Summary The ability to create and maintain a specialized organelle that supports bacterial replication is an important virulence property for many intracellular pathogens. Living in a membrane-bound vacuole presents inherent challenges including the need to remodel a plasma membrane-derived organelle into a novel structure that will expand and provide essential nutrients to support replication, while also having the vacuole avoid membrane transport pathways that target bacteria for destruction in lysosomes. It is clear that pathogenic bacteria use different strategies to accomplish these tasks. The dynamics by which host Rab GTPases associate with pathogen-occupied vacuoles provides insight into the mechanisms used by different bacteria to manipulate host membrane transport. In this review we highlight some of the strategies bacteria use to maintain a pathogen-occupied vacuole by focusing on the Rab proteins involved in biogenesis and maintenance of these novel organelles.

Summary Autophagy is a conserved membrane transport pathway used to destroy pathogenic microbes that access the cytosol of cells. The intracellular pathogen Legionella pneumophila interferes with autophagy by delivering an effector protein, RavZ, into the host cytosol. RavZ acts by cleaving membrane-conjugated Atg8/LC3 proteins from preautophagosomal structures. Its remarkable efficiency allows minute quantities of RavZ to block autophagy throughout the cell. To understand how RavZ targets preautophagosomes and specifically acts only on membrane-associated Atg8 proteins, we elucidated its structure. Revealed is a catalytic domain related in fold to Ulp-family deubiquitinase-like enzymes and a C-terminal PI3P-binding module. RavZ targets the autophagosome via the PI3P-binding module and a catalytic domain helix and preferentially binds high-curvature membranes, intimating localization to highly curved domains in autophagosome intermediate membranes. RavZ-membrane interactions enhance substrate affinity, providing a mechanism for interfacial activation that may also be used by host autophagy proteins engaging only lipidated Atg8 proteins.

The gram-negative bacterial pathogen Legionella pneumophila creates a novel organelle inside of eukaryotic host cells that supports intracellular replication. The L. pneumophila-containing vacuole evades fusion with lysosomes and interacts intimately with the host endoplasmic reticulum (ER). Although the natural hosts for L. pneumophila are free-living protozoa that reside in freshwater environments, the mechanisms that enable this pathogen to replicate intracellularly also function when mammalian macrophages phagocytose aerosolized bacteria, and infection of humans by L. pneumophila can result in a severe pneumonia called Legionnaires' disease. A bacterial type IVB secretion system called Dot/Icm is essential for intracellular replication of L. pneumophila. The Dot/Icm apparatus delivers over 300 different bacterial proteins into host cells during infection. These bacterial proteins have biochemical activities that target evolutionarily conserved host factors that control membrane transport processes, which results in the formation of the ER-derived vacuole that supports L. pneumophila replication. This review highlights research discoveries that have defined interactions between vacuoles containing L. pneumophila and the host ER. These studies reveal how L. pneumophila creates a vacuole that supports intracellular replication by subverting host proteins that control biogenesis and fusion of early secretory vesicles that exit the ER and host proteins that regulate the shape and dynamics of the ER. In addition to recruiting ER-derived membranes for biogenesis of the vacuole in which L. pneumophila replicates, these studies have revealed that this pathogen has a remarkable ability to interfere with the host's cellular process of autophagy, which is an ancient cell autonomous defense pathway that utilizes ER-derived membranes to target intracellular pathogens for destruction. Thus, this intracellular pathogen has evolved multiple mechanisms to control membrane transport processes that center on the involvement of the host ER.

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