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

Abstract

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...

Formation and Maturation of the Phagosome: A Key Mechanism in Innate Immunity against Intracellular Bacterial Infection

“…Similar to M. tuberculosis , Neisseria gonorrhoeae PorB, the major outer membrane porin, permits Rab5 to be continuously expressed on the early phagosome, resulting in the blockade of phagosome maturation [ 143 ]. Coxiella burnetii also arrests phagosome maturation by consistently expressing Rab5 on early phagosomes [ 144 ]. Marcelo G Binker et al found that N. gonorrhoeae expresses an IgA1 protease that specifically cleaves LAMP-1 to arrest phagosome maturation [ 145 ].…”

Modulation of Host Lipid Pathways by Pathogenic Intracellular Bacteria

“…Specifically, this study showed a correlation of an increase in host cholesterol within the PCV membrane and increases in PCV acidity during later stages of mouse embryonic fibroblast C. burnetii infection [ 56 , 57 ] ( Figure 1 ). Inhibition of bacterial protein synthesis followed by a decrease in sterol recruitment suggests that host sterol composition within the PCV is actively regulated by Coxiella effector proteins, which likely modulate endosomal trafficking and cholesterol acquisition [ 58 , 59 ]. C. burnetii also contains an active sterol reductase, which catalyzes the last step in the cholesterol biosynthesis pathway, implying a possible use of host sterol intermediates for bacterial completion of cholesterol production [ 60 ].…”

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

“…Additionally, HeLa cells overexpressing the transcription factor EB (TFEB), which coordinates expression of lysosomal genes for lysosomal proteases and hydrolases, led to increased Cts B activity, indicating an increase in the number of proteolyticallyactive lysosomes. TFEB-induced lysosome biogenesis significantly reduced C. burnetii CCV formation and intracellular growth due to acidification and increased protease activity (Samanta et al, 2019). These data underscore the importance of Cts B in the control of C. burnetii intracellularly and present a mechanism where a bacterial intracellular niche is created by regulating the acidity of the vacuoles-containing bacteria and blocking endosomal maturation.…”