Quantification of EnterohemorrhagicEscherichia coliO157:H7 proteome using TMT-Based Analysis

Abstract

25Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a human pathogen responsible for 26 diarrhea, hemorrhagic colitis and hemolytic uremic syndrome (HUS). EHEC infection is 27 distributed worldwide and numerous outbreaks of diseases caused by enterohemorrhagic 28 have been reported. To promote a comprehensive insight into the molecular basis of EHEC 29 O157:H7 physiology and pathogenesis, the combined proteome of EHEC O157:H7 strains, 30 Clade 8 and Clade 6 isolated from cattle in Argentina, and the standard EDL933 (clade 3) 31 strain has been analyzed. TMT (Tandem Mass Tags)-based quantitative proteomic and 32 emPAI analyses were performed to estimate the protein abundance in EHEC proteome. 33 2,234 non-redundant proteins of EHEC O157:H7 were identified. A comparison of this 34 result with in silico data of EHEC O157:H7 genome showed that approximately 40% of the 35 predicted proteome of this pathogen were covered. According to the emPAI analysis, 85 36 proteins were among the most abundant (e.g. GAPDH, FliC H-antigen, Enolase, and 37 GroEL). Tellurite resistance proteins were also highly abundant. COG analysis showed that 38 although most of the identified proteins are related to cellular metabolism, the majority of 39 the most abundant proteins are associated with translation processes. A KEGG enrichment 40 analysis revealed that Glycolysis / Gluconeogenesis was the most significant pathway. On 41 the other hand, the less abundant detected proteins are those related to DNA processes, cell 42 respiration and prophage. Among the proteins that composed the Type III Secretion 43 System, the most abundant protein was EspA. Altogether, the results show a subset of 44 important proteins that contribute to physiology and pathogenicity of EHEC O157:H7. 45 IMPORTANCE 46 3 The study of the abundance of proteins present within a complex mixture of proteins in a 47 cell, under different conditions, can provide important information about the activities of 48 individual protein components and protein networks that are cornerstones for the 49 comprehension of physiological adaptations in response to biological demands promoted 50 by environmental changes. We generated a comprehensive and accurate quantitative list of 51 EHEC O157:H7 proteome, which provides a description of the most abundant proteins 52 produced by this pathogen that were related to physiology and pathogenesis of EHEC. This 53 study provides information and extends the understanding on functional genomics and the 54 biology of this pathogen.55 Keywords: EHEC, Tandem mass tags (TMT), protein quantitation, bacterial proteomic 56 57 58 Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a zoonotic pathogen belonging to 59 Shiga toxin-producing E. coli (STEC) and responsible for different diseases as diarrhea, 60 hemorrhagic colitis and hemolytic uremic syndrome (HUS). HUS is distributed worldwide 61 and considered to be a public health problem in several countries (1,2). Unfortunately, 62 Argentina is the country with the highest incidence of HUS i...

Comparison of the Proteome of Staphylococcus aureus Planktonic Culture and 3-Day Biofilm Reveals Potential Role of Key Proteins in Biofilm

“…Whilst the proteomics of S. aureus biofilm have previously been investigated [13,14,16,24], we have utilised highly powerful TMT-MS in this study. TMT-labelling combined with tandem MS can label and analyse up to 10 protein samples simultaneously in high-resolution in the low mass region [25,26]. This powerful proteomic strategy can be helpful for a deeper understanding of biological mechanisms as well as a screening of biomarkers by examining the variations in protein expression levels.…”