Genetic Manipulation of Non-pneumophila Legionella: Protocols Developed for Legionella longbeachae

Korevaar, Elizabeth; Khoo, Chen Ai; Newton, Hayley J

doi:10.1007/978-1-4939-9048-1_9

Cited by 2 publications

(3 citation statements)

References 17 publications

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“…longbeachae Δ ravZ LLO , and L . longbeachae Δ dotB were generated by allelic exchange as described previously [ 33 , 34 ]. To determine the translocation of RavZ LLO , fragments amplified from L .…”

Section: Methodsmentioning

confidence: 99%

“…When necessary, thymidine was added at 200 μg/mL. The in-frame deletion mutants L. pneumophila ΔravZ, L. longbeachae ΔravZ LLO , and L. longbeachae ΔdotB were generated by allelic exchange as described previously [33,34]. To determine the translocation of RavZ LLO , fragments amplified from L. longbeachae genomic DNA were inserted into pXDC61m [35], and the resulting plasmid was introduced into relevant Legionella strains by electroporation (2.5 kV; 200 O; 0.25 μF).…”

Section: Bacterial Strains Plasmids and Growth Conditionsmentioning

confidence: 99%

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Legionella longbeachae effector protein RavZ inhibits autophagy and regulates phagosome ubiquitination during infection

Shi

Liu

et al. 2023

PLoS ONE

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Legionella organisms are ubiquitous environmental bacteria that are responsible for human Legionnaires’ disease, a fatal form of severe pneumonia. These bacteria replicate intracellularly in a wide spectrum of host cells within a distinct compartment termed the Legionella-containing vacuole (LCV). Effector proteins translocated by the Dot/Icm apparatus extensively modulate host cellular functions to aid in the biogenesis of the LCV and intracellular proliferation. RavZ is an L. pneumophila effector that functions as a cysteine protease to hydrolyze lipidated LC3, thereby compromising the host autophagic response to bacterial infection. In this study, we characterized the RavZ (RavZLP) ortholog in L. longbeachae (RavZLLO), the second leading cause of Legionella infections in the world. RavZLLO and RavZLP share approximately 60% sequence identity and a conserved His-Asp-Cys catalytic triad. RavZLLO is recognized by the Dot/Icm systems of both L. pneumophila and L. longbeachae. Upon translocation into the host, it suppresses autophagy signaling in cells challenged with both species, indicating the functional redundancy of RavZLLO and RavZLP. Additionally, ectopic expression of RavZLLO but not RavZLP in mammalian cells reduces the levels of cellular polyubiquitinated and polyneddylated proteins. Consistent with this process, RavZLLO regulates the accumulation of polyubiquitinated species on the LCV during L. longbeachae infection.

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Section: Methodsmentioning

confidence: 99%

Section: Bacterial Strains Plasmids and Growth Conditionsmentioning

confidence: 99%

Legionella longbeachae effector protein RavZ inhibits autophagy and regulates phagosome ubiquitination during infection

Shi

Liu

et al. 2023

PLoS ONE

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“…If needed, Legionella cultures were supplemented with appropriate antibiotics at the following concentrations: 20 μg/mL kanamycin, 50 μg/mL streptomycin, and 5 μg/mL chloramphenicol. In-frame deletion mutants of L. longbeachae were constructed as previously described methods ( 46 ). In brief, the up- and downstream flanking regions of the target gene were amplified from the genomic DNA of L. longbeachae .…”

Section: Methodsmentioning

confidence: 99%

Legionella longbeachae Regulates the Association of Polyubiquitinated Proteins on Bacterial Phagosome with Multiple Deubiquitinases

Shi

Liu

et al. 2023

Microbiol Spectr

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Legionella spp. are opportunistic intracellular bacterial pathogens that cause Legionnaires’ disease. Legionella utilizes the Dot/Icm type IV secretion system to deliver effector protein into host cells to modulate various cellular functions. At least 26 L. pneumophila effectors are known to hijack the host ubiquitin system via diverse mechanisms. L. longbeachae is the second leading cause of Legionnaires’ disease worldwide.

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Genetic Manipulation of Non-pneumophila Legionella: Protocols Developed for Legionella longbeachae

Cited by 2 publications

References 17 publications

Legionella longbeachae effector protein RavZ inhibits autophagy and regulates phagosome ubiquitination during infection

Legionella longbeachae effector protein RavZ inhibits autophagy and regulates phagosome ubiquitination during infection

Legionella longbeachae Regulates the Association of Polyubiquitinated Proteins on Bacterial Phagosome with Multiple Deubiquitinases

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