The enteric bacterial pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium), utilizes two type III secretion systems (T3SSs) to invade host cells, survive and replicate intracellularly. T3SS1 and its dedicated effector proteins are required for bacterial entry into non-phagocytic cells and establishment and trafficking of the nascent Salmonella-containing vacuole (SCV). Here we identify the first T3SS1 effector required to maintain the integrity of the nascent SCV as SopF. SopF associates with host cell membranes, either when translocated by bacteria or ectopically expressed. Recombinant SopF binds to multiple phosphoinositides in protein-lipid overlays, suggesting that it targets eukaryotic cell membranes via phospholipid interactions. In yeast, the subcellular localization of SopF is dependent on the activity of Mss4, a phosphatidylinositol 4-phosphate 5-kinase that generates PI(4,5)P₂ from PI(4)P, indicating that membrane recruitment of SopF requires specific phospholipids. Ectopically expressed SopF partially colocalizes with specific phosphoinositide pools present on the plasma membrane in mammalian cells and with cytoskeletal-associated markers at the leading edge of cells. Translocated SopF concentrates on plasma membrane ruffles and around intracellular bacteria, presumably on the SCV. SopF is not required for bacterial invasion of non-phagocytic cells but is required for maintenance of the internalization vacuole membrane as infection with a S. Typhimurium ΔsopF mutant led to increased lysis of the SCV compared to wild type bacteria. Our structure-function analysis shows that the carboxy-terminal seven amino acids of SopF are essential for its membrane association in host cells and to promote SCV membrane stability. We also describe that SopF and another T3SS1 effector, SopB, act antagonistically to modulate nascent SCV membrane dynamics. In summary, our study highlights that a delicate balance of type III effector activities regulates the stability of the Salmonella internalization vacuole. Pathogenic bacteria that adopt an intracellular lifestyle must create a specialized niche that supports their replication while avoiding detection and killing by the host. The foodborne pathogen, S. Typhimurium, invades epithelial cells lining the intestine and establishes residence within a host-derived membrane-bound compartment called the Salmonella-containing vacuole (SCV). To create this intracellular niche, S. Typhimurium relies on two type III secretion systems (T3SS). These are specialized needle-like devices that inject bacterial effector proteins into host cells, and thereby manipulate host cell signalling and immune responses. T3SS1 delivers effectors that are required for early events in the infectious cycle. In this study we describe a new T3SS1 effector called SopF that is unique to Salmonella spp. We show that SopF associates with host cell membranes by binding to phosphoinositides, which are specialized lipids present in eukaryotic cellular membranes, and that SopF is required for maintaining the integrity of the nascent SCV membrane. Salmonella has therefore evolved to reside within a membrane-bound compartment by acquiring a unique type III effector whose actions promote vacuole stability.

Coxiella burnetii is an intracellular bacterial pathogen that infects alveolar macrophages and replicates within a unique lysosome-derived vacuole. When Coxiella is trafficked to a host cell lysosome the essential Dot/Icm type IV secretion system is activated allowing over 130 bacterial effector proteins to be translocated into the host cytosol. This cohort of effectors is believed to manipulate host cell functions to facilitate Coxiella-containing vacuole (CCV) biogenesis and bacterial replication. Transposon mutagenesis has demonstrated that the Dot/Icm effector Cig57 is required for CCV development and intracellular replication of Coxiella. Here, we demonstrate a role for Cig57 in subverting clathrin-mediated traffic through its interaction with FCHO2, an accessory protein of clathrin coated pits. A yeast two-hybrid screen identified FCHO2 as a binding partner of Cig57 and this interaction was confirmed during infection using immunoprecipitation experiments. The interaction between Cig57 and FCHO2 is dependent on one of three endocytic sorting motif encoded by Cig57. Importantly, complementation analysis demonstrated that this endocytic sorting motif is required for full function of Cig57. Consistent with the intracellular growth defect in cig57-disrupted Coxiella, siRNA gene silencing of FCHO2 or clathrin (CLTC) inhibits Coxiella growth and CCV biogenesis. Clathrin is recruited to the replicative CCV in a manner that is dependent on the interaction between Cig57 and FCHO2. Creation of an FCHO2 knockout cell line confirmed the importance of this protein for CCV expansion, intracellular replication of Coxiella and clathrin recruitment to the CCV. Collectively, these results reveal Cig57 to be a significant virulence factor that co-opts clathrin-mediated trafficking, via interaction with FCHO2, to facilitate the biogenesis of the fusogenic Coxiella replicative vacuole and enable intracellular success of this human pathogen. Human Q fever is caused by the intracellular bacterium Coxiella burnetii. Successful infection of human cells relies on a Dot/Icm secretion system and the translocation of effector proteins into the host cell cytosol. The functions of many Coxiella effector proteins, and their contribution to bacterial growth and host manipulation, remain unknown. We show that a unique effector, Cig57, has an important role in manipulation of host cellular clathrin-mediated trafficking. In particular, Cig57 binds FCHO2, a protein involved in formation of clathrin-coated vesicles, in a manner that is dependent on a tyrosine-based endocytic sorting motif. Through engaging proteins in the clathrin pathway, Cig57 facilitates expansion of the Coxiella replicative vacuole and enables the pathogen to replicate to large numbers. Thus, we identify a relationship between a host process and a key virulence protein that are required for pathogen success.

Coxiella burnetii is an intracellular bacterial pathogen which causes Q fever, a human infection with the ability to cause chronic disease with potentially life-threatening outcomes. In humans, Coxiella infects alveolar macrophages where it replicates to high numbers in a unique, pathogen-directed lysosome-derived vacuole. This compartment, termed the Coxiella-containing vacuole (CCV), has a low internal pH and contains markers both of lysosomes and autophagosomes. The CCV membrane is also enriched with CLTC (clathrin heavy chain) and this contributes to the success of the CCV. Here, we describe a role for CLTC, a scaffolding protein of clathrin-coated vesicles, in facilitating the fusion of autophagosomes with the CCV. During gene silencing of CLTC, CCVs are unable to fuse with each other, a phenotype also seen when silencing genes involved in macroautophagy/autophagy. MAP1LC3B/LC3B, which is normally observed inside the CCV, is excluded from CCVs in the absence of CLTC. Additionally, this study demonstrates that autophagosome fusion contributes to CCV size as cell starvation and subsequent autophagy induction leads to further CCV expansion. This is CLTC dependent, as the absence of CLTC renders autophagosomes no longer able to contribute to the expansion of the CCV. This investigation provides a functional link between CLTC and autophagy in the context of Coxiella infection and highlights the CCV as an important tool to explore the interactions between these vesicular trafficking pathways.

Legionella organisms are environmental bacteria and accidental human pathogens that can cause severe pneumonia, termed Legionnaires' disease. These bacteria replicate within a pathogen-derived vacuole termed the Legionella -containing vacuole (LCV). Our understanding of the development and dynamics of this vacuole is based on extensive analysis of Legionella pneumophila . Here, we have characterized the Legionella longbeachae replicative vacuole ( longbeachae -LCV) and demonstrated that, despite important genomic differences, key features of the replicative LCV are comparable to those of the LCV of L. pneumophila ( pneumophila -LCV). We constructed a Dot/Icm-deficient strain by deleting dotB and demonstrated the inability of this mutant to replicate inside THP-1 cells. L. longbeachae does not enter THP-1 cells as efficiently as L. pneumophila , and this is reflected in the observation that translocation of BlaM-RalF _LLO (where RalF _LLO is the L. longbeachae homologue of RalF) into THP-1 cells by the L. longbeachae Dot/Icm system is less efficient than that by L. pneumophila . This difference is negated in A549 cells where L. longbeachae and L. pneumophila infect with similar entry dynamics. A β-lactamase assay was employed to demonstrate the translocation of a novel family of proteins, the R ab- l ike e ffector (Rle) proteins. Immunofluorescence analysis confirmed that these proteins enter the host cell during infection and display distinct subcellular localizations, with RleA and RleC present on the longbeachae -LCV. We observed that the host Rab GTPase, Rab1, and the v-SNARE Sec22b are also recruited to the longbeachae -LCV during the early stages of infection, coinciding with the LCV avoiding endocytic maturation. These studies further our understanding of the L. longbeachae replicative vacuole, highlighting phenotypic similarities to the vacuole of L. pneumophila as well as unique aspects of LCV biology.

The entry of pathogens into nonphagocytic host cells has received much attention in the past three decades, revealing a vast array of strategies employed by bacteria and viruses. A method of internalization that has been extensively studied in the context of viral infections is the use of the clathrin-mediated pathway.

Investigating the interactions between bacterial pathogens and their hosts is an important area of biological research. Here, we...