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"Inc protein"
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Embrace the suck : what I learned at the box about hard work, (very) sore muscles, and burpees before sunrise
A lifelong amateur athlete and former editor of \"Bicycling\" magazine chronicles the year he devoted to trying to master all of the basic CrossFit exercises and immersing himself in the paleo diet.
Book
A Co-infection Model System and the Use of Chimeric Proteins to Study Chlamydia Inclusion Proteins Interaction
is an obligate intracellular bacterium associated with trachoma and sexually transmitted diseases. During its intracellular developmental cycle,
resides in a membrane bound compartment called the inclusion. A subset of Type III secreted effectors, the inclusion membrane proteins (Inc), are inserted into the inclusion membrane. Inc proteins are strategically positioned to promote inclusion interaction with host factors and organelles, a process required for bacterial replication, but little is known about Inc proteins function or host interacting partners. Moreover, it is unclear whether each Inc protein has a distinct function or if a subset of Inc proteins interacts with one another to perform their function. Here, we used IncD as a model to investigate Inc/Inc interaction in the context of Inc protein expression in
. We developed a co-infection model system to display different tagged Inc proteins on the surface of the same inclusion. We also designed chimeric Inc proteins to delineate domains important for interaction. We showed that IncD can self-interact and that the full-length protein is required for dimerization and/or oligomerization. Altogether our approach can be generalized to any Inc protein and will help to characterize the molecular mechanisms by which
Inc proteins interact with themselves and/or host factors, eventually leading to a better understanding of
interaction with the mammalian host.
Journal Article
Inc/GFP chimera protein-based interactomics reveals host cellular interactions of Cps0558, a novel Chlamydia psittaci inclusion protein
Abstract
The obligate intracellular Gram-negative bacterium Chlamydia psittaci, a zoonotic pathogen transmissible between birds and humans, has played a pioneering role in research on its membrane-bound replicative niche termed the inclusion. Inclusion membrane proteins (Inc proteins) are crucial for Chlamydia–host interactions and were first identified in C. psittaci. This study investigates putative C. psittaci Inc proteins by a combination of in silico analyses, immunofluorescence and, strikingly, a new Inc/GFP chimera protein-based interactomics approach to identify host cellular interaction partners. Here, we report a novel C. psittaci Inc protein, Cps0558, along with respective host cellular interaction partners, in particular ACAD11, which is involved in lipid metabolism. We confirm their physical interaction in the native infection context, supporting the physiological relevance of our chimera-based screen. Furthermore, new interaction partners for the known Inc protein IncA are identified, revealing a potential role of IncA as modulator of the host ubiquitylation system. These results provide further insights into the biology of C. psittaci and present a novel tool for studying Inc proteins under conditions closely resembling their natural tertiary structure.
This study investigates putative Chlamydia psittaci Inc proteins by a combination of in silico analyses, immunofluorescence, and a new Inc/GFP chimera protein-based interactomics approach to identify host cellular interaction partners.
Journal Article
Conserved properties of Drosophila Insomniac link sleep regulation and synaptic function
Sleep is an ancient animal behavior that is regulated similarly in species ranging from flies to humans. Various genes that regulate sleep have been identified in invertebrates, but whether the functions of these genes are conserved in mammals remains poorly explored. Drosophila insomniac (inc) mutants exhibit severely shortened and fragmented sleep. Inc protein physically associates with the Cullin-3 (Cul3) ubiquitin ligase, and neuronal depletion of Inc or Cul3 strongly curtails sleep, suggesting that Inc is a Cul3 adaptor that directs the ubiquitination of neuronal substrates that impact sleep. Three proteins similar to Inc exist in vertebrates-KCTD2, KCTD5, and KCTD17-but are uncharacterized within the nervous system and their functional conservation with Inc has not been addressed. Here we show that Inc and its mouse orthologs exhibit striking biochemical and functional interchangeability within Cul3 complexes. Remarkably, KCTD2 and KCTD5 restore sleep to inc mutants, indicating that they can substitute for Inc in vivo and engage its neuronal targets relevant to sleep. Inc and its orthologs localize similarly within fly and mammalian neurons and can traffic to synapses, suggesting that their substrates may include synaptic proteins. Consistent with such a mechanism, inc mutants exhibit defects in synaptic structure and physiology, indicating that Inc is essential for both sleep and synaptic function. Our findings reveal that molecular functions of Inc are conserved through ~600 million years of evolution and support the hypothesis that Inc and its orthologs participate in an evolutionarily conserved ubiquitination pathway that links synaptic function and sleep regulation.
Journal Article
Reconceptualizing the chlamydial inclusion as a pathogen-specified parasitic organelle: an expanded role for Inc proteins
Chlamydia is an obligate intracellular pathogen that develops in the host cell in a vacuole termed the chlamydial inclusion. The prevailing concept of the chlamydial inclusion is of a parasitophorous vacuole. Here, the inclusion is the recipient of one-way host-pathogen interactions thus draining nutrients from the cell and negatively impacting it. While Chlamydia orchestrates some aspects of cell function, recent data indicate host cells remain healthy up until, and even after, chlamydial egress. Thus, while Chlamydia relies on the host cell for necessary metabolites, the overall function of the host cell, during chlamydial growth and development, is not grossly disturbed. This is consistent with the obligate intracellular organism's interest to maintain viability of its host. To this end, Chlamydia expresses inclusion membrane proteins, Incs, which serve as molecular markers for the inclusion membrane. Incs also contribute to the physical structure of the inclusion membrane and facilitate host-pathogen interactions across it. Given the function of Incs and the dynamic interactions that occur at the inclusion membrane, we propose that the inclusion behaves similarly to an organelle-albeit one that benefits the pathogen. We present the hypothesis that the chlamydial inclusion acts as a pathogen-specified parasitic organelle. This representation integrates the inclusion within existing subcellular trafficking pathways to divert a subset of host-derived metabolites thus maintaining host cell homeostasis. We review the known interactions of the chlamydial inclusion with the host cell and discuss the role of Inc proteins in the context of this model and how this perspective can impact the study of these proteins. Lessons learnt from the chlamydial pathogen-specified parasitic organelle can be applied to other intracellular pathogens. This will increase our understanding of how intracellular pathogens engage the host cell to establish their unique developmental niches.
Journal Article
The Hypothetical Inclusion Membrane Protein CPSIT_0846 Regulates Mitochondrial-Mediated Host Cell Apoptosis via the ERK/JNK Signaling Pathway
Chlamydia psittaci is an important zoonotic factor associated with human and animal atypical pneumonia. Resisting host cell apoptosis is central to sustaining Chlamydia infection in vivo . Chlamydia can secrete inclusion membrane proteins (Incs) that play important roles in their development cycle and pathogenesis. CPSIT_0846 is an Inc protein in C. psittaci identified by our team in previous work. In the current study, we investigated the regulatory role of CPSIT_0846 in HeLa cell apoptosis, and explored potential mechanisms. The results showed that HeLa cells treated with CPSIT_0846 contained fewer apoptotic bodies and exhibited a lower apoptotic rate than untreated cells either with Hoechst 33258 fluorescence staining or flow cytometry with or without induction by staurosporine (STS). CPSIT_0846 could increase the phosphorylation of the extracellular signal-regulated kinases 1/2 (ERK1/2) or stress-activated protein kinases/c-Jun amino-terminal kinases (SAPK/JNK) signaling pathways, and the Bcl-2 associated X protein (Bax)/B cell lymphoma 2 (Bcl-2) ratio, levels of cleaved caspase-3/9 and cleaved Poly-ADP-ribose polymerase (PARP) were significantly up-regulated following inhibition of ERK1/2 or SAPK/JNK pathways with U0126 or SP600125. After carbonyl cyanide 3-chlorophenylhydrazone (CCCP) treatment, the mitochondrial membrane potential (MMP) of cells was significantly decreased in control group, but stable in the CPSIT_0846 treated one, and less cytochrome c (Cyt.c) was released into the cytoplasm. Inhibition of the ERK1/2 or SAPK/JNK pathway significantly decreased the JC-1 red-green fluorescence signal, and promoted Cyt.c discharge into the cytoplasm in HeLa cells treated with CPSIT_0846. In conclusion, CPSIT_0846 can regulate mitochondrial pathway-mediated apoptosis in HeLa cells by activating the ERK/JNK signaling pathway.
Journal Article
Characterization of interactions between inclusion membrane proteins from Chlamydia trachomatis
Chlamydiae are obligate intracellular pathogens of eukaryotes. The bacteria grow in an intracellular vesicle called an inclusion, the membrane of which is heavily modified by chlamydial proteins called Incs (Inclusion membrane proteins). Incs represent 7-10% of the genomes of Chlamydia and, given their localization at the interface between the host and the pathogen, likely play a key role in the development and pathogenesis of the bacterium. However, their functions remain largely unknown. Here, we characterized the interaction properties between various Inc proteins of C. trachomatis, using a bacterial two-hybrid (BACTH) method suitable for detecting interactions between integral membrane proteins. To validate this approach, we first examined the oligomerization properties of the well-characterized IncA protein and showed that both the cytoplasmic domain and the transmembrane region independently contribute to IncA oligomerization. We then analyzed a set of Inc proteins and identified novel interactions between these components. Two small Incs, IncF, and Ct222, were found here to interact with many other Inc proteins and may thus represent interaction nodes within the inclusion membrane. Our data suggest that the Inc proteins may assemble in the membrane of the inclusion to form specific multi-molecular complexes in an hierarchical and temporal manner. These studies will help to better define the putative functions of the Inc proteins in the infectious process of Chlamydia.
Journal Article
Murine Endometrial Organoids to Model Chlamydia Infection
The obligate intracellular bacterium
is the leading cause of bacterial sexually transmitted infections. Once internalized in host cells,
undergoes a biphasic developmental cycle within a membrane-bound compartment, known as the inclusion. Successful establishment of the intracellular niche relies on bacterial Type III effector proteins, such as Inc proteins.
and
systems have contributed to elucidating the intracellular lifestyle of
, but additional models combining the archetypal environment of infection with the advantages of
systems are needed. Organoids are three-dimensional structures that recapitulate the microanatomy of an organ's epithelial layer, bridging the gap between
and
systems. Organoids are emerging as relevant model systems to study interactions between bacterial pathogens and their hosts. Here, we took advantage of recently developed murine endometrial organoids (EMOs) and present a
-murine EMO infection model system. Confocal microscopy of EMOs infected with fluorescent protein-expressing bacteria revealed that inclusions are formed within the cytosol of epithelial cells. Moreover, infection with a
strain that allows for the tracking of RB to EB transition indicated that the bacteria undergo a full developmental cycle, which was confirmed by harvesting infectious bacteria from infected EMOs. Finally, the inducible gene expression and cellular localization of a
Inc protein within infected EMOs further demonstrated that this model is compatible with the study of Type III secreted effectors. Altogether, we describe a novel and relevant system for the study of
-host interactions.
Journal Article
Recombinant protein CPSIT_0846 induces protective immunity against Chlamydia psittaci infection in BALB/c mice
Chlamydia psittaci is an obligate intracellular bacteria that causes respiratory disease in poultry and humans. Currently, there are no licensed vaccines against chlamydial infection in humans. The transmembrane head protein CPSIT_0846 of C. psittaci is a putative member of the larger Inc protein family. In this study, we investigated immunogenicity and protective efficacy of the recombinant CPSIT_0846 protein in BALB/c mice. Mice immunized with CPSIT_0846 developed strong T-lymphocyte responses that were recalled by the immunogen CPSIT_0846 in an in vitro restimulation assay. These T cells displayed a strong Th1-biased cytokine profile with high levels of IFN-γ. At the same time, a strong humoral immune response was also detected in the immunized mice with high titers of Chlamydia psittaci-specific serum IgG antibodies. More importantly, the robust immune responses correlated well with significantly reduced chlamydial burden and inflammatory pathology in the mouse lungs upon an airway challenge infection. The above results together suggest that the CPSIT_0846 protein may be a potential vaccine candidate antigen for inducing protection against C. psittaci infection and disease in the airway.
Journal Article
The Human Centrosomal Protein CCDC146 Binds Chlamydia trachomatis Inclusion Membrane Protein CT288 and Is Recruited to the Periphery of the Chlamydia-Containing Vacuole
is an obligate intracellular human pathogen causing mainly ocular and genital infections of significant clinical and public health impact.
multiplies intracellularly in a membrane bound vacuole, known as inclusion. Both extracellularly and from within the inclusion,
uses a type III secretion system to deliver several effector proteins into the cytoplasm of host cells. A large proportion of these effectors, the inclusion membrane (Inc) proteins, are exposed to the host cell cytosol but possess a characteristic hydrophobic domain mediating their insertion in the inclusion membrane. By yeast two-hybrid, we found that
Inc CT288 interacts with the human centrosomal protein CCDC146 (coiled-coil domain-containing protein 146). The interaction was also detected by co-immunoprecipitation in mammalian cells either ectopically expressing CCDC146 and CT288 or ectopically expressing CCDC146 and infected by a
strain expressing epitope-tagged and inclusion membrane-localized CT288. In uninfected mammalian cells, ectopically expressed full-length CCDC146 (955 amino acid residues) localized at the centrosome; but in cells infected by wild-type
, its centrosomal localization was less evident and CCDC146 accumulated around the inclusion. Recruitment of CCDC146 to the inclusion periphery did not require intact host Golgi, microtubules or microfilaments, but was dependent on chlamydial protein synthesis. Full-length CCDC146 also accumulated at the periphery of the inclusion in cells infected by a
mutant; however, a C-terminal fragment of CCDC146 (residues 692-955), which interacts with CT288, showed differences in localization at the periphery of the inclusion in cells infected by wild-type or
mutant
. This suggests a model in which chlamydial proteins other than CT288 recruit CCDC146 to the periphery of the inclusion, where the CT288-CCDC146 interaction might contribute to modulate the function of this host protein.
Journal Article