Measuring pH of the <i>Coxiella burnetii</i> Parasitophorous Vacuole

Samanta, Dhritiman; Gilk, Stacey D.

doi:10.1002/cpmc.38

Cited by 6 publications

(9 citation statements)

References 24 publications

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“…Therefore, in contrast to many other intracellular bacteria which block phagosome-lysosome fusion, including Legionella pneumophila, Mycobacterium tuberculosis, Anaplasma sp., and Yersinia pestis [21][22][23][24][25], C. burnetii survives in the phagolysosomal environment. We recently developed a ratiometric microscopy-based method to measure CCV pH using the pH-sensitive fluorophore Oregon Green 488 [26] and determined the CCV pH to bẽ 5.2 in both HeLa cells and cholesterol-free mouse embryonic fibroblasts (MEFs) [27]. In agreement with our results, a study with Chinese Hamster Ovary (CHO) cells measured pH of intact CCVs to be~5.2 [28].…”

Section: Introductionsupporting

confidence: 75%

“…Given this apparent narrow pH tolerance inside the host cell, we hypothesized that C. burnetii regulates the CCV pH at a less acidic pH than host lysosomes. To confirm that the CCV is indeed less acidic than lysosomes, we compared the CCV pH from wild type (WT) C. burnetii-infected HeLa and murine alveolar macrophage (MH-S) cells to mature endosomal/lysosomal pH of mock-infected cells using a ratiometric microscopy-based method with pH-sensitive Oregon Green 488 dextran and pH-stable Alexa fluor 647 dextran [26]. Oregon Green fluorescence is quenched as the vesicular pH becomes more acidic.…”

Section: Burnetii Regulates CCV and Endosomal Ph In Infected Host mentioning

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: Introductionsupporting

confidence: 75%

Section: Burnetii Regulates CCV and Endosomal Ph In Infected Host mentioning

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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“…Therefore, in contrast to many other intracellular bacteria which block phagosome-lysosome fusion, including Legionella pneumophila, Mycobacterium tuberculosis, Anaplasma sp., and Yersinia pestis [18–22], C. burnetii survives in the phagolysosomal environment. We recently developed a ratiometric microscopy-based method to measure CCV pH using Oregon Green 488, a pH-sensitive fluorophore [23] and determined the CCV pH to be ~5.2 in both HeLa cells and cholesterol-free mouse embryonic fibroblasts (MEFs) [24]. In agreement with our results, a study with Chinese hamster ovary (CHO) cells measured pH of intact CCVs to be ~5.2 [25].…”

Section: Introductionsupporting

confidence: 70%

“…Given this apparent narrow pH tolerance inside the host cell, we hypothesized that C. burnetii regulates the CCV pH at a less acidic pH than host lysosomes. To test this hypothesis, we compared the CCV pH from wild type (WT) C. burnetii -infected HeLa cells at 3 days post infection (dpi) to mature endosomal/lysosomal pH of mock-infected cells using a ratiometric microscopy-based method using pH-sensitive Oregon Green 488 dextran and pH-stable Alexa fluor 647 dextran [23]. Dextran is internalized through fluid-phase endocytosis and delivered to the CCV lumen through CCV-endosome fusion (Fig 1A).…”

Section: Resultsmentioning

confidence: 99%

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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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Coxiella burnetii-Infected NK Cells Release Infectious Bacteria by Degranulation

et al. 2020

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Natural killer (NK) cells are critically involved in the early immune response against various intracellular pathogens, including Coxiella (C.) burnetii and Chlamydia (C.) psittaci. Chlamydia-infected NK cells functionally mature, induce cellular immunity, and protect themselves by killing the bacteria in secreted granules. Here, we report that infected NK cells do not allow intracellular multi-day growth of Coxiella, as is usually observed in other host cell types. C. burnetii-infected NK cells display maturation and IFN-γ secretion, as well as the release of Coxiella-containing lytic granules. Thus, NK cells possess a potent program to restrain and expel different types of invading bacteria via degranulation. Strikingly though, in contrast to Chlamydia, expulsed Coxiella largely retain their infectivity and, hence, escape the cell-autonomous self-defense mechanism in NK cells.

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Measuring pH of the Coxiella burnetii Parasitophorous Vacuole

Cited by 6 publications

References 24 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

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

Coxiella burnetii-Infected NK Cells Release Infectious Bacteria by Degranulation

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