Uropathogenic Escherichia coli (UPEC), the primary causative agent of urinary tract infections, typically express filamentous adhesive organelles called type 1 pili that mediate both bacterial attachment to and invasion of bladder urothelial cells. Several host proteins have previously been identified as receptors for type 1 pili, but none have been conclusively shown to promote UPEC entry into host bladder cells. Using overlay assays with FimH, the purified type 1 pilus adhesin, and mass spectroscopy, we have identified β1 and α3 integrins as key host receptors for UPEC. FimH recognizes N-linked oligosaccharides on these receptors, which are expressed throughout the urothelium. In a bladder cell culture system, β1 and α3 integrin receptors co-localize with invading type 1–piliated bacteria and F-actin. FimH-mediated bacterial invasion of host bladder cells is inhibited by β1 and α3 integrin–specific antibodies and by disruption of the β1 integrin gene in the GD25 fibroblast cell line. Phosphorylation site mutations within the cytoplasmic tail of β1 integrin that alter integrin signaling also variably affect UPEC entry into host cells, by either attenuating or boosting invasion frequencies. Furthermore, focal adhesion and Src family kinases, which propagate integrin-linked signaling and downstream cytoskeletal rearrangements, are shown to be required for FimH-dependent bacterial invasion of target host cells. Cumulatively, these results indicate that β1 and α3 integrins are functionally important receptors for type 1 pili–expressing bacteria within the urinary tract and possibly at other sites within the host.

SummaryThe FimH adhesin, localized at the distal tips of type 1 pili, binds mannose-containing glycoprotein receptors like a3b1 integrins and stimulates bacterial entry into target host cells. Strains of uropathogenic Escherichia coli (UPEC), the major cause of urinary tract infections, utilize FimH to invade bladder epithelial cells. Here we set out to define the mechanism by which UPEC enters host cells by investigating four of the major entry routes known to be exploited by invasive pathogens: caveolae, clathrin, macropinocytosis and secretory lysosomes. Using pharmacological inhibitors in combination with RNA interference against specific endocytic pathway components, mutant host cell lines and a mouse infection model system, we found that type 1 pili-dependent bacterial invasion of host cells occurs via a cholesterol-and dynamin-dependent phagocytosis-like mechanism. This process did not require caveolae or secretory lysosomes, but was modulated by calcium levels, clathrin, and cooperative input from the primary clathrin adaptor AP-2 and a subset of alternate adaptors comprised of Numb, ARH and Dab2. These alternate clathrin adaptors recognize NPXY motifs, as found within the cytosolic tail of b1 integrin, suggesting a functional link between the engagement of integrin receptors by FimH and the clathrin-dependent uptake of type 1-piliated bacteria.

Branching morphogenesis, the process by which cells or tissues generate tree-like networks that function to increases surface area or in contacting multiple targets, is a common developmental motif in multicellular organisms. We use Drosophila tracheal terminal cells, a component of the insect respiratory system, to investigate branching morphogenesis that occurs on the single cell level. Here, we show that the exocyst, a conserved protein complex that facilitates docking and tethering of vesicles at the plasma membrane, is required for terminal cell branch outgrowth. We find that exocyst-deficient terminal cells have highly truncated branches and show an accumulation of vesicles within their cytoplasm and are also defective in subcellular lumen formation. We also show that vesicle trafficking pathways mediated by the Rab GTPases Rab10 and Rab11 are redundantly required for branch outgrowth. In terminal cells, the PAR-polarity complex is required for branching, and we find the PAR complex is required for proper membrane localization of the exocyst, thus identifying a molecular link between the branching and outgrowth programs. Together, our results suggest a model where exocyst mediated vesicle trafficking facilitates branch outgrowth, while de novo branching requires cooperation between the PAR and exocyst complexes.

The processes that generate cellular morphology are not well understood. To investigate this problem, we use Drosophila melanogaster tracheal terminal cells, which undergo two distinct morphogenetic processes: subcellular branching morphogenesis and subcellular apical lumen formation. Here we show these processes are regulated by components of the PAR-polarity complex. This complex, composed of the proteins Par-6, Bazooka (Par-3), aPKC, and Cdc42, is best known for roles in asymmetric cell division and apical/basal polarity. We find Par-6, Bazooka, and aPKC, as well as known interactions between them, are required for subcellular branch initiation, but not for branch outgrowth. By analysis of single and double mutants, and isolation of two novel alleles of Par-6, one of which specifically truncates the Par-6 PDZ domain, we conclude that dynamic interactions between apical PAR-complex members control the branching pattern of terminal cells. These data suggest that canonical apical PAR-complex activity is required for subcellular branching morphogenesis. In addition, we find the PAR proteins are downstream of the FGF pathway that controls terminal cell branching. In contrast, we find that while Par-6 and aPKC are both required for subcellular lumen formation, neither Bazooka nor a direct interaction between Par-6 and aPKC is needed for this process. Thus a novel, noncanonical role for the polarity proteins Par-6 and aPKC is used in formation of this subcellular apical compartment. Our results demonstrate that proteins from the PAR complex can be deployed independently within a single cell to control two different morphogenetic processes. FOR most cell types, morphology is key to cell function. A dramatic example of this association is seen in cells that undergo subcellular branching morphogenesis. In this process, cells send out extensions from their plasma membranes, which grow and undergo bifurcation events to form complex, branched networks. Examples of subcellular branching morphogenesis are seen in glial oligodendrocytes (Bauer et al. 2009) and in dendritic cells of the mammalian immune system (Makala and Nagasawa 2002), but by far the best studied examples of this process are in neurons (reviewed by Gibson and Ma 2011;Jan and Jan 2010). Indeed, neurons are frequently categorized entirely by differences in their branching morphologies (see Puelles 2009). However, despite the importance of subcellular branching morphogenesis, little is known about the molecular mechanisms that organize distinctive subcellular branching patterns.We are studying the process of subcellular branching morphogenesis in Drosophila tracheal terminal cells, a component of the insect respiratory system. Terminal cells reside at the ends of a network of cellular tubes that functions in delivering air to internal tissues (Guillemin et al. 1996). The cells are specified during embryogenesis, primarily through a process of competitive FGF signaling and lateral inhibition among tracheal precursors (Llimargas 1999;Ghabrial and Krasno...

Gut microbiota facilitate many aspects of human health and development, but dysbiotic microbiota can promote hyperplasia and inflammation and contribute to human diseases such as cancer. Human patients infected with the gastric cancer-causing bacterium Helicobacter pylori have altered microbiota; however, whether dysbiosis contributes to disease in this case is unknown. Many H. pylori human disease phenotypes are associated with a potent virulence protein, CagA, which is translocated into host epithelial cells where it alters cell polarity and manipulates host-signaling pathways to promote disease. We hypothesized that CagA alone could contribute to H. pylori pathogenesis by inducing microbial dysbiosis that promotes disease. Here we use a transgenic Drosophila model of CagA expression to genetically disentangle the effects of the virulence protein CagA from that of H. pylori infection. We found that expression of CagA within Drosophila intestinal stem cells promotes excess cell proliferation and is sufficient to alter host microbiota. Rearing CagA transgenic flies germ-free revealed that the dysbiotic microbiota contributes to cell proliferation phenotypes and also elicits expression of innate immune components, Diptericin and Duox. Further investigations revealed interspecies interactions are required for this dysbiotic CagA-dependent microbiota to promote proliferation in CagA transgenic and healthy control Drosophila. Our model establishes that CagA can alter gut microbiota and exacerbate cell proliferation and immune phenotypes previously attributed to H. pylori infection. This work provides valuable new insights into the mechanisms by which interactions between a specific virulence factor and the resident microbiota can contribute to the development and progression of disease.