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. 2016 Jul 21:7:1103.
doi: 10.3389/fmicb.2016.01103. eCollection 2016.

The Exposed Proteomes of Brachyspira hyodysenteriae and B. pilosicoli

Vanessa Casas  1 , Santiago Vadillo  2 , Carlos San Juan  2 , Montserrat Carrascal  1 , Joaquin Abian  1
Affiliations

The Exposed Proteomes of Brachyspira hyodysenteriae and B. pilosicoli

Vanessa Casas et al. Front Microbiol. .

Abstract

Brachyspira hyodysenteriae and Brachyspira pilosicoli are well-known intestinal pathogens in pigs. B. hyodysenteriae is the causative agent of swine dysentery, a disease with an important impact on pig production while B. pilosicoli is responsible of a milder diarrheal disease in these animals, porcine intestinal spirochetosis. Recent sequencing projects have provided information for the genome of these species facilitating the search of vaccine candidates using reverse vaccinology approaches. However, practically no experimental evidence exists of the actual gene products being expressed and of those proteins exposed on the cell surface or released to the cell media. Using a cell-shaving strategy and a shotgun proteomic approach we carried out a large-scale characterization of the exposed proteins on the bacterial surface in these species as well as of peptides and proteins in the extracellular medium. The study included three strains of B. hyodysenteriae and two strains of B. pilosicoli and involved 148 LC-MS/MS runs on a high resolution Orbitrap instrument. Overall, we provided evidence for more than 29,000 different peptides pointing to 1625 and 1338 different proteins in B. hyodysenteriae and B. pilosicoli, respectively. Many of the most abundant proteins detected corresponded to described virulence factors and vaccine candidates. The level of expression of these proteins, however, was different among species and strains, stressing the value of determining actual gene product levels as a complement of genomic-based approaches for vaccine design.

Keywords: Brachyspira; membrane shaving; shotgun proteomics; surface proteins; virulence factors.

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Figures

Figure 1
Figure 1
Increase in the total number of identified peptides and proteins with the number of combined analyses. Values were obtained by searching each independent file with Proteome Discoverer 1.4.
Figure 2
Figure 2
Distribution of sequence matches in the different Brachyspira annotated proteomes for the collection of peptides identified in the B. hyodysenteriae and B. pilosicoli samples. PILO, B. pilosicoli; HYO, B. hyodysenteriae; OTHERS, other Brachyspira species.
Figure 3
Figure 3
Distribution of identified proteins (unique accessions) among compartments. Only proteins with more than 15 validated PSM are shown in the figure. Proteins considered exclusive must have more than 80% of total validated spectra in one compartment, and any other compartment, considering each species separately, must contain <5% of the total validated spectra.
Figure 4
Figure 4
Cellular location of the compartment-specific proteins obtained using STRAP. GO annotation, Cell component.
Figure 5
Figure 5
Molecular mass distribution for the proteins annotated in the exopeptidome compared with those specific to the surfaceome.
Figure 6
Figure 6
Cytoscape biological network 47 of B. pilosicoli surface-exclusive proteins. GOA Cellular Component Annotation (node color): green, membrane, and cell periphery; red, cytoplasm, and intracellular; yellow, unknown. PSORTb prediction (node shape): triangle, cytoplasmic; trapezoid, cytoplasmic membrane; octagon, outer membrane; hexagon, periplasmic; arrow, extracellular; circle, unknown. STRING Database: B. pilosicoli, Interaction Score ≥0.8.
Figure 7
Figure 7
Motifs in the N- and C-terminal regions of peptides in the exopeptidome fraction. Size and top to bottom position of the amino acid letters are related with the frequency at which the amino acid is observed on that position. Frequencies are corrected for the amino acid frequency in the full Brachyspira database. An asterisk denotes the absence of an amino acid (off-sequence positions in protein terminal peptides).
Figure 8
Figure 8
Comparison of the Brachyspira strains. (Left) Number of proteins identified in the different Brachyspira strains analyzed. Only proteins with more than 15 PeptideShaker-validated spectra were considered. (Right) Hierarchical clustering of the peptide sequences identified in the different fractions from each Brachyspira strain. Only sequences showing a standard deviation >10 for the PSM observed in the different strains were considered for clustering. The cluster images are highly compressed because they include 240 and 853 peptides for the peptidome and surfaceome, respectively. Full-size cluster figures, including peptide sequence clusters, are shown in Figure S5.
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