Clickable methionine as a universal probe for labelling intracellular bacteria

Atwal, Sharanjeet; Giengkam, Suparat; Jaiyen, Yanin; Feaga, Heather A.; Dworkin, Jonathan; Salje, Jeanne

doi:10.1016/j.mimet.2019.105812

Cited by 7 publications

(5 citation statements)

References 10 publications

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“…Giemsa staining is traditionally used to image bacteria in tissue sections and clinical samples 267 , whilst immunofluorescence microscopy is widely used to study fixed samples of Rickettsiales in cultured cells and animal models although this suffers the limitation that antibodies need to be custom generated for particular organisms and strains. A clickable methionine probe that specifically labels bacteria that undergoing protein synthesis has been used to label intracellular Rickettsiales species and identify individual cells that are metabolically active 268 . Commercially available fluorescent probes have been used to carry out live cell imaging of Orientia tsutsugamushi, 269 Wolbachia 270 271,272 and Ehrlichia chaffeensis 273 whilst fluorescence in situ hybridization (FISH) has been used to label Wolbachia and Rickettsia in fixed cells and tissues 274,275 276 277 .…”

Section: Discussionmentioning

confidence: 99%

Cells within cells: Rickettsiales and the obligate intracellular bacterial lifestyle

Salje

2021

Nat Rev Microbiol

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124

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The Rickettsiales are a group of obligate intracellular vector-borne Gram-negative bacteria that include many organisms of clinical and agricultural importance, including Anaplasma spp, Ehrlichia chaffeensis, Wolbachia, Rickettsia spp, and Orientia tsutsugamushi. This review provides an overview of the current state of knowledge of the biology of these organisms and their interactions with host cells, with a particular focus on pathogenic species or those that are otherwise important for human health. This includes a description of rickettsial genomics, bacterial cell biology, the intracellular lifestyles of Rickettsiales and the mechanisms by which they induce and evade the innate immune response.

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Section: Discussionmentioning

confidence: 99%

Cells within cells: Rickettsiales and the obligate intracellular bacterial lifestyle

Salje

2021

Nat Rev Microbiol

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124

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“…These observations led us to hypothesize that EB bacteria are metabolically inactive owing to their extracellular location. To test this, we used a microscopy-based assay whereby growing bacteria are exposed to a clickable methionine analog L-Homopropargylglycine (HPG) that is subsequently labeled with a fluorophore 15 (Fig. 1F).…”

Section: Resultsmentioning

confidence: 99%

“…To test this, we used a microscopy-based assay whereby growing bacteria are exposed to a clickable methionine analog L-Homopropargylglycine (HPG). that is subsequently labeled with a fluorophore 15 (Fig. 1F).…”

Section: Ib and Eb Forms Of Ot Differ In Physical Properties And Metabolic Activitymentioning

confidence: 99%

The obligate intracellular bacteriumOrientia tsutsugamushidifferentiates into a developmentally distinct extracellular state

Atwal

Wongsantichon

Giengkam

et al. 2020

Preprint

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Developmental differentiation has been described for several vacuole-dwelling obligate intracellular bacteria but never for an obligate intracellular bacterium that resides in the cytoplasm. Here, we show that the cytoplasm-dwelling obligate intracellular bacterium Orientia tsutsugamushi (Ot) exists in five distinct subpopulations. We show that Ot differentiates into a distinct, metabolically inactive, extracellular state upon budding from the surface of host cells and that this stage is preceded by a surface-associated maturation stage. We identify proteins that are differentially expressed in intracellular, replicative bacteria and extracellular, metabolically inactive bacteria. Metabolic activity resumes rapidly upon entry into the cytoplasm and is triggered by the host cell reducing environment. This example of developmental differentiation in a species of Rickettsiaceae provides a new model system for studying synchronized differentiation in a bacterium that has a minimal genome and where the interactions between bacterium and host cell are more direct than they are for bacteria separated from the eukaryote cell host by a vacuolar membrane.Author SummaryScrub typhus is a life-threatening human infection that is caused by the bacterium Orientia tsutsugamushi and spread by mites. Although the disease is estimated to affect at least one million people annually and is often fatal, the infectious agent is much less well understood than many other pathogens. O. tsutsugamushi is an intracellular bacterium that can only grow and divide within eukaryotic cells. During infection, it is found primarily in the cells that make up the lining of blood vessels and in certain immune cell types. O. tsutsugamushi bacteria can remain inside a single infected cell for seven days or more before budding out. The ways in which the bacterium itself changes during the course of an intracellular infection cycle have not been studied. In the current work, we used a range of techniques to show that O. tsutsugamushi differentiates into five distinct subpopulations, and that these are associated with measurable differences in metabolic activity, replication and infectivity. This work opens new avenues of research into the regulation and mechanisms of differentiation of O. tsutsugamushi, which could lead to improved diagnosis and treatments. It also provides a new model system for studying fundamental questions about bacterial development.

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“…As described (Atwal et al., 2020), 300 µl of cells were transferred to a 5‐ml glass tube, and 0.25 µl of 20‐mM Click‐IT OPP reagent (Thermofisher) was added. Cells were placed on a roller drum at 37°C for 10 min to allow OPP incorporation, after which the reaction was quenched and cells were fixed with 3.7% formaldehyde in phosphate‐buffered saline (PBS).…”

Section: Methodsmentioning

confidence: 99%

“…As described (Atwal et al, 2020), 300 µl of cells were transferred to a 5-ml glass tube, and 0.25 µl of 20-mM Click-IT OPP reagent (Thermofisher) was added. Cells were placed on a roller drum at 37°C…”

Section: Detection Of Protein Synthesis In Vivo Using Click Chemistrymentioning

confidence: 99%

Transcription regulates ribosome hibernation

Feaga

Dworkin

2021

Molecular Microbiology

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Most bacteria are quiescent, typically as a result of nutrient limitation. In order to minimize energy consumption during this potentially prolonged state, quiescent bacteria substantially attenuate protein synthesis, the most energetically costly cellular process. Ribosomes in quiescent bacteria are present as dimers of two 70S ribosomes.Dimerization is dependent on a single protein, hibernation promoting factor (HPF), that binds the ribosome in the mRNA channel. This interaction indicates that dimers are inactive, suggesting that HPF inhibits translation. However, we observe that HPF does not significantly affect protein synthesis in vivo suggesting that dimerization is a consequence of inactivity, not the cause. The HPF-dimer interaction further implies that re-initiation of translation when the bacteria exit quiescence requires dimer resolution. We show that ribosome dimers quickly resolve in the presence of nutrients, and this resolution is dependent on transcription, indicating that mRNA synthesis is required for dimer resolution. Finally, we observe that ectopic HPF expression in growing cells where mRNA is abundant does not significantly affect protein synthesis despite stimulating dimer formation, suggesting that dimerization is dynamic. Thus, the extensive transcription that occurs in response to nutrient availability rapidly reactivates the translational apparatus of a quiescent cell and induces dimer resolution.

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Clickable methionine as a universal probe for labelling intracellular bacteria

Cited by 7 publications

References 10 publications

Cells within cells: Rickettsiales and the obligate intracellular bacterial lifestyle

Cells within cells: Rickettsiales and the obligate intracellular bacterial lifestyle

The obligate intracellular bacteriumOrientia tsutsugamushidifferentiates into a developmentally distinct extracellular state

Transcription regulates ribosome hibernation

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