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 has been the lack of ideal reporters to identify T4SS translocated effectors and study T4SS function. Most reporter systems make use of fusions to reporter proteins, in particular, β-lactamase (TEM) and calmodulin-dependent adenylate cyclase (CYA), that allow detection of translocated enzymatic activity inside host cells. However, both systems require costly reagents and use complex, multistep procedures for loading host cells with substrate (TEM) or for analysis (CYA). Therefore, we have 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 Legionella pneumophila T4SS in an IcmSW chaperone-dependent manner via an N-terminal translocation signal. Extensive mutagenesis in the NLuc N terminus suggested the importance of an α-helical domain spanning D5 to V9, as mutations predicted to disrupt this structure, with one exception, 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 transfected with GFPopt, thereby resulting in in vivo assembly of superfolder green fluorescent protein (GFP). This provided a bifunctional assay in which translocation could be assayed by fluorescence microplate, confocal microscopy, and/or luciferase assays. 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, these results indicate that NLuc provides both new insight into and also tools for studying T4SS biology. IMPORTANCE Type 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 identified nanoluciferase (NLuc) as an efficient IcmSW-dependent translocated T4SS substrate, and we 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 found that NLuc provides a powerful, cost-effective new tool to address these limitations and facilitate high-throughput exploration of secretion system biology.

• Authors: Yoon-Suk Kang, James E Kirby
• Journal: Microbiology spectrum (Microbiol Spectr) Pg. e0233822 (Feb 23 2023) ISSN: 2165-0497 [Electronic] United States
• PMID: 36815834 (Publication Type: Journal Article)