Abstract
The type 3 secretion system is essential for pathogenesis of several human and animal Gram-negative bacterial pathogens. The T3SS comprises a transmembrane injectisome, providing a conduit from the bacterial cytoplasm to the host cell cytoplasm for the direct delivery of effectors (including toxins). Functional studies of T3SS commonly monitor the extracellular secretion of proteins by SDS-PAGE and western blot analysis, which are slow and semi-quantitative in nature. Here, we describe an enzymatic reporter-based quantitative and rapid in vivo assay for T3SS secretion studies in enteropathogenic E. coli (EPEC). The assay monitors the secretion of the fusion protein SctA-PhoA through the injectisome based on a colorimetric assay that quantifies the activity of alkaline phosphatase. We validated the usage of this reporter system by following the secretion in the absence of various injectisome components, including domains of the gatekeeper essential for T3SS function. This platform can now be used for the isolation of mutations, functional analysis and anti-virulence compound screening.
Highlights
The type three protein secretion system (T3SS) is essential for the pathogenic potential of many Gram-negative bacteria [1,2]
The injectisome is divided into three parts: (i) the cytoplasmic part, composed of the ATPase complex and its regulators, which peripherally associate with the inner membrane embedded translocase or export apparatus [5], (ii) the basal body, which contains stacks of inner and outer membrane rings, which encircle the inner rod and contribute to the formation of a tubular conduit through the periplasmic space linking the export apparatus at one end and the external needle (iii) at the other [6,7]
The M9 medium previously optimized for T3SS secretion by Enteropathogenic E. coli (EPEC) (M9-mod1; [43]) was further modified in order to grow cells that could be assayed by the alkaline phosphatase activity (M9-mod2; Table S1)
Summary
The type three protein secretion system (T3SS) is essential for the pathogenic potential of many Gram-negative bacteria [1,2]. The secretion switches to that of middle substrates [14] comprising the filament protein (SctA) and the translocon components (SctB and E) [3,9] that complete the assembly of the injectisome (Figure 1A). This enables the secretion/injection of the late T3S substrates (effectors) in responseMticorosoprgeacniisfimcs e20n2v0,ir8o, xnFmOeRnPtEaElRstRimEVuIEliW[9]
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