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|Title:||Secretion and interaction of the heat-stable enterotoxin b (STb) with the gut hormone secreting STC-1 Cells|
|Keywords:||Cell biology;biochemistry;microbiology;genetics;Escherichia coli;mutation;site-directed mutagenesis;protein engineering;protein folding;signal peptide;protein secretion;heat-stable enterotoxin b;STb;SecB;Signal recognition particle (SRP);FFh;mRNA;AU richness;tight junction;type two secretion system;mRNA secondary structure|
|Abstract:||Enterotoxigenic Escherichia coli (ETEC) employs a number of secretion systems for efficient translocation of a number of virulence factors during infection. Studies of the relationship between substrates and specific secretion pathways suggested that secretion substrates are identified and are targeted to their proper secretion pathway via inherent amino acid sequences, or secretion signals, within each substrate. ETEC utilizes the Sec-dependent pathway of the type two secretion system (T2SS) to secrete the Heat-Stable Enterotoxin B (STb), in addition to several other toxins, to the extracellular milieu. In this work, the nature of the nucleotide sequence of STb’s mRNA, rather than the amino acid sequence of its signal sequence, was investigated for its effect on STb secretion. Additionally, the interaction of STb with the gut hormone secreting cells (STC-1 cells) was investigated to elucidate the nature and consequences of such interaction. In the first study, the N-terminal signal sequence of premature STb (at the mRNA level) was subjected to various mutations (a number of accumulative silent mutations and a number of non-silent point mutations) to test whether or not its mRNA secondary structure affects its targeting for secretion. In the second study, the two main key secretion factors of Secdependant pathway, ffh and secb, were separately knocked out and overexpressed to uncover their potential involvement in pro-STb targeting and secretion processes. In the final study, an in vitro approach was devised to identify and characterize the STb cell surface receptor on STC-1 cells (mouse intestinal enteroendocrine cell line). The results indicated that the nucleotide sequence of the STb mRNA (within the N-terminal amino acid signal sequence of the toxin) can affect toxin secretion. Reducing the AU richness within the 5′ end of the STb mRNA (without altering the translated amino acid sequence of this region) significantly reduced toxin secretion. From the second study, deletion of FFh, the protein component of the signal recognition particle in E. coli, resulted in significant effects on targeting and translocation of pro-STb and STb to the cytoplasmic membrane and the extracellular milieu more so than SecB deletion. Overexpression of FFh, not SecB, was shown to enhance STb synthesis and secretion. Finally, the results of the third study confirm a direct disruption to the tight junction of STC-1 cells. The results strongly suggest that STb interaction with these cells is mediated, at least in part, by the tight junction associated protein occludin. This interaction ultimately led to Caspase 3-mediated apoptosis.|
|Appears in Collections:||Biomolecular Sciences - Doctoral Theses|
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