Identification Of Salmonella enterica serovar Typhi Putative Virulence Factors Using A Yeast Growth Inhibition Assay
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Date
2018-02
Authors
Koay, Ley Teng
Journal Title
Journal ISSN
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Publisher
Universiti Sains Malaysia
Abstract
Pathogenic bacteria such as Salmonella enterica serovar Typhi (S. Typhi) evade the host immune system and cause disease in their host by producing proteins known as virulence factors (VFs). When VFs are expressed in yeast Saccharomyces cerevisiae (S. cerevisiae), they have been observed to be detrimental to yeast cells (e.g. causing growth inhibition). In this sense, yeast can act as a sensitive indicator to identify VFs. There are still unidentified VFs and for those already reported, their roles in pathogenesis are still not completely understood. This study aims to identify putative S. Typhi VFs by expressing them in S. cerevisiae and examining their effects on yeast growth. First, putative VFs were identified by bioinformatics analysis using online databases and literature review. A total of 192 potential putative VFs were selected from the proteome of S. Typhi CT18. Then, the selected VF genes were cloned and expressed in yeast in a 96-well plate format. Of the 192 putative VFs selected, 173 putative VFs were successfully cloned and expressed in yeast. By using a microplate growth assay that measures optical density of liquid culture and by confirmation of cell viability on solid medium, 23 putative VFs were found to inhibit the growth of yeast and were subjected to further characterization studies. Two recombinant putative VFs were purified to investigate the targeted host proteins through the study of VF-yeast protein interactions using a pull-down assay. Pulled down host proteins were identified by mass spectrometry analysis. Two putative VFs were identified in this study, a virulence-associated secretory protein (STY3018) and SopD possible secreted protein (STY3073) which were possibly interacting and targeting conserved yeast proteins such as guanine nucleotide binding proteins (G proteins) that function as signal transduction molecules and ribosomal proteins which are crucial for important cellular processes. In conclusion, this workflow can be used for the identification of S. Typhi putative VFs or yeast growth inhibiting proteins and their target interactions in yeast cell.
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Keywords
Salmonella enterica serovar Typhi putative virulence factors , using a yeast growth inhibition assay