Role of the dynein/dynactin motor complex of the host cell in the biogenesis of the vacuole containing Coxiella burnetii

En el trasporte retrógrado participa el complejo motor dineína/dinactina. Coxiella burnetii (Cb) es un patógeno intracelular obligado que transita a través de la vía fagocítica para formar la vacuola replicativa que contiene a Cb (VCC). Existe poca evidencia acerca de la interrelación entre el tráfico intracelular de Cb y proteínas motoras. Para estudiar esa interrelación se analizó la sobreexpresión de las formas WT de las subunidades del complejo motor dineína/dinactina, y la sobreexpresión de las mutantes no funcionales respectivas, alterando la formación de la VCC. Nuestros resultados sugieren que los fagosomas que contienen C. burnetii, se transportan utilizando dineína/dinactina para formar la VCC, donde Cb se multiplica.In the retrograde transport, the dynein/dynactin motor complex is involved. Coxiella burnetii (Cb) is an obligate intracellular pathogen that transits through the phagocytic pathway to form the replicative vacuole containing Cb (VCC). There is little evidence about the interrelationship between intracellular Cb trafficking and motor proteins. To study this interrelation, we analysed the overexpression of the WT forms of the dynein/dynactin motor complex subunits, and overexpression of the respective non-functional mutants, altering the formation of the VCC. Our results suggest that C. burnetiicontaining phagosomes are transported using dynein/ dinactin to form the VCC, where Cb is multiplied.Fil: Ortiz Flores, Rodolfo M.. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histologia y Embriología Mendoza. "Dr. Mario H. Burgos"Fil: Distel, Jesús S.. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histologia y Embriología Mendoza. "Dr. Mario H. Burgos"Fil: Aguilera, Milton O.. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histologia y Embriología Mendoza. "Dr. Mario H. Burgos"Fil: Berón, Walter. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histologia y Embriología Mendoza. "Dr. Mario H. Burgos

Cortactin Is Involved in the Entry of Coxiella burnetii into Non-Phagocytic Cells

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

Zonda is a novel early component of the autophagy pathway in Drosophila

Autophagy is an evolutionary conserved process by which eukaryotic cells undergo self-digestion of cytoplasmic components. Here we report that a novel Drosophila immunophilin, which we have named Zonda, is critically required for starvation-induced autophagy. We show that Zonda operates at early stages of the process, specifically for Vps34-mediated phosphatidylinositol 3-phosphate (PI3P) deposition. Zonda displays an even distribution under basal conditions, and soon after starvation nucleates in endoplasmic reticulum-associated foci that colocalize with omegasome markers. Zonda nucleation depends on Atg1, Atg13 and Atg17 but does not require Vps34, Vps15, Atg6 or Atg14. Zonda interacts physically with ATG1 through its kinase domain, as well as with ATG6 and Vps34. We propose that Zonda is an early component of the autophagy cascade necessary for Vps34-dependent PI3P deposition and omegasome formation

Coxiella burnetii Phagocytosis Is Regulated by GTPases of the Rho Family and the RhoA Effectors mDia1 and ROCK

The GTPases belonging to the Rho family control the actin cytoskeleton rearrangements needed for particle internalization during phagocytosis. ROCK and mDia1 are downstream effectors of RhoA, a GTPase involved in that process. Coxiella burnetii, the etiologic agent of Q fever, is internalized by the host´s cells in an actin-dependent manner. Nevertheless, the molecular mechanism involved in this process has been poorly characterized. This work analyzes the role of different GTPases of the Rho family and some downstream effectors in the internalization of C. burnetii by phagocytic and non-phagocytic cells. The internalization of C. burnetii into HeLa and RAW cells was significantly inhibited when the cells were treated with Clostridium difficile Toxin B which irreversibly inactivates members of the Rho family. In addition, the internalization was reduced in HeLa cells that overexpressed the dominant negative mutants of RhoA, Rac1 or Cdc42 or that were knocked down for the Rho GTPases. The pharmacological inhibition or the knocking down of ROCK diminished bacterium internalization. Moreover, C. burnetii was less efficiently internalized in HeLa cells overexpressing mDia1-N1, a dominant negative mutant of mDia1, while the overexpression of the constitutively active mutant mDia1-ΔN3 increased bacteria uptake. Interestingly, when HeLa and RAW cells were infected, RhoA, Rac1 and mDia1 were recruited to membrane cell fractions. Our results suggest that the GTPases of the Rho family play an important role in C. burnetii phagocytosis in both HeLa and RAW cells. Additionally, we present evidence that ROCK and mDia1, which are downstream effectors of RhoA, are involved in that processFil: Salinas Ojeda, Romina Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Ortiz Flores, Rodolfo Matias. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Distel, Jesús Sebastián. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Aguilera, Milton Osmar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Colombo, Maria Isabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Beron, Walter. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentin

Chronic Infections: A Possible Scenario for Autophagy and Senescence Cross-Talk

Multiple tissues and systems in the organism undergo modifications during aging due to an accumulation of damaged proteins, lipids, and genetic material. To counteract this process, the cells are equipped with specific mechanisms, such as autophagy and senescence. Particularly, the immune system undergoes a process called immunosenescence, giving rise to a chronic inflammatory status of the organism, with a decreased ability to counteract antigens. The obvious result of this process is a reduced defence capacity. Currently, there is evidence that some pathogens are able to accelerate the immunosenescence process for their own benefit. Although to date numerous reports show the autophagy–senescence relationship, or the connection between pathogens with autophagy or senescence, the link between the three actors remains unexplored. In this review, we have summarized current knowledge about important issues related to aging, senescence, and autophagy

Chronic Infections: A Possible Scenario for Autophagy and Senescence Cross-Talk

Multiple tissues and systems in the organism undergo modifications during aging due to an accumulation of damaged proteins, lipids, and genetic material. To counteract this process, the cells are equipped with specific mechanisms, such as autophagy and senescence. Particularly, the immune system undergoes a process called immunosenescence, giving rise to a chronic inflammatory status of the organism, with a decreased ability to counteract antigens. The obvious result of this process is a reduced defence capacity. Currently, there is evidence that some pathogens are able to accelerate the immunosenescence process for their own benefit. Although to date numerous reports show the autophagy–senescence relationship, or the connection between pathogens with autophagy or senescence, the link between the three actors remains unexplored. In this review, we have summarized current knowledge about important issues related to aging, senescence, and autophagy

The overexpression of the dominant negative mutants of mDia1 inhibits internalization of C. <i>burnetii</i>.

<p>(A) HeLa cells were transfected with pEGFP (panels a-e), pEGFP-mDia1 WT (panels f-j), pEGFP-mDia1-N1 (dominant negative form) (panels k-o) or pEGFP-mDia1-ΔN3 (constitutively active form) (panels p-t). Transfected cells were infected for 4 h at 37°C with <i>C</i>. <i>burnetii</i>. Cells were fixed and processed for immunofluorescence to determine <i>C</i>. <i>burnetii</i> internalization as described in Materials and Methods. Cells were analyzed by confocal microscopy. Representative micrographs of cells are presented. As indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145211#pone.0145211.g001" target="_blank">Fig 1</a>, extracellular and total bacteria were stained in white pseudo color (panels c, h, m, and r) and red pseudo color (panels b, g, l, and q), respectively. In the merged images (panels d, i, n, and s) and the insets of merged images (panels e, j, o, and t), extracellular <i>C</i>. <i>burnetii</i> is shown in white and red pseudo colors (arrows), while intracellular <i>C</i>. <i>burnetii</i> is shown in red pseudo color (yellow arrowheads). Scale bar: 5 μm. (B) Quantification of <i>C</i>. <i>burnetii</i> internalized by transfected HeLa cells. (C) Quantification of total <i>C</i>. <i>burnetii</i> associated to HeLa cells. Between 40 and 60 cells and between 400 and 600 bacteria were counted in each experiment. Results are expressed as means ± SE of three independent experiments. *p < 0.05, **p < 0.01 compared to the EGFP control (one-way ANOVA and Dunnett's <i>post hoc</i> test). ns: non-significant differences between groups (p > 0.05).</p

The specific inhibitor of ROCK, Y27632, diminishes <i>C</i>. <i>burnetii</i> internalization by HeLa or RAW cells.

<p>(A) HeLa or (D) RAW cells were infected with <i>C</i>. <i>burnetii</i> for 4 h at 37°C in the presence of 0.05% DMSO (control, A, panel a; D, panel a) or different concentrations of Y27632 (A, panels b-d; D, panels b-d). Cells were fixed and processed for indirect immunofluorescence to determine <i>C</i>. <i>burnetii</i> internalization and F-actin distribution as described in Materials and Methods. Cells were analyzed by confocal microscopy. Representative micrographs of cells are presented. F-actin was labeled with phalloidin-FITC (green). Representative micrographs of cells are presented. As indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145211#pone.0145211.g001" target="_blank">Fig 1</a>, in the merged images (A, panels a, b, c, and d; D, panels a, b, c, and d), extracellular <i>C</i>. <i>burnetii</i> is shown in white and red pseudo colors (arrows), while intracellular <i>C</i>. <i>burnetii</i> is shown in red pseudo color (yellow arrowheads). Between 100 and 120 cells and between 1200 and 1600 bacteria were counted in each experiment. Scale bar: 5 μm (A); 10 μm (D). Quantification of <i>C</i>. <i>burnetii</i> internalized by Y27632-treated HeLa (B) or RAW (E) cells. Quantification of total <i>C</i>. <i>burnetii</i> associated to HeLa (C) or RAW (F) cells. Results are expressed as means ± SE of three independent experiments. ***p < 0.001, compared to DMSO treatment (one-way ANOVA and Dunnett's <i>post hoc</i> test). ns: non-significant differences between groups (p > 0.05).</p

The overexpression of the dominant negative mutants of mDia1 inhibits internalization of C. <i>burnetii</i>.

<p>(A) HeLa cells were transfected with pEGFP (panels a-e), pEGFP-mDia1 WT (panels f-j), pEGFP-mDia1-N1 (dominant negative form) (panels k-o) or pEGFP-mDia1-ΔN3 (constitutively active form) (panels p-t). Transfected cells were infected for 4 h at 37°C with <i>C</i>. <i>burnetii</i>. Cells were fixed and processed for immunofluorescence to determine <i>C</i>. <i>burnetii</i> internalization as described in Materials and Methods. Cells were analyzed by confocal microscopy. Representative micrographs of cells are presented. As indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145211#pone.0145211.g001" target="_blank">Fig 1</a>, extracellular and total bacteria were stained in white pseudo color (panels c, h, m, and r) and red pseudo color (panels b, g, l, and q), respectively. In the merged images (panels d, i, n, and s) and the insets of merged images (panels e, j, o, and t), extracellular <i>C</i>. <i>burnetii</i> is shown in white and red pseudo colors (arrows), while intracellular <i>C</i>. <i>burnetii</i> is shown in red pseudo color (yellow arrowheads). Scale bar: 5 μm. (B) Quantification of <i>C</i>. <i>burnetii</i> internalized by transfected HeLa cells. (C) Quantification of total <i>C</i>. <i>burnetii</i> associated to HeLa cells. Between 40 and 60 cells and between 400 and 600 bacteria were counted in each experiment. Results are expressed as means ± SE of three independent experiments. *p < 0.05, **p < 0.01 compared to the EGFP control (one-way ANOVA and Dunnett's <i>post hoc</i> test). ns: non-significant differences between groups (p > 0.05).</p

Knockdown of Rho GTPases and Rock inhibits internalization of <i>C</i>. <i>burnetii</i>.

<p>(A) HeLa cells were co-transfected with pEGFP and a scramble (panels a and b), Rac1 (panels c and d), RhoA (panels e and f) or ROCK (panels g and h) siRNAs or the RhoA/Rac1 siRNA combination (panels i and j). Cells were infected for 4 h at 37°C with <i>C</i>. <i>burnetii</i> and then fixed and processed for immunofluorescence to determine <i>C</i>. <i>burnetii</i> internalization as described in Materials and Methods. Cells were analyzed by confocal microscopy. Representative micrographs of cells are presented. As indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145211#pone.0145211.g001" target="_blank">Fig 1</a>, in the merged images (panels a, c, e, g, and i) and the insets of the merged images (panels d, d, f, h, and j), extracellular <i>C</i>. <i>burnetii</i> is shown in white and red pseudo colors (arrows), while intracellular <i>C</i>. <i>burnetii</i> is shown in red pseudo color (yellow arrowheads). Scale bar: 5 μm. (B) Quantification of <i>C</i>. <i>burnetii</i> internalized by transfected HeLa cells. (C) Quantification of total <i>C</i>. <i>burnetii</i> associated to HeLa cells. Between 40 and 60 cells and between 400 and 600 bacteria were counted in each experiment. Results are expressed as means ± SE of three independent experiments. ***p < 0.001, compared to scramble siRNA (one-way ANOVA and Dunnett's <i>post hoc</i> test). ns: non-significant differences between groups (p > 0.05). (D) Lysates of cotransfected HeLa cells were analyzed by SDS-PAGE and Western blot using antibodies against Rac1, RhoA and ROCK. An anti-actin antibody was used as loading control. Scr: scramble siRNA.</p