The Toolbox for Uncovering the Functions of Legionella Dot/Icm Type IVb Secretion System Effectors: Current State and Future Directions

Abstract

The defective in organelle trafficking/intracellular multiplication (Dot/Icm) Type IVb secretion system (T4SS) is the essential virulence factor for the intracellular life style and pathogenicity of Legionella species. Screens demonstrated that an individual L. pneumophila strain can use the Dot/Icm T4SS to translocate an unprecedented number of more than 300 proteins into host cells, where these, so called Icm/Dot-translocated substrates (IDTS) or effectors, manipulate host cell functions to the benefit of the bacteria. Bioinformatic analysis of the pan-genus genome predicts at least 608 orthologous groups of putative effectors. Deciphering the function of these effectors is key to understanding Legionella pathogenesis; however, the analysis is challenging. Substantial functional redundancy renders classical, phenotypic screening of single gene deletion mutants mostly ineffective. Here, I review experimental approaches that were successfully used to identify, validate and functionally characterize T4SS effectors and highlight new methods, which promise to facilitate unlocking the secrets of Legionella's extraordinary weapons arsenal.

Keywords: Dot/Icm; Legionella; Type IVb secretion system; effectors; functional genomics; host targets; infection models; toolbox.

Figure 1

(A) Scheme of the typical architecture of Dot/Icm T4SS effectors. Effectors often show a modular structure consisting of a translocation signal and localization, target binding and enzymatic activity domains. (B) Reporter systems for determining the translocation and localization of Dot/Icm T4SS effectors. (1) Fluorophore- or gold particle-conjugated antibodies specific for an effector or an epitope tag are used to detect the effector in host cell lysates by immunoblot or visualize it by immunofluorescence- or electron microscopy. (2) Cre/loxP recombinase system: After delivery of Cre-effector fusions into recipient cells the recombinase removes a loxP-flanked disruptor cassette from a gene reporter conferring antibiotic resistance. (3) β-lactamase (TEM-1) assay: Translocation of TEM-1-effector fusions results in hydrolysis of a green fluorescent β-lactam FRET substrate, separating FRET donor and acceptor, generating a blue fluorescent product. (4) Calmodulin-dependent adenylate-cyclase (Cya) assay: Upon arrival of a Cya-effector fusion in the host, Cya gets activated by binding calmodulin and turns over ATP to cyclic AMP, which can be quantified by ELISA. (5) Split-GFP reporter system: Effectors are fused to the small (GFP11) fragment of a split GFP. Upon delivery into cells expressing the non-fluorescent large (GFP1-10) GFP fragment, spontaneous reassembly of effector-fused GFP11 and GFP1-10 occurs, restoring fluorescence emission which can be visualized by fixed or live imaging fluorescence microscopy. (6) Halo-tag reporter: After translocation into the host the Halo-tagged effector can be ligated with versatile fluorophores for detection by conventional, super-resolution- or electron microscopy.

Figure 2

Genetics and proteomics methods for the functional characterization of Dot/Icm T4SS effectors. (A) Insertional mutagenesis and depletion (iMAD) disentangles the complex network of effector-host manipulations: (1) Characterization of a Legionella mutant lacking a single effector (E1), which acts through host protein A, does not result in reduced intracellular growth, because a second effector (E2) induces a redundant process through host protein B. (2) Following the iMAD strategy, screening of single effector mutants in host cells, which are also depleted for host factors, eliminates redundant pathways, resulting in attenuation of the strain. (B) Nucleic Acid-Programmable Protein Array (NAPPA) for profiling of host cell targets of effectors: Human genes are printed as array on slides and translated in vitro. Recombinant Halo-tagged effector is added and after washes reacted with a fluorophore-ligand for the Halo-tag, allowing detection of the human proteins, which bound and retained the effector on the array. (C) Proteomics approaches for the characterization of effectors: (1) BirA/Bio-tag system: Bio-tagged effector is translocated into biotin ligase BirA expressing cells leading to biotinlylation of the Bio-tag. After optional cross-linking the bioinylated effector and bound host proteins are isolated by tandem-affinity purification for interactome analysis by MS. (2) Proximity biotinylation: Translocation of an effector fused to e.g., the peroxidase APEX or a promiscuous biotin ligase (BioID) results in biotinylation of host proteins in the proximity of the effector, which can be processed as for (1) to identify potential interactors. (3) Identifying post-translational modifications (PTMs): Infected cells are infused or metabolically-labeled with a chemical substrate analog (CSA) for a PTM-catalyzing enzyme. Host proteins and effectors, which are modified with the CSA, can be isolated after ligation of an affinity handle such as biotin to the CSA and characterized by MS. (4) Profiling the enzymatic activities of effectors with activity-based probes (ABPs): Infected cells or lysates are treated with a chemical ABP, which irreversibly binds to a specific enzyme class and contains or allows addition of an affinity handle. Effectors, which possess such enzymatic activity, react with the probe, are isolated and identified by MS.