A combined approach for comparative exoproteome analysis of Corynebacterium pseudotuberculosis

Abstract

BackgroundBacterial 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.ResultsAn 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.ConclusionsComparative 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.

Identification of a Novel Zinc Metalloprotease through a Global Analysis of Clostridium difficile Extracellular Proteins

“…The presence of cytoplasmic proteins, which is a common indicator of lysis, is found in several proteomic studies examining the exoproteomes and complicates the interpretation of the cellular localization of proteins [17,18]. Our work suggests that the proteins we detect in culture supernatants are indeed extracellular, as the preparations we used almost completely lacked proteins of predicted cytoplasmic origin.…”

“…Proteomics have been previously employed effectively to identify new pathogenic determinants in various pathogens and to understand differences between various clinical strains [17,18]. Although previous studies have tried to characterize extracellular protein profiles of C. difficile [19-21], the precise cellular localization of the proteins identified was unclear due to the presence of cytoplasmic proteins.…”

Humoral and cellular immune responses in mice against secreted and somatic antigens from a Corynebacterium pseudotuberculosis attenuated strain: Immune response against a C. pseudotuberculosis strain

“…Moreover, the physiology, pathogenicity and virulence mechanisms of C. pseudotuberculosis strains have been elucidated using genomics [8, 10], transcriptomics and proteomics methodologies [11, 12]. …”

Global relative and absolute quantitation in microbial proteomics

“…Implementations of this concept, either from academics [43,44] or as part of a complete proteomics technology [34,42,45] have greatly succeeded in label-free absolute proteomics. In microbial proteomics this was used, for example, for the analysis of different mutants of S. aureus SCV [46] and the probing of the exoproteome of Corynebacterium pseudotuberculosis [47].…”