A colorimetric assay for studying effector secretion through the bacterial type III secretion system

Abstract

We have devised a colorimetric method that monitors secretion of effector proteins into host cytoplasm through the bacterial type III secretion machinery. Here we used constructs of effectors fused with Bordetella adenylate cyclase as a reporter, but evaluated the effector translocation by quantifying cell viability, rather than by measuring the intracellular cAMP concentration. This is based on our findings that cells infected by a secretion-competent bacterium expressing the fusion protein lost their viability under our experimental conditions. Cell death was quantified using commercially available reagents and basic research equipment. An observation that cell death was potentiated when the infected cells were treated with 2-deoxyglucose and sodium azide suggests that the depletion of intracellular ATP is partly involved in the process. Using enteropathogenic Escherichia coli, we demonstrated that the method was applicable to at least three effectors of bacteria, Tir, EspF, and Map, and was useful for studying a secretion signal sequence for Tir. This technically simple and inexpensive method is a good alternative to the existing procedure for studying the mechanism by which effectors are secreted through the type III secretion system in a high-throughput format.

Pertussis Toxin and Adenylate Cyclase Toxin: Key Virulence Factors of Bordetella Pertussis and Cell Biology Tools

“…In particular, this has been used to study protein targeting by type III secretion machinery in several bacteria. Examples of this from the recent literature include identification of type III-secreted effector proteins of Salmonella enterica serovar Typhimurium [126], type III-secreted proteins of Yersinia pestis that are putatively insecticidal [127], type III-secreted effectors of the plant pathogen Xanthomonas oryzae [128] and development of a colorimetric assay of cell viability to analyze type III secretion [129]. Another use of ACT is in genetic screens for protein–protein interaction in E. coli .…”

Advances and Challenges in Studying Type III Secretion Effectors of Attaching and Effacing Pathogens

“…A common approach, developed in 2004 by Charpentier and Oswald, is to C-terminally tag the effector with the TEM-1 β-lactamase and infect CCF2-loaded cells (Charpentier and Oswald, 2004 ); alternative and refined protocols have since been developed that decrease the tag size, minimize cell toxicity and offer single cell resolution. Collectively, these approaches benefit from their capacity to support different modes of analysis depending on the infection setup, such as enzymatic assays, optical readouts in a 96-well plate, flow cytometry and immunofluorescence microscopy (Mills et al, 2008 ; Miyake et al, 2008 ; Gawthorne et al, 2016 ; O'Boyle et al, 2018 ).…”