Figure 1. Reverse vaccinology approach. Proteome data (blue) and in silico protein characterization (yellow) were combined to predict potential vaccine and drug targets (green).

Figure 1. Reverse vaccinology approach. Proteome data (blue) and in silico protein characterization (yellow) were combined to predict potential vaccine and drug targets (green).

Figure 2. A maximum-likelihood tree from the 16S rRNA gene sequence alignment. The scale bar represents nucleotide substitution per site. Strain 12CS0282 is highlighted in red.

Figure 2. A maximum-likelihood tree from the 16S rRNA gene sequence alignment. The scale bar represents nucleotide substitution per site. Strain 12CS0282 is highlighted in red.

Figure 3. Maximum-likelihood tree from nucleotide sequence alignment of the core genome calculated from 131 C. pseudotuberculosis strains. Clades with the average branch distance to their leaves below 0.0001 were collapsed. Nine out of 11 strains in the clade marked by * possess the tox gene. The scale bar represents nucleotide substitution per site (see Supplementary Figure S1 for the all strain details). Figure 3. Maximum-likelihood tree from nucleotide sequence alignment of the core genome calculated from 131 C. pseudotuberculosis strains. Clades with the average branch distance to their leaves below 0.0001 were collapsed. Nine out of 11 strains in the clade marked by * possess the tox gene. The scale bar represents nucleotide substitution per site (see Supplementary Figure S1 for the all strain details).

Figure 4. C. pseudotuberculosis 12CS0282 proteins identified in this study. Distribution and overlap of proteins in the different fractions analyzed. Data sets were curated using localization prediction approaches and cytoplasmic proteins were eliminated from surface and secreted proteome fractions.

Figure 4. C. pseudotuberculosis 12CS0282 proteins identified in this study. Distribution and overlap of proteins in the different fractions analyzed. Data sets were curated using localization prediction approaches and cytoplasmic proteins were eliminated from surface and secreted proteome fractions.

Figure 5. Metabolic pathways of identified proteins in the whole cell proteome (left), surface fraction (middle) and extracellular proteome (right). Each area displays one protein and equal to the relative abundance. Pathways are separated in metabolism (green), information storage and processing (purple), cellular processes and signaling (blue), environmental information processing (orange), genetic information processing (yellow), poorly characterized (grey), uncharacterized (black), and in pathogenicity (red).

Figure 5. Metabolic pathways of identified proteins in the whole cell proteome (left), surface fraction (middle) and extracellular proteome (right). Each area displays one protein and equal to the relative abundance. Pathways are separated in metabolism (green), information storage and processing (purple), cellular processes and signaling (blue), environmental information processing (orange), genetic information processing (yellow), poorly characterized (grey), uncharacterized (black), and in pathogenicity (red).

Figure 6. Protein-protein interaction networks of putative vaccine and drug targets. (a) The two probable vaccine and drug targets SecD (cp12CS0282_01259) and YidC1 (cp12CS0282_00093), have direct interactions and are interacting with components of bacterial protein secretion (Sec proteins) and cell division (Fts proteins). (b,c) Pdp4 (cp12CS0282_00932) and FtsI (cp12CS0282_01491) show interactions with DacA, Fts proteins and Mur proteins, which are involved in cell wall synthesis and cell division. Putative vaccine and drug targets identified here are indicated by arrows.

Figure 6. Protein-protein interaction networks of putative vaccine and drug targets. (a) The two probable vaccine and drug targets SecD (cp12CS0282_01259) and YidC1 (cp12CS0282_00093), have direct interactions and are interacting with components of bacterial protein secretion (Sec proteins) and cell division (Fts proteins). (b,c) Pdp4 (cp12CS0282_00932) and FtsI (cp12CS0282_01491) show interactions with DacA, Fts proteins and Mur proteins, which are involved in cell wall synthesis and cell division. Putative vaccine and drug targets identified here are indicated by arrows.

Figure 7. Quantitative analysis of C. pseudotuberculosis–macrophage interaction. THP-1 cells were infected with C. ulcerans 809 (2), C. silvaticum W25 (3), C. pseudotuberculosis 12CS0282 (4) and C. pseudotuberculosis FRC41 (5) at MOI 1 for 30 min. Infection with the non-pathogenic C. glutamicum ATCC13032 (1) served as a negative control. In order to kill extracellular bacteria, cells were incubated with medium containing gentamicin and after 2 (blue), 4 (orange) and 20 h (grey), cells were harvested, lysed and lysates were plated on blood agar plates to recover intracellular CFU. (a) Intracellular CFU in percent referred to the inoculum. (b) Intracellular survival in percent referred to the bacteria that were taken up after 2 h.

Figure 7. Quantitative analysis of C. pseudotuberculosis–macrophage interaction. THP-1 cells were infected with C. ulcerans 809 (2), C. silvaticum W25 (3), C. pseudotuberculosis 12CS0282 (4) and C. pseudotuberculosis FRC41 (5) at MOI 1 for 30 min. Infection with the non-pathogenic C. glutamicum ATCC13032 (1) served as a negative control. In order to kill extracellular bacteria, cells were incubated with medium containing gentamicin and after 2 (blue), 4 (orange) and 20 h (grey), cells were harvested, lysed and lysates were plated on blood agar plates to recover intracellular CFU. (a) Intracellular CFU in percent referred to the inoculum. (b) Intracellular survival in percent referred to the bacteria that were taken up after 2 h.

Table 1. Digital DNA-DNA hybridization values between strain 12CS0282 and representative strains of closely related species.

Table 1. Digital DNA-DNA hybridization values between strain 12CS0282 and representative strains of closely related species.

Reference GenomeDDHDistanceG + C DifferenceC. belfanti DSM 105776 T20.60.21291.44C. diphtheriae DSM44123 T20.80.21161.35C. pseudotuberculosis ATCC 19410 T99.90.00030C. pseudotuberculosis DSM 20689 T99.90.00020C. silvaticum KL0182 T28.50.15092.26C. silvaticum W2528.50.15092.25C. ulcerans FRC1127.60.15631.17C. ulcerans NCTC 7910 T27.50.15681.13

Table 2. Presence of known virulence genes in strain 12CS0282.

Table 2. Presence of known virulence genes in strain 12CS0282.

Virulence GeneFunctionIdentifier in Strain 12CS0282cpfrc_00029 (pld)phospholipase D (sphingomyelin-degrading enzyme)cp12CS0282_00124cpfrc_00128 (nor)nitric oxide reductasecp12CS0282_00230cpfrc_00386 (nanH)neuraminidase H (sialidase)cp12CS0282_00502cpfrc_00397secreted subtilisin-like serine proteasecp12CS0282_00513cpfrc_00491 (dtsR2)acyl-CoA carboxylase b-subunit involved in mycolic acid synthesiscp12CS0282_00606cpfrc_00492 (dtsR1)acetyl-CoA carboxylase b-subunit involved in fatty acid synthesiscp12CS0282_00607cpfrc_00536secreted SGNH-hydrolasecp12CS0282_00649cpfrc_00562secreted trypsin-like serine proteasecp12CS0282_00678cpfrc_00565 (nrpS1)nonribosomal peptide synthetase 1cp12CS0282_00682cpfrc_00594 (rpfA)resuscitation-promoting factor A (muralytic enzyme)cp12CS0282_02168cpfrc_00679 (rpfB)resuscitation-promoting factor B (muralytic enzyme)cp12CS0282_02083cpfrc_01079 (rpfI)resuscitation-promoting factor interacting protein (D,L-endopeptidase)cp12CS0282_01163cpfrc_01634secreted subtilisin-like serine proteasecp12CS0282_01728cpfrc_01801nonribosomal peptide synthetase 2cp12CS0282_00926cpfrc_01895 (cpp)corynebacterial protease CP40 (serine protease)cp12CS0282_00833cpfrc_01953 (accD3)acyl-CoA carboxylase b-subunit involved in mycolic acid synthesiscp12CS0282_00773

Table 3. Metabolic pathway analysis.

Table 3. Metabolic pathway analysis.

PathwayTheoretical ProteomeIdentified Proteinsn = 3Cellular processes and signaling235 [11.1%]141 [9.8%]97 [9.5%]Environmental information processing67 [3.2%]37 [2.6%]25 [2.4%]Genetic information processing52 [2.5%]38 [2.6%]24 [2.3%]Information storage and processing224 [10.6%]142 [9.8%]100 [9.8%]Metabolism563 [26.6%]382 [26.5%]267 [26.1%]Pathogenicity53 [3.1%]45 [3.1%]40 [3.9%]Poorly characterized253 [11.9%]187 [13.0%]142 [13.9%]Uncharacterized671 [31.7%]472 [32.7%]328 [32.1%]Total211814441023 Table 4. Validation and distribution of C. pseudotuberculosis 12CS0282 virulence factors. Proteins encoded by virulence genes in Table 1 were analyzed in respect to presence, localization (E, extracellular, S, surface; W, whole cell fraction) and relative abundance (% of protein content in fraction). Table 4. Validation and distribution of C. pseudotuberculosis 12CS0282 virulence factors. Proteins encoded by virulence genes in Table 1 were analyzed in respect to presence, localization (E, extracellular, S, surface; W, whole cell fraction) and relative abundance (% of protein content in fraction). DesignationFunctionLocalization and Relative Abundancecp12CS0282_00124 (pld)phospholipase DE (0.5%), W (0.4%)cp12CS0282_00230 (nor)nitric oxide reductase-cp12CS0282_00502 (nanH)neuraminidase HS (0.6%)cp12CS0282_00513secreted subtilisin-like serine protease-cp12CS0282_00606 (dtsR2)acyl-CoA carboxylase b-subunit involved in mycolic acid synthesisW (0.5%)cp12CS0282_00607 (dtsR1)acetyl-CoA carboxylase b-subunit involved in fatty acid synthesisW (0.4%)cp12CS0282_00649secreted SGNH-hydrolaseS (6.3%), W (0.2%)cp12CS0282_00678secreted trypsin-like serine proteaseE, S, Wcp12CS0282_00682 (nrpS1)nonribosomal peptide synthetase 1-cp12CS0282_02168 (rpfA)resuscitation-promoting factor A (muralytic enzyme)E (4.2%)cp12CS0282_02083 (rpfB)resuscitation-promoting factor B (muralytic enzyme)S (0.3%)cp12CS0282_01163 (rpfI)resuscitation-promoting factor interacting protein (D,L-endopeptidase)-cp12CS0282_01728secreted subtilisin-like serine proteaseE (0.04%), S (0.3%), W (0.2%)cp12CS0282_0092nonribosomal peptide synthetase 2-cp12CS0282_00833 (cpp)corynebacterial protease CP40S (0.2%)cp12CS0282_00773 (accD3)acyl-CoA carboxylase b-subunit involved in mycolic acid synthesisW (0.6%)

Table 5. Predicted vaccine and drug targets. The table shows the protein ID of the corresponding protein from C. pseudotuberculosis strain 12CS0282, the protein name, molecular weight (MW) and predicted stability (+: stable; −: unstable).

Table 5. Predicted vaccine and drug targets. The table shows the protein ID of the corresponding protein from C. pseudotuberculosis strain 12CS0282, the protein name, molecular weight (MW) and predicted stability (+: stable; −: unstable).

Protein IDProtein NameMW (Da)Stabilitycp12CS0282_00093membrane protein insertase36,402.13−cp12CS0282_00370signal-transduction histidine kinase44,858.97+cp12CS0282_00394cytochrome C biogenesis protein60,045.54+cp12CS0282_00666hypothetical protein109,088.9+cp12CS0282_00740putative cell wall biosynthesis protein46,819.89−cp12CS0282_00766diacylglycerol acyltransferase/mycolyltransferase36,582.47+cp12CS0282_00770hypothetical protein32,832.33+cp12CS0282_00932hypothetical protein41,309.86+cp12CS0282_00991adaptive-response sensory-kinase54,403.91−cp12CS0282_01097NADH dehydrogenase-like protein49,062.65+cp12CS0282_01233endolytic murein transglycosylase41,096.36+cp12CS0282_01244FMN reductase19,296.33−cp12CS0282_01259protein translocase subunit65,933.18+cp12CS0282_01491penicillin-binding protein73,053.21+cp12CS0282_01513cytochrome Bc1 complex cytochrome B subunit110,964+cp12CS0282_01515cytochrome Bc1 complex cytochrome C subunit31,394.71+cp12CS0282_01518cytochrome C oxidase subunit 240,192.72+cp12CS0282_01584bifunctional protein41,939.33+cp12CS0282_01590hypothetical protein34,917.49−cp12CS0282_01839hypothetical protein34,482.87−cp12CS0282_02102signal transduction histidine-protein kinase/phosphatase56,585.5−cp12CS0282_02120hypothetical protein24,721.76+