cytochalasin-d and Chlamydia-Infections

Chlamydia spp. are major causes of important human diseases, but dissecting the host-pathogen interactions has been hampered by the lack of bacterial genetics and the difficulty in carrying out forward genetic screens in mammalian hosts. RNA interference (RNAi)-based methodologies for gene inactivation can now be easily carried out in genetically tractable model hosts, such as Drosophila melanogaster, and offer a new approach to identifying host genes required for pathogenesis. We tested whether Chlamydia trachomatis infection of D. melanogaster S2 cells recapitulated critical aspects of mammalian cell infections. As in mammalian cells, C. trachomatis entry was greatly reduced by heparin and cytochalasin D. Inclusions were formed in S2 cells, acquired Golgi-derived sphingolipids, and avoided phagolysosomal fusion. Elementary body (EB) to reticulate body (RB) differentiation was observed, however, no RB to EB development or host cell killing was observed. RNAi-mediated inactivation of Rac, a Rho GTPase recently shown to be required for C. trachomatis entry in mammalian cells, inhibits C. trachomatis infection in S2 cells. We conclude that Drosophila S2 cells faithfully mimic early events in Chlamydia host cell interactions and provides a bona fide system to systematically dissect host functions important in the pathogenesis of obligate intracellular pathogens.

Topics: Actins; Animals; Cells, Cultured; Chlamydia Infections; Chlamydia trachomatis; Cytochalasin D; Disease Models, Animal; Drosophila melanogaster; Golgi Apparatus; Heparin; Humans; Lysosomes; Microscopy, Electron, Transmission; Sphingomyelins

Chlamydia trachomatis L2 is an obligate intracellular microorganism with a unique biphasic life cycle. The extracellular form, the elementary body (EB), is infectious but metabolically inactive. Attachment of EBs to host cells is medicated by a heparan sulfate-like glycosaminoglycan. Following attachment, the EB is internalized within a membrane-bound vesicle, and during the first 8 h of infection the vesicles are transported to a perinuclear location where they aggregate and fuse. By use of a monoclonal antibody against phosphotyrosine, we showed that three classes of proteins are tyrosine phosphorylated: a triple band of 68, 66, and 64 kDa, a 97-kDa band, and a 140-kDa band. The phosphorylation could be detected by immunoblotting from 15 min after infection of HeLa cells. We followed the movement of the EBs and the tyrosine phosphorylation of proteins by double-labelling immunofluorescence microscopy with the same monoclonal anti-phosphotyrosine antibody and a polyclonal antibody against the C. trachomatis L2 outer membrane complex. During the first 8 h of infection, the phosphorylation colocalized with EBs. Sixteen hours after infection, EBs have reorganized to the replicating reticulate bodies, forming an inclusion. At this time, phosphorylation was seen as dotted spots in the periphery of the inclusion.

Topics: Actins; Chlamydia Infections; Chlamydia trachomatis; Cytochalasin D; ErbB Receptors; Fluorescent Antibody Technique; HeLa Cells; Humans; Molecular Weight; Phosphoproteins; Phosphotyrosine; Protein-Tyrosine Kinases; Tyrosine

The microfilament-disrupting drug cytochalasin D and, initially, inoculation at 20 degrees C were used to differentiate between phagocytosis (sensitive to both treatments) and pinocytosis (resistant to both treatments) to assess whether chlamydial uptake into McCoy cells occurred by one or both mechanisms and whether each could contribute to productive infection. Both treatments suppressed the infectivity of Chlamydia trachomatis L2/434/Bu and C. psittaci GPIC (the guinea pig inclusion conjunctivitis strain) following static inoculation by only 50%, indicating that there was simultaneous operation of both phagocytosis and pinocytosis during uptake that led to productive infection. Measurement of the entry of organisms by two separate assays established that both strains predominantly used a cytochalasin D-resistant (pinocytic) mechanism, implying that phagocytic uptake was coupled to a higher frequency of productive infection. Integration of the data on infectivity and entry allowed the potential for an organism to infect a host cell to be quantified. This synthesis revealed that for both strains the infectivity potential following phagocytic entry was ca. 10-fold greater than that following pinocytic entry. However, both entry mechanisms were exploited more efficiently by strain L2/434/Bu than by strain GPIC (unless the latter was inoculated with centrifugation), indicating that intrinsic strain properties are more important for infectivity potential than the endocytic mechanism utilized.

Topics: Cell Line; Chlamydia Infections; Chlamydia trachomatis; Chlamydophila psittaci; Colony Count, Microbial; Cytochalasin D; Fluorescent Antibody Technique; Phagocytosis; Pinocytosis

Immunofluorescence was used to examine the distribution of Chlamydia trachomatis serovars L2 and E, F-actin, and clathrin in infected McCoy and HeLa cells. After incubation at 4 degrees C, C. trachomatis serovar L2 was randomly distributed on the McCoy cell surface. After a temperature shift to 37 degrees C, chlamydiae redistributed, within 30 min, to one local aggregate in the central or perinuclear region of individual cells. About 90% of these aggregated chlamydiae were intracellularly localized, but some remained randomly distributed on the cell surface. Similar results were obtained with HeLa cells and C. trachomatis serovar E, except that the redistribution was slower in HeLa cells than in McCoy cells and fewer cells infected with serovar E exhibited a local aggregate than those infected with serovar L2. Cytochalasin D inhibited more than 90% of this local aggregation. Instead, in cytochalasin D-treated cells, the entry of chlamydiae was inhibited and the organisms became localized on the cell surface in a peripheral local aggregate that distributed in a manner similar to that of phalloidin-stained actin. In a double immunofluorescence assay, F-actin and clathrin aggregated correspondingly in time and position with central or perinuclear aggregation of chlamydiae. These results indicate that polymerized actin and clathrin participate in a rapid redistribution of chlamydiae to an intracellular aggregate.

Topics: Actins; Bacterial Adhesion; Cell Line; Chlamydia Infections; Chlamydia trachomatis; Clathrin; Cytochalasin D; Endocytosis; HeLa Cells; Humans