Dot/Icm-Translocated Proteins Important for Biogenesis of theCoxiella burnetii-Containing Vacuole Identified by Screening of an Effector Mutant Sublibrary

ABSTRACTCoxiella burnetiiis an intracellular pathogen that replicates in a lysosome-derived vacuole. A determinant necessary forC. burnetiivirulence is the Dot/Icm type IVB secretion system (T4SS). The Dot/Icm system delivers more than 100 proteins, called type IV effectors (T4Es), across the vacuolar membrane into the host cell cytosol. Several T4Es have been shown to be important for vacuolar biogenesis. Here, transposon (Tn) insertion sequencing technology (INSeq) was used to identifyC. burnetiiNine Mile phase II mutants in an arrayed library, which facilitated the identification and clonal isolation of mutants deficient in 70 different T4E proteins. These effector mutants were screened in HeLa cells for deficiencies inCoxiella-containing vacuole (CCV) biogenesis. This screen identified and validated seven new T4Es that were important for vacuole biogenesis. Loss-of-function mutations incbu0414(coxH1),cbu0513,cbu0978(cem3),cbu1387(cem6),cbu1524(caeA),cbu1752, orcbu2028resulted in a small-vacuole phenotype. These seven mutant strains produced small CCVs in all cells tested, which included macrophage-like cells. Thecbu2028::Tn mutant, though unable to develop large CCVs, had intracellular replication rates similar to the rate of the parental strain ofC. burnetii, whereas the other six effector mutants defective in CCV biogenesis displayed significant reductions in intracellular replication. Vacuoles created by thecbu0513::Tn mutant did not accumulate lipidated microtubule-associated protein 1A/1B light chain 3 (LC3-II), suggesting a failure in fusion of the CCV with autophagosomes. These seven T4E proteins add to the growing repertoire ofC. burnetiifactors that contribute to CCV biogenesis.

Multiple methods of visualizing the yeast vacuole permit evaluation of its morphology and inheritance during the cell cycle.

The vacuole of the yeast Saccharomyces cerevisiae was visualized with three unrelated fluorescent dyes: FITC-dextran, quinacrine, and an endogenous fluorophore produced in ade2 yeast. FITC-dextran, which enters cells by endocytosis, had been previously developed as a vital stain for yeast vacuoles. Quinacrine, which diffuses across membranes and accumulates in acidic compartments in mammalian cells, can also be used as a marker for yeast vacuoles. ade2 yeast accumulate an endogenous fluorophore in their vacuoles. Using these stains, yeast were examined for vacuole morphology throughout the cell division cycle. In both the parent cell and the bud, a single vacuole was the most common morphology at every stage. Two or more vacuoles could also be found in the mother cell or in the bud; however, this morphology was not correlated with any stage of the cell division cycle. Even small buds (in early S phase) often contained a small vacuole. By the time the bud was half the diameter of the mother cell, it almost always bore a vacuole. This picture of vacuole division and segregation differs from what is seen with synchronized cultures. In ade2 yeast, the bud usually inherits a substantial portion of its vacuole contents from the mother cell. We propose that vacuolar segregation is accomplished by vesicular traffic between the parent cell and the bud.