scholarly journals Septins Arrange F-Actin-Containing Fibers on the Chlamydia trachomatis Inclusion and Are Required for Normal Release of the Inclusion by Extrusion

mBio ◽  
2014 ◽  
Vol 5 (5) ◽  
Author(s):  
Larisa Volceanov ◽  
Katharina Herbst ◽  
Martin Biniossek ◽  
Oliver Schilling ◽  
Dirk Haller ◽  
...  
Keyword(s):  
Chlamydia Trachomatis ◽  
Mammalian Cells ◽  
Binding Proteins ◽  
Higher Order ◽  
Host Cells ◽  
Developmental Cycle ◽  
Content Type ◽  
Gtp Binding Proteins ◽  
Gtp Binding ◽  
Chlamydial Inclusion

ABSTRACTChlamydia trachomatisis an obligate intracellular human pathogen that grows inside a membranous, cytosolic vacuole termed an inclusion. Septins are a group of 13 GTP-binding proteins that assemble into oligomeric complexes and that can form higher-order filaments. We report here that the septins SEPT2, -9, -11, and probably -7 form fibrillar structures around the chlamydial inclusion. Colocalization studies suggest that these septins combine with F actin into fibers that encase the inclusion. Targeting the expression of individual septins by RNA interference (RNAi) prevented the formation of septin fibers as well as the recruitment of actin to the inclusion. At the end of the developmental cycle ofC. trachomatis, newly formed, infectious elementary bodies are released, and this release occurs at least in part through the organized extrusion of intact inclusions. RNAi against SEPT9 or against the combination of SEPT2/7/9 substantially reduced the number of extrusions from a culture of infected HeLa cells. The data suggest that a higher-order structure of four septins is involved in the recruitment or stabilization of the actin coat around the chlamydial inclusion and that this actin recruitment by septins is instrumental for the coordinated egress ofC. trachomatisfrom human cells. The organization of F actin around parasite-containing vacuoles may be a broader response mechanism of mammalian cells to the infection by intracellular, vacuole-dwelling pathogens.IMPORTANCEChlamydia trachomatisis a frequent bacterial pathogen throughout the world, causing mostly eye and genital infections.C. trachomatiscan develop only inside host cells; it multiplies inside a membranous vacuole in the cytosol, termed an inclusion. The inclusion is covered by cytoskeletal “coats” or “cages,” whose organization and function are poorly understood. We here report that a relatively little-characterized group of proteins, septins, is required to organize actin fibers on the inclusion and probably through actin the release of the inclusion. Septins are a group of GTP-binding proteins that can organize into heteromeric complexes and then into large filaments. Septins have previously been found to be involved in the interaction of the cell with bacteria in the cytosol. Our observation that they also organize a reaction to bacteria living in vacuoles suggests that they have a function in the recognition of foreign compartments by a parasitized human cell.

scholarly journals Single-Inclusion Kinetics of Chlamydia trachomatis Development

mSystems ◽  
2020 ◽  
Vol 5 (5) ◽  
Author(s):  
Travis J. Chiarelli ◽  
Nicole A. Grieshaber ◽  
Anders Omsland ◽  
Christopher H. Remien ◽  
Scott S. Grieshaber
Keyword(s):  
Chlamydia Trachomatis ◽  
Mathematical Models ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Host Cells ◽  
Developmental Cycle ◽  
Cell Type ◽  
Content Type ◽  
Obligate Intracellular

ABSTRACT The obligate intracellular bacterial pathogen Chlamydia trachomatis is reliant on a developmental cycle consisting of two cell forms, termed the elementary body (EB) and the reticulate body (RB). The EB is infectious and utilizes a type III secretion system and preformed effector proteins during invasion, but it does not replicate. The RB replicates in the host cell but is noninfectious. This developmental cycle is central to chlamydial pathogenesis. In this study, we developed mathematical models of the developmental cycle that account for potential factors influencing RB-to-EB cell type switching during infection. Our models predicted that two categories of regulatory signals for RB-to-EB development could be differentiated experimentally, an “intrinsic” cell-autonomous program inherent to each RB and an “extrinsic” environmental signal to which RBs respond. To experimentally differentiate between mechanisms, we tracked the expression of C. trachomatis development-specific promoters in individual inclusions using fluorescent reporters and live-cell imaging. These experiments indicated that EB production was not influenced by increased multiplicity of infection or by superinfection, suggesting the cycle follows an intrinsic program that is not directly controlled by environmental factors. Additionally, live-cell imaging revealed that EB development is a multistep process linked to RB growth rate and cell division. The formation of EBs followed a progression with expression from the euo and ihtA promoters evident in RBs, while expression from the promoter for hctA was apparent in early EBs/IBs. Finally, expression from the promoters for the true late genes, hctB, scc2, and tarp, was evident in the maturing EB. IMPORTANCE Chlamydia trachomatis is an obligate intracellular bacterium that can cause trachoma, cervicitis, urethritis, salpingitis, and pelvic inflammatory disease. To establish infection in host cells, Chlamydia must complete a multiple-cell-type developmental cycle. The developmental cycle consists of specialized cells, the EB cell, which mediates infection of new host cells, and the RB cell, which replicates and eventually produces more EB cells to mediate the next round of infection. By developing and testing mathematical models to discriminate between two competing hypotheses for the nature of the signal controlling RB-to-EB cell type switching, we demonstrate that RB-to-EB development follows a cell-autonomous program that does not respond to environmental cues. Additionally, we show that RB-to-EB development is a function of chlamydial growth and division. This study serves to further our understanding of the chlamydial developmental cycle that is central to the bacterium’s pathogenesis.

scholarly journals A rab protein regulates the localization of secretory granules in AtT-20 cells.

1993 ◽  
Vol 4 (7) ◽  
pp. 747-756 ◽  
Author(s):  
J K Ngsee ◽  
A M Fleming ◽  
R H Scheller
Keyword(s):  
Mammalian Cells ◽  
Binding Proteins ◽  
Secretory Granules ◽  
Release Site ◽  
Secretory Vesicles ◽  
Gtp Binding Proteins ◽  
Rab Protein ◽  
Gtp Binding ◽  
Cell Processes ◽  
Intracellular Vesicles

Low molecular weight (LMW) GTP-binding proteins are hypothesized to play a role in the vectorial transport of intracellular vesicles. Mutational studies in yeast and subcellular localization in mammalian cells suggest that a family of LMW GTP-binding proteins, termed rab, target intracellular vesicles to their appropriate acceptor compartment. In this report, we demonstrate that an elasmobranch homologue of rab3A, o-rab3, plays a significant role in the sequestration of regulated secretory vesicles. When transfected into the murine endocrine cell line AtT-20, the wild-type o-rab3 protein is localized exclusively to the tips of the processes, a region of the cell known to accumulate proteins associated with regulated secretory vesicles. Two mutations, Gln81 to Leu (Q81L) and Asn135 Ile (N135I), which alter GTP binding or rate of hydrolysis, blocked the localization of the o-rab3 protein to the tips of cell processes. These mutations also hindered the sequestration of ACTH-containing secretory vesicles to the process tips but did not affect the basal or stimulated release of ACTH. Moreover, the sequestration of the protein VAMP to the process tip was also hindered by the mutation. The results demonstrate a role for the rab3 proteins in localization, sequestration, and storage of secretory vesicles near their release site.

scholarly journals Structural Basis of the Proteolytic and Chaperone Activity of Chlamydia trachomatis CT441

2014 ◽  
Vol 197 (1) ◽  
pp. 211-218 ◽  
Author(s):  
Friedrich Kohlmann ◽  
Kensuke Shima ◽  
Rolf Hilgenfeld ◽  
Werner Solbach ◽  
Jan Rupp ◽  
...  
Keyword(s):  
Chlamydia Trachomatis ◽  
Protein Quality ◽  
Mutational Analysis ◽  
Chaperone Activity ◽  
Host Cells ◽  
Estrogen Signaling ◽  
Structural Basis ◽  
Developmental Cycle ◽  
Content Type

Chlamydia trachomatisis the most prevalent cause of preventable blindness worldwide and a major reason for infectious infertility in females. Several bacterial factors have been implicated in the pathogenesis ofC. trachomatis. Combining structural and mutational analysis, we have shown that the proteolytic function of CT441 depends on a conserved Ser/Lys/Gln catalytic triad and a functional substrate-binding site within a flexible PDZ (postsynaptic density of 95 kDa,discs large, andzonula occludens) domain. Previously, it has been suggested that CT441 is involved in modulating estrogen signaling responses of the host cell. Our results show that althoughin vitroCT441 exhibits proteolytic activity against SRAP1, a coactivator of estrogen receptor α, CT441-mediated SRAP1 degradation is not observed during the intracellular developmental cycle before host cells are lysed and infectious chlamydiae are released. Most compellingly, we have newly identified a chaperone activity of CT441, indicating a role of CT441 in prokaryotic protein quality control processes.

scholarly journals In Contrast to Chlamydia trachomatis, Waddlia chondrophila Grows in Human Cells without Inhibiting Apoptosis, Fragmenting the Golgi Apparatus, or Diverting Post-Golgi Sphingomyelin Transport

2015 ◽  
Vol 83 (8) ◽  
pp. 3268-3280 ◽  
Author(s):  
Stephanie Dille ◽  
Eva-Maria Kleinschnitz ◽  
Collins Waguia Kontchou ◽  
Thilo Nölke ◽  
Georg Häcker
Keyword(s):  
Chlamydia Trachomatis ◽  
Golgi Apparatus ◽  
Bacterial Species ◽  
Human Cells ◽  
Host Cells ◽  
Developmental Cycle ◽  
Important Species ◽  
Content Type ◽  
Obligate Intracellular

TheChlamydialesare an order of obligate intracellular bacteria sharing a developmental cycle inside a cytosolic vacuole, with very diverse natural hosts, from amoebae to mammals. The clinically most important species isChlamydia trachomatis. Many uncertainties remain as to howChlamydiaorganizes its intracellular development and replication. The discovery of newChlamydialesspecies from other families permits the comparative analysis of cell-biological events and may indicate events that are common to all or peculiar to some species and more or less tightly linked to “chlamydial” development. We used this approach in the infection of human cells withWaddlia chondrophila, a species from the familyWaddliaceaewhose natural host is uncertain. Compared toC. trachomatis,W. chondrophilahad slightly different growth characteristics, including faster cytotoxicity. The embedding in cytoskeletal structures was not as pronounced as for theC. trachomatisinclusion.C. trachomatisinfection generates proteolytic activity by the proteaseChlamydiaprotease-like activity factor (CPAF), which degrades host substrates upon extraction; these substrates were not cleaved in the case ofW. chondrophila. UnlikeChlamydia,W. chondrophiladid not protect against staurosporine-induced apoptosis.C. trachomatisinfection causes Golgi apparatus fragmentation and redirects post-Golgi sphingomyelin transport to the inclusion; both were absent fromW. chondrophila-infected cells. When host cells were infected with both species, growth of both species was reduced. This study highlights differences between bacterial species that both depend on obligate intracellular replication inside an inclusion. Some features seem principally dispensable for intracellular development ofChlamydialesin vitrobut may be linked to host adaptation ofChlamydiaand the higher virulence ofC. trachomatis.

Small GTP-binding Protein RalA Associates with Weibel-Palade Bodies in Endothelial Cells

1999 ◽  
Vol 82 (09) ◽  
pp. 1177-1181 ◽  
Author(s):  
Hubert de Leeuw ◽  
Pauline Wijers-Koster ◽  
Jan van Mourik ◽  
Jan Voorberg
Keyword(s):  
Endothelial Cells ◽  
Binding Proteins ◽  
Binding Protein ◽  
Control Release ◽  
Small Gtpase ◽  
Gtp Binding Protein ◽  
Two Dimensional Gel Electrophoresis ◽  
Gtp Binding Proteins ◽  
Dense Granules ◽  
Gtp Binding

SummaryIn endothelial cells von Willebrand factor (vWF) and P-selectin are stored in dense granules, so-called Weibel-Palade bodies. Upon stimulation of endothelial cells with a variety of agents including thrombin, these organelles fuse with the plasma membrane and release their content. Small GTP-binding proteins have been shown to control release from intracellular storage pools in a number of cells. In this study we have investigated whether small GTP-binding proteins are associated with Weibel-Palade bodies. We isolated Weibel-Palade bodies by centrifugation on two consecutive density gradients of Percoll. The dense fraction in which these subcellular organelles were highly enriched, was analysed by SDS-PAGE followed by GTP overlay. A distinct band with an apparent molecular weight of 28,000 was observed. Two-dimensional gel electrophoresis followed by GTP overlay revealed the presence of a single small GTP-binding protein with an isoelectric point of 7.1. A monoclonal antibody directed against RalA showed reactivity with the small GTP-binding protein present in subcellular fractions that contain Weibel-Palade bodies. The small GTPase RalA was previously identified on dense granules of platelets and on synaptic vesicles in nerve terminals. Our observations suggest that RalA serves a role in regulated exocytosis of Weibel-Palade bodies in endothelial cells.

Rap1b Association with the Platelet Cytoskeleton Occurs in the Absence of Glycoproteins IIb/IIIa

1998 ◽  
Vol 79 (04) ◽  
pp. 832-836 ◽  
Author(s):  
Thomas Fischer ◽  
Christina Duffy ◽  
Gilbert White
Keyword(s):  
Molecular Weight ◽  
Divalent Cation ◽  
Binding Proteins ◽  
Binding Protein ◽  
Platelet Membrane ◽  
Low Molecular Weight ◽  
Membrane Glycoproteins ◽  
Gtp Binding Protein ◽  
Gtp Binding Proteins ◽  
Gtp Binding

SummaryPlatelet membrane glycoproteins (GP) IIb/IIIa and rap1b, a 21 kDa GTP binding protein, associate with the triton-insoluble, activation-dependent platelet cytoskeleton with similar rates and divalent cation requirement. To examine the possibility that GPIIb/IIIa was required for rap1b association with the cytoskeleton, experiments were performed to determine if the two proteins were linked under various conditions. Chromatography of lysates from resting platelets on Sephacryl S-300 showed that GPIIb/IIIa and rap1b were well separated and distinct proteins. Immunoprecipitation of GPIIb/IIIa from lysates of resting platelets did not produce rap1b or other low molecular weight GTP binding proteins and immunoprecipitation of rap1b from lysates of resting platelets did not produce GPIIb/IIIa. Finally, rap1b was associated with the activation-dependent cytoskeleton of platelets from a patient with Glanzmann’s thrombasthenia who lacks surface expressed glycoproteins IIb and IIIa. Based on these findings, we conclude that no association between GPIIb/IIIa and rap1b is found in resting platelets and that rap1b association with the activation-dependent cytoskeleton is at least partly independent of GPIIb/IIIa.

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