scholarly journals A NanoLuc luciferase‐based assay enabling the real‐time analysis of protein secretion and injection by bacterial type III secretion systems

2020 ◽  
Vol 113 (6) ◽  
pp. 1240-1254 ◽  
Author(s):  
Sibel Westerhausen ◽  
Melanie Nowak ◽  
Claudia E. Torres‐Vargas ◽  
Ursula Bilitewski ◽  
Erwin Bohn ◽  
...  
2019 ◽  
Author(s):  
Sibel Westerhausen ◽  
Melanie Nowak ◽  
Claudia Torres-Vargas ◽  
Ursula Bilitewski ◽  
Erwin Bohn ◽  
...  

AbstractThe elucidation of the molecular mechanisms of secretion through bacterial protein secretion systems is impeded by a lack of assays to quantitatively assess secretion kinetics. Also the analysis of the biological role of these secretion systems as well as the identification of inhibitors targeting these systems would greatly benefit from the availability of a simple, quick and quantitative assay to monitor principle secretion and injection into host cells. Here we present a versatile solution to this need, utilizing the small and very bright NanoLuc luciferase to assess secretion and injection through the type III secretion system encoded by Salmonella pathogenicity island 1. The NanoLuc-based secretion assay features a very high signal-to-noise ratio and sensitivity down to the nanoliter scale. The assay enables monitoring of secretion kinetics and is adaptable to a high throughput screening format in 384-well microplates. We further developed NanoLuc and split-NanoLuc-based assays that enable the monitoring of type III secretion-dependent injection of effector proteins into host cells.ImportanceThe ability to secrete proteins to the bacterial cell surface, to the extracellular environment, or even into target cells is one of the foundations of interbacterial as well as pathogen-host interaction. While great progress has been made in elucidating assembly and structure of secretion systems, our understanding of their secretion mechanism often lags behind, not last because of the challenge to quantitatively assess secretion function. Here, we developed a luciferase-based assay to enable the simple, quick, quantitative, and high throughput-compatible assessment of secretion and injection through virulence-associated type III secretion systems. The assay allows detection of minute amounts of secreted substrate proteins either in the supernatant of the bacterial culture or within eukaryotic host cells. It thus provides an enabling technology to elucidate the mechanisms of secretion and injection of type III secretion systems and is likely adaptable to assay secretion through other bacterial secretion systems.


2008 ◽  
Vol 283 (49) ◽  
pp. 34062-34068 ◽  
Author(s):  
Anastasia D. Gazi ◽  
Marina Bastaki ◽  
Spyridoula N. Charova ◽  
Eirini A. Gkougkoulia ◽  
Efthymios A. Kapellios ◽  
...  

2003 ◽  
Vol 101 (1) ◽  
pp. 70-75 ◽  
Author(s):  
V. E. Fadouloglou ◽  
A. P. Tampakaki ◽  
N. M. Glykos ◽  
M. N. Bastaki ◽  
J. M. Hadden ◽  
...  

1998 ◽  
Vol 62 (2) ◽  
pp. 379-433 ◽  
Author(s):  
Christoph J. Hueck

SUMMARY Various gram-negative animal and plant pathogens use a novel, sec-independent protein secretion system as a basic virulence mechanism. It is becoming increasingly clear that these so-called type III secretion systems inject (translocate) proteins into the cytosol of eukaryotic cells, where the translocated proteins facilitate bacterial pathogenesis by specifically interfering with host cell signal transduction and other cellular processes. Accordingly, some type III secretion systems are activated by bacterial contact with host cell surfaces. Individual type III secretion systems direct the secretion and translocation of a variety of unrelated proteins, which account for species-specific pathogenesis phenotypes. In contrast to the secreted virulence factors, most of the 15 to 20 membrane-associated proteins which constitute the type III secretion apparatus are conserved among different pathogens. Most of the inner membrane components of the type III secretion apparatus show additional homologies to flagellar biosynthetic proteins, while a conserved outer membrane factor is similar to secretins from type II and other secretion pathways. Structurally conserved chaperones which specifically bind to individual secreted proteins play an important role in type III protein secretion, apparently by preventing premature interactions of the secreted factors with other proteins. The genes encoding type III secretion systems are clustered, and various pieces of evidence suggest that these systems have been acquired by horizontal genetic transfer during evolution. Expression of type III secretion systems is coordinately regulated in response to host environmental stimuli by networks of transcription factors. This review comprises a comparison of the structure, function, regulation, and impact on host cells of the type III secretion systems in the animal pathogens Yersinia spp., Pseudomonas aeruginosa, Shigella flexneri, Salmonella typhimurium, enteropathogenic Escherichia coli, and Chlamydia spp. and the plant pathogens Pseudomonas syringae, Erwinia spp., Ralstonia solanacearum, Xanthomonas campestris, and Rhizobium spp.


2014 ◽  
Vol 77 (12) ◽  
pp. 2633-2640 ◽  
Author(s):  
Rohan A. Davis ◽  
Karren D. Beattie ◽  
Min Xu ◽  
Xinzhou Yang ◽  
Sheng Yin ◽  
...  

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