Tracking elusive cargo: Illuminating spatio-temporal Type 3 effector protein dynamics using reporters

Abstract

Type 3 secretion systems form an integral part of the arsenal of many pathogenic bacteria. These injection machines, together with their cargo of subversive effector proteins, are capable of manipulating the cellular environment of the host in order to ensure persistence of the pathogen. In order to fully appreciate the functions of Type 3 effectors, it is necessary to gain spatio‐temporal knowledge of each effector during the process of infection. A number of genetic modifications have been exploited in order to reveal effector protein secretion, translocation and subsequent activity, and localisation within host cells. In this review, we will discuss the many available approaches for tracking effector protein dynamics and discuss the challenges faced to improve the current technologies and gain a clearer picture of effector protein function.

Fluorescent secreted bacterial effectors reveal active intravacuolar proliferation of Listeria monocytogenes in epithelial cells

“…In recent years, several strategies have emerged for fluorescent labelling of Sec–or T3SS–dependent substrates [ 26 ]. Tagging bacterial effectors with Split-GFP provides a possible solution that has been successfully applied for live detection of Salmonella T3SS-dependent effectors or Listeria Sec-dependent secreted substrates [ 27 , 28 ]; however, the reconstitution process is slow compared with microbial growth, and requires the stable expression of GFP 1-10 in recipient cells, which limits its application in most biological systems.…”

Recent Advancements in Tracking Bacterial Effector Protein Translocation

“…As extensively reviewed in [28], fluorescent chaperones provide another elegant alternative method to circumvent the FP fusion problem. In this case, effectors are not tagged, but instead, fluorescent chaperone reporters are expressed inside host cells.…”

“…However, this method is less suited for high-throughput studies and does not allow for real-time monitoring of effector translocation. Consequently, several enzyme-linked or fluorescent reporter methods have been developed aiming to track secretion in a spatiotemporal manner, which have extensively been reviewed in [27,28]. Importantly, the type of translational fusion (N-or C-terminal tagging) of the effectors under study with a reporter has to be carefully considered in function of the type of effectors being studied.…”

“…developed aiming to track secretion in a spatio-temporal manner, which have exte been reviewed in [27,28]. Importantly, the type of translational fusion (N-or C-te tagging) of the effectors under study with a reporter has to be carefully consid function of the type of effectors being studied.…”

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 ).…”