A combined approach for comparative exoproteome analysis of Corynebacterium pseudotuberculosis

Abstract

Background: Bacterial exported proteins represent key components of the host-pathogen interplay. Hence, we sought to implement a combined approach for characterizing the entire exoproteome of the pathogenic bacterium Corynebacterium pseudotuberculosis, the etiological agent of caseous lymphadenitis (CLA) in sheep and goats.

Results: An optimized protocol of three-phase partitioning (TPP) was used to obtain the C. pseudotuberculosis exoproteins, and a newly introduced method of data-independent MS acquisition (LC-MSE) was employed for protein identification and label-free quantification. Additionally, the recently developed tool SurfG+ was used for in silico prediction of sub-cellular localization of the identified proteins. In total, 93 different extracellular proteins of C. pseudotuberculosis were identified with high confidence by this strategy; 44 proteins were commonly identified in two different strains, isolated from distinct hosts, then composing a core C. pseudotuberculosis exoproteome. Analysis with the SurfG+ tool showed that more than 75% (70/93) of the identified proteins could be predicted as containing signals for active exportation. Moreover, evidence could be found for probable non-classical export of most of the remaining proteins.

Conclusions: Comparative analyses of the exoproteomes of two C. pseudotuberculosis strains, in addition to comparison with other experimentally determined corynebacterial exoproteomes, were helpful to gain novel insights into the contribution of the exported proteins in the virulence of this bacterium. The results presented here compose the most comprehensive coverage of the exoproteome of a corynebacterial species so far.

Figure 1

Analysis of the extracellular proteins of two different C. pseudotuberculosis strains allowed for identification of the core and variant exoproteomes. TPP-extracted extracellular proteins of the strains 1002 and C231 of C. pseudotuberculosis were submitted to LC-MS^E analysis. The Venn-diagram shows the numbers of commonly identified and variant exoproteins between the strains. The number of replicates in which a given protein was observed, the average peptides identified per protein, and the average sequence coverage of the proteins in each exoproteome studied, are shown as frequency distributions for comparison purposes.

Figure 2

Most of the identified C. pseudotuberculosis exoproteins were predicted by the SurfG+ program as having an extracytoplasmic localization. The proteins identified in the exoproteomes of each C. pseudotuberculosis strain were analyzed by SurfG+ and attributed a probable final sub-cellular localization. Proteins classified as having a cytoplasmic localization were further analyzed with the SecretomeP tool for prediction of non-classical (leaderless) secretion. Besides, literature evidence for exportation by non-classical pathways was also used to re-classify the cytoplasmic proteins (see text for details). SE = secreted; PSE = potentially surface exposed; C = cytoplasmic; M = membrane; NCS = non-classically secreted.

Figure 3

Differential expression of the proteins composing the core C. pseudotuberculosis exoproteome, evaluated by label-free relative quantification using LC-MS^E. Results are shown as natural log scale of the relative quantifications (1002:C231) for each protein. Only proteins that were given a variation score higher than 250 by PLGS quantification algorithm are presented. Proteins regulated more than 2-fold in each strain are indicated. Protein identification numbers correspond to additional files 2 and 7: Tables S1 and S4.

Figure 4

Comparative analysis of corynebacterial exoproteomes. Numbers of extracellular proteins identified in previous corynebacterial exoproteome analyses [17,37,69,70] in comparison to those identified in this study with the two strains of C. pseudotuberculosis.

Figure 5

Distribution of orthologous proteins of the C. pseudotuberculosis experimental exoproteins throughout other experimentally confirmed corynebacterial exoproteomes. Pathogenic species: C. diphtheriae C7s(-)^tox- and C. jeikeium K411 [17,69]; non-pathogenic species: C. glutamicum ATCC13032 and C. efficiens YS-314 [37,70]. Pie charts show Gene Ontology (GO) functional annotations for the 93 different C. pseudotuberculosis exoproteins identified (24 commonly identified in pathogenic and non-pathogenic corynebacteria; 19 commonly identified only in pathogenic corynebacteria; and 50 only identified in C. pseudotuberculosis). Annotations were obtained following analyses with the Blast2GO tool [84], used through the web application available at http://www.blast2go.org/start_blast2go.