Role for myosin II in regulating positioning of Salmonella-containing vacuoles and intracellular replication.

Salmonella enterica serovar Typhimurium grows within host cells in a permissive compartment termed the Salmonella-containing vacuole (SCV). These bacteria use two distinct type III secretion systems (T3SS) to deliver virulence proteins (effectors) into cells. Effectors secreted by the Salmonella pathogenicity island 1 (SPI-1)-encoded T3SS mediate invasion and early SCV maturation steps, while those secreted by the SPI-2 T3SS affect the SCV at later stages postinfection. Some SPI-2 effectors modulate microtubule motor activity on the SCV. Here, we show that the actin-based motor myosin II also affects SCV dynamics during infection. Following invasion, myosin II is required for SCV positioning near the nucleus of host cells. Later, myosin II counteracts the activities of the SPI-2 effectors PipB2 and SseJ to maintain SCV positioning and stability, respectively. Myosin II activity was required for maximal bacterial growth in macrophages. Rho kinase activity was required for SCV positioning. The effector SopB, a known activator of Rho GTPases, was found to be required for SCV positioning, and transfection of cells with SopB was sufficient to induce myosin II phosphorylation. These studies reveal a novel role for myosin II in controlling SCV dynamics during infection and suggest that SopB activates myosin II.

Evidence that embryonic neurons regulate the onset of cortical gliogenesis via cardiotrophin-1.

Precursor cells of the embryonic cortex sequentially generate neurons and then glial cells, but the mechanisms regulating this neurogenic-to-gliogenic transition are unclear. Using cortical precursor cultures, which temporally mimic this in vivo differentiation pattern, we demonstrate that cortical neurons synthesize and secrete the neurotrophic cytokine cardiotrophin-1, which activates the gp130-JAK-STAT pathway and is essential for the timed genesis of astrocytes in vitro. Our data indicate that a similar phenomenon also occurs in vivo. In utero electroporation of neurotrophic cytokines in the environment of embryonic cortical precursors causes premature gliogenesis, while acute perturbation of gp130 in cortical precursors delays the normal timed appearance of astrocytes. Moreover, the neonatal cardiotrophin-1-/- cortex contains fewer astrocytes. Together, these results describe a neural feedback mechanism; newly born neurons produce cardiotrophin-1, which instructs multipotent cortical precursors to generate astrocytes, thereby ensuring that gliogenesis does not occur until neurogenesis is largely complete.

Aspergillus fumigatus conidia survive and germinate in acidic organelles of A549 epithelial cells.

Aspergillus fumigatus is an environmental mould that can cause invasive disease in the immunocompromised host. Previous work has shown that conidia can be internalized by lung epithelial cells (A549) and murine macrophages (J774) in vitro. Therefore, the purpose of this study was to determine the fate of A. fumigatus conidia within the endosomal network of these cells. Co-localization of conidia expressing green fluorescent protein with proteins present in the early endosomal (CD71) and lysosomal (CD63, LAMP-1) membrane was assessed using confocal microscopy. In J774 cells, 75% of internalized conidia were found in phagosomes containing LAMP-1 120 minutes post-infection. In A549 cells, 55% and 58% of internalized conidia were found to co-localize with LAMP-1 and CD63 by 24 hours. Cathepsin D also co-localized with internalized conidia in A549 cells. Phagosomes containing conidia were shown to be acidified in both cell types. Less than 1% of the initial inoculum survived in J774 cells by 12 hours post-infection. After 24 hours, 3% of internalized conidia survived in A549 cells and 34% of these had germinated. By 36 hours, the germlings were able to escape the phagosome and form extracellular hyphae without lysis of the host cell.

Uptake of Aspergillus fumigatus Conidia by phagocytic and nonphagocytic cells in vitro: quantitation using strains expressing green fluorescent protein.

Several pathogenic fungal organisms enter eukaryotic cells and manipulate the host cell environment to favor their own growth and survival. Aspergillus fumigatus is a saprophytic fungus that causes invasive lung disease in the immunocompromised host. To determine whether A. fumigatus could enter eukaryotic cells, we studied the uptake of two different GFP-expressing A. fumigatus strains into A549 lung epithelial cells, human umbilical vein endothelial (HUVE) cells, and J774 murine macrophages in vitro. A549 cells internalized 30% of the bound conidia whereas HUVE and J774 cells internalized 50 and 90%, respectively. Conidia within A549 cells remained viable for 6 h; however, 60 to 80% of conidia within J774 cells were killed after only 4 h. Live and heat-killed conidia were internalized to the same extent by A549 cells. After 6 h, almost none of the conidia inside A549 cells had germinated, whereas extracellular conidia had developed germ tubes. Internalization of conidia by A549 cells was a temperature-dependent process and required rearrangement of the underlying host cell cytoskeleton; uptake was inhibited by 75% with 0.5 microM cytochalasin D and by 65% with 5 microM colchicine. Fluorescent labeling of infected A549 cells with rhodamine phalloidin provided visible evidence of cytoskeletal alteration as many of the intracellular conidia were contained in actin-coated phagosomes. These data provide evidence that significant numbers of A. fumigatus conidia can be internalized by nonprofessional phagocytes in vitro and these cells may serve as reservoirs for immune cell evasion and dissemination throughout the host.

Differences in sialic acid density in pathogenic and non-pathogenic Aspergillus species.

ASPERGILLUS: fumigatus is a ubiquitous soil fungus that causes invasive lung disease in the immunocompromised host. The structure of the conidial wall has not been well characterized although it is thought that adhesins present on the surface are involved in attachment of the conidia to host lung cells and proteins, which is a prerequisite for the establishment of infection. Negatively charged carbohydrates on the conidial surface have been previously identified as the molecules responsible for attachment of conidia to extracellular matrix proteins. The aim of this research was to identify carbohydrates on the conidial surface that contribute to its negative charge. Direct chemical analysis and indirect binding assays have demonstrated that A. fumigatus possesses sialic acids on the conidial surface. Pre-treatment of A. fumigatus conidia with sialidase decreased binding of a sialic acid-specific lectin, Limax flavus agglutinin (LFA), to the conidial surface and decreased adhesion of conidia to the positively charged polymer poly L-lysine. Two other sialic acid-specific lectins, Maackia amurensis agglutinin and Sambucus nigra agglutinin, exhibited negligible binding to A. fumigatus conidia indicating that 2,3-alpha- and 2,6-alpha-linked sialic acids are not the major structures found on the conidial surface. Mild acid hydrolysis and purification of conidial wall carbohydrates yielded a product that had the same R(F) as the Neu5Ac standard when analysed by high-performance thin-layer chromatography. A density of 6.7 x 10(5) sialic acid residues per conidium was estimated using a colorimetric assay. Conidia grown on a minimal medium lacking sialic acid also reacted with LFA, indicating that sialic acid biosynthesis occurs de novo. Sialic acid biosynthesis was shown to be regulated by nutrient composition: the density of sialic acids on the surface of conidia grown in minimal media was lower than that observed when conidia were grown on rich, complex media. It has previously been shown that pathogenic Aspergillus species adhere to basal lamina proteins to a greater extent than non-pathogenic Aspergillus species. To determine whether the expression of sialic acid on the conidial surface was correlated with adhesion to basal lamina, conidia from other non-pathogenic Aspergillus species were tested for their reactivity towards LFA. Flow cytometric analysis demonstrated that A. fumigatus had a significantly greater sialic acid density than three non-pathogenic Aspergillus species. Sialic acids on the conidial wall may be involved in adhesion to fibronectin, a component of the basal lamina, as binding of A. fumigatus conidia to fibronectin was strongly inhibited in the presence of a sialylated glycoprotein.