Biomedical subjects
Publications and source records attributed to T Hackstadt.
Chlamydiae dissociate themselves from the endocytic pathway shortly after internalization by actively modifying the vacuole to become fusogenic with sphingomyelin-containing exocytic vesicles. Interaction with this secretory pathway appears to provide a pathogenic mechanism that allows chlamydiae to establish themselves in a site that is not destined to fuse with lysosomes.
Chlamydiae are obligate intracellular bacteria that replicate within a non-acidified vacuole called an inclusion. Chlamydia psittaci (strain GPIC) produces a 39 kDa protein (IncA) that is localized to the inclusion membrane. While IncA is present as a single 39 kDa species in purified reticulate bodies, two additional higher M(r) forms are found in C. psittaci-infected cells. This finding suggested that IncA may be post-translationally modified in the host cell. Here we present evidence that IncA is a serine/threonine phosphoprotein that is phosphorylated by host cell enzymes. This conclusion is supported by the following experimental findings: (i) treatment of infected cells with inhibitors of host cell phosphatases or kinases altered the electrophoretic migration pattern of IncA; (ii) treatment with calf intestinal alkaline phosphatase eliminated the multiple-banding pattern of IncA, leaving only the protein band with the lowest relative molecular weight; and (iii) radioimmunoprecipitation of lysates of [32P]-orthophosphate-labelled infected HeLa cells with anti-IncA antisera demonstrated that the two highest M(r) IncA bands were phosphorylated. A vaccinia-virus recombinant expressing incA was used to determine if HeLa cells can phosphorylate IncA in the absence of a chlamydial background. IncA in lysates of these cells migrated identically to that seen in C. psittaci-infected cells, indicating the host cell was responsible for the phosphorylation of the protein. Microinjection of fluorescently labelled anti-IncA antibodies into C. psittaci-infected HeLa cells resulted in immunostaining of the outer face of the inclusion membrane. Collectively, these results demonstrate that IncA is phosphorylated by the host cell, and regions of IncA are exposed at the cytoplasmic face of the inclusion.
Rickettsia rickettsii has limited adverse effects on its arthropod vector, but causes severe disease in man. To model differences in host-parasite interaction, R. rickettsii growth and protein expression were examined at temperatures reflective of host environment in the tick cell lines DALBE3 and IDE2, the human endothelial cell line ECV304, and the African green monkey kidney cell line Vero76. At low multiplicities of infection, rickettsial titres increased 10(2)-10(3)-fold in all cell lines after incubation for 3 days at 34 degrees C. At higher multiplicities and with extended incubation, R. rickettsii showed enhanced survival in tick versus mammalian cells. No difference in rickettsial ultrastructure or protein profiles was detected between different host cell types. Rickettsial proteins of 42, 43, 48, 75 and 100 kDa are induced in tick cells shifted from 28 degrees to 34 degrees C, but not in cells maintained at 28 degrees C. This temperature response may be associated with expression of rickettsial determinants that are pathogenic to mammalian hosts.
Chlamydia trachomatis is an obligately intracellular bacterial parasite of eucaryotic cells that undergoes a biphasic life cycle within a parasitophorous vacuole (PV) called an inclusion. The parasitophorous vacuolar membrane (PVM) constitutes a barrier between the replicating bacteria and the nutrient-rich environment of the host cytoplasm. To determine whether the chlamydial PVM contains pores that allow passive diffusion of metabolites between the host cytoplasm and the PV, fluorescent tracer molecules were introduced directly into the cytoplasm of infected cells by transfection or microinjection. Fluorescence microscopy and laser scanning confocal microscopy were subsequently employed to determine whether equilibration of the fluorescent tracers between the cytoplasm and the PV occurred. No movement of tracer molecules as small as 520 Da from the cytoplasm to the PV was observed. These data suggest that the chlamydial PV is not passively permeable to small molecules through open channels in the PVM.
Chlamydial attachment to columnar conjunctival or urogenital epithelial cells is an initial and critical step in the pathogenesis of chlamydial mucosal infections. The chlamydial major outer membrane protein (MOMP) has been implicated as a putative chlamydial cytoadhesin; however, direct evidence supporting this hypothesis has not been reported. The function of MOMP as a cytoadhesin was directly investigated by expressing the protein as a fusion with the Escherichia coli maltose binding protein (MBP-MOMP) and studying its interaction with human epithelial cells. The recombinant MBP-MOMP bound specifically to HeLa cells at 4 degrees C but was not internalized after shifting the temperature to 37 degrees C. The MBP-MOMP competitively inhibited the infectivity of viable chlamydiae for epithelial cells, indicating that the MOMP and intact chlamydiae bind the same host receptor. Heparan sulfate markedly reduced binding of the MBP-MOMP to cells, whereas chondroitin sulfate had no effect on binding. Enzymatic treatment of cells with heparitinase but not chondroitinase inhibited the binding of MBP-MOMP. These same treatments were also shown to reduce the infectivity of chlamydiae for epithelial cells. Mutant cell lines defective in heparan sulfate synthesis but not chondroitin sulfate synthesis showed a marked reduction in the binding of MBP-MOMP and were also less susceptible to infection by chlamydiae. Collectively, these findings provide strong evidence that the MOMP functions as a chlamydial cytoadhesin and that heparan sulfate proteoglycans are the host-cell receptors to which the MOMP binds.
Chlamydia trachomatis acquires C6-NBD-sphingomyelin endogenously synthesized from C6-NBD-ceramide and transported to the vesicle (inclusion) in which they multiply. Here we explore the mechanisms of this unusual trafficking and further characterize the association of the chlamydial inclusion with the Golgi apparatus. Endocytosed chlamydiae are trafficked to the Golgi region and begin to acquire sphingolipids from the host within a few hours following infection. The transport of NBD-sphingolipid to the inclusion is energy- and temperature-dependent with the characteristics of an active, vesicle-mediated process. Photo-oxidation of C5-DMB-ceramide, in the presence of diaminobenzidine, identified DMB-lipids in vesicles in the process of fusing to the chlamydial inclusion membrane. C6-NBD-sphingomyelin incorporated into the plasma membrane is not trafficked to the inclusion to a significant degree, suggesting the pathway for sphingomyelin trafficking is direct from the Golgi apparatus to the chlamydial inclusion. Lectins and antibody probes for Golgi-specific glycoproteins demonstrate the close association of the chlamydial inclusion with the Golgi apparatus but do not detect these markers in the inclusion membrane. Collectively, the data are consistent with a model in which C.trachomatis inhabits a unique vesicle which interrupts an exocytic pathway to intercept host sphingolipids in transit from the Golgi apparatus to the plasma membrane.
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Chlamydia trachomatis is an obligate intracellular pathogen that multiples within the confines of a membrane-bound vacuole called an inclusion. Approximately 40-50% of the sphingomyelin synthesized from exogenously added NBD-ceramide is specifically transported from the Golgi apparatus to the chlamydial inclusion (Hackstadt, T., M.A. Scidmore, and D.D. Rockey. 1995. Proc. Natl. Acad. Sci. USA. 92: 4877-4881). Given this major disruption of a cellular exocytic pathway and the similarities between glycolipid and glycoprotein exocytosis, we wished to determine whether the processing and trafficking of glycoproteins through the Golgi apparatus to the plasma membrane in chlamydia-infected cells was also disrupted. We analyzed the processing of several model glycoproteins including vesicular stomatitis virus G-protein, transferrin receptor, and human histocompatibility leukocyte class I antigen. In infected cells, the posttranslational processing and trafficking of these specific proteins through the Golgi apparatus and subsequent transport to the plasma membrane was not significantly impaired, nor were these glycoproteins found associated with the chlamydial inclusion membrane. Studies of receptor recycling from endocytic vesicles employing fluorescently and HRP-tagged transferrin and anti-transferrin receptor antibody revealed an increased local concentration of transferrin and transferrin receptor around but never within the chlamydial inclusion. However, Scatchard analysis failed to show either an increased intracellular accumulation of transferrin receptor or a decreased number of plasma membrane receptors in infected cells. Furthermore, the rate of exocytosis from the recycling endosomes to the plasma membrane was not altered in chlamydia-infected cells. Thus, although C. trachomatis disrupts the exocytosis of sphingolipids and the Golgi apparatus appears physically distorted, glycosylation and exocytosis of representative secreted and endocytosed proteins are not disrupted. These results suggest the existence of a previously unrecognized sorting of sphingolipids and glycoproteins in C. trachomatis-infected cells.
Coxiella burnetii undergoes a poorly defined developmental cycle within phagolysosomes of eukaryotic host cells. Two distinct developmental forms are part of this cycle: a small-cell variant (SCV) and large-cell variant (LCV). Ultrastructurally, the SCV is distinguished from the LCV by its smaller size and condensed chromatin. At a molecular level, little is known about morphogenesis in C. burnetii, and no proteins specific to the SCV have been identified. Preparative isoelectric focusing was conducted to purify basic proteins possibly involved in SCV chromatin structure. A predominant protein of low M(r) was present in the most basic fraction, eluting with a pH of approx. 11. Degenerate deoxyoligonucleotides corresponding to the N-terminal sequence of this protein were used to recover a cosmid clone from a C. burnetii genomic library. Nucleotide sequencing of insert DNA revealed an open reading frame designated scvA (Small-Cell-variant protein A) with coding potential for a 30 amino acid protein (ScvA) with a predicted M(r) of 3610. ScvA is 46% arginine plus 46% glutamine with a predicted pl of 12.6. SDS-PAGE and silver staining of lysates of SCV and LCV purified by caesium chloride-equilibrium density centrifugation revealed a number of proteins unique to each cell type. Immunoblot analysis with ScvA antiserum demonstrated the presence of ScvA only in the SCV. By Immunoelectron microscopy, ScvA antiserum labelled only the SCV, with the label concentrated on the condensed nucleoid. In addition, ScvA bound double-stranded DNA in gel mobility-shift assays. A 66% reduction in the mean number of gold particles per Coxiella call was observed at 12 h post-infection when compared with the starting inoculum. Collectively, these data suggest that synthesis of ScvA is developmentally regulated, and that the protein may serve a structural or functional role as an integral component of the SCV chromatin. Moreover, degradation of this protein may be a necessary prerequisite for morphogenesis from SCV to LCV.
The chlamydiae are obligate intracellular parasites that develop and multiply within a vacuole (termed an inclusion) that does not fuse with lysosomes. Inclusion morphology varies dramatically among the different chlamydiae, particularly within the species Chlamydia psittaci. Some strains develop within a single vacuole, while the mature inclusion of other strains consists of several distinct lobes, each filled with chlamydial developmental forms. The development of this lobed structure was investigated in HeLa cells infected with the guinea pig inclusion conjunctivitis (GPIC) strain of C. psittaci. We employed two recently described probes for the chlamydial inclusion to study the development of these unique lobed structures. The novel probes were an antiserum directed at a protein localized to the GPIC inclusion membrane (anti-IncA) and the fluorescent sphingolipid (N-[7-(4-nitrobenzo-2-oxa-1,3-)]) aminocaproyl sphingosine (NBD-ceramide). Lobed inclusions developed in cells infected at very low multiplicities of infection, suggesting that the structure is not a function of infection by more than one elementary body (EB). Double-label fluorescent-antibody analysis with anti-IncA and an antibody directed at a chlamydial outer membrane protein showed that, prior to 18 h postinfection (p.i.), the inclusion membrane and the chlamydial membrane were tightly associated. After 18 to 20 h p.i., the lobes began to expand and fill with developmental forms and the inclusion membrane and chlamydial membrane became distinct. At times from 8 to 48 h p.i., GPIC inclusions were shown to receive fluorescent derivatives of NBD-ceramide and to be localized to the perinuclear region of the host cell. Labeled lectins with affinity for carbohydrate moieties localized to the Golgi apparatus showed that the lobes of mature inclusions surround the Golgi apparatus. Labeling with NBD-ceramide and the Golgi apparatus-specific lectins therefore demonstrated a functional and physical association of the inclusion with the Golgi apparatus throughout the developmental cycle. Collectively, these results lead to a model for the development of the lobed chlamydial inclusion. We propose that the lobed structure is a result of division of inclusions occurring in parallel with the multiplication of reticulate bodies (RB) early in the developmental cycle. The division of inclusions slows or stops in mid-cycle, and dividing RB accumulate within the enlarging lobes. The RB then differentiate to EBs, the inclusion and cell are lysed, and EBs are freed to infect another cell.
Chlamydiae replicate intracellularly within a vacuole that has recently been characterized as intersecting an exocytic pathway. One of the initial events during chlamydial infection is the expression of a chlamydial early gene product(s) that effectively isolates the inclusion from the endocytic-lysosomal pathway and makes it fusogenic with sphingomyelin-containing exocytic vesicles. Associated with this change in vesicular interaction is the delivery of the vacuole to the peri-Golgi region of the host cell. Inhibition of chlamydial early transcription or translation causes Chlamydia trachomatis-containing vesicles to remain dispersed throughout the cytoplasm, where they eventually fuse with lysosomes. Chlamydiae that have been internalized by Fc-mediated endocytosis also avoid lysosomal digestion by a mechanism that requires chlamydial protein synthesis. These results suggest that the vesicular interactions of the chlamydial inclusion are defined by parasite-directed modification of the endocytic vesicle rather than by the route of internalization.
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, and fluid-phase markers in conjunction with electron, confocal, and conventional microscopy to characterize the vacuolar environments of these pathogens. The acidic nature of the C. burnetii-containing vacuole was confirmed by its acquisition of the acidotropic base acridine orange (AO). The presence of the vacuolar-type (H+) ATPase (V-ATPase) within the Coxiella vacuolar membrane was demonstrated by indirect immunofluorescence, and growth of C. burnetii was inhibited by bafilomycin A1 (Baf A), a specific inhibitor of the V-ATPase. In contrast, AO did not accumulate in C. trachomatis inclusions nor was the V-ATPase found in the inclusion membrane. Moreover, chlamydial growth was not inhibited by Baf A or the lysosomotropic amines methylamine, ammonium chloride, and chloroquine. Vacuoles harboring C. burnetii incorporated the fluorescent fluid- phase markers, fluorescein isothiocyanate-dextran (FITC-dex) and Lucifer yellow (LY), indicating trafficking between that vacuole and the endocytic pathway. Neither FITC-dex nor LY was sequestered by chlamydial inclusions. The late endosomal-prelysosomal marker cation-independent mannose 6-phosphate receptor was not detectable in the vacuolar membranes encompassing either parasite. However, the lysosomal enzymes acid phosphatase and cathepsin D and the lysosomal glycoproteins LAMP-1 and LAMP-2 localized to the C. burnetii vacuole but not the chlamydial vacuole. Interaction of C. trachomatis inclusions with the Golgi-derived vesicles was demonstrated by the transport of sphingomyelin, endogenously synthesized from C6-NBD-ceramide, to the chlamydial inclusion and incorporation into the bacterial cell wall. Similar trafficking of C-NBD-ceramide was not evident in C. burnetii-infected cells. Collectively, the data indicate that C. trachomatis replicates within a nonacidified vacuole that is disconnected from endosome-lysosome trafficking but may receive lipid from exocytic vesicles derived from the trans-Golgi network. These observations are in sharp contrast to those for C. burnetii, which by all criteria resides in a typical phagolysosome.
Two DNA-binding proteins have been detected in Coxiella burnetii by southwestern (DNA-protein) blotting. One of these, termed Hq1, is enriched in the small cell variant stage of the developmental cycle and displays compositional and primary amino acid sequence similarities to eukaryotic histone H1. C. burnetii appears to be another example of an intracellular parasite with morphologically distinct developmental forms whose nucleoid structure may be controlled by histone H1 homologs.
Typhus group rickettsiae, including Rickettsia prowazekii and R. typhi, produce visible plaques on primary chick embryo fibroblasts and low-passage mouse embryo fibroblasts but do not form reproducible plaques on continuous cell culture lines. We tested medium overlay modifications for plaque formation of typhus group rickettsiae on the continuous fibroblast cell line Vero76. A procedure involving primary overlay with medium at pH 6.8, which was followed 2 to 3 days later with secondary overlay at neutral pH containing 1 microgram of emetine per ml and 20 micrograms of NaF per ml, resulted in visible plaques at 7 to 10 days postinfection. A single-step procedure involving overlay with medium containing 50 ng of dextran sulfate per ml also resulted in plaque formation within 8 days postinfection. These assays represent reproducible and inexpensive methods for evaluating the infectious titers of typhus group rickettsiae, cloning single plaque isolates, and testing the susceptibilities of rickettsiae to antibiotics.
Rickettsiae are bacterial obligate intracellular parasites ranging from harmless endosymbionts to the etiologic agents of some of the most devastating diseases known to mankind. Rickettsiae are primarily associated with arthropod vectors in which they may exist commensally and, in most cases, only accidentally infect humans. These fascinating microbes are the prototypical obligate intracellular parasites. Other than being extremely fastidious in their growth requirements, however, rickettsiae are typical gram-negative bacteria. Only a few intracellular parasites multiply within the cytoplasm of eukaryotic cells. In this environment, rickettsiae are provided with a rich source of biosynthetic precursors not normally encountered by free-living bacteria and have evolved a number of unique mechanisms to transport such metabolites as nucleotides and nucleotide sugars. The physiologic basis for their obligate parasitism, however, has remained elusive for > 90 years. Other than the obvious property of replicating inside eukaryotic cells, the molecular mechanisms of cellular damage are ill defined. The typhus-group rickettsiae multiply within host cells to great numbers without profound damage until lysis occurs. In contrast, the spotted fever-group rickettsiae spread rapidly from cell to cell by an actin-based motility. This property, in itself, is not sufficient to cause cell death, because avirulent spotted fever-group rickettsiae also spread by actin-based movement but do not cause lysis of the host cell. Despite the obvious limitations imposed by their obligate intracellular lifestyle and the current lack of methods for genetic manipulation, there are enough interesting biological properties of rickettsiae to offer an attractive area for research.
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 with conventional fluorescence or confocal microscopy to identify interactions between the Golgi apparatus and the chlamydial inclusion. We observed not only a close physical association between the Golgi apparatus and the chlamydial inclusion but the eventual presence of a metabolite of this fluorescent probe associated with the chlamydiae themselves. Sphingomyelin, endogenously synthesized from C6-NBD-Cer, was specifically transported to the inclusion and incorporated into the cell wall of the intracellular chlamydiae. Incorporation of the fluorescent sphingolipid by chlamydiae was inhibited by brefeldin A. Chlamydiae therefore occupy a vesicle distal to the Golgi apparatus that receives anterograde vesicular traffic from the Golgi normally bound for the plasma membrane. Collectively, the data suggest that the chlamydial inclusion may represent a unique compartment within the trans-Golgi network.
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 the chlamydial inclusion membrane. To identify chlamydial proteins which are unique to the intracellular phase of the life cycle, an expression library of Chlamydia psittaci DNA was screened with convalescent antisera from infected animals and hyperimmune antisera generated against formalin-killed purified chlamydiae. Overlapping genomic clones were identified which expressed a 39 kDa protein only recognized by the convalescent sera. Sequence analysis of the clones identified two open reading frames (ORFs), one of which (ORF1) coded for a predicted 39 kDa gene product. The ORF1 sequence was amplified and fused to the malE gene of Escherichia coli and antisera were raised against the resulting fusion protein. Immunoblotting with these antisera demonstrated that the 39 kDa protein was present in lysates of infected cells and in reticulate bodies (RBs), but was at the limit of detection in lysates of purified C. psittaci elementary bodies. Fluorescence microscopy experiments demonstrated that this protein was localized in the inclusion membrane of infected HeLa cells, but was not detected on the developmental forms within the inclusion. Because the protein produced by ORF1 is deposited on the inclusion membrane of infected cells, this gene has been designated incA, (inclusion membrane protein A) and its gene product, IncA. In addition to the inclusion membrane, these antisera labelled structures that extended from the inclusion over the nucleus or into the cytoplasm of infected cells. Immunoblotting also demonstrated that IncA, in lysates of infected cells, had a migration pattern that seemed indicative of post-translational modification. This pattern was not observed in immunoblots of RBs or in the E. coli expressing IncA. Collectively, these data identify a chlamydial gene which codes for a protein that is released from RB and is localized in the inclusion membrane of infected cells.
The outer membrane of the Gram-negative obligate intracellular parasite Rickettsia rickettsii contains two large surface protein antigens with approximate molecular masses of 200 and 135 kDa termed rOmpA and rOmpB, respectively. rOmpB is the most abundant protein in the outer membrane, while rOmpA is a relatively minor constituent. Densitometry of intrinsically radiolabelled protein profiles from R. rickettsii-infected Vero cells indicated a molar ratio of approximately 1:9 between rOmpA and rOmpB. The putative promoter-5' untranslated regions (5' UTR) from their recently characterized genes (rompA and rompB) were placed in the promoter assay vector pKK232-8 to test whether these elements conserve aspects of differential expression in a heterologous host-reporter system. Primer extension analysis of RNA from Escherichia coli clones containing the constructs indicated that E. coli RNA polymerase faithfully utilizes rompA and rompB transcription start sites identified previously in R. rickettsii. The rompB insert directs 28-fold higher levels of chloramphenicol acetyl transferase activity than the rompA insert.