Optimized Fluorescence Complementation Platform for Visualizing <i>Salmonella</i> Effector Proteins Reveals Distinctly Different Intracellular Niches in Different Cell Types

Young, Alexandra M.; Minson, Michael; McQuate, Sarah E.; Palmer, Amy E.

doi:10.1021/acsinfecdis.7b00052

Cited by 18 publications

(36 citation statements)

References 48 publications

(140 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Poorly expressed or diffusely localised effectors present a greater challenge for microscopic tracking, particularly in real time where photobleaching is a concern. The application of tandem repeat fluorescent tags is sometimes not sufficient to overcome issues with low signal from such effectors (Park et al, ; Young et al, ). Multimerisation of tags comes at the cost of increased propensity for aggregate formation and an increased risk of interfering with functionality.…”

Section: Discussionmentioning

confidence: 99%

“…Interestingly, a recent study demonstrated that 3×GFP 11 labelling of the Salmonella SPI2 effector proteins SseF, SseG, and SlrP resulted in higher fluorescence intensity; however, due to low levels of expression from their endogenous promoters, an experimental set‐up involving plasmid‐based expression driven by the stronger steA promoter was favoured over chromosomal tagging. The resultant signal amplification allowed for real‐time identification of differential effector localisation in infected primary macrophages and HeLa cells (Young, Minson, McQuate, & Palmer, ). A tripartite split‐GFP labelling method has also been developed to allow the study of protein–protein interactions and to reduce background levels due to aggregation and spontaneous reassociation that are sometimes observed with the GFP 1–10 /GFP 11 system.…”

Section: Effector‐tagging Methodologiesmentioning

confidence: 99%

See 1 more Smart Citation

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

O’Boyle

Connolly

Roe

2017

Cellular Microbiology

View full text Add to dashboard Cite

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.

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Effector‐tagging Methodologiesmentioning

confidence: 99%

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

O’Boyle

Connolly

Roe

2017

Cellular Microbiology

View full text Add to dashboard Cite

show abstract

“…Effectors tagged with a fluorescent peptide allow following the secretion process from the intracellular bacteria by the loss of fluorescence. Another approach consists on an adaptation of the split‐green fluorescent protein (GFP) assay, fusing the FP11 GFP domain to the effector and expressing the rest of the GFP protein (FP1‐10) in the host cell (Batan et al, ; Young, Minson, McQuate, & Palmer, ).…”

Section: Effector‐mediated Subversion Of Host Cellsmentioning

confidence: 99%

Bacterial injection machines: Evolutionary diverse but functionally convergent

Bleves

Galán

Llosa

2020

Cellular Microbiology

View full text Add to dashboard Cite

Many human pathogens use Type III, Type IV, and Type VI secretion systems to deliver effectors into their target cells. The contribution of these secretion systems to microbial virulence was the main focus of a workshop organised by the International University of Andalusia in Spain. The meeting addressed structure–function, substrate recruitment, and translocation processes, which differ widely on the different secretion machineries, as well as the nature of the translocated effectors and their roles in subverting the host cell. An excellent panel of worldwide speakers presented the state of the art of the field, highlighting the involvement of bacterial secretion in human disease and discussing mechanistic aspects of bacterial pathogenicity, which can provide the bases for the development of novel antivirulence strategies.

show abstract

“…Live-fluorescence-microscopy approaches are ideal to dissect dynamics of bacterial infections on a single-cell level. We and others previously developed live fluorescence tools to visualize and quantify effector proteins secreted from different Gram-negative pathogens in infection, including Salmonella, Shigella, and Escherichia coli (33)(34)(35)(36)(37)(38)(39)(40). The effector proteins labeled by different fluorescent tags in these studies are secreted by the type III secretion system (41), a well-studied and common protein secretion pathway across the inner and outer membrane in Gram-negative bacteria.…”

Section: Introductionmentioning

confidence: 99%

“…The effector proteins labeled by different fluorescent tags in these studies are secreted by the type III secretion system (41), a well-studied and common protein secretion pathway across the inner and outer membrane in Gram-negative bacteria. A particularly powerful tool is the split-green fluorescent protein (GFP) system, in which the last b-strand of GFP (GFP11, 16 amino acids) is genetically fused to the protein of interest via a short and flexible linker (34,35) (see also Fig. 1 a).…”

Section: Introductionmentioning

confidence: 99%

A Multicolor Split-Fluorescent Protein Approach to Visualize Listeria Protein Secretion in Infection

Batan

Braselmann

Minson

et al. 2018

Biophysical Journal

Self Cite

View full text Add to dashboard Cite

Listeria monocytogenes is an intracellular food-borne pathogen that has evolved to enter mammalian host cells, survive within them, spread from cell to cell, and disseminate throughout the body. A series of secreted virulence proteins from Listeria are responsible for manipulation of host-cell defense mechanisms and adaptation to the intracellular lifestyle. Identifying when and where these virulence proteins are located in live cells over the course of Listeria infection can provide valuable information on the roles these proteins play in defining the host-pathogen interface. These dynamics and protein levels may vary from cell to cell, as bacterial infection is a heterogeneous process both temporally and spatially. No assay to visualize virulence proteins over time in infection with Listeria or other Gram-positive bacteria has been developed. Therefore, we adapted a live, long-term tagging system to visualize a model Listeria protein by fluorescence microscopy on a single-cell level in infection. This system leverages split-fluorescent proteins, in which the last strand of a fluorescent protein (a 16-amino-acid peptide) is genetically fused to the virulence protein of interest. The remainder of the fluorescent protein is produced in the mammalian host cell. Both individual components are nonfluorescent and will bind together and reconstitute fluorescence upon virulence-protein secretion into the host cell. We demonstrate accumulation and distribution within the host cell of the model virulence protein InlC in infection over time. A modular expression platform for InlC visualization was developed. We visualized InlC by tagging it with red and green split-fluorescent proteins and compared usage of a strong constitutive promoter versus the endogenous promoter for InlC production. This split-fluorescent protein approach is versatile and may be used to investigate other Listeria virulence proteins for unique mechanistic insights in infection progression.

show abstract

Optimized Fluorescence Complementation Platform for Visualizing Salmonella Effector Proteins Reveals Distinctly Different Intracellular Niches in Different Cell Types

Cited by 18 publications

References 48 publications

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

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

Bacterial injection machines: Evolutionary diverse but functionally convergent

A Multicolor Split-Fluorescent Protein Approach to Visualize Listeria Protein Secretion in Infection

Contact Info

Product

Resources

About