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Anopheles gambiae PRS1 modulates Plasmodium development at both midgut and salivary gland steps.

Chertemps T, Mitri C, Perrot S, Sautereau J, Jacques JC, Thiery I, Bourgouin C, Rosinski-Chupin I - PLoS ONE (2010)

Bottom Line: Furthermore, this induction is observed using either the rodent parasite Plasmodium berghei or the human pathogen Plasmodium falciparum.In the midgut, PRS1 overexpression is associated with a relocalization of the protein at the periphery of invaded cells.While providing insights into potential functions of DM9 proteins, our results reveal that PRS1 likely contributes to fundamental interactions between Plasmodium and mosquito epithelia, which do not depend on the specific Anopheles/P. falciparum coevolutionary history.

View Article: PubMed Central - PubMed

Affiliation: Unité de Biochimie et Biologie Moléculaire des Insectes, Département de Parasitologie et Mycologie, Centre National de la Recherche Scientifique URA 3012, Institut Pasteur, Paris, France.

ABSTRACT

Background: Invasion of the mosquito salivary glands by Plasmodium is a critical step for malaria transmission. From a SAGE analysis, we previously identified several genes whose expression in salivary glands was regulated coincident with sporozoite invasion of salivary glands. To get insights into the consequences of these salivary gland responses, here we have studied one of the genes, PRS1 (Plasmodium responsive salivary 1), whose expression was upregulated in infected glands, using immunolocalization and functional inactivation approaches.

Methodology/principal findings: PRS1 belongs to a novel insect superfamily of genes encoding proteins with DM9 repeat motifs of uncharacterized function. We show that PRS1 is induced in response to Plasmodium, not only in the salivary glands but also in the midgut, the other epithelial barrier that Plasmodium has to cross to develop in the mosquito. Furthermore, this induction is observed using either the rodent parasite Plasmodium berghei or the human pathogen Plasmodium falciparum. In the midgut, PRS1 overexpression is associated with a relocalization of the protein at the periphery of invaded cells. We also find that sporozoite invasion of salivary gland cells occurs sequentially and induces intra-cellular modifications that include an increase in PRS1 expression and a relocalization of the corresponding protein into vesicle-like structures. Importantly, PRS1 knockdown during the onset of midgut and salivary gland invasion demonstrates that PRS1 acts as an agonist for the development of both parasite species in the two epithelia, highlighting shared vector/parasite interactions in both tissues.

Conclusions/significance: While providing insights into potential functions of DM9 proteins, our results reveal that PRS1 likely contributes to fundamental interactions between Plasmodium and mosquito epithelia, which do not depend on the specific Anopheles/P. falciparum coevolutionary history.

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Confocal microscopy of triple-stained, infected and non-infected A. gambiae midguts.Midgut sections were incubated with preimmune serum (A) or anti-PRS1 immune serum (B and C) in the same way in parallel experiments and analyzed by confocal microscopy; the image acquisition parameters of the confocal microscope were maintained the same. In the left panels, Z-stack projections of PRS1 staining are shown in a false color representation (labeling intensity increasing from blue to yellow). In the right panels, a merge image of the three channels (red: PRS1; green: GFP-expressing ookinetes; blue: DAPI-counterstaining of the nuclei) is given according three planes: z-stack projection of the xy planes (main image), xz and yz cross-sections according to the directions marked by the white lanes on the main image. A: Control midgut stained with preimmune serum. B. Mosquito midgut, 24 h after a non-infective blood-meal. C. Mosquito midgut 24 h after feeding on a P. berghei-infected mouse. Note the higher intensity in PRS1 labeling in the proximity of ookinetes, as well as the relocalization of PRS1 in the cell periphery of cells protruding towards midgut lumen. Bar: 20 µm.
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pone-0011538-g003: Confocal microscopy of triple-stained, infected and non-infected A. gambiae midguts.Midgut sections were incubated with preimmune serum (A) or anti-PRS1 immune serum (B and C) in the same way in parallel experiments and analyzed by confocal microscopy; the image acquisition parameters of the confocal microscope were maintained the same. In the left panels, Z-stack projections of PRS1 staining are shown in a false color representation (labeling intensity increasing from blue to yellow). In the right panels, a merge image of the three channels (red: PRS1; green: GFP-expressing ookinetes; blue: DAPI-counterstaining of the nuclei) is given according three planes: z-stack projection of the xy planes (main image), xz and yz cross-sections according to the directions marked by the white lanes on the main image. A: Control midgut stained with preimmune serum. B. Mosquito midgut, 24 h after a non-infective blood-meal. C. Mosquito midgut 24 h after feeding on a P. berghei-infected mouse. Note the higher intensity in PRS1 labeling in the proximity of ookinetes, as well as the relocalization of PRS1 in the cell periphery of cells protruding towards midgut lumen. Bar: 20 µm.

Mentions: PRS1 localization during ookinete invasion of midgut cells was then investigated by immunofluorescence and confocal microscopy using anti-PRS1 antibodies and GFP-expressing P. berghei. The fluorescence intensity was compared between midgut sections prepared and analyzed in the same conditions. As observed in Fig. 3, the fluorescent labeling is higher on infected than non-infected midgut sections. To obtain a more precise quantification of this difference, the fluorescence intensity was estimated and found to be in mean 7-fold higher in infected midguts (relative fluorescence intensities compared to sections labeled with preimmune serum: 1.7 +/− 1.6 (non infected midgut sections); 13.6 +/− 10.3 (infected midgut sections). N = 3). In non-infected midguts, the anti-PRS1 labeling was relatively homogeneous (Fig. 3B). In contrast, in the Plasmodium-infected midguts, a more heterogeneous fluorescence pattern was detected (Fig. 3C), with 80% of the regions with higher fluorescence intensities found in the proximity of an ookinete. Furthermore, 90% of the ookinetes were detected near more intensely labeled cells, showing that the increase in PRS1 expression is associated with midgut cell invasion by the ookinetes.


Anopheles gambiae PRS1 modulates Plasmodium development at both midgut and salivary gland steps.

Chertemps T, Mitri C, Perrot S, Sautereau J, Jacques JC, Thiery I, Bourgouin C, Rosinski-Chupin I - PLoS ONE (2010)

Confocal microscopy of triple-stained, infected and non-infected A. gambiae midguts.Midgut sections were incubated with preimmune serum (A) or anti-PRS1 immune serum (B and C) in the same way in parallel experiments and analyzed by confocal microscopy; the image acquisition parameters of the confocal microscope were maintained the same. In the left panels, Z-stack projections of PRS1 staining are shown in a false color representation (labeling intensity increasing from blue to yellow). In the right panels, a merge image of the three channels (red: PRS1; green: GFP-expressing ookinetes; blue: DAPI-counterstaining of the nuclei) is given according three planes: z-stack projection of the xy planes (main image), xz and yz cross-sections according to the directions marked by the white lanes on the main image. A: Control midgut stained with preimmune serum. B. Mosquito midgut, 24 h after a non-infective blood-meal. C. Mosquito midgut 24 h after feeding on a P. berghei-infected mouse. Note the higher intensity in PRS1 labeling in the proximity of ookinetes, as well as the relocalization of PRS1 in the cell periphery of cells protruding towards midgut lumen. Bar: 20 µm.
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Related In: Results  -  Collection

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pone-0011538-g003: Confocal microscopy of triple-stained, infected and non-infected A. gambiae midguts.Midgut sections were incubated with preimmune serum (A) or anti-PRS1 immune serum (B and C) in the same way in parallel experiments and analyzed by confocal microscopy; the image acquisition parameters of the confocal microscope were maintained the same. In the left panels, Z-stack projections of PRS1 staining are shown in a false color representation (labeling intensity increasing from blue to yellow). In the right panels, a merge image of the three channels (red: PRS1; green: GFP-expressing ookinetes; blue: DAPI-counterstaining of the nuclei) is given according three planes: z-stack projection of the xy planes (main image), xz and yz cross-sections according to the directions marked by the white lanes on the main image. A: Control midgut stained with preimmune serum. B. Mosquito midgut, 24 h after a non-infective blood-meal. C. Mosquito midgut 24 h after feeding on a P. berghei-infected mouse. Note the higher intensity in PRS1 labeling in the proximity of ookinetes, as well as the relocalization of PRS1 in the cell periphery of cells protruding towards midgut lumen. Bar: 20 µm.
Mentions: PRS1 localization during ookinete invasion of midgut cells was then investigated by immunofluorescence and confocal microscopy using anti-PRS1 antibodies and GFP-expressing P. berghei. The fluorescence intensity was compared between midgut sections prepared and analyzed in the same conditions. As observed in Fig. 3, the fluorescent labeling is higher on infected than non-infected midgut sections. To obtain a more precise quantification of this difference, the fluorescence intensity was estimated and found to be in mean 7-fold higher in infected midguts (relative fluorescence intensities compared to sections labeled with preimmune serum: 1.7 +/− 1.6 (non infected midgut sections); 13.6 +/− 10.3 (infected midgut sections). N = 3). In non-infected midguts, the anti-PRS1 labeling was relatively homogeneous (Fig. 3B). In contrast, in the Plasmodium-infected midguts, a more heterogeneous fluorescence pattern was detected (Fig. 3C), with 80% of the regions with higher fluorescence intensities found in the proximity of an ookinete. Furthermore, 90% of the ookinetes were detected near more intensely labeled cells, showing that the increase in PRS1 expression is associated with midgut cell invasion by the ookinetes.

Bottom Line: Furthermore, this induction is observed using either the rodent parasite Plasmodium berghei or the human pathogen Plasmodium falciparum.In the midgut, PRS1 overexpression is associated with a relocalization of the protein at the periphery of invaded cells.While providing insights into potential functions of DM9 proteins, our results reveal that PRS1 likely contributes to fundamental interactions between Plasmodium and mosquito epithelia, which do not depend on the specific Anopheles/P. falciparum coevolutionary history.

View Article: PubMed Central - PubMed

Affiliation: Unité de Biochimie et Biologie Moléculaire des Insectes, Département de Parasitologie et Mycologie, Centre National de la Recherche Scientifique URA 3012, Institut Pasteur, Paris, France.

ABSTRACT

Background: Invasion of the mosquito salivary glands by Plasmodium is a critical step for malaria transmission. From a SAGE analysis, we previously identified several genes whose expression in salivary glands was regulated coincident with sporozoite invasion of salivary glands. To get insights into the consequences of these salivary gland responses, here we have studied one of the genes, PRS1 (Plasmodium responsive salivary 1), whose expression was upregulated in infected glands, using immunolocalization and functional inactivation approaches.

Methodology/principal findings: PRS1 belongs to a novel insect superfamily of genes encoding proteins with DM9 repeat motifs of uncharacterized function. We show that PRS1 is induced in response to Plasmodium, not only in the salivary glands but also in the midgut, the other epithelial barrier that Plasmodium has to cross to develop in the mosquito. Furthermore, this induction is observed using either the rodent parasite Plasmodium berghei or the human pathogen Plasmodium falciparum. In the midgut, PRS1 overexpression is associated with a relocalization of the protein at the periphery of invaded cells. We also find that sporozoite invasion of salivary gland cells occurs sequentially and induces intra-cellular modifications that include an increase in PRS1 expression and a relocalization of the corresponding protein into vesicle-like structures. Importantly, PRS1 knockdown during the onset of midgut and salivary gland invasion demonstrates that PRS1 acts as an agonist for the development of both parasite species in the two epithelia, highlighting shared vector/parasite interactions in both tissues.

Conclusions/significance: While providing insights into potential functions of DM9 proteins, our results reveal that PRS1 likely contributes to fundamental interactions between Plasmodium and mosquito epithelia, which do not depend on the specific Anopheles/P. falciparum coevolutionary history.

Show MeSH
Related in: MedlinePlus