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

ABSTRACT: 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.

SUBMITTER: Batan D

PROVIDER: S-EPMC6050711 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

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

Batan Dilara D Braselmann Esther E Minson Michael M Nguyen Dieu My Thanh DMT Cossart Pascale P Palmer Amy E AE

Biophysical journal 20180410 2

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 in ...[more]

PMID: 29653838

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Project description:Fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes is a key method for the detection of (uncultured) microorganisms in environmental and medical samples. A major limitation of standard FISH protocols, however, is the small number of phylogenetically distinct target organisms that can be detected simultaneously. In this study, we introduce a multicolor FISH approach that uses eight fluorophores with distinct spectral properties, which can unambiguously be distinguished by confocal laser scanning microscopy combined with white light laser technology. Hybridization of rRNA-targeted DNA oligonucleotide probes, which were mono-labeled with these fluorophores, to Escherichia coli cultures confirmed that the fluorophores did not affect probe melting behavior. Application of the new multicolor FISH method enabled the differentiation of seven (potentially up to eight) phylogenetically distinct microbial populations in an artificial community of mixed pure cultures (five bacteria, one archaeon, and one yeast strain) and in activated sludge from a full-scale wastewater treatment plant. In contrast to previously published multicolor FISH approaches, this method does not rely on combinatorial labeling of the same microorganisms with different fluorophores, which is prone to biases. Furthermore, images acquired by this method do not require elaborate post-processing prior to analysis. We also demonstrate that the newly developed multicolor FISH method is compatible with an improved cell fixation protocol for FISH targeting Gram-negative bacterial populations. This fixation approach uses agarose embedding during formaldehyde fixation to better preserve the three-dimensional structure of spatially complex samples such as biofilms and activated sludge flocs. The new multicolor FISH approach should be highly suitable for studying structural and functional aspects of microbial communities in virtually all types of samples that can be analyzed by conventional FISH methods.

Dataset Information

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

Publications

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

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