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Systems biology of tissue-specific response to Anaplasma phagocytophilum reveals differentiated apoptosis in the tick vector Ixodes scapularis.

Ayllón N, Villar M, Galindo RC, Kocan KM, Šíma R, López JA, Vázquez J, Alberdi P, Cabezas-Cruz A, Kopáček P, de la Fuente J - PLoS Genet. (2015)

Bottom Line: All apoptosis pathways described in other organisms were identified in I. scapularis, except for the absence of the Perforin ortholog.Functional characterization using RNA interference showed that Porin knockdown significantly increases tick colonization by A. phagocytophilum.In tick nymphs, the results suggested a possible effect of bacterial infection on the inhibition of tick immune response.

View Article: PubMed Central - PubMed

Affiliation: SaBio. Instituto de Investigación en Recursos Cinegéticos IREC, CSIC-UCLM-JCCM, Ciudad Real, Spain.

ABSTRACT
Anaplasma phagocytophilum is an emerging pathogen that causes human granulocytic anaplasmosis. Infection with this zoonotic pathogen affects cell function in both vertebrate host and the tick vector, Ixodes scapularis. Global tissue-specific response and apoptosis signaling pathways were characterized in I. scapularis nymphs and adult female midguts and salivary glands infected with A. phagocytophilum using a systems biology approach combining transcriptomics and proteomics. Apoptosis was selected for pathway-focused analysis due to its role in bacterial infection of tick cells. The results showed tissue-specific differences in tick response to infection and revealed differentiated regulation of apoptosis pathways. The impact of bacterial infection was more pronounced in tick nymphs and midguts than in salivary glands, probably reflecting bacterial developmental cycle. All apoptosis pathways described in other organisms were identified in I. scapularis, except for the absence of the Perforin ortholog. Functional characterization using RNA interference showed that Porin knockdown significantly increases tick colonization by A. phagocytophilum. Infection with A. phagocytophilum produced complex tissue-specific alterations in transcript and protein levels. In tick nymphs, the results suggested a possible effect of bacterial infection on the inhibition of tick immune response. In tick midguts, the results suggested that A. phagocytophilum infection inhibited cell apoptosis to facilitate and establish infection through up-regulation of the JAK/STAT pathway. Bacterial infection inhibited the intrinsic apoptosis pathway in tick salivary glands by down-regulating Porin expression that resulted in the inhibition of Cytochrome c release as the anti-apoptotic mechanism to facilitate bacterial infection. However, tick salivary glands may promote apoptosis to limit bacterial infection through induction of the extrinsic apoptosis pathway. These dynamic changes in response to A. phagocytophilum in I. scapularis tissue-specific transcriptome and proteome demonstrated the complexity of the tick response to infection and will contribute to characterize gene regulation in ticks.

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Immunohistochemical localization of tick Porin and Cytochrome c.The results showed that Porin levels were lower in infected than in uninfected tick salivary glands but demonstrated that differences in Cytochrome c levels were not at the protein level but in the localization of Cytochrome c, which was distributed in the cell cytoplasm of uninfected tick salivary glands but was mainly localized within organelles that probably corresponded to mitochondria in A. phagocytophilum-infected tick salivary glands. (A) Representative images of immunofluorescence analysis of uninfected and A. phagocytophilum-infected adult female tick salivary glands. Tick tissues were stained with rabbit anti-tick proteins antibodies (green, FITC). Arrows show positive staining in response to anti-Porin and anti-Cytochrome c IgG not present after incubation with preimmune IgG. (a-b) preimmune control serum-treated cells, which gave similar results between uninfected and infected ticks. (c-d) uninfected and infected cells stained with anti-Porin antibodies. (e-f) uninfected and infected cells stained with anti-Cytochrome c antibodies. (g-h) uninfected and infected cells stained with DAPI. Bars, 10 μm. (B) Sections in the red squares in e-h were magnified 5X and superimposed to illustrate the localization of the Cytochrome c in the cytoplasm of uninfected and infected adult female tick salivary glands. Arrows show positive staining. (C) Model of the Porin-mediated inhibition of Cytochrome c release as the anti-apoptotic mechanism to facilitate bacterial infection of tick salivary glands.
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pgen.1005120.g008: Immunohistochemical localization of tick Porin and Cytochrome c.The results showed that Porin levels were lower in infected than in uninfected tick salivary glands but demonstrated that differences in Cytochrome c levels were not at the protein level but in the localization of Cytochrome c, which was distributed in the cell cytoplasm of uninfected tick salivary glands but was mainly localized within organelles that probably corresponded to mitochondria in A. phagocytophilum-infected tick salivary glands. (A) Representative images of immunofluorescence analysis of uninfected and A. phagocytophilum-infected adult female tick salivary glands. Tick tissues were stained with rabbit anti-tick proteins antibodies (green, FITC). Arrows show positive staining in response to anti-Porin and anti-Cytochrome c IgG not present after incubation with preimmune IgG. (a-b) preimmune control serum-treated cells, which gave similar results between uninfected and infected ticks. (c-d) uninfected and infected cells stained with anti-Porin antibodies. (e-f) uninfected and infected cells stained with anti-Cytochrome c antibodies. (g-h) uninfected and infected cells stained with DAPI. Bars, 10 μm. (B) Sections in the red squares in e-h were magnified 5X and superimposed to illustrate the localization of the Cytochrome c in the cytoplasm of uninfected and infected adult female tick salivary glands. Arrows show positive staining. (C) Model of the Porin-mediated inhibition of Cytochrome c release as the anti-apoptotic mechanism to facilitate bacterial infection of tick salivary glands.

Mentions: Although Porin and Cytochrome c expression was down-regulated in infected tick salivary glands, differences in protein levels between uninfected and A. phagocytophilum-infected tick salivary glands were not found (Fig 5C). These results were corroborated by immunocytochemistry (Fig 8A), demonstrating that differences between infected and uninfected tick salivary glands were not at the protein level but in the localization of Cytochrome c (Fig 8B). While Cytochrome c was distributed in the cell cytoplasm of uninfected tick salivary glands, in A. phagocytophilum-infected tick salivary glands Cytochrome c was mainly localized within organelles that probably correspond to mitochondria (Fig 8C). Although the mechanism(s) regulating mitochondrial permeability and the release of Cytochrome c during apoptosis are not fully understood, Bcl-2 may acts through the voltage-dependent anion channel or Porin, which in turn may play a role in regulating Cytochrome c release [34]. Taken together, these results demonstrated that A. phagocytophilum infection results in down-regulation of Porin expression in tick salivary gland but not midgut cells, which did not translate in different protein levels but resulted in the inhibition of Cytochrome c release as the anti-apoptotic mechanism to facilitate bacterial infection (Fig 8C).


Systems biology of tissue-specific response to Anaplasma phagocytophilum reveals differentiated apoptosis in the tick vector Ixodes scapularis.

Ayllón N, Villar M, Galindo RC, Kocan KM, Šíma R, López JA, Vázquez J, Alberdi P, Cabezas-Cruz A, Kopáček P, de la Fuente J - PLoS Genet. (2015)

Immunohistochemical localization of tick Porin and Cytochrome c.The results showed that Porin levels were lower in infected than in uninfected tick salivary glands but demonstrated that differences in Cytochrome c levels were not at the protein level but in the localization of Cytochrome c, which was distributed in the cell cytoplasm of uninfected tick salivary glands but was mainly localized within organelles that probably corresponded to mitochondria in A. phagocytophilum-infected tick salivary glands. (A) Representative images of immunofluorescence analysis of uninfected and A. phagocytophilum-infected adult female tick salivary glands. Tick tissues were stained with rabbit anti-tick proteins antibodies (green, FITC). Arrows show positive staining in response to anti-Porin and anti-Cytochrome c IgG not present after incubation with preimmune IgG. (a-b) preimmune control serum-treated cells, which gave similar results between uninfected and infected ticks. (c-d) uninfected and infected cells stained with anti-Porin antibodies. (e-f) uninfected and infected cells stained with anti-Cytochrome c antibodies. (g-h) uninfected and infected cells stained with DAPI. Bars, 10 μm. (B) Sections in the red squares in e-h were magnified 5X and superimposed to illustrate the localization of the Cytochrome c in the cytoplasm of uninfected and infected adult female tick salivary glands. Arrows show positive staining. (C) Model of the Porin-mediated inhibition of Cytochrome c release as the anti-apoptotic mechanism to facilitate bacterial infection of tick salivary glands.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4376793&req=5

pgen.1005120.g008: Immunohistochemical localization of tick Porin and Cytochrome c.The results showed that Porin levels were lower in infected than in uninfected tick salivary glands but demonstrated that differences in Cytochrome c levels were not at the protein level but in the localization of Cytochrome c, which was distributed in the cell cytoplasm of uninfected tick salivary glands but was mainly localized within organelles that probably corresponded to mitochondria in A. phagocytophilum-infected tick salivary glands. (A) Representative images of immunofluorescence analysis of uninfected and A. phagocytophilum-infected adult female tick salivary glands. Tick tissues were stained with rabbit anti-tick proteins antibodies (green, FITC). Arrows show positive staining in response to anti-Porin and anti-Cytochrome c IgG not present after incubation with preimmune IgG. (a-b) preimmune control serum-treated cells, which gave similar results between uninfected and infected ticks. (c-d) uninfected and infected cells stained with anti-Porin antibodies. (e-f) uninfected and infected cells stained with anti-Cytochrome c antibodies. (g-h) uninfected and infected cells stained with DAPI. Bars, 10 μm. (B) Sections in the red squares in e-h were magnified 5X and superimposed to illustrate the localization of the Cytochrome c in the cytoplasm of uninfected and infected adult female tick salivary glands. Arrows show positive staining. (C) Model of the Porin-mediated inhibition of Cytochrome c release as the anti-apoptotic mechanism to facilitate bacterial infection of tick salivary glands.
Mentions: Although Porin and Cytochrome c expression was down-regulated in infected tick salivary glands, differences in protein levels between uninfected and A. phagocytophilum-infected tick salivary glands were not found (Fig 5C). These results were corroborated by immunocytochemistry (Fig 8A), demonstrating that differences between infected and uninfected tick salivary glands were not at the protein level but in the localization of Cytochrome c (Fig 8B). While Cytochrome c was distributed in the cell cytoplasm of uninfected tick salivary glands, in A. phagocytophilum-infected tick salivary glands Cytochrome c was mainly localized within organelles that probably correspond to mitochondria (Fig 8C). Although the mechanism(s) regulating mitochondrial permeability and the release of Cytochrome c during apoptosis are not fully understood, Bcl-2 may acts through the voltage-dependent anion channel or Porin, which in turn may play a role in regulating Cytochrome c release [34]. Taken together, these results demonstrated that A. phagocytophilum infection results in down-regulation of Porin expression in tick salivary gland but not midgut cells, which did not translate in different protein levels but resulted in the inhibition of Cytochrome c release as the anti-apoptotic mechanism to facilitate bacterial infection (Fig 8C).

Bottom Line: All apoptosis pathways described in other organisms were identified in I. scapularis, except for the absence of the Perforin ortholog.Functional characterization using RNA interference showed that Porin knockdown significantly increases tick colonization by A. phagocytophilum.In tick nymphs, the results suggested a possible effect of bacterial infection on the inhibition of tick immune response.

View Article: PubMed Central - PubMed

Affiliation: SaBio. Instituto de Investigación en Recursos Cinegéticos IREC, CSIC-UCLM-JCCM, Ciudad Real, Spain.

ABSTRACT
Anaplasma phagocytophilum is an emerging pathogen that causes human granulocytic anaplasmosis. Infection with this zoonotic pathogen affects cell function in both vertebrate host and the tick vector, Ixodes scapularis. Global tissue-specific response and apoptosis signaling pathways were characterized in I. scapularis nymphs and adult female midguts and salivary glands infected with A. phagocytophilum using a systems biology approach combining transcriptomics and proteomics. Apoptosis was selected for pathway-focused analysis due to its role in bacterial infection of tick cells. The results showed tissue-specific differences in tick response to infection and revealed differentiated regulation of apoptosis pathways. The impact of bacterial infection was more pronounced in tick nymphs and midguts than in salivary glands, probably reflecting bacterial developmental cycle. All apoptosis pathways described in other organisms were identified in I. scapularis, except for the absence of the Perforin ortholog. Functional characterization using RNA interference showed that Porin knockdown significantly increases tick colonization by A. phagocytophilum. Infection with A. phagocytophilum produced complex tissue-specific alterations in transcript and protein levels. In tick nymphs, the results suggested a possible effect of bacterial infection on the inhibition of tick immune response. In tick midguts, the results suggested that A. phagocytophilum infection inhibited cell apoptosis to facilitate and establish infection through up-regulation of the JAK/STAT pathway. Bacterial infection inhibited the intrinsic apoptosis pathway in tick salivary glands by down-regulating Porin expression that resulted in the inhibition of Cytochrome c release as the anti-apoptotic mechanism to facilitate bacterial infection. However, tick salivary glands may promote apoptosis to limit bacterial infection through induction of the extrinsic apoptosis pathway. These dynamic changes in response to A. phagocytophilum in I. scapularis tissue-specific transcriptome and proteome demonstrated the complexity of the tick response to infection and will contribute to characterize gene regulation in ticks.

Show MeSH
Related in: MedlinePlus