Abstract
Gram negative pathogenic bacteria Yersinia pseudotuberculosis use a type III secretion system (T3SS) to translocate toxic proteins into the eukaryotic cell. Once these proteins are inside the host cell, they interfere with the cell signalling pathways and alter the cellular response. The genes for the bacterial T3SS are located on the 70-kbp virulence plasmid that is common in all pathogenic species of Yersinia. A tetracysteine tag (FLNCCPGCCMEP) has been introduced in the C terminal end of Yersinia outer membrane protein E (YopE) as a translational fusion to observe secretion of these proteins into host cell that is seen in naturally occurring proteins which allows the expressed fusion protein to be specifically recognised by a biarsenical compound. The Lumio/tetracysteine and FLAsH/tetracysteine labelling system was used to fluorescently label YopE-Tc tag in Y. pseudotuberculosis to observe the secretion and quantification of these specific proteins. In this experiment, different bacterial strains (YPIIIpIB102, YPIIIpIB102(ETC12), YPIIIpIB155(ETC12), YPIIIpIB526, YPIIIpIB529(ETC12), YPIIIpIB604 (ETC12), YPIIIpIB621(ETC12), YPIIIpIB625(ETC12) and YPIII) with different proteins deletion (YopK, YopB, YopE, YopDΔ4-303, YopDΔ4-20 and YopED54-75), with or without tetracysteine tag were used to quantify the expression of YopE. Here, we demonstrated that the proteins YopE exhibit different secretion pattern with the deletion of other proteins. This may allow the possible role of these particular deleted proteins to activate or suppress the secretion of YopE. Overall, data from this experiment suggest that the total YopE expression depends on the conformation of regulator. Key words: Yersinia pseudotuberculosis, T3SS, tetracysteine tag, YopE quantification.
Highlights
Preoteomics studies on Yersinia pseudotuberculosis - a Gram negative gastrointestinal pathogen has given insight on how bacteria invade and survive in mammalian cells
The genes for the bacterial T3SS are located on the 70-kbp virulence plasmid that is common in all pathogenic species of Yersinia
A tetracysteine tag (FLNCCPGCCMEP) has been introduced in the C terminal end of Yersinia outer membrane protein E (YopE) as a translational fusion to observe secretion of these proteins into host cell that is seen in naturally occurring proteins which allows the expressed fusion protein to be recognised by a biarsenical compound
Summary
Preoteomics studies on Yersinia pseudotuberculosis - a Gram negative gastrointestinal pathogen has given insight on how bacteria invade and survive in mammalian cells. Yersinia species rarely enters the bloodstream and exhibit high affinity to lymphatic system via interactions between adhesins and integrin receptors on the surface of M cells of the Peyer’s patches. Successful colonisation of the Peyer’s patches causes local inflammation, enterocolitis When further spread from the Peyer’s patches to mesenteric lymph node an inflammation that mimic appendicitis result. To survive inside the host and escape the human immune system, the bacteria utilises a set of proteins denoted Yops that are secreted by T3SS. All pathogenic Yersinia species carry a 70 kd virulence plasmid (Costa et al, 2012; Lindler, 2004) pYV (pIB in Y. pseudotuberculosis) which is encoded by the T3SS and
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