Browse
Submit Data
Databases
API
Help

Dataset Information

16 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

The Chlamydia effector chlamydial outer protein N (CopN) sequesters tubulin and prevents microtubule assembly.

ABSTRACT: Chlamydia species are obligate intracellular pathogens that utilize a type three secretion system to manipulate host cell processes. Genetic manipulations are currently not possible in Chlamydia, necessitating study of effector proteins in heterologous expression systems and severely complicating efforts to relate molecular strategies used by Chlamydia to the biochemical activities of effector proteins. CopN is a chlamydial type three secretion effector that is essential for virulence. Heterologous expression of CopN in cells results in loss of microtubule spindles and metaphase plate formation and causes mitotic arrest. CopN is a multidomain protein with similarity to type three secretion system "plug" proteins from other organisms but has functionally diverged such that it also functions as an effector protein. We show that CopN binds directly to ??-tubulin but not to microtubules (MTs). Furthermore, CopN inhibits tubulin polymerization by sequestering free ??-tubulin, similar to one of the mechanisms utilized by stathmin. Although CopN and stathmin share no detectable sequence identity, both influence MT formation by sequestration of ??-tubulin. CopN displaces stathmin from preformed stathmin-tubulin complexes, indicating that the proteins bind overlapping sites on tubulin. CopN is the first bacterial effector shown to disrupt MT formation directly. This recognition affords a mechanistic understanding of a strategy Chlamydia species use to manipulate the host cell cycle.

SUBMITTER: Archuleta TL

PROVIDER: S-EPMC3190796 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Chlamydia pneumoniae effector chlamydial outer protein N sequesters fructose bisphosphate aldolase A, providing a benefit to bacterial growth.

Project description:BackgroundPathogenic chlamydiae are obligate intracellular pathogens and have adapted successfully to human cells, causing sexually transmitted diseases or pneumonia. Chlamydial outer protein N (CopN) is likely a critical effector protein secreted by the type III secretion system in chlamydiae, which manipulates host cells. However, the mechanisms of its action remain to be clarified. In this work, we aimed to identify previously unidentified CopN effector target in host cells.ResultsWe first performed a pull-down assay with recombinant glutathione S-transferase (GST) fusion CopN proteins (GST-CpCopN: Chlamydia pneumoniae TW183, GST-CtCopN: Chlamydia trachomatis D/UW-3/CX) as "bait" and soluble lysates obtained from human immortal epithelial HEp-2 cells as "prey", followed by SDS-PAGE with mass spectroscopy (MS). We found that a host cell protein specifically bound to GST-CpCopN, but not GST-CtCopN. MS revealed the host protein to be fructose bisphosphate aldolase A (aldolase A), which plays a key role in glycolytic metabolism. We also confirmed the role of aldolase A in chlamydia-infected HEp-2 cells by using two distinct experiments for gene knockdown with an siRNA specific to aldolase A transcripts, and for assessment of glycolytic enzyme gene expression levels. As a result, both the numbers of chlamydial inclusion-forming units and RpoD transcripts were increased in the chlamydia-infected aldolase A knockdown cells, as compared with the wild-type HEp-2 cells. Meanwhile, chlamydial infection tended to enhance expression of aldolase A.ConclusionsWe discovered that one of the C. pneumoniae CopN targets is the glycolytic enzyme aldolase A. Sequestering aldolase A may be beneficial to bacterial growth in infected host cells.

| S-EPMC4302594 | biostudies-literature

A Chlamydia effector recruits CEP170 to reprogram host microtubule organization.

Project description:The obligate intracellular bacterial pathogen Chlamydia trachomatis deploys virulence effectors to subvert host cell functions enabling its replication within a specialized membrane-bound compartment termed an inclusion. The control of the host cytoskeleton is crucial for Chlamydia uptake, inclusion biogenesis and cell exit. Here, we demonstrate how a Chlamydia effector rearranges the microtubule (MT) network by initiating organization of the MTs at the inclusion surface. We identified an inclusion-localized effector that is sufficient to interfere with MT assembly, which we named inclusion protein acting on MTs (IPAM). We established that IPAM recruits and stimulates the centrosomal protein 170 kDa (CEP170) to hijack the MT organizing functions of the host cell. We show that CEP170 is essential for chlamydial control of host MT assembly, and is required for inclusion morphogenesis and bacterial infectivity. Together, we demonstrate how a pathogen effector reprograms the host MT network to support its intracellular development.

| S-EPMC4582400 | biostudies-literature

Nucleotide-dependent bending flexibility of tubulin regulates microtubule assembly.

Project description:The atomic structure of tubulin in a polymerized, straight protofilament is clearly distinct from that in a curved conformation bound to a cellular depolymerizer. The nucleotide contents are identical, and in both cases the conformation of the GTP-containing, intra-dimer interface is indistinguishable from the GDP-containing, inter-dimer contact. Here we present two structures corresponding to the start and end points in the microtubule polymerization and hydrolysis cycles that illustrate the consequences of nucleotide state on longitudinal and lateral assembly. In the absence of depolymerizers, GDP-bound tubulin shows distinctive intra-dimer and inter-dimer interactions and thus distinguishes the GTP and GDP interfaces. A cold-stable tubulin polymer with the non-hydrolysable GTP analogue GMPCPP, containing semi-conserved lateral interactions, supports a model in which the straightening of longitudinal interfaces happens sequentially, starting with a conformational change after GTP binding that straightens the dimer enough for the formation of lateral contacts into a non-tubular intermediate. Closure into a microtubule does not require GTP hydrolysis.

| S-EPMC1386036 | biostudies-literature

Tubulin carbamoylation. Functional amino groups in microtubule assembly.

Project description:The characteristics of the carbamoylation of pig brain tubulin were examined by using the modification conditions with cyanate described previously [Mellado, Slebe + Maccioni (1980) Biochem. Int. I, 584--590]. The carbamoylation reaction resulted in an inhibition of microtubule assembly, which was dependent on the concentration of the modifying agent. This tubulin modification appears to inhibit the growth of microtubules. The presence of GTP did not protect tubulin against this inhibition. Electron microscopy showed a marked decrease in the number of tubules after carbamoylation, but no alterations were observed in the microtubule morphology. The incorporation of KN14CO into alpha- and beta-subunits with similar kinetics was also shown, and the carbamoylated residues were identified as epsilon-N-carbamoyl-lysine residues.

| S-EPMC1158283 | biostudies-other

Recombinant outer membrane vesicles carrying Chlamydia muridarum HtrA induce antibodies that neutralize chlamydial infection in vitro.

Project description:BACKGROUND:Outer membrane vesicles (OMVs) are spheroid particles released by all Gram-negative bacteria as a result of the budding out of the outer membrane. Since they carry many of the bacterial surface-associated proteins and feature a potent built-in adjuvanticity, OMVs are being utilized as vaccines, some of which commercially available. Recently, methods for manipulating the protein content of OMVs have been proposed, thus making OMVs a promising platform for recombinant, multivalent vaccines development. METHODS:Chlamydia muridarum DO serine protease HtrA, an antigen which stimulates strong humoral and cellular responses in mice and humans, was expressed in Escherichia coli fused to the OmpA leader sequence to deliver it to the OMV compartment. Purified OMVs carrying HtrA (CM rHtrA-OMV) were analyzed for their capacity to induce antibodies capable of neutralizing Chlamydia infection of LLC-MK2 cells in vitro. RESULTS:CM rHtrA-OMV immunization in mice induced antibodies that neutralize Chlamydial invasion as judged by an in vitro infectivity assay. This was remarkably different from what observed with an enzymatically functional recombinant HtrA expressed in, and purified from the E. coli cytoplasm (CM rHtrA). The difference in functionality between anti-CM rHtrA and anti-CM rHtrA-OMV antibodies was associated to a different pattern of protein epitopes recognition. The epitope recognition profile of anti-CM HtrA-OMV antibodies was similar to that induced in mice during Chlamydial infection. CONCLUSIONS:When expressed in OMVs HtrA appears to assume a conformation similar to the native one and this results in the elicitation of functional immune responses. These data further support the potentiality of OMVs as vaccine platform.

| S-EPMC3760637 | biostudies-literature

Assembly of chick brain MAP2-tubulin microtubule protein. Analysis of tubulin subunit flux rates by immunofluorescence microscopy.

Project description:A filter-based immunofluorescence-microscopy method for obtaining microtubule lengths has been developed and evaluated. Kinetic constants and mean lengths obtained show close agreement with those obtained by complementary methods applied to chick brain MAP2-tubulin microtubule protein in NaCl-supplemented buffer. The filter-based method has been used to estimate tubulin subunit flux (Jon) resulting from isothermal dilution of microtubule populations to various free tubulin concentrations, (c). This experimental Jon(c) plot is significantly different from that predicted by a variety of theoretical models, but is consistent with a 'lateral cap' model of dynamic instability [Bayley, Schilstra & Martin (1990) J. Cell. Sci. 95, 33-48] adapted to accommodate the observed vectorial GTP hydrolysis.

| S-EPMC1151216 | biostudies-other

Activated actin-depolymerizing factor/cofilin sequesters phosphorylated microtubule-associated protein during the assembly of alzheimer-like neuritic cytoskeletal striations.

Project description:In Alzheimer's disease (AD), rod-like cofilin aggregates (cofilin-actin rods) and thread-like inclusions containing phosphorylated microtubule-associated protein (pMAP) tau form in the brain (neuropil threads), and the extent of their presence correlates with cognitive decline and disease progression. The assembly mechanism of these respective pathological lesions and the relationship between them is poorly understood, yet vital to understanding the causes of sporadic AD. We demonstrate that, during mitochondrial inhibition, activated actin-depolymerizing factor (ADF)/cofilin assemble into rods along processes of cultured primary neurons that recruit pMAP/tau and mimic neuropil threads. Fluorescence resonance energy transfer analysis revealed colocalization of cofilin-GFP (green fluorescent protein) and pMAP in rods, suggesting their close proximity within a cytoskeletal inclusion complex. The relationship between pMAP and cofilin-actin rods was further investigated using actin-modifying drugs and small interfering RNA knockdown of ADF/cofilin in primary neurons. The results suggest that activation of ADF/cofilin and generation of cofilin-actin rods is required for the subsequent recruitment of pMAP into the inclusions. Additionally, we were able to induce the formation of pMAP-positive ADF/cofilin rods by exposing cells to exogenous amyloid-beta (Abeta) peptides. These results reveal a common pathway for pMAP and cofilin accumulation in neuronal processes. The requirement of activated ADF/cofilin for the sequestration of pMAP suggests that neuropil thread structures in the AD brain may be initiated by elevated cofilin activation and F-actin bundling that can be caused by oxidative stress, mitochondrial dysfunction, or Abeta peptides, all suspected initiators of synaptic loss and neurodegeneration in AD.

| S-EPMC2821881 | biostudies-literature

The lattice as allosteric effector: structural studies of alphabeta- and gamma-tubulin clarify the role of GTP in microtubule assembly.

Project description:GTP-dependent microtubule polymerization dynamics are required for cell division and are accompanied by domain rearrangements in the polymerizing subunit, alphabeta-tubulin. Two opposing models describe the role of GTP and its relationship to conformational change in alphabeta-tubulin. The allosteric model posits that unpolymerized alphabeta-tubulin adopts a more polymerization-competent conformation upon GTP binding. The lattice model posits that conformational changes occur only upon recruitment into the growing lattice. Published data support a lattice model, but are largely indirect and so the allosteric model has prevailed. We present two independent solution probes of the conformation of alphabeta-tubulin, the 2.3 A crystal structure of gamma-tubulin bound to GDP, and kinetic simulations to interpret the functional consequences of the structural data. These results (with our previous gamma-tubulin:GTPgammaS structure) support the lattice model by demonstrating that major domain rearrangements do not occur in eukaryotic tubulins in response to GTP binding, and that the unpolymerized conformation of alphabeta-tubulin differs significantly from the polymerized one. Thus, geometric constraints of lateral self-assembly must drive alphabeta-tubulin conformational changes, whereas GTP plays a secondary role to tune the strength of longitudinal contacts within the microtubule lattice. alphabeta-Tubulin behaves like a bent spring, resisting straightening until forced to do so by GTP-mediated interactions with the growing microtubule. Kinetic simulations demonstrate that resistance to straightening opposes microtubule initiation by specifically destabilizing early assembly intermediates that are especially sensitive to the strength of lateral interactions. These data provide new insights into the molecular origins of dynamic microtubule behavior.

| S-EPMC2291134 | biostudies-literature

Assembly of chick brain MAP2-tubulin microtubule protein. Characterization of the protein and the MAP2-dependent addition of tubulin dimers.

Project description:The principle proteins present in twice-cycled chick brain microtubule protein were characterized. The protein consists of a stoichiometric mixture of MAP2 and tubulin, together with a number of minor components. Its composition remains unaltered after a third cycle of assembly in a buffer supplemented with 67 mM-NaCl, with the exception of the phosphorylation of MAP2 to a low level (congruent to 1 mol.mol-1). The inclusion of 67 mM-NaCl dissociates the MAP2-tubulin oligomers, and restricts the assembly to the MAP2-dependent addition and loss of tubulin dimers, such that the assembly kinetics approximate to a simple pseudo-first-order reaction. The assembled microtubules exhibit dynamic instability, with no evidence for end-to-end annealing.

| S-EPMC1151214 | biostudies-other

Systematic identification of tubulin-interacting fragments of the microtubule-associated protein Tau leads to a highly efficient promoter of microtubule assembly.

Project description:Tau is a microtubule-associated protein that stabilizes microtubules and stimulates their assembly. Current descriptions of the tubulin-interacting regions of Tau involve microtubules as the target and result mainly from deletions of Tau domains based on sequence analysis and from NMR spectroscopy experiments. Here, instead of microtubules, we use the complex of two tubulin heterodimers with the stathmin-like domain of the RB3 protein (T(2)R) to identify interacting Tau fragments generated by limited proteolysis. We show that fragments in the proline-rich region and in the microtubule-binding repeats domain each interact on their own not only with T(2)R but also with microtubules, albeit with moderate affinity. NMR analysis of the interaction with T(2)R of constructs in these two regions leads to a fragment, composed of adjacent parts of the microtubule-binding repeat domain and of the proline-rich region, that binds tightly to stabilized microtubules. This demonstrates the synergy of the two Tau regions we identified in the Tau-microtubule interaction. Moreover, we show that this fragment, which binds to two tubulin heterodimers, stimulates efficiently microtubule assembly.

| S-EPMC3190875 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data