LAMP proteins account for the maturation delay during the establishment of theCoxiella burnetii-containing vacuole

Abstract

The obligate intracellular pathogen Coxiella burnetii replicates in a large phagolysosomal-like vacuole. Currently, both host and bacterial factors required for creating this replicative parasitophorous C. burnetii-containing vacuole (PV) are poorly defined. Here, we assessed the contributions of the most abundant proteins of the lysosomal membrane, LAMP-1 and LAMP-2, to the establishment and maintenance of the PV. Whereas these proteins were not critical for uptake of C. burnetii, they influenced the intracellular replication of C. burnetii. In LAMP-1/2 double-deficient fibroblasts as well as in LAMP-1/2 knock-down cells, C. burnetii establishes a significantly smaller, yet faster maturing vacuole, which harboured more bacteria. The accelerated maturation of PVs in LAMP double-deficient fibroblasts, which was partially or fully reversed by ectopic expression of LAMP-1 or LAMP-2, respectively, was characterized by an increased fusion rate with endosomes, lysosomes and bead-containing phagosomes, but not by different fusion kinetics with autophagy vesicles. These findings establish that LAMP proteins are critical for the maturation delay of PVs. Unexpectedly, neither the creation of the spacious vacuole nor the delay in maturation was found to be prerequisites for the intracellular replication of C. burnetii.

Elevated Cholesterol in the Coxiella burnetii Intracellular Niche Is Bacteriolytic

“…We utilized the fluorescein derivative Oregon Green 488, given the improved pH sensitivity of Oregon Green 488 in acidic environments (41). A recent study on the role of lysosome-associated membrane glycoprotein (LAMP) proteins in PV maturation found the pH of the PV to be between 4.0 and 4.5 using LysoSensor Yellow/Blue DND-160 (50). In our hands, this reagent stained the bacteria and was not free in the PV lumen, prompting us to utilize the dual dextran labeling approach (41).…”

Right on Q: Genetics begin to Unravel Coxiella Burnetii host cell Interactions

“…Following uptake, Coxiella is confined to a nascent vacuole that transits through the default endocytic maturation pathway from early endosome to phagolysosome [31,56,57]. Full maturation of the Coxiella-containing vacuole (CCV) to fusion with lysosomes is indicated by the presence of active cathepsins [31,[56][57][58][59][60].…”

“…Full maturation of the Coxiella-containing vacuole (CCV) to fusion with lysosomes is indicated by the presence of active cathepsins [31,[56][57][58][59][60]. Coxiella is uniquely adapted among intravacuolar bacterial pathogens to replicate in an acidic phagolysosome-like compartment that retains significant hydrolytic activity [31,57].…”

“…Several lines of evidence indicate the CCV fuses with autophagosomes, and that this interaction benefits Coxiella. The CCV clearly decorates with the autophagy markers LC3 [56,57,59,60,[67][68][69], Beclin 1 [59,70], Rab24 [70] and p62 [67]. Autophagosome interactions with the CCV occur within a few hours of infection and require Coxiella protein synthesis and an active type 4B secretion system (T4BSS) [56,67].…”

“…Clathrin-mediated vesicular trafficking is an important component of CCV endosome recruitment as depletion of clathrin or the clathrin-adaptor protein AP2, inhibits vacuole expansion and Coxiella replication [73]. Through an unknown mechanism, the major lysosmal membrane proteins LAMP-1 and LAMP-2 also participate in CCV formation by increasing the rate of endosome fusion [57].…”