Masanta, Wycliffe Omurwa

English

Campylobacter jejuni is the leading cause of diarrhoea among human beings worldwide. Epidemiological investigations have shown that it affects over 500 million people per year. 
C. jejuni is mainly transmitted to human through consumption of cross contaminated chicken. In most cases, the diarrhoea clears by itself within 3 to 5 days. But it causes a big discomfort in the affected individuals. In addition, it has a huge economic impact due to sick leaves. Because of this, efforts are being put into understanding how C. jejuni interacts with human beings and other hosts. An indepth understanding of how this pathogen interacts with its hosts will lead to development of appropriate diagnosis tools and prevention measures. 
Bile acids are a major component of the gut fluid in all the hosts of C. jejuni. However, the interaction of C. jejuni and different types of bile acids at human body temperature of 37oC is poorly understood. Consequently, this study was designed to unearth the proteomic response in C. jejuni reference strain 81-176 to sublethal concentrations of cholic acid (CA), deoxycholic acid (DCA), lithocholic acid (LCA), taurocholic acid (TCA), chenodeoxycholic acid (CDCA), ursodeoxycholic acid (UDCA) and glycocholic acid (GCA). The specific objectives were: (i) to investigate the response in 81-176 to DCA 0.05% at 37oC for 12h and 24h using both stable isotope labeling with amino acids in cell culture (SILAC) and label-free analysis with sequantial window acquisation of all theoretical mass spectra (SWATH); and determine a suitable quantitative method for the study. (ii) To use the method selected quantitative method to investigate global protein expression in 81-176 in response to sublethal concentrations of CA, LCA, TCA, CDCA, UDCA and GCA cultured at 37oC for 12h under microaerophilic conditions. (iii) To identify and characterize a currently uncharacterized and widely induced protein (iv) To use label-free analysis with SWATH to investigate protein expression in 81-176 cultured in temperatures of 37oC (human) and 42oC (chicken) without bile acids..
Intially, the capability of C. jejuni to adhere and invade Caco-2 cells in the presence of various concentrations of bile acids was investigated using gentamicin protection assay (GPA). The results showed that DCA, CDCA and GCA promoted adherence and invasion in a dose depandant fashion. LCA and UDCA didn’t neither promote nor suppress adherence and invasion. Subsequently, IC50 of each bile acid was obtained. Half of this concentration of each bile acid corresponded to the concentrations that are present in the large intestines of human beings. Hence half IC50 concentrations were taken to be sublethal concentration. The concentrations were: CA 0.1%, DCA 0.05%, LCA 0.05%, TCA 0.5%, CDCA 0.05%, UDCA 0.5% and GCA 0.4%. Quantitative proteomic analysis of the response of 81-176 to DCA 0.05% showed that SILAC generated 500 quantifiable proteins and label-free analysis with SWATH generated 957 quantifiable proteins. The difference was attributed to poor incorporation of arginine and lysine in 81-176. As a result, SWATH analysis was used to quantify the response in 81-176 to different bile acids. These analyses found that CA significantly upregulated 19 proteins and downregulated 28 proteins; DCA significantly upregulated 113 proteins and downregulated 79 proteins; LCA significantly upregulated 4 proteins and downregulated 13 proteins; TCA significantly upregulated 51 proteins and downregulated 60 proteins; CDCA  significantly upregulated 89 proteins and downregulated 79 proteins; UDCA significantly upregulated 2 proteins and downregulated 4 proteins; GCA significantly upregulated 139 proteins and downregulated 20 proteins. Among the significantly upregulated proteins, MazF was selected for further characterization. The mutant showed significant reduction in adhering onto Caco-2 cells in the presence of CA 0.1% (p<0.05). Also, the mutant showed significant reduction in invading Caco-2 cells in the presence of CA 0.1% and TCA 0.5% (p<0.05). Similarly, the muatnt showed decline in growth after 20 hr in broth supplemented with CA 0.01%, DCA 0.05%, TCA 0.05%, CDCA 0.05% and GCA 0.4%. Separately, 83 proteins were significantly upregulated and 65 proteins were significantly downregulated between 81-176 that was cultured at 37oC for 12h and 24h. While 83 proteins were significantly upregulated and 50 proteins were significantly downregulated between 81-176 that was cultured at 37oC for 24h and 42oC for 24h. All the differentially expressed proteins belonged to the following biological processes: (i) cell division and cell cycle (ii) maintenance of integrity of outer, periplasmic and inner membranes (iii) DNA replication and transcription (iv) metabolism (v) chemotaxis and motility (vi) stress response and 291 uncharacterized proteins.
 
In conclusion, SWATH analysis is a more suitable quantitative method for wide scale Campylobacter proteomic research. However, other methods such as SILAC should be concurrently included to complement its weaknesses. DCA, CDCA and GCA had the highest number of differentially expressed proteins. Equally, CA differentially expressed a reasonable number of proteins but not as high as DCA, CDCA and GCA. CA, DCA, LCA, TCA, CDCA, UDCA and GCA promote adherence and invasion of epithelial cells. Majority of the proteins which are promoted adherence and invasion are involved in metabolic processes. Also all the bile acids that were examined in this study are toxic to 81-176. The results show that 81-176 has a well built adaptation system to both bile acid antimicrobial activities and changes in temperatures. This system involves activation and deactivation of a set of genes involved in metabolism, stress response, maintenance of integrity of outer, periplasmic and inner membranes, chemotaxis and motility. Undoubtedly, the findings of this study will enhance the understanding of the biology on the interaction of C. jejuni and bile acids

Georg-August-University Göttingen

Quantitative Proteomics Analysis of Global Protein Expression in Campylobacter jejuni Cultured in Sublethal Concentrations of Bile Acids and Varying Temperatures

https://ediss.uni-goettingen.de/bitstream/11858/00-1735-0000-0023-3E9B-4/1/WM_Thesis_Submitted_Copy_28th%20June%20%202017_UG2.pdf

Quantitative Proteomics Analysis of Global Protein Expression in Campylobacter jejuni Cultured in Sublethal Concentrations of Bile Acids and Varying Temperatures

Abstract

Similar works

Full text

Available Versions

Georg-August-University Göttingen