typhi6, Staphylococcus aureus7–9, and Escherichia coli10–12.
The combination of several variable biomarker masses corresponding to allelic isoforms offers the option for deeper subtyping. Previously, we successfully implemented a method to convert these variations in mass profiles into meaningful and reproducible phyloproteomic relations called mass spectrometry based phyloproteomics (MSPP) on a C. jejuni ssp. jejuni isolate collection13. MSPP can be used a mass spectrometric equivalent to DNA sequence based subtyping techniques like multilocus sequence typing (MLST).
Campylobacter species are the leading cause of bacterial gastroenteritis worldwide14,15. As a consequence of Campylobacteriosis post-infectious sequela, namely, Guillain Barré Syndrome, reactive arthritis and inflammatory bowel disease can arise16. The main sources of infection are contaminated livestock meat from chicken, turkey, swine, cattle, sheep and ducks, milk and surface water15,17. Therefore, regular epidemiological surveillance studies in the context of food safety are necessary. MLST is the "gold standard" in molecular typing for Campylobacter species18. Because the Sanger-sequencing based MLST method is labor intensive, time consuming and relatively expensive, MLST typing is restricted to relatively small isolate cohorts. Therefore, there is a need for cheaper and faster subtyping methods. This need could be met by mass spectrometric methods like MSPP.
This paper presents a detailed protocol for MSPP-typing using a collection of Campylobacter jejuni ssp. doylei isolates and comparison of its potential with MLST.
1. Prepare a Safe Workplace by Considering Biosafety Conditions
2. Select Reference and Collection Isolates
3. Prepare a MALDI Target Plate
CAUTION: TFA is a strong acid. Improper use of TFA bears the risk of severe skin burns, eye damage and severe irritation of the upper respiratory tract if inhaled. Therefore, stringent safety measures must be respected and proper personal protective equipment (PPE) including safety goggles, face shields, appropriate gloves, boots, or even a full protective suit is needed, while handling TFA. Possible exposure to TFA must be controlled by handling the substance under adequate ventilation with an effective exhaust ventilation system. In case of insufficient ventilation, a respirator with approved filter must be used. Additionally, TFA is harmful to aquatic life with long lasting effects. Any release of TFA in waste water to the environment must be avoided.
Note: Before spotting the samples onto a MALDI target, clean the target plate thoroughly if the plate was used previously.
4. Preparation of an α-Cyano-4-hydroxy-cinnamic Acid Matrix Solution Containing an Internal Calibrant
5. MALDI-TOF Mass Spectrometry
Note: Culture conditions specific for the organisms of interest must be used. Samples for MALDI-TOF MS can be prepared either by smear preparation or extraction, depending on the organism (see section 8.4.1). While the ethanol-formic acid extraction method provides sufficient inactivation of pathogens, smear preparation has to be performed under sufficient biosafety conditions as required (see section 1). Usually, there is no risk of infection after the application of the matrix, but for specific pathogens specific inactivation protocols are required. Thus, for example MALDI-TOF MS of Nocardia species requires previous lysis of the bacteria in boiling water, following by ethanol precipitation of proteins23. EI Khéchine et al. developed a procedure for inactivation of Mycobacteria, heating the bacterial colonies at 95 °C for 1 hr in screw-cap tubes containing water and 0.5% Tween 2024.
6. Verify the Internal Calibration Procedure
7. Identify Biomarker Ions in the Reference Strain
8. Assess Biomarker Variability in the Population
9. Calculate a MSPP-based Phylogeny and Compare to the Gold Standard
Previously, we successfully established a MSPP scheme for C. jejuni ssp. jejuni13. Here, we aimed to extend the method to the sibling subspecies C. jejuni ssp. doylei. In this specific setting, seven C. jejuni ssp. doylei isolates were acquired from the Belgian collection of microorganisms/Laboratory of Microbiology UGent BCCM/LMG Ghent, Belgium. All seven isolates used for our analyses were of human origin. The genome-sequenced strain ATCC 49349 (LMG 8843), originally isolated from feces of a 2-year-old Australian child with diarrhea served as the reference. From the collection, six additional strains were available: LMG 9143, LMG 9243, and LMG 9255 isolated from feces of children suffering diarrhea and living in Brussels, Belgium; LMG 7790 (ATCC 49350) isolated from a gastric biopsy sample of an individual from Germany; and LMG 8870 (NCTC A613/87) as well as LMG 8871 (NCTC A603/87), both isolated from different blood samples drawn from South African children.
Tutti C. jejuni ssp. doylei isolates were stored at -80 °C as cryobank stocks, freshly thawed for each analysis and cultured by using Columbia agar base supplemented with 5% sheep blood and incubation for 48 hr at 37 °C under microaerophilic conditions (5% O2, 10% CO2, 85% N2).
In contrast to the establishment of the MSPP technique for C. jejuni ssp. jejuni13 it was not possible to base the method on an isoform database derived from a larger sequence collection. Only a single C. jejuni ssp. doylei entry was present in the rMLST database, and this corresponded to the reference strain C. jejuni ssp. doylei ATCC 4934927. Therefore, each biomarker mass shift detected in comparison to strain C. jejuni ssp. doylei ATCC 49349 was a new entry in the isoform database and needed to be reconfirmed by Sanger sequencing.
To analyze the diversity of the obtained C. jejuni ssp. doylei isolates, MLST was performed and a MLST-based UPGMA-dendrogram including the seven examined C. jejuni ssp. doylei isolates and C. jejuni ssp. jejuni strain 81-176 as the outgroup calculated (Figure 1). Each C. jejuni ssp. doylei isolate belonged to a different MLST-sequence type, falling into two subclusters. Strain LMG7790 had the longest phylogenetic distance as compared to the remaining C. jejuni ssp. doylei isolates.
The recordable MALDI-TOF reference mass spectrum of C. jejuni ssp. doylei ATCC 49349 contained 14 singularly charged biomarker ions that could be identified by comparison of the calculated molecular masses with the reference spectrum (Figure 2).
Within the collection, varying isoforms were detected for L32-M, L33, the hypothetical protein encoded by gi|152939117, S20-M, and S15-M (Figure 3). The remaining masses assigned to biomarker ions were invariable in the tested C. jejuni ssp. doylei isolates. The amino acid substitution corresponding to the mass shifts have been identified by PCR-amplification and Sanger sequencing of the particular gene using the primers listed in Table 3. Table 4 lists the amino acid sequences of all biomarker ions included in the MSPP scheme as well as the detected allelic isoforms.
A MSPP-based phyloproteomic UPGMA-tree (Figure 4) was calculated for the same seven C. jejuni ssp. doylei isolates and C. jejuni ssp. jejuni strain 81-176 as the outgroup using the concatenated amino acid sequences of all 14 biomarker ions in order of their molecular weight in the reference strain. Each isolate represented an individual MSPP-sequence type. Although the obtained phyloproteomic relations were not fully identical to the ones obtained by MLST, the C. jejuni ssp. doylei isolates again arranged in two subclusters. The first subcluster was formed by LMG 8843, LMG 8870, and LMG 9243, the second by LMG 9255, LMG 9143, LMG 8871 and LMG 7790. As seen with the MLST-analysis, isolate LMG7790 showed the longest phyloproteomic distance in the MSPP-analysis.
Figure 1: MLST-based phylogenetic UPGMA-tree. Balanced MLST-based UPGMA-dendrogram constructed from seven C. jejuni ssp. doylei isolates and C. jejuni ssp. jejuni strain 81-176. Strain color code: LMG 8843 (black), LMG 8870 (grey), LMG 9243 (blue), LMG 9255 (turquoise), LMG 9143 (green), and LMG 8871 (pink), LMG 7790 (yellow), and 81-176 (white). The MLST-sequence type is given behind the name of each isolate. Every C. jejuni ssp. doylei isolate belongs to a different MLST-sequence type. X-axis indicates the linkage distances. Strain LMG 7790 shows the longest phylogenetic distance compared to the remaining six isolates. Strains LMG 8843, LMG 8870, and LMG 9243 as well as LMG 9255, LMG 9143, and LMG 8871 form two different subclusters within the C. jejuni ssp. doylei cluster. Please click here to view a larger version of this figure.
Figure 2: Tentative assignment of genomic correlates of C. jejuni ssp. doylei ATCC 49349 (LMG 8843) to observed biomarker masses. Based on the calculated masses (average isotopic composition) of predicted ORFs from the whole genome sequence of C. jejuni ssp. doylei strain ATCC 49349, biomarker masses were assigned to the corresponding protein coding sequences. However, within measurement range, the biomarker masses for the ribosomal subunits L31 (MW = 7,315 Da), S17 (MW = 9,549 Da), and S18 (MW = 10,285 Da) as well as their de-methioninated isoforms (inset m/z = 7,000-7,200 Da) could not be unambiguously assigned. Therefore, L31, S17, and S18 were not included in the C. jejuni ssp. doylei MSPP scheme. Please click here to view a larger version of this figure.
Figure 3: Specific biomarker mass peaks of the C. jejuni ssp. doylei MSPP scheme. In each panel the mass spectra of all seven tested C. jejuni ssp. doylei isolates (color code as in Figure 1) corresponding to the particular MSPP types have been overlaid to indicate biomarker mass shifts due to allelic isoforms: (A) L36+H+; (B) L34+H+; (C) L32-M+H+; (D) L33+H+; (E) S14-M+H+; (F) L29+H+, L28-M+H+, and L35-M+H+; (G) L24-M+H+; (H) gi|152939117+H+; (I) L27-M+H+; (J) S20-M+H+; (K) S15-M+H+; (L) S19-M+H+; (M) an intense mass obscuring variable potential biomarker peak of ribosomal protein S18; (N) S20-M+2H+; (O) L15-M+2H+; X-Axes: mass [Da]·charge-1 ratio, scale 200 Da. Y-Axes: intensity [arbitrary units], spectra were individually adjusted to a comparable noise level for better visualization of low-intensity peaks. "-M" after the name of a ribosomal subunit indicates the de-methioninated isoform. Please click here to view a larger version of this figure.
Figure 4: MSPP-based phyloproteomic UPGMA-tree. The depicted phyloproteomic UPGMA-tree includes the same seven C. jejuni ssp. doylei isolates and C. jejuni ssp. jejuni strain 81-176 as in Figure 1. A colored spot (color code as in Figure 1) indicates each strain. For better comparison the isolates are arranged in input order, which was the order obtained from the MLST-based tree. The axis below the dendrogram indicates the linkage distances. Although the obtained phyloproteomic relations are not completely identical to the phylogenetic MLST-based tree, the global picture is comparable. The population splits into two groups: the three isolates LMG 8843, LMG 8870, and LMG 9243 form one cluster, while LMG 9255, LMG 9143, LMG 8871 and LMG 7790 form a second cluster. Within this cluster LMG 7790 shows the longest phyloproteomic distance compared to the subcluster formed by LMG 9255, LMG 9143, and LMG 8871. Within this subcluster LMG 9143 switches position in relation to LMG 9255. However, even using the MSPP-method each isolate represents an individual MSSP-sequence type. Please click here to view a larger version of this figure.
Table 1: Calculated masses of all annotated ORFs of the C. jejuni ssp. doylei ATCC 49349 strain. List of all calculated monoisotopic molecular weights of each protein encoded in the C. jejuni ssp. doylei ATCC 49349 genome. The ExPASy Bioinformatics Resource Portal was used for calculation of the particular molecular masses. Column B lists the methioninated isoforms whereas column C lists the de-methioninated isoforms. Biomarker masses included in the C. jejuni ssp. doylei MSPP scheme are highlighted in red. Note: The biomarker protein L36 is not annotated in the genome sequence of C. jejuni ssp. doylei ATCC49349. Please click here to download this file.
Table 2: Mass changes induced by amino acid changes due to single SNPs. All potential single nucleotide polymorphisms were checked for the resulting amino acid exchange using the standard genetic code. All silent mutations were discarded, and nonsynonymous mutations compiled together with the resulting mass change. Please click here to download this file.
Table 3: Oligonucleotide primers used for sequencing of the C. jejuni ssp. doylei genes included in the MSPP-scheme Please click here to download this file.
Table 4: Overview of all isoforms included in the C. jejuni ssp. doylei MSPP-scheme. This table lists all biomarker ions included in the C. jejuni ssp. doylei MSPP scheme. The amino acid sequence of the reference strain ATCC 49349 is given completely. For further detectable isoforms, only specific amino acid substitutions are listed. The amino acid numbering always includes the start-methionine; if mass spectrometry indicates its absence, it is written in brackets (M). For each isoform molecular mass, mass difference to reference strain ATCC 49349 isoforms and frequency within the isoform dataset is indicated. Please click here to download this file.
The most critical step in the establishment of an MSPP scheme is the unequivocal genetic determination of biomarker ion identities. If it is not possible to identify a biomarker undoubtedly, then it should be excluded from the scheme13.
The C. jejuni ssp. doylei scheme includes 14 different biomarker ions. These are 5 less compared to the C. jejuni ssp. jejuni MSPP scheme13.The most significant difference between the detectable C. jejuni ssp. jejuni and C. jejuni ssp. doylei biomarkers was the posttranslational removal of the amino-terminal methionine in case of L31(-M) and L35(-M)13.
As shown in the tested C. jejuni ssp. doylei isolate collection, MSPP was able to discriminate all seven different MLST sequence types. This means that every tested isolate belonged to a specific MSPP sequence type. Additionally, MSPP allows subspecies differentiation between C. jejuni ssp. jejuni and C. jejuni ssp. doylei.
In general, the potential to discriminate isolates at the below-strain level by MSPP is lower compared to DNA sequence-based methods like MLST. Assuming a well-established isoform database, the subtyping of bacterial isolates is much faster and much cheaper compared to DNA sequencing methods4,13.
The biggest advantage of MSPP in comparison to hierarchical clustering methods of ICMS-spectra such as using the principal component analysis (PCA)4,10,28 or UPGMA analysis of the mathematical matrix that results of the comparison of binary peak matching profiles29,30 is its high intrinsic reproducibility. While different cultural conditions such as different culture media, different agar charges, different incubation temperature, and different incubation periods significantly affect the phyloproteomic relations calculated by PCA, they do not affect the MSPP deduced relations 6,13. The main reason for this high reproducibility is the independence of the peak intensity and mass spectrum quality in general13. If a MSPP relevant peak in the mass spectrum of an isolate cannot be identified indubitably, it is necessary to record the mass spectrum of the individual isolate again.
The MSPP method can be adapted, in principle, to every microbial species. Especially, the subtyping of clinical relevant microbial species like Clostridium difficile, Escherichia coli, Staphylococcus aureus, and Salmonella enterica ssp. enterica are potential future applications. If the expression of virulence factors or resistance genes correlates with the phyloproteomic relationship, MSPP can become an innovative tool in routine clinical diagnostics. The number of detectable peaks and thereby the number of biomarker ions included in the MSPP scheme could potentially be increased by using specific extraction and/or lysis protocols, especially in the case of fungi31.
The recently used MALDI-TOF techniques can mainly detect only the highly abundant ribosomal proteins. These highly abundant proteins interfere with nearly all other smaller proteins such as virulence factors in the mass spectrum. Smaller proteins could be detected by linking ESI-TOF-MS (Electrospray ionization time of flight mass spectrometry) and HPLC (high performance liquid chromatography)32. However, currently this method is too labor and cost intensive to characterize larger isolate cohorts.
The authors have nothing to disclose.
We are grateful to Hannah Kleinschmidt for excellent technical support. This paper was funded by the Open Access support program of the Deutsche Forschungsgemeinschaft and the publication fund of the Georg August Universität Göttingen.
acetonitrile | Sigma-Aldrich, Taufkirchen, Germany | 34967 | |
Autoflex III TOF/TOF 200 system | Bruker Daltonics, Bremen, Germany | GT02554 G201 | Mass spectrometer |
bacterial test standard BTS | Bruker Daltonics, Bremen, Germany | 604537 | |
BioTools 3.2 SR1 | Bruker Daltonics, Bremen, Germany | 263564 | Software Package |
Bruker IVD Bakterial Test Standard | Bruker Daltonics, Bremen, Germany | 8290190 | 5 tubes |
Campylobacter jejuni subsp. doylei isolate | Belgium coordinated collection of microorganisms/Laboratory of Microbiology UGent BCCM/LMG Ghent, Belgium | LMG8843 | ATCC 49349;IMVS 1141;NCTC 11951;strain 093 |
Campylobacter jejuni subsp. doylei isolate | Belgium coordinated collection of microorganisms/Laboratory of Microbiology UGent BCCM/LMG Ghent, Belgium | LMG9143 | Goossens Z90 |
Campylobacter jejuni subsp. doylei isolate | Belgium coordinated collection of microorganisms/Laboratory of Microbiology UGent BCCM/LMG Ghent, Belgium | LMG7790 | ATCC 49350;CCUG 18265;Kasper 71;LMG 8219;NCTC 11847 |
Campylobacter jejuni subsp. doylei isolate | Belgium coordinated collection of microorganisms/Laboratory of Microbiology UGent BCCM/LMG Ghent, Belgium | LMG9243 | Goossens N130 |
Campylobacter jejuni subsp. doylei isolate | Belgium coordinated collection of microorganisms/Laboratory of Microbiology UGent BCCM/LMG Ghent, Belgium | LMG8871 | NCTC A603/87 |
Campylobacter jejuni subsp. doylei isolate | Belgium coordinated collection of microorganisms/Laboratory of Microbiology UGent BCCM/LMG Ghent, Belgium | LMG9255 | Goossens B538 |
Campylobacter jejuni subsp. doylei isolate | Belgium coordinated collection of microorganisms/Laboratory of Microbiology UGent BCCM/LMG Ghent, Belgium | LMG8870 | NCTC A613/87 |
Columbia agar base | Merck, Darmstadt, Germany | 1.10455 .0500 | 500 g |
Compass for FlexSeries 1.2 SR1 | Bruker Daltonics, Bremen, Germany | 251419 | Software Package |
defibrinated sheep blood | Oxoid Deutschland GmbH, Wesel, Germany | SR0051 | |
ethanol | Sigma-Aldrich, Taufkirchen, Germany | 02854 Fluka | |
formic acid | Sigma-Aldrich, Taufkirchen, Germany | F0507 | |
HCCA matrix | Bruker Daltonics, Bremen, Germany | 604531 | |
Kimwipes paper tissue | Kimtech Science via Sigma-Aldrich, Taufkirchen, Germany | Z188956 | |
MALDI Biotyper 2.0 | Bruker Daltonics, Bremen, Germany | 259935 | Software Package |
Mast Cryobank vials | Mast Diagnostica, Reinfeld, Germany | CRYO/B | |
MSP 96 polished steel target | Bruker Daltonics, Bremen, Germany | 224989 | |
QIAamp DNA Mini Kit | Qiagen, Hilden, Germany | 51304 | |
recombinant human insulin | Sigma-Aldrich, Taufkirchen, Germany | I2643 | |
trifluoroacetic acid | Sigma-Aldrich, Taufkirchen, Germany | T6508 | |
water, molecular biology-grade | Sigma-Aldrich, Taufkirchen, Germany | W4502 |