Quantitative Dextran Trafficking to the <i>Coxiella burnetii</i> Parasitophorous Vacuole

Winfree, Seth; Gilk, Stacey D.

doi:10.1002/cpmc.34

Cited by 4 publications

(7 citation statements)

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“…Thus, it is possible that the decrease in lysosomal content is due to increasing fusion between the CCV and endosomes/lysosomes during CCV expansion. To address this possibility, we measured CCV fusogenicity using a quantitative dextran trafficking assay [47]. HeLa cells infected with WT mCherry-C. burnetii were pulsed with fluorescent Alexa fluor 488 dextran for 10 min, followed by imaging every 4 min for 28 minutes using live-cell confocal microscopy ( Fig 5A).…”

Section: Burnetii T4bss Reduces Lysosomal Content In Infected Cellsmentioning

confidence: 99%

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Coxiella burnetii Type 4B Secretion System-dependent manipulation of endolysosomal maturation is required for bacterial growth

et al. 2019

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Upon host cell infection, the obligate intracellular bacterium Coxiella burnetii resides and multiplies within the Coxiella-Containing Vacuole (CCV). The nascent CCV progresses through the endosomal maturation pathway into a phagolysosome, acquiring endosomal and lysosomal markers, as well as acidic pH and active proteases and hydrolases. Approximately 24-48 hours post infection, heterotypic fusion between the CCV and host endosomes/lysosomes leads to CCV expansion and bacterial replication in the mature CCV. Initial CCV acidification is required to activate C. burnetii metabolism and the Type 4B Secretion System (T4BSS), which secretes effector proteins required for CCV maturation. However, we found that the mature CCV is less acidic (pH~5.2) than lysosomes (pH~4.8). Further, inducing CCV acidification to pH~4.8 causes C. burnetii lysis, suggesting C. burnetii actively regulates pH of the mature CCV. Because heterotypic fusion with host endosomes/ lysosomes may influence CCV pH, we investigated endosomal maturation in cells infected with wildtype (WT) or T4BSS mutant (ΔdotA) C. burnetii. In WT-infected cells, we observed a significant decrease in proteolytically active, LAMP1-positive endolysosomal vesicles, compared to mock or ΔdotA-infected cells. Using a ratiometric assay to measure endosomal pH, we determined that the average pH of terminal endosomes in WT-infected cells was pH~5.8, compared to pH~4.75 in mock and ΔdotA-infected cells. While endosomes progressively acidified from the periphery (pH~5.5) to the perinuclear area (pH~4.7) in both mock and ΔdotA-infected cells, endosomes did not acidify beyond pH~5.2 in WT-infected cells. Finally, increasing lysosomal biogenesis by overexpressing the transcription factor EB resulted in smaller, more proteolytically active CCVs and a significant decrease in C. burnetii growth, indicating host lysosomes are detrimental to C. burnetii. Overall, our data suggest that C. burnetii inhibits endosomal maturation to reduce the number of proteolytically active lysosomes available for heterotypic fusion with the CCV, possibly as a mechanism to regulate CCV pH.The obligate intracellular bacterium Coxiella burnetii causes human Q fever, which manifests as a flu-like illness but can develop into a life-threatening and difficult to treat endocarditis. C. burnetii, in contrast to many other intracellular bacteria, thrives within a lysosome-like vacuole in host cells. However, we previously found that the C. burnetii vacuole is not as acidic as lysosomes and increased acidification kills the bacteria, suggesting that C. burnetii regulates the pH of its vacuole. Here, we discovered that C. burnetii blocks endolysosomal maturation and acidification during host cell infection, resulting in fewer lysosomes in the host cell. Moreover, increasing lysosomes in the host cells inhibited C. burnetii growth. Together, our study suggests that C. burnetii regulates vacuole acidity and blocks endosomal maturation in order to produce a permissive intracellular niche.Coxiella manipulates endosom...

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Section: Burnetii T4bss Reduces Lysosomal Content In Infected Cellsmentioning

confidence: 99%

“…Dextran trafficking and fusion with CCVs was measured as described previously [47]. Briefly, HeLa cells were infected with mCherry-WT C. burnetii in six-well plates (5X10 4 cells/well; two wells per condition).…”

Section: Dextran Traffickingmentioning

confidence: 99%

Coxiella burnetii Type 4B Secretion System-dependent manipulation of endolysosomal maturation is required for bacterial growth

et al. 2019

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“…Refer to Winfree and Gilk (2017;UNIT 6C.2) for information on working with HeLa cells and Coxiella burnetii.…”

Section: Critical Parametersmentioning

confidence: 99%

Measuring pH of the Coxiella burnetii Parasitophorous Vacuole

Samanta

Gilk

2017

CP Microbiology

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Coxiella burnetii is the causative agent of human Q fever, a zoonotic disease that can cause a debilitating, flu-like illness in acute cases, or a life-threatening endocarditis in chronic patients. An obligate intracellular bacterial pathogen, Coxiella survives and multiplies in a large lysosome-like vacuole known as the Coxiella parasitophorous vacuole (CPV). A unique characteristic of the CPV is the acidic environment (pH ∼5.0), which is required to activate Coxiella metabolism and the Coxiella type 4 secretion system (T4SS), a major virulence factor required for intracellular survival. Further, inhibiting or depleting vacuolar ATPase, a host cell protein that regulates lysosomal pH, inhibits intracellular Coxiella growth. Together, these data suggest that CPV pH is an important limiting factor for Coxiella growth and virulence. This unit describes a method to determine CPV pH using live cell microscopy of a pH-sensitive fluorophore conjugated to dextran. This technique is useful to measure changes in CPV pH during infection or in response to drug treatment. © 2017 by John Wiley & Sons, Inc.

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“…Thus, it is possible that the decrease in lysosomal content between 2 and 3 dpi is due to increasing CCV-lysosome fusion. To address this possibility, we measured CCV fusogenicity using a quantitative dextran trafficking assay [38]. HeLa cells infected with WT mCherry- C. burnetii were pulsed with fluorescent Alexa fluor 488 dextran for 10 min, followed by imaging every 4 min for 28 minutes using live-cell confocal microscopy (Fig 4A).…”

Section: Resultsmentioning

confidence: 99%

“…Dextran trafficking and fusion with CCVs was measured as described previously [38]. Briefly, HeLa cells were infected with mCherry-WT C. burnetii in six-well plates (5X10 4 cells/well; two wells per condition).…”

Section: Methodsmentioning

confidence: 99%

Coxiella burnetiiType 4B Secretion System-dependent manipulation of endolysosomal maturation is required for bacterial growth

Samanta

Clemente

Gilk

2019

Preprint

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29Upon host cell infection, the obligate intracellular bacterium C. burnetii resides and multiplies 30 within the Coxiella-Containing Vacuole (CCV). The nascent CCV progresses through the 31 endosomal maturation pathway into a phagolysosome, acquiring lysosomal markers as well as 32 acidic pH and active proteases and hydrolases. Approximately 24-48 hours post infection, 33 heterotypic fusion between the CCV and host endosomes/lysosomes leads to CCV expansion 34 and subsequent bacterial replication in the mature CCV. Initial CCV acidification is required to 35 activate C. burnetii metabolism and the Type 4B Secretion System (T4BSS), which secretes 36 effector proteins required for CCV maturation. However, we recently found that the mature CCV 37 is less acidic (pH~5.2) than lysosomes (pH~4.8). Further, CCV acidification to pH~4.8 causes C. 38 burnetii lysis, suggesting C. burnetii actively regulates CCV pH. Because heterotypic fusion with 39 host endosomes/lysosomes may influence CCV pH, we investigated endosomal maturation in 40 cells infected with wildtype (WT) or T4BSS mutant (dotA) C. burnetii. We observed significantly 41 fewer LAMP1-positive lysosomes, along with less acidic "mature" endosomes (pH~5.8), in WT-42 infected cells, compared to mock or dotA-infected cells. Further, while endosomes progressively 43 acidified from the periphery (pH~5.5) to the perinuclear area (pH~4.7) in both mock and dotA-44 infected cells, endosomes did not acidify beyond pH~5.2 in WT-infected cells, indicating that the 45 C. burnetii T4BSS inhibits endosomal maturation. Finally, increasing the number of acidic 46 lysosomes by overexpressing the transcription factor EB inhibited C. burnetii growth, indicating 47 lysosomes are detrimental to C. burnetii. Overall, our data suggest that C. burnetii regulates CCV 48 pH, possibly by reducing the number of host lysosomes available for heterotypic fusion. 49 50 51 3 52 Author summary 53 54 The obligate intracellular bacterium Coxiella burnetii causes human Q fever, which manifests as 55 a flu-like illness but can develop into a life-threatening and difficult to treat endocarditis. C. burnetii, 56in contrast to many other intracellular bacteria, thrives within a lysosome-like vacuole in host cells. 57However, we previously found that the C. burnetii vacuole is not as acidic as lysosomes and 58 increased acidification kills the bacteria, suggesting that C. burnetii regulates the pH of its vacuole. 59Here, we discovered that C. burnetii blocks endosomal maturation and acidification during hostresulting in fewer lysosomes in the host cell. Moreover, increasing lysosomes in the 61 host cells blocked C. burnetii growth. Together, our study suggests that C. burnetii regulates 62 vacuole acidity and blocks endosomal acidification in order to produce a permissive intracellular 63 niche. 64 65 79 infection, phagosome expansion, presumably through fusion with the endocytic pathway, gives 80 rise to a large acidic phagolysosomal-like compartment termed the Coxiella-containing vacuole 81 (CCV) [...

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Quantitative Dextran Trafficking to the Coxiella burnetii Parasitophorous Vacuole

Cited by 4 publications

References 20 publications

Coxiella burnetii Type 4B Secretion System-dependent manipulation of endolysosomal maturation is required for bacterial growth

Coxiella burnetii Type 4B Secretion System-dependent manipulation of endolysosomal maturation is required for bacterial growth

Measuring pH of the Coxiella burnetii Parasitophorous Vacuole

Coxiella burnetiiType 4B Secretion System-dependent manipulation of endolysosomal maturation is required for bacterial growth

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