Overall, our results hint at the importance of monoubiquitination of AVM-associated proteins throughout the A. phagocytophilum infection cycle in promyelocytic HL-60 cells as well as endothelial cells, as a comparable degree of ubiquitination of the AVM was observed for infected RF/6A cells. Considerably, fewer ApVs of infected ISE6 cells exhibited ubiquitination than infected mammalian cells. Either AVM ubiquitination does not play a prominent role in A. phagocytophilum infection of ISE6 cells or association of ubiquitinated proteins with the AVM may be temporally regulated during infection of ISE6

cells. By accruing monoubiquitinated click here proteins that localize and direct traffic to endocytic compartments, A. phagocytophilum conceivably camouflages its vacuolar membrane as a means for avoiding lysosomal targeting. Support for this possibility comes from the precedent that the ApV selectively recruits Rab GTPases that are predominantly associated with recycling endosomes while concomitantly check details blocking recruitment of Rabs that are important for lysosomal delivery. Tetracycline treatment of infected cells culminates in the dissociation of recycling endosome-associated Rabs with the concomitant association of the lysosomal markers Rab7

and LAMP-1 (Huang et al., 2010a). Confocal microscopic analysis of fixed cells reveals that no more than 52.6% ± 4.2% or 61.0% ± 6.2% ApVs in HL-60 cells or RF/6A cells, respectively, are positive for ubiquitin at any time point examined. A highly similar trend occurs when one examines the percentages of ApVs to which GFP-tagged recycling endosome-associated Rab GTPases localize (Huang et al., 2010a). Ubiquitin machinery, like Rab GTPases, dynamically cycles on- and off-target organelle membranes (Grabbe et al., 2011; Segev, 2011). Thus, examining fixed A. phagocytophilum-infected cells provides a snapshot of the AVMs that are monoubiquitinated or have associated Rab GTPases at the instant at which preservative was added. Protein kinase N1 Several bacterial effectors have been shown to exploit the host cell’s ubiquitination system to diversify or regulate their biological functions. Several effectors secreted by intracellular bacterial pathogens

mimic the activities of E3 ubiquitin ligases to spatially or temporally regulate host or bacterial proteins (Kubori & Galan, 2003; Kubori et al., 2010). Alternatively, the ubiquitination of other bacterial effectors regulates their activities and subcellular localization rather than serve as a signal for their proteasomal degradation (Marcus et al., 2002; Knodler et al., 2009; Patel et al., 2009). As AVM monoubiquitination is bacterial protein synthesis-dependent, it is plausible that A. phagocytophilum encodes one or more effectors that either may recruit monoubiquitinated host proteins to the AVM or may be monoubiquitinated themselves. To date, only three A. phagocytophilum-encoded AVM proteins – APH_1387, APH_0032, and AptA – have been identified (Huang et al.