Proteomic Analysis and Virulence Assessment of Granulicatella adiacens Secretome

Maribasappa Karched 1, Radhika G Bhardwaj 1, Ali Tiss 2, Sirkka Asikainen 1

Affiliations

doi: 10.3389/fcimb.2019.00104

Abstract

Despite reports on the occurrence of Granulicatella adiacens in infective endocarditis, few mechanistic studies on its virulence characteristics or pathogenicity are available. Proteins secreted by this species may act as determinants of host-microbe interaction and play a role in virulence. Our aim in this study was to investigate and functionally characterize the secretome of G. adiacens. Proteins in the secretome preparation were digested by trypsin and applied to nanoLC-ESI-MS/MS. By using a combined mass spectrometry and bioinformatics approach, we identified 101 proteins. Bioinformatics tools predicting subcellular localization revealed that 18 of the secreted proteins possessed signal sequence. More than 20% of the secretome proteins were putative virulence proteins including serine protease, superoxide dismutase, aminopeptidase, molecular chaperone DnaK, and thioredoxin. Ribosomal proteins, molecular chaperones, and glycolytic enzymes, together known as "moonlighting proteins," comprised fifth of the secretome proteins. By Gene Ontology analysis, more than 60 proteins of the secretome were grouped in biological processes or molecular functions. KEGG pathway analysis disclosed that the secretome consisted of enzymes involved in biosynthesis of antibiotics. Cytokine profiling revealed that secreted proteins stimulated key cytokines, such as IL-1β, MCP-1, TNF-α, and RANTES from human PBMCs. In summary, the results from the current investigation of the G. adiacens secretome provide a basis for understanding possible pathogenic mechanisms of G. adiacens.

Keywords: Granulicatella; infective endocarditis; oral; periodontitis; secretome; virulence.

Figures

Figure 1 Analysis of the secretome of G. adiacens. (A) SDS-PAGE gel showing protein bands from secretome preparation. (B) Protein sequences from LC-MS analysis of the secretome were analyzed by an in silico 2DE tool. (C) Western blot analysis showing the absence of the cytoplasmic marker protein FtsZ in the secretome preparation.

Figure 1 Analysis of the secretome of G. adiacens. (A) SDS-PAGE gel showing protein bands from secretome preparation. (B) Protein sequences from LC-MS analysis of the secretome were analyzed by an in silico 2DE tool. (C) Western blot analysis showing the absence of the cytoplasmic marker protein FtsZ in the secretome preparation.

Figure 2 Gene Ontology analysis of G. adiacens secreted proteins. Gene ontology annotation was achieved using Blast2GO and an online software “WEGO.” Protein sequences were grouped into 3 categories based on their properties and functions.

Figure 2 Gene Ontology analysis of G. adiacens secreted proteins. Gene ontology annotation was achieved using Blast2GO and an online software “WEGO.” Protein sequences were grouped into 3 categories based on their properties and functions.

Figure 3 KEGG pathway analysis of the G. adiacens secretome. All protein sequences from the secretome were analyzed by KEGG pathway tool, which was included as a “plugin” tool within Blast2GO software.

Figure 3 KEGG pathway analysis of the G. adiacens secretome. All protein sequences from the secretome were analyzed by KEGG pathway tool, which was included as a “plugin” tool within Blast2GO software.

Figure 4 Functional protein association networks of G. adiacens secretome. The online tool STRING was used for grouping the secreted proteins on the basis of functional networks. Minimum interaction scores were set at a strong confidence level of 0.7. The three major network groups formed are shown in dotted circles. Seven different colored link a number of nodes and represent seven types of evidence used in predicting associations. A red line indicates the presence of fusion evidence; a green line represents neighborhood evidence, a blue line represents co-occurrence evidence; a purple line represents experimental evidence; a yellow line represents textmining evidence; a light blue line represents database evidence and a black line represents coexpression evidence.

Figure 4 Functional protein association networks of G. adiacens secretome. The online tool STRING was used for grouping the secreted proteins on the basis of functional networks. Minimum interaction scores were set at a strong confidence level of 0.7. The three major network groups formed are shown in dotted circles. Seven different colored link a number of nodes and represent seven types of evidence used in predicting associations. A red line indicates the presence of fusion evidence; a green line represents neighborhood evidence, a blue line represents co-occurrence evidence; a purple line represents experimental evidence; a yellow line represents textmining evidence; a light blue line represents database evidence and a black line represents coexpression evidence.

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