Date of Graduation

5-2024

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Cell & Molecular Biology (PhD)

Degree Level

Graduate

Department

Cell & Molecular Biology

Advisor/Mentor

Sun, Xiaolun

Committee Member

Alrubaye, Adnan A.

Second Committee Member

Kwon, Young Min

Third Committee Member

Shi, Ainong

Keywords

Bile acid; Bile salt hydrolase; C. perfringens; Cloning; Protein expression; Toxic gas

Abstract

Clostridium perfringens is a predominant intestinal pathogen affecting both humans and animals, including chickens resulting in significant economic losses. In chapter I, I have overviewed the dynamics of the intestinal immune system, the composition of the intestinal microbiome, and how the microbiome influences inflammation in the development of Clostridium infection. The intricate interplay between immunity, microbiota, and their metabolites is likely critical in the pathogenesis of C. perfringens and uncovering these interactions may unveil new avenues for prevention and treatment. The objective of this chapter is to provide updated insights into the role of host-microbe interactions and their potential therapeutic applications in addressing C. perfringens infection. In chapter II, I focused on investigating one of the prominent Clostridium infections of chicken necrotic enteritis (NE), mainly caused by C. perfringens. The classical sign of NE is the foul smell gas in the ballooned small intestine. We hypothesized that deoxycholic acid (DCA) reduces NE by inhibiting C. perfringens virulence signaling pathways. To evaluate the hypothesis, C. perfringens strains CP1 and wild-type (WT) HN13 and its mutants were cultured with different bile acids, including DCA and isoallolithocholic acid (isoalloLCA). Growth, hydrogen sulfide (H2S) production, and virulence gene expression were measured. Notably, isoalloLCA was more potent in reducing growth, H2S production, and virulence gene expression in CP1 and WT HN13 compared to DCA, while other bile acids were less potent compared to DCA. Interestingly, there was a slightly different impact between DCA and isoalloLCA on the growth, H2S production, and virulence gene expression in the three HN13 mutants, suggesting possibly different signaling pathways modulated by the two bile acids. In chapter III, I investigated the effect of deconjugating taurodeoxycholic acids (TDCA) on reducing C. perfringens virulence. Although dietary secondary bile acid DCA reduces chicken NE, the accumulation of conjugated TDCA raised the concerns of its dietary efficacy. In this study, we aimed to deconjugate TDCA by recombinant bile salt hydrolase (BSH) to increase DCA efficacy against NE pathogen C. perfringens. Assays were conducted to evaluate the inhibition of C. perfringens growth, hydrogen sulfide (H2S) production, and virulence gene expression by TDCA and DCA. BSH activity and sequence alignment were conducted to select bsh gene for cloning. The bsh gene from Bifidobacterium longum was PCR-amplified and cloned into plasmids pET-28a (pET-BSH) and pDR111 (pDR-BSH) for expressing BSH protein in E. coli BL21 and Bacillus subtilis 168 (B-sub-BSH), respectively. His-tag purified BSH from BL21 cells was evaluated by SDS-PAGE, Coomassie blue staining, and Western Blot assays. Secretory BSH from B. subtilis was analyzed by Dot-Blot. B-sub-BSH was evaluated for the inhibition of C. perfringens growth. C. perfringens growth reached 7.8 log10 CFU/ml after 24 h culture. C. perfringens growth was at 8 vs. 7.4, 7.8 vs. 2.6, and 6 vs. 0 log10 CFU/ml in 0.2, 0.5, and 1 mM TDCA vs. DCA, respectively. Compared to TDCA, DCA reduced C. perfringens H2S production and virulence gene expression of asrA1, netB, colA, and virT. BSH activity was observed in L. jonsonii and B. longum under anaerobic conditions but not L. jonsonii under 10% CO2. After sequence alignment of bsh from ten bacteria, bsh from B. longum was selected, cloned into pET-BSH, and sequenced at 951 bp. After pET-BSH was transformed in BL21, BSH expression was assessed around 35 kDa using Coomassie staining and verified for His-tag using WB. After subcloned bsh and amylase signal peptide sequence into pDR-BSH, B. subtilis was transformed and named B-sub-BSH. The transformation was evaluated using PCR with B. subtilis around 3 kb and B-sub-BSH around 5 kb. Secretory BSH expressed from B-sub-BSH was determined for His-tag using Dot-Blot. Importantly, C. perfringens growth was reduced greater than 59% log10 CFU/ml in the B-sub-BSH media precultured with 1 vs. 0 mM TDCA. In conclusion, despite the urgent need for antimicrobial free alternatives in agricultural and healthcare industries, few interventions are available. The findings from my research could be used to design new strategies to prevent and treat C. perfringens-induced enteritis or other diseases.

Download

Included in

Microbiology Commons

Share

COinS