Surface and Extracellular Proteome of the Emerging Pathogen Corynebacterium ulcerans

Abstract

Corynebacterium ulcerans is an emerging pathogen, which is increasingly recognized as an etiological agent of diphtheria, but can also evoke ulcers of the skin and systemic infections in humans. Besides man, the bacteria can colonize a wide variety of different animals, including cattle and pet animals, which might serve as a reservoir for human infections. In this study, surface-located proteins and the exoproteome of two Corynebacterium ulcerans strains were analyzed, since these may have key roles in the interaction of the pathogen with host cells. Strain 809 was isolated from a fatal case of human respiratory tract infection, while strain BR-AD22 was isolated from a nasal swap of an asymptomatic dog. While a very similar pattern of virulence factors was observed in the culture supernatant and surface protein fractions of the two strains, proteome analyses revealed a higher stability of 809 cells compared to strain BR-AD22. During exponential growth, 17% of encoded proteins of strain 809 were detectable in the medium, while 38% of the predicted proteins encoded by the BR-AD22 chromosome were found. Furthermore, the data indicate differential expression of phospholipase D and a cell wall-associated hydrolase, since these were only detected in strain BR-AD22.

Cellular and Extracellular Proteome of the Animal Pathogen Corynebacterium silvaticum, a Close Relative of Zoonotic Corynebacterium ulcerans and Corynebacterium pseudotuberculosis

“…Extracellular proteins were prepared as described [ 31 ]. Cells were removed by centrifugation (10 min, 4 °C, 4000× g ) and the supernatant was subsequently filtered using a 0.2 µm pore size filter (Minisart, Sartorius, Göttingen, Germany).…”

“…For isolation of cell surface proteins by tryptic shaving [ 31 , 45 ] independent batches of cells were harvested and treated with 1.5 µg sequencing grade trypsin (Promega, Madison, WI, USA) for 1.5 h at 37 °C.…”

“…Since we are interested in the analysis of pathogenicity determinants of corynebacteria [ 24 , 25 , 26 , 27 ] and different aspects of bacterial proteomics [ 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 ], we started a characterization of the cellular and extracellular proteome of C. silvaticum isolate W25, which is presented here. In total, 1305 proteins were identified out of 2013 proteins encoded by the W25 genome sequence including a number of putative virulence factors such as phospholipase D and sialidase.…”

The Genus Corynebacterium in the Genomic Era

“…In the case of pathogenic corynebacterial species, the repertoire of secreted and surfaceexposed proteins, the exoproteome, have been documented because of their potential to act as antigenicity and virulence factors, since these molecules are promptly exposed to the host cells, making they are suitable for the use of vaccine and drug targets. A recent study investigated both surface and extracellular proteome of two C. ulcerans strains, where NanoLC-MS/MS was performed to analyze the set of proteins, which were similar expression patterns of putative virulence factors [72].…”

Secreted and cell surface proteome analysis of pathogenicCorynebacterium diphtheriaereveals proteins relevant to virulence

“…From a 2006 proteomics study 9 of C. diphtheriae using 2D gel electrophoresis separation of C. diphtheriae proteins, 85 surface and secreted proteins were identified. However, more recent studies on Corynebacterium ulcerans and Corynebacterium glutamicum identified many more proteins on or predicted to be localized to the cell surface, [10][11][12] suggesting that more coverage of the secreted and cell surface C. diphtheriae proteome should be obtainable. We used a high resolution HPLC mass spectrometry experimental strategy (e.g., trypsin digestion of precipitated supernatant and intact cells) to obtain an updated secretome and surfaceome of C. diphtheriae.…”