The obligate intracellular bacterium Chlamydia trachomatis rapidly induces its own entry into host cells. Initial attachment is mediated by electrostatic interactions to heparan sulfate moieties on the cell, followed irreversible binding an unknown secondary receptor. This leads recruitment of actin site attachment, formation actin-rich, pedestallike structure, and finally internalization bacteria. How chlamydiae induce this process unknown. We have identified a high-molecular-mass...

Coxiella burnetii and Chlamydia trachomatis are bacterial obligate intracellular parasites that occupy distinct vacuolar niches within eucaryotic host cells. We have employed immunofluorescence, cytochemistry, fluorescent vital stains, fluid-phase markers in conjunction with electron, confocal, conventional microscopy to characterize the environments of these pathogens. The acidic nature C. burnetii-containing vacuole was confirmed by its acquisition acidotropic base acridine orange (AO)....

Coxiella burnetti, the etiologic agent of Q fever, is an oligate intracellular parasite eukaryotes. Unlike majority successful bacterial parasites, which escape bactericidal environment phagolysosome by various means, C. burnetii multiplies only in phagolysosome. In view relatively harsh inhabited burnetii, we have examined (i) vitro metabolism glucose and glutamate whole cells under conditions designed to approximate pH within (ii) effect manipulation phagolysosomal lysosomotropic amines on...

The acquisition of host-derived lipids is essential for the pathogenesis obligate intracellular bacteria Chlamydia trachomatis . Current models chlamydial lipid center on fusion Golgi-derived exocytic vesicles and endosomal multivesicular bodies with bacteria-containing parasitophorous vacuole (“inclusion”). In this study, we describe a mechanism organelle subversion by C. We show live cell fluorescence microscopy electron that droplets (LDs), neutral storage organelles, are translocated...

Chlamydia trachomatis undergoes its entire life cycle within an uncharacterized intracellular vesicle that does not fuse with lysosomes. We used a fluorescent Golgi-specific probe, (N-[7-(4-nitrobenzo-2-oxa-1,3-diazole)]) aminocaproylsphingosine (C6-NBD-Cer), in conjunction conventional fluorescence or confocal microscopy to identify interactions between the Golgi apparatus and chlamydial inclusion. observed only close physical association inclusion but eventual presence of metabolite this...

The obligate intracellular bacterium Chlamydia trachomatis has a unique developmental cycle that involves functionally and morphologically distinct cell types adapted for extracellular survival multiplication. Infection is initiated by an environmentally resistant type called elementary body (EB). Over the first several hours of infection, EBs differentiate into larger replicative form, termed reticulate (RB). Late in infectious process, RBs asynchronously begin to back EBs, which accumulate...

Cholesterol, a lipid not normally found in prokaryotes, was identified purified Chlamydia trachomatis elementary bodies and the chlamydial parasitophorous vacuole (inclusion) membrane of infected HeLa cells. Chlamydiae obtained eukaryotic host cell cholesterol both from de novo synthesis or low-density lipoprotein. Acquisition either novo-synthesized lipoprotein-derived microtubule-dependent brefeldin A-sensitive, indicating requirement for Golgi apparatus. Transport also required protein...

Members of the spotted fever group (SFG) rickettsiae spread rapidly from cell to by an unknown mechanism(s). Staining Rickettsia rickettsii-infected Vero cells with rhodamine phalloidin demonstrated unique actin filaments associated one pole intracellular rickettsiae. F-actin tails greater than 70 microns in length were seen extending Treatment infected chloramphenicol eliminated rickettsia-associated tails, suggesting that de novo protein synthesis or more rickettsial proteins is required...

Chlamydiae replicate within an intracellular vacuole, termed inclusion, that is non-fusogenic with vesicles of the endosomal or lysosomal compartments. Instead, inclusion appears to intersect exocytic pathway from which chlamydiae intercept sphingomyelin en route Golgi apparatus plasma membrane. Chlamydial protein synthesis required establish this interaction. In effort identify those chlamydial proteins controlling vesicle fusion, we have prepared polyclonal antibodies against several...

We compared the relative infectivity and virulence of lipopolysaccharide (LPS) variants Nine Mile strain Coxiella burnetii with those Priscilla strain, a representative endocarditis-type strains. In agreement results previous studies, phase I (9mi/I) organisms were highly infectious, eliciting seroconversion fever inocula containing as few four organisms. Viable 9mi/I was recovered from spleens infected animals 30 days postinfection. II (9mi/II) did not elicit or except very large inocula,...

Chlamydia trachomatis entry into host cells results from a parasite-directed remodeling of the actin cytoskeleton. A type III secreted effector, TARP (translocated recruiting phosphoprotein), has been implicated in recruitment to site internalization. To elucidate role recruitment, we identified cell proteins that associated with recombinant GST-TARP fusions. directly actin, and this interaction promoted nucleation as determined by vitro polymerization assays. Domain analysis an...

We isolated lipopolysaccharides (LPSs) from phase variants of Coxiella burnetii Nine Mile and compared the LPS C. cells by sodium dodecyl sulfate-polyacrylamide gel electrophoresis immunoblotting. The LPSs were found to be predominant component which varied structurally antigenically between virulent I avirulent II. A comparison techniques historically used extract antigenic revealed that aqueous phenol-water, trichloroacetic acid, dimethyl sulfoxide extractions burnettii all contained LPS,...

Chlamydia trachomatis LGV-434 was grown in HeLa 229 cells. Benzylpenicillin completely inhibited the formation of infectious elementary bodies (EBs) at a concentration 19 pmol/ml or higher and produced abnormally large reticulate (RBs) inclusions 30 higher. The possible targets for penicillin C. were three penicillin-binding proteins (PBPs) which identified Sarkosyl-soluble fractions both RBs EBs. apparent subunit molecular weights 88,000 (PBP 1), 61,000 (BPB 2), 36,000 3). 50% binding...

Chlamydiae are obligate intracellular bacteria which occupy a non-acidified vacuole (the inclusion) throughout their developmental cycle. Little is known about events leading to the establishment and maintenance of chlamydial inclusion membrane. To identify proteins unique phase life cycle, an expression library Chlamydia psittaci DNA was screened with convalescent antisera from infected animals hyperimmune generated against formalin-killed purified chlamydiae. Overlapping genomic clones...

Chlamydia trachomatis is among the most clinically significant human pathogens, yet their obligate intracellular nature places severe restrictions upon research. Chlamydiae undergo a biphasic developmental cycle characterized by an infectious cell type known as elementary body (EB) and replicative form called reticulate (RB). EBs have historically been described metabolically dormant. A cell-free (axenic) culture system was developed, which showed high levels of metabolic biosynthetic...

Rickettsii rickettsii, the etiologic agent of Rocky Mountain spotted fever, replicates within cytosol infected cells and uses actin-based motility to spread inter- intracellularly. Although ultrastructure actin tail host proteins associated with it are distinct from those Listeria or Shigella, comparatively little is known regarding rickettsial involved in its organization. Here, we have used random transposon mutagenesis R. rickettsii generate a small-plaque mutant that defective does not...

ABSTRACT Chlamydia trachomatis is an obligate intracellular pathogen that replicates in a vacuole termed the inclusion. Many of interactions chlamydiae with host cell are dependent upon bacterial protein synthesis and presumably exposure these proteins to cytosol. Because dearth genetic tools for chlamydiae, previous studies examining secreted required use heterologous systems. Recent advances manipulation chlamydia now allow transformation bacteria plasmids. We describe here shuttle vector...

Chlamydiae are Gram-negative obligate intracellular bacteria that cause diseases with significant medical and economic impact. Chlamydia trachomatis replicates within a vacuole termed an inclusion, which is extensively modified by the insertion of number bacterial effector proteins known as inclusion membrane (Incs). Once modified, trafficked in dynein-dependent manner to microtubule-organizing centre (MTOC), where it associates host centrosomes. Here we describe novel structure on comprised...

Chlamydia spp. are obligate intracellular pathogens that replicate within a vacuole termed the inclusion. Chlamydiae extensively modify inclusion membrane via insertion of chlamydial proteins (Incs) which decorate cytosolic face We have assessed overall relatedness and phylogeny Incs in order to identify potential evolutionary trends. Despite high degree conservation among C. trachomatis serovars, phylogenetic analysis showed some cluster according clinical groupings suggesting certain may...

Chlamydia trachomatis replicates within a membrane-bound compartment termed an inclusion. The inclusion membrane is modified by the insertion of multiple proteins known as Incs. In yeast two-hybrid screen, interaction was found between protein CT228 and MYPT1, subunit myosin phosphatase. MYPT1 recruited peripherally around inclusion, whereas phosphorylated, inactive form localized to active Src-family kinase-rich microdomains. Phosphorylated light chain 2 (MLC2), kinase (MLCK), IIA, IIB also...

Chlamydia trachomatis is a human pathogen associated with significant morbidity worldwide. As obligate intracellular parasites, chlamydiae must survive within eukaryotic cells for sufficient time to complete their developmental cycle. To promote host cell survival, express poorly understood anti-apoptotic factors. Using recently developed genetic tools, we show that three inclusion membrane proteins (Incs) out of eleven examined are required stability and avoidance death pathways. In the...