A Versatile Nanoluciferase Reporter Reveals Structural Properties Associated With a Highly Efficient, N-Terminal Legionella pneumophila type IV Secretion Translocation Signal

Abstract

Many gram-negative pathogens rely on type IV secretion systems (T4SS) for infection. One limitation in the field has been the lack of ideal reporters to identify T4SS translocated effectors and study T4SS function. Most existing reporter systems make use of fusions to reporter proteins, for example, {beta}-lactamase, to detect translocated enzymatic activity inside the host cell. However, these systems require costly substrates, complex procedures to separate eukaryotic cytoplasm for analysis, and/or are insensitive. Here, we developed and characterized a novel reporter system using nanoluciferase (NLuc) fusions to address these limitations. Serendipitously, we discovered that Nluc itself is efficiently translocated by L. pneumophila T4SS in an IcmSW chaperone-dependent manner via an N-terminal translocation signal. Extensive directed and random mutagenesis in the NLuc N-terminus revealed a critical -helical domain spanning D5 to V9, as mutations that are predicted to disrupt this -helix were translocation defective. Notably, NLuc was capable of translocating several proteins examined when fused to the N or C-terminus, while maintaining robust luciferase activity. In particular, it delivered the split GFP11 fragment into J774 macrophages permanently transfected with GFPopt, thereby resulting in in vivo assembly of superfolder GFP. This provided a bifunctional assay in which translocation could be assayed in by fluorescence microplate, confocal microscopy, and/or luciferase assay. We further identified an optimal NLuc substrate, which allowed a robust, inexpensive, one-step, high throughput screening assay to identify T4SS translocation substrates and inhibitors. Taken, together NLuc provides both new insight into and tools for studying T4SS biology. ImportanceType IV secretion systems (T4SS) are used by gram-negative pathogens to coopt host cell function. However, the translocation signals recognized by T4SS are not fully explained by primary amino acid sequence, suggesting yet to be defined contributions of secondary and tertiary structure. Here, we unexpectedly identify nanoluciferase (NLuc) as an efficient IcmSW-dependent translocated T4SS substrate and provide extensive mutagenesis data suggesting that the first N-terminal, alpha helix domain is a critical translocation recognition motif. Notably, most existing reporter systems for studying translocated proteins make use of fusions to reporters to permit detection of translocated enzymatic activity inside the host cell. However, existing systems require extremely costly substrates, complex technical procedures to isolate eukaryotic cytoplasm for analysis, and/or are insensitive. Importantly, we find that NLuc provides a powerful, cost-effective new tool to address these limitations and facilitate high throughput exploration of secretion system biology.