Replication of Coxiella burnetii in a Lysosome-Like Vacuole Does Not Require Lysosomal Hydrolases

Miller, Heather; Hoyt, Forrest; Heinzen, Robert A.

doi:10.1128/iai.00493-19

Cited by 10 publications

(10 citation statements)

References 48 publications

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“…Lysosomal enzymes such as proteases and cathepsins are most active against their substrates in the pH range 3-5 [64,65] and optimally active at pH 4.5 [64]. While the CCV is known to be proteolytically active [5,6], lysosomal hydrolases are not required for growth [66]. Based on our findings that the CCV is less acidic than lysosomes, the mature CCV may not be optimal for lysosomal enzyme activity.…”

Section: Discussionmentioning

confidence: 81%

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: Discussionmentioning

confidence: 81%

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

et al. 2019

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“…However, these prior findings are contradicted by Howe et al (2010) who reported that CCV harboring virulent or avirulent variants of C. burnetii mature similarly through the endolysosomal route and reach the phagolysosome stage containing proteolytically active cathepsins. Another study supports the notion that lysosomal hydrolases, including Cts D, are not required for C. burnetii growth or viability in CCV (Miller et al, 2019). Specific antibodies found in chronic Q fever patients likely favor C. burnetii replication in human MDM, because opsonization of bacteria with these antibodies prevented the phagosome conversion.…”

Section: Coxiella Burnetiimentioning

confidence: 53%

Cathepsins in Bacteria-Macrophage Interaction: Defenders or Victims of Circumstance?

Szulc-Dąbrowska

Bossowska-Nowicka

Struzik

et al. 2020

Front. Cell. Infect. Microbiol.

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Macrophages are the first encounters of invading bacteria and are responsible for engulfing and digesting pathogens through phagocytosis leading to initiation of the innate inflammatory response. Intracellular digestion occurs through a close relationship between phagocytic/endocytic and lysosomal pathways, in which proteolytic enzymes, such as cathepsins, are involved. The presence of cathepsins in the endo-lysosomal compartment permits direct interaction with and killing of bacteria, and may contribute to processing of bacterial antigens for presentation, an event necessary for the induction of antibacterial adaptive immune response. Therefore, it is not surprising that bacteria can control the expression and proteolytic activity of cathepsins, including their inhibitors – cystatins, to favor their own intracellular survival in macrophages. In this review, we summarize recent developments in defining the role of cathepsins in bacteria-macrophage interaction and describe important strategies engaged by bacteria to manipulate cathepsin expression and activity in macrophages. Particularly, we focus on specific bacterial species due to their clinical relevance to humans and animal health, i.e., Mycobacterium, Mycoplasma, Staphylococcus, Streptococcus, Salmonella, Shigella, Francisella, Chlamydia, Listeria, Brucella, Helicobacter, Neisseria, and other genera.

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“…[9] and Miller et al . [10] with our GNPTAB −/− cells. Despite the absence of GlcNAc‐1‐phosphotransferase, confocal immunofluorescence microscopy showed that the pro‐cathepsin D in the GNPTAB −/− cells colocalized with the lysosomal membrane protein, LAMP1, in enlarged late endosomes/lysosomes, indicating transport of the aspartyl protease to this organelle (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…This indicated that cathepsin D protein expression was markedly elevated in the GNPTAB À/À HeLa cells. Moreover, we and others noted that these cells accumulate only procathepsin D while failing to convert this single-chain molecule to the two-chain form [8][9][10]. Here, we demonstrate that in the absence of the M-6-P pathway, cathepsin D gene expression in HeLa cells is elevated fivefold at the mRNA level but this upregulation occurs independent of either Transcription Factor EB (TFEB) or Transcription Factor E3 (TFE3), two transcription factors that function as master regulators of lysosomal homeostasis [11].…”

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confidence: 78%

Elevated mRNA expression and defective processing of cathepsin D in HeLa cells lacking the mannose 6‐phosphate pathway

Liu

Doray

2021

FEBS Open Bio

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Disruption of the mannose 6‐phosphate (M‐6‐P) pathway in HeLa cells by inactivation of the GNPTAB gene, which encodes the α/β subunits of GlcNAc‐1‐phosphotransferase, results in missorting of newly synthesized lysosomal acid hydrolases to the cell culture media instead of transport to the endolysosomal system. We previously demonstrated that the majority of the lysosomal aspartyl protease, cathepsin D, is secreted in these GNPTAB−/− HeLa cells. However, the intracellular content of cathepsin D in these cells was still greater than that of WT HeLa cells which retained most of the protease, indicating a marked elevation of cathepsin D expression in response to abrogation of the M‐6‐P pathway. Here, we demonstrate that HeLa cells lacking GlcNAc‐1‐phosphotransferase show a fivefold increase in cathepsin D mRNA expression over control cells, accounting for the increase in cathepsin D at the protein level. Further, we show that this increase at the mRNA level occurs independent of the transcription factors TFEB and TFE3. The intracellular cathepsin D can still be trafficked to lysosomes in the absence of the M‐6‐P pathway, but fails to undergo proteolytic processing into the fully mature heavy and light chains. Uptake experiments performed by feeding GNPTAB−/− HeLa cells with various phosphorylated cathepsins reveal that only cathepsin B is capable of partially restoring cleavage, providing evidence for a role for cathepsin B in the proteolytic processing of cathepsin D.

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Replication of Coxiella burnetii in a Lysosome-Like Vacuole Does Not Require Lysosomal Hydrolases

Cited by 10 publications

References 48 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

Cathepsins in Bacteria-Macrophage Interaction: Defenders or Victims of Circumstance?

Elevated mRNA expression and defective processing of cathepsin D in HeLa cells lacking the mannose 6‐phosphate pathway

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