RESUMO
Cyclic Adenosine 3',5'-monophosphate (cAMP) is a key second messenger known to directly regulate not only the protein kinase A (PKA) activity but also other important molecules such as the exchange protein activated by cAMP (EPAC), which is as a guanine nucleotide exchange factor (GEF) of the low molecular weight GTPase, Rap2. Coxiella burnetii is a Gram negative bacterium that survives and grows in a large Coxiella replicative vacuole (CRV), which displays lysosomal and autophagic features. In this report, we present evidence that both, EPAC and its downstream effector Rap2b, were recruited to the CRV. The transient over-expression of the Rap2b wt protein, but not its inactive mutant Rap2b ΔAAX, markedly inhibited the development of the large CRV. Additionally, Rap2b wtinhibited the fusion of early Coxiella phagosomes with the fully developed CRV, indicating that homotypic fusion events are altered in the presence of high levels of Rap2b wt. Likewise, the fusion of endosome/lysosomal compartments (heterotypic fusions) with the large CRV was also affected by the over-expression of this GTPase. Interestingly, cell overexpression of Rap2b wt markedly decreased the levels of the v-SNARE, Vamp7, suggesting that this down-regulation impairs the homotypic and heterotypic fusions events of the Coxiella vacuole.
Assuntos
Coxiella burnetii/fisiologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Febre Q/metabolismo , Vacúolos/microbiologia , Proteínas rap de Ligação ao GTP/metabolismo , Animais , Células CHO , Chlorocebus aethiops , Cricetulus , AMP Cíclico/metabolismo , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Fusão de Membrana , Fagossomos/metabolismo , Fagossomos/microbiologia , Febre Q/microbiologia , Vacúolos/metabolismo , Células VeroRESUMO
Microtubules (Mts) are dynamic cytoskeleton structures that play a key role in vesicular transport. The Mts-mediated transport depends on motor proteins named kinesins and the dynein/dynactin motor complex. The Rab7 adapter protein FYCO1 controls the anterograde transport of the endocytic compartments through the interaction with the kinesin KIF5. Rab7 and its partner RILP induce the recruitment of dynein/dynactin to late endosomes regulating its retrograde transport to the perinuclear area to fuse with lysosomes. The late endosomal-lysosomal fusion is regulated by the HOPS complex through its interaction with RILP and the GTPase Arl8. Coxiella burnetii (Cb), the causative agent of Q fever, is an obligate intracellular pathogen, which generates a large compartment with autophagolysosomal characteristics named Cb-containing vacuole (CCV). The CCV forms through homotypic fusion between small non-replicative CCVs (nrCCV) and through heterotypic fusion with other compartments, such as endosomes and lysosomes. In this work, we characterise the role of Mts, motor proteins, RILP/Rab7 and Arl8 on the CCV biogenesis. The formation of the CCV was affected when either the dynamics and/or the acetylation state of Mts were modified. Similarly, the overexpression of the dynactin subunit non-functional mutants p150Glued and RILP led to the formation of small nrCCVs. This phenomenon is not observed in cells overexpressing WT proteins, the motor KIF5 or its interacting protein FYCO1. The formation of the CCV was normal in infected cells that overexpressed Arl8 alone or together with hVps41 (a HOPS subunit) or in cells co-overexpressing hVps41 and RILP. The dominant negative mutant of Arl8 and the non-functional hVps41 inhibited the formation of the CCV. When the formation of CCV was affected, the bacterial multiplication diminished. Our results suggest that nrCCVs recruit the molecular machinery that regulate the Mts-dependent retrograde transport, Rab7/RILP and the dynein/dynactin system, as well as the tethering processes such as HOPS complex and Arl8 to finally originate the CCV where C. burnetii multiplies.
Assuntos
Coxiella burnetii/metabolismo , Dineínas/metabolismo , Microtúbulos/metabolismo , Citoesqueleto de Actina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Transporte Biológico , Chlorocebus aethiops , Coxiella burnetii/patogenicidade , Citoesqueleto/metabolismo , Complexo Dinactina/metabolismo , Endossomos/metabolismo , Células HeLa , Humanos , Lisossomos/metabolismo , Microtúbulos/fisiologia , Transporte Proteico/fisiologia , Febre Q/metabolismo , Vacúolos/metabolismo , Células Vero , Proteínas rab de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7RESUMO
BACKGROUND: Cortactin is a key regulator of the actin cytoskeleton and is involved in pathogen-host cell interactions. Numerous pathogens exploit the phagocytic process and actin cytoskeleton to infect host cells. Coxiella burnetii, the etiologic agent of Q fever, is internalized by host cells through a molecular mechanism that is poorly understood. METHODOLOGY/PRINCIPAL FINDING: Here we analyzed the role of different cortactin motifs in the internalization of C. burnetii by non-phagocytic cells. C. burnetii internalization into HeLa cells was significantly reduced when the cells expressed GFP-cortactin W525K, which carries a mutation in the SH3 domain that renders the protein unable to bind targets such as N-WASP. However, internalization was unaffected when the cells expressed the W22A mutant, which has a mutation in the N-terminal acidic region that destroys the protein's ability to bind and activate Arp2/3. We also determined whether the phosphorylation status of cortactin is important for internalization. Expression of GFP-cortactin 3F, which lacks phosphorylatable tyrosines, significantly increased internalization of C. burnetii, while expression of GFP-cortactin 3D, a phosphotyrosine mimic, did not affect it. In contrast, expression of GFP-cortactin 2A, which lacks phosphorylatable serines, inhibited C. burnetii internalization, while expression of GFP-cortactin SD, a phosphoserine mimic, did not affect it. Interestingly, inhibitors of Src kinase and the MEK-ERK kinase pathway blocked internalization. In fact, both kinases reached maximal activity at 15 min of C. burnetii infection, after which activity decreased to basal levels. Despite the decrease in kinase activity, cortactin phosphorylation at Tyr421 reached a peak at 1 h of infection. CONCLUSIONS/SIGNIFICANCE: Our results suggest that the SH3 domain of cortactin is implicated in C. burnetii entry into HeLa cells. Furthermore, cortactin phosphorylation at serine and dephosphorylation at tyrosine favor C. burnetii internalization. We present evidence that ERK and Src kinases play a role early in infection by this pathogen.
Assuntos
Citoesqueleto de Actina/metabolismo , Cortactina/metabolismo , Coxiella burnetii/metabolismo , Febre Q/microbiologia , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Endocitose , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células HeLa , Humanos , Fagócitos/metabolismo , Fosforilação , Febre Q/metabolismo , Quinases da Família src/metabolismoRESUMO
Coxiella burnetii is the etiological agent of the human disease, Q fever, and is an obligate intracellular bacterium that invades and multiplies in a vacuole with lysosomal characteristics. We have previously shown that Coxiella interacts with the autophagic pathway as a strategy for its survival and replication. In addition, recent studies have shown that Coxiella exerts anti-apoptotic activity to maintain the host cell viability, thus generating a persistent infection. In the present report, we have explored the role of Beclin 1 and Bcl-2 in C. burnetii infection to elucidate how this bacterium modulates autophagy and apoptosis to its own benefit. Beclin 1, a Bcl-2 interacting protein, is required for autophagy. In this study, we show that Beclin 1 is recruited to the Coxiella-membrane vacuole, favoring its development and bacterial replication. In contrast, the anti-apoptotic protein Bcl-2 alters the normal development of the Coxiella-replicative compartment, in spite of also being recruited to the vacuole membrane. Furthermore, both vacuole development and the anti-apoptotic effect of C. burnetii are affected by Beclin 1 depletion and by the expression of a Beclin 1 mutant defective in Bcl-2 binding. Overall, these findings indicate that C. burnetii infection modulates autophagy and apoptotic pathways through Beclin 1/Bcl-2 interplay to establish a successful infection in the host cell.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose/fisiologia , Coxiella burnetii , Proteínas de Membrana/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Febre Q/metabolismo , Proteínas Reguladoras de Apoptose/genética , Autofagia/fisiologia , Proteína Beclina-1 , Coxiella burnetii/metabolismo , Coxiella burnetii/patogenicidade , Células HeLa , Humanos , Proteínas de Membrana/genética , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Vacúolos/metabolismoRESUMO
The obligate intracellular bacterium Coxiella burnetii, the agent of Q fever in humans and of coxiellosis in other animals, survives and replicates within large, acidified, phagolysosome-like vacuoles known to fuse homo- and heterotypically with other vesicles. To further characterize these vacuoles, HeLa cells were infected with C. burnetii phase II; 48 h later, bacteria-containing vacuoles were labeled by LysoTracker, a marker of acidic compartments, and accumulated monodansylcadaverine and displayed protein LC3, both markers of autophagic vacuoles. Furthermore, 3-methyladenine and wortmannin, agents known to inhibit early stages in the autophagic process, each blocked Coxiella vacuole formation. These autophagosomal features suggest that Coxiella vacuoles interact with the autophagic pathway. The localization and role of wild-type and mutated Rab5 and Rab7, markers of early and late endosomes, respectively, were also examined to determine the role of these small GTPases in the trafficking of C. burnetii phase II. Green fluorescent protein (GFP)-Rab5 and GFP-Rab7 constructs were overexpressed and visualized by fluorescence microscopy. Coxiella-containing large vacuoles were labeled with wild-type Rab7 (Rab7wt) and with GTPase-deficient mutant Rab7Q67L, whereas no colocalization was observed with the dominant-negative mutant Rab7T22N. The vacuoles were also decorated by GFP-Rab5Q79L but not by GFP-Rab5wt. These results suggest that Rab7 participates in the biogenesis of the parasitophorous vacuoles.