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1.
Int J Med Microbiol ; 308(1): 197-205, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29122514

RESUMO

Chlamydia trachomatis is an important human pathogen. This obligate intracellular bacterium grows inside the eukaryotic cell in a membrane-bound compartment, the inclusion. Recent global approaches describe the interactions of C. trachomatis with its host cell and indicate the inclusion is an intracellular trafficking hub embedded into the cellular vesicular trafficking pathways recruiting subunits of the retromer protein complex of the host cell. Here we review these recent developments in deciphering Chlamydia-host cell interactions with emphasis on the role of the retromer complex.


Assuntos
Infecções por Chlamydia/microbiologia , Chlamydia trachomatis/metabolismo , Interações Hospedeiro-Patógeno , Animais , Proteínas de Bactérias/metabolismo , Infecções por Chlamydia/metabolismo , Chlamydia trachomatis/crescimento & desenvolvimento , Humanos , Corpos de Inclusão/metabolismo , Corpos de Inclusão/microbiologia , Transporte Proteico , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo
2.
Cell Microbiol ; 19(10)2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28544656

RESUMO

Chlamydiaceae are bacterial pathogens that cause diverse diseases in humans and animals. Despite their broad host and tissue tropism, all Chlamydia species share an obligate intracellular cycle of development and have evolved sophisticated mechanisms to interact with their eukaryotic host cells. Here, we have analysed interactions of the zoonotic pathogen Chlamydia psittaci with a human epithelial cell line. We found that C. psittaci recruits the ceramide transport protein (CERT) to its inclusion. Chemical inhibition and CRISPR/Cas9-mediated knockout of CERT showed that CERT is a crucial factor for C. psittaci infections thereby affecting different stages of the infection including inclusion growth and infectious progeny formation. Interestingly, the uptake of fluorescently labelled sphingolipids in bacteria inside the inclusion was accelerated in CERT-knockout cells indicating that C. psittaci can exploit CERT-independent sphingolipid uptake pathways. Moreover, the CERT-specific inhibitor HPA-12 strongly diminished sphingolipid transport to inclusions of infected CERT-knockout cells, suggesting that other HPA-12-sensitive factors are involved in sphingolipid trafficking to C. psittaci. Further analysis is required to decipher these interactions and to understand their contributions to bacterial development, host range, tissue tropism, and disease outcome.


Assuntos
Chlamydophila psittaci/metabolismo , Chlamydophila psittaci/patogenicidade , Proteínas Serina-Treonina Quinases/metabolismo , Esfingolipídeos/metabolismo , Transporte Biológico/fisiologia , Linhagem Celular , Ceramidas/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/fisiologia , Humanos
3.
Nature ; 483(7391): 608-12, 2012 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-22460906

RESUMO

Deregulated expression of the MYC oncoprotein contributes to the genesis of many human tumours, yet strategies to exploit this for a rational tumour therapy are scarce. MYC promotes cell growth and proliferation, and alters cellular metabolism to enhance the provision of precursors for phospholipids and cellular macromolecules. Here we show in human and murine cell lines that oncogenic levels of MYC establish a dependence on AMPK-related kinase 5 (ARK5; also known as NUAK1) for maintaining metabolic homeostasis and for cell survival. ARK5 is an upstream regulator of AMPK and limits protein synthesis via inhibition of the mammalian target of rapamycin 1 (mTORC1) signalling pathway. ARK5 also maintains expression of mitochondrial respiratory chain complexes and respiratory capacity, which is required for efficient glutamine metabolism. Inhibition of ARK5 leads to a collapse of cellular ATP levels in cells expressing deregulated MYC, inducing multiple pro-apoptotic responses as a secondary consequence. Depletion of ARK5 prolongs survival in MYC-driven mouse models of hepatocellular carcinoma, demonstrating that targeting cellular energy homeostasis is a valid therapeutic strategy to eliminate tumour cells that express deregulated MYC.


Assuntos
Regulação Neoplásica da Expressão Gênica , Genes myc/genética , Proteínas Quinases/metabolismo , Proteínas Repressoras/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Apoptose , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Respiração Celular , Sobrevivência Celular , Transformação Celular Neoplásica/genética , Modelos Animais de Doenças , Doxiciclina/farmacologia , Transporte de Elétrons , Glutamina/metabolismo , Homeostase , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Mitocôndrias/metabolismo , Complexos Multiproteicos , Proteína Oncogênica p55(v-myc)/genética , Proteína Oncogênica p55(v-myc)/metabolismo , Biossíntese de Proteínas , Proteínas Quinases/deficiência , Proteínas Quinases/genética , Proteínas/antagonistas & inibidores , Proteínas/metabolismo , Interferência de RNA , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/deficiência , Proteínas Repressoras/genética , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo
4.
PLoS Pathog ; 11(6): e1004883, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26042774

RESUMO

Chlamydia trachomatis is an important human pathogen that replicates inside the infected host cell in a unique vacuole, the inclusion. The formation of this intracellular bacterial niche is essential for productive Chlamydia infections. Despite its importance for Chlamydia biology, a holistic view on the protein composition of the inclusion, including its membrane, is currently missing. Here we describe the host cell-derived proteome of isolated C. trachomatis inclusions by quantitative proteomics. Computational analysis indicated that the inclusion is a complex intracellular trafficking platform that interacts with host cells' antero- and retrograde trafficking pathways. Furthermore, the inclusion is highly enriched for sorting nexins of the SNX-BAR retromer, a complex essential for retrograde trafficking. Functional studies showed that in particular, SNX5 controls the C. trachomatis infection and that retrograde trafficking is essential for infectious progeny formation. In summary, these findings suggest that C. trachomatis hijacks retrograde pathways for effective infection.


Assuntos
Infecções por Chlamydia/metabolismo , Chlamydia trachomatis/patogenicidade , Vacúolos/metabolismo , Separação Celular , Chlamydia trachomatis/metabolismo , Citometria de Fluxo , Células HeLa , Humanos , Corpos de Inclusão/metabolismo , Transporte Proteico , Proteoma , Proteômica , RNA Interferente Pequeno , Espectrometria de Massas em Tandem , Transfecção
5.
Mol Microbiol ; 90(4): 744-55, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24033548

RESUMO

Mitochondrial translation in the parasitic protozoan Trypanosoma brucei relies on imported eukaryotic-type tRNAs as well as on bacterial-type ribosomes that have the shortest known rRNAs. Here we have identified the mitochondrial translation elongation factors EF-Tu, EF-Ts, EF-G1 and release factor RF1 of trypanosomatids and show that their ablation impairs growth and oxidative phosphorylation. In vivo labelling experiments and a SILAC-based analysis of the global proteomic changes induced by EF-Tu RNAi directly link EF-Tu to mitochondrial translation. Moreover, EF-Tu RNAi reveals downregulation of many nuclear encoded subunits of cytochrome oxidase as well as of components of the bc1-complex, whereas most cytosolic ribosomal proteins were upregulated. Interestingly, T. brucei EF-Tu has a 30-amino-acid-long, highly charged subdomain, which is unique to trypanosomatids. A combination of RNAi and complementation experiments shows that this subdomain is essential for EF-Tu function, but that it can be replaced by a similar sequence found in eukaryotic EF-1a, the cytosolic counterpart of EF-Tu. A recent cryo-electron microscopy study revealed that trypanosomatid mitochondrial ribosomes have a unique intersubunit space that likely harbours the EF-Tu binding site. These findings suggest that the trypanosomatid-specific EF-Tu subdomain serves as an adaption for binding to these unusual mitochondrial ribosomes.


Assuntos
Motivos de Aminoácidos , Mitocôndrias/metabolismo , Proteínas Mitocondriais/química , Fator Tu de Elongação de Peptídeos/química , Proteínas de Protozoários/química , Ribossomos/metabolismo , Trypanosoma brucei brucei/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Regulação da Expressão Gênica , Mitocôndrias/genética , Proteínas Mitocondriais/fisiologia , Dados de Sequência Molecular , Mutação , Fosforilação Oxidativa , Fator G para Elongação de Peptídeos/genética , Fator G para Elongação de Peptídeos/metabolismo , Fator Tu de Elongação de Peptídeos/genética , Fator Tu de Elongação de Peptídeos/fisiologia , Fatores de Alongamento de Peptídeos/genética , Fatores de Alongamento de Peptídeos/metabolismo , Fatores de Terminação de Peptídeos/genética , Fatores de Terminação de Peptídeos/metabolismo , Proteômica , Proteínas de Protozoários/genética , Proteínas de Protozoários/fisiologia , Interferência de RNA , Alinhamento de Sequência , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/crescimento & desenvolvimento
6.
Antimicrob Agents Chemother ; 58(9): 5537-46, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25001308

RESUMO

Chlamydia trachomatis is a medically important human pathogen causing different diseases, including trachoma, the leading cause of preventable blindness in developing countries, and sexually transmitted infections that can lead to infertility and ectopic pregnancies. There is no vaccine against C. trachomatis at present. Broad-spectrum antibiotics are used as standard therapy to treat the infection but have unwanted side effects, such as inducing persistent or recurring infections and affecting the host microbiome, necessitating the development of novel anti-Chlamydia therapies. Here, we describe the establishment of a robust, fast, and simple plaque assay using liquid overlay medium (LOM) for the identification of anti-Chlamydia compounds. Using the LOM plaque assay, we identified nitrobenzoxadiazole (NBD)-labeled 1-O-methyl-ceramide-C16 as a compound that efficiently inhibits C. trachomatis replication without affecting the viability of the host cell. Further detailed analyses indicate that 1-O-methyl-NBD-ceramide-C16 acts outside the inclusion. Thereby, 1-O-methyl-NBD-ceramide-C16 represents a lead compound for the development of novel anti-Chlamydia drugs and furthermore constitutes an agent to illuminate sphingolipid trafficking pathways in Chlamydia infections.


Assuntos
Antibacterianos/farmacologia , Ceramidas/farmacologia , Chlamydia trachomatis/efeitos dos fármacos , Técnicas de Laboratório Clínico/métodos , Animais , Antibacterianos/uso terapêutico , Transporte Biológico , Linhagem Celular Tumoral , Ceramidas/uso terapêutico , Infecções por Chlamydia/tratamento farmacológico , Descoberta de Drogas/métodos , Células HeLa , Humanos , Camundongos , Testes de Sensibilidade Microbiana , Esfingolipídeos/metabolismo
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