Browsing by Author "Kinkar, Eyad"

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CROSH COVID Conversations: Breaking the Chain of Infection in the Workplace
(Centre for Research in Occupational Safety and Health, 2020-10-15) Dorman, Sandra; Kinkar, Eyad
The secondary structure of the 5’-end of the STb mRNA affects its secretion efficiency
(2016-12-15) Kinkar, Eyad
The majority of secreted proteins in E. coli are targeted to and translocated across the cytoplasmic membrane through a signal sequence within the N-terminus of the protein. The major membrane complex facilitating this process is the general secretory or Sec-dependent membrane complex, also known as T2SS secretion. Research with proteins that are processed for export through a different pathway, called the T3SS secretion system, has recently challenged this widely accepted hypothesis. This research has provided evidence that the T3SS secretion system substrates may use mRNA sequences as a secondary means of protein targeting in addition to the N-terminus of the synthesized protein. The main objective of this study was to test whether or not the mRNA of the T2SS secretion system substrates may have structural information sufficient for efficient targeting of the protein for secretion. A well-studied substrate of the T2SS secretion system in E. coli is the heat-stable enterotoxin B (STb). The STb gene was cloned and a number of mutations were introduced within the 5' end of its mRNA to look at the effect of such mutations on protein secretion. The results showed that mutations that lead to changes in the AT/GC ratio within this 5’ region of the gene, while conserving the amino acid sequence of that region, affected toxin secretion. Bioinformatic analysis showed that these mutations affected the secondary structure of the 5' end of the mRNA and indicated that significant changes can be produced in the secondary structure of the mRNA when the AT/GC ratio is modified. This data shows that alterations in the secondary structure of the 5' end of the mRNA, without changes in the amino acid sequence of the N-terminus of the protein, can affect the efficiency with which the toxin is secreted and hints to a possible role for the mRNA in targeting proteins for export across the cytoplasmic membrane.
Secretion and interaction of the heat-stable enterotoxin b (STb) with the gut hormone secreting STC-1 Cells
(2021-08-31) Kinkar, Eyad
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.