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Specific expression and export of the Plasmodium falciparum Gametocyte EXported Protein-5 marks the gametocyte ring stage.

Tibúrcio M, Dixon MW, Looker O, Younis SY, Tilley L, Alano P - Malar. J. (2015)

Bottom Line: PfGEXP5 represents the earliest post-invasion sexual stage marker described to date.This provides a tool that can be used to identify sexually committed ring stage parasites in natural infections.The fact that regulation of PfGEXP5 does not depend on the AP2-G master regulator of parasite sexual development suggests that, after sexual commitment, differentiation progresses through multiple checkpoints in the early phase of gametocytogenesis.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Malattie Infettive, Parassitarie e Immunomediate, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161, Rome, Italy. martatiburcio@gmail.com.

ABSTRACT

Background: Plasmodium falciparum sexual development plays a fundamental role in the transmission and spread of malaria. The ability to generate gametocytes can be lost during culture in vitro, often associated with the loss of a subtelomeric region of chromosome 9. Gametocytogenesis starts with erythrocyte invasion by a sexually committed merozoite, but the first available specific marker of sexual differentiation appears only from 24 h post invasion.

Methods: Specific antibodies and gene fusions were produced to study the timing of expression and the sub-cellular localization of the P. falciparum Gametocyte EXported Protein-5 (PfGEXP5), encoded in the subtelomeric region of chromosome 9. Expression patterns were examined in wild-type parasites and in parasite lines mutated in the Apetala2-G (AP2-G) transcription factor, governing a cascade of early sexual stage specific genes.

Results: PfGEXP5 is highly expressed in early sexual stages and it is actively exported to the infected erythrocyte cytoplasm from as early as 14 h post-invasion in haemozoin-free, ring stage-like parasites. The pattern of PfGEXP5 expression and export is similar in wild-type parasites and in independent AP2-G defective parasite lines unable to produce gametocytes.

Conclusions: PfGEXP5 represents the earliest post-invasion sexual stage marker described to date. This provides a tool that can be used to identify sexually committed ring stage parasites in natural infections. This early gametocyte marker would enable the identification and mapping of malaria transmission reservoirs in human populations and the study of gametocyte sequestration dynamics in infected individuals. The fact that regulation of PfGEXP5 does not depend on the AP2-G master regulator of parasite sexual development suggests that, after sexual commitment, differentiation progresses through multiple checkpoints in the early phase of gametocytogenesis.

No MeSH data available.


Related in: MedlinePlus

A PfGEXP5-GFP chimera is exported to the RBC cytoplasm. a Western blot analysis of the 3D7 parent line and the PfGEXP5-GFP transfectant. Stage III gametocytes were treated with Eqt-II and the pellet (P) and supernatant (SN) fractions subjected to Western blot and probed with anti-PfGEXP5 and anti-GFP antibodies. b Expression of PfGEXP5-GFP at different stages of gametocyte development; GFP fluorescence signal and DIC images (BF). The GFP fluorescence is present in the RBC cytoplasm. Arrows in the top panel indicate accumulation of PfGEXP5-GFP at the PV. Scale bars 5 μm. c Immunofluorescence microscopy of sexually induced PfGEXP5-GFP transfectants (14 h pi), probed with rabbit anti-GFP (green) and mouse anti-Pfs16 (red) antibodies. d Immunofluorescence microscopy of sexually induced PfGEXP5-GFP transfectants (14 h pi, top) and an asexual trophozoite from the same line (bottom), probed with anti-GFP (green) and anti-KAHRP (red) antibodies. Scale bars 3 μm
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Fig3: A PfGEXP5-GFP chimera is exported to the RBC cytoplasm. a Western blot analysis of the 3D7 parent line and the PfGEXP5-GFP transfectant. Stage III gametocytes were treated with Eqt-II and the pellet (P) and supernatant (SN) fractions subjected to Western blot and probed with anti-PfGEXP5 and anti-GFP antibodies. b Expression of PfGEXP5-GFP at different stages of gametocyte development; GFP fluorescence signal and DIC images (BF). The GFP fluorescence is present in the RBC cytoplasm. Arrows in the top panel indicate accumulation of PfGEXP5-GFP at the PV. Scale bars 5 μm. c Immunofluorescence microscopy of sexually induced PfGEXP5-GFP transfectants (14 h pi), probed with rabbit anti-GFP (green) and mouse anti-Pfs16 (red) antibodies. d Immunofluorescence microscopy of sexually induced PfGEXP5-GFP transfectants (14 h pi, top) and an asexual trophozoite from the same line (bottom), probed with anti-GFP (green) and anti-KAHRP (red) antibodies. Scale bars 3 μm

Mentions: To investigate PfGEXP5 expression and export in an independent approach, a P. falciparum transfectant line was generated expressing full-length PfGEXP5 C-terminally tagged with green fluorescent protein (GFP), autologously regulated by 1.5 kb of the pfgexp5 genomic upstream region. The parent (3D7) and transfectant (PfGEXP5-GFP) lines were induced to undergo sexual differentiation and stage II–III gametocytes were harvested and selectively lysed with Eqt-II to release the host cell content. Anti-PfGEXP5 antibodies recognized the endogenous protein in the Eqt-II supernatant and an additional band (~60 kDa) corresponding to the PfGEXP5-GFP (Fig. 3a). The relative intensity of the upper band indicates that PfGEXP5-GFP is expressed at a similar level to the endogenous PfGEXP5. Some of the PfGEXP5-GFP chimera is still detectable in the parasite fraction, indicating a less efficient export of the chimera beyond the parasitophorous vacuole (PV), which is not breached by Eqt-II, compared to that of the endogenous protein. When PfGEXP5-GFP transfectants were probed with anti-GFP, only the 62 kDa protein species was detected, with no reactivity in the 3D7 parent (Fig. 3a).Fig. 3


Specific expression and export of the Plasmodium falciparum Gametocyte EXported Protein-5 marks the gametocyte ring stage.

Tibúrcio M, Dixon MW, Looker O, Younis SY, Tilley L, Alano P - Malar. J. (2015)

A PfGEXP5-GFP chimera is exported to the RBC cytoplasm. a Western blot analysis of the 3D7 parent line and the PfGEXP5-GFP transfectant. Stage III gametocytes were treated with Eqt-II and the pellet (P) and supernatant (SN) fractions subjected to Western blot and probed with anti-PfGEXP5 and anti-GFP antibodies. b Expression of PfGEXP5-GFP at different stages of gametocyte development; GFP fluorescence signal and DIC images (BF). The GFP fluorescence is present in the RBC cytoplasm. Arrows in the top panel indicate accumulation of PfGEXP5-GFP at the PV. Scale bars 5 μm. c Immunofluorescence microscopy of sexually induced PfGEXP5-GFP transfectants (14 h pi), probed with rabbit anti-GFP (green) and mouse anti-Pfs16 (red) antibodies. d Immunofluorescence microscopy of sexually induced PfGEXP5-GFP transfectants (14 h pi, top) and an asexual trophozoite from the same line (bottom), probed with anti-GFP (green) and anti-KAHRP (red) antibodies. Scale bars 3 μm
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4552133&req=5

Fig3: A PfGEXP5-GFP chimera is exported to the RBC cytoplasm. a Western blot analysis of the 3D7 parent line and the PfGEXP5-GFP transfectant. Stage III gametocytes were treated with Eqt-II and the pellet (P) and supernatant (SN) fractions subjected to Western blot and probed with anti-PfGEXP5 and anti-GFP antibodies. b Expression of PfGEXP5-GFP at different stages of gametocyte development; GFP fluorescence signal and DIC images (BF). The GFP fluorescence is present in the RBC cytoplasm. Arrows in the top panel indicate accumulation of PfGEXP5-GFP at the PV. Scale bars 5 μm. c Immunofluorescence microscopy of sexually induced PfGEXP5-GFP transfectants (14 h pi), probed with rabbit anti-GFP (green) and mouse anti-Pfs16 (red) antibodies. d Immunofluorescence microscopy of sexually induced PfGEXP5-GFP transfectants (14 h pi, top) and an asexual trophozoite from the same line (bottom), probed with anti-GFP (green) and anti-KAHRP (red) antibodies. Scale bars 3 μm
Mentions: To investigate PfGEXP5 expression and export in an independent approach, a P. falciparum transfectant line was generated expressing full-length PfGEXP5 C-terminally tagged with green fluorescent protein (GFP), autologously regulated by 1.5 kb of the pfgexp5 genomic upstream region. The parent (3D7) and transfectant (PfGEXP5-GFP) lines were induced to undergo sexual differentiation and stage II–III gametocytes were harvested and selectively lysed with Eqt-II to release the host cell content. Anti-PfGEXP5 antibodies recognized the endogenous protein in the Eqt-II supernatant and an additional band (~60 kDa) corresponding to the PfGEXP5-GFP (Fig. 3a). The relative intensity of the upper band indicates that PfGEXP5-GFP is expressed at a similar level to the endogenous PfGEXP5. Some of the PfGEXP5-GFP chimera is still detectable in the parasite fraction, indicating a less efficient export of the chimera beyond the parasitophorous vacuole (PV), which is not breached by Eqt-II, compared to that of the endogenous protein. When PfGEXP5-GFP transfectants were probed with anti-GFP, only the 62 kDa protein species was detected, with no reactivity in the 3D7 parent (Fig. 3a).Fig. 3

Bottom Line: PfGEXP5 represents the earliest post-invasion sexual stage marker described to date.This provides a tool that can be used to identify sexually committed ring stage parasites in natural infections.The fact that regulation of PfGEXP5 does not depend on the AP2-G master regulator of parasite sexual development suggests that, after sexual commitment, differentiation progresses through multiple checkpoints in the early phase of gametocytogenesis.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Malattie Infettive, Parassitarie e Immunomediate, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161, Rome, Italy. martatiburcio@gmail.com.

ABSTRACT

Background: Plasmodium falciparum sexual development plays a fundamental role in the transmission and spread of malaria. The ability to generate gametocytes can be lost during culture in vitro, often associated with the loss of a subtelomeric region of chromosome 9. Gametocytogenesis starts with erythrocyte invasion by a sexually committed merozoite, but the first available specific marker of sexual differentiation appears only from 24 h post invasion.

Methods: Specific antibodies and gene fusions were produced to study the timing of expression and the sub-cellular localization of the P. falciparum Gametocyte EXported Protein-5 (PfGEXP5), encoded in the subtelomeric region of chromosome 9. Expression patterns were examined in wild-type parasites and in parasite lines mutated in the Apetala2-G (AP2-G) transcription factor, governing a cascade of early sexual stage specific genes.

Results: PfGEXP5 is highly expressed in early sexual stages and it is actively exported to the infected erythrocyte cytoplasm from as early as 14 h post-invasion in haemozoin-free, ring stage-like parasites. The pattern of PfGEXP5 expression and export is similar in wild-type parasites and in independent AP2-G defective parasite lines unable to produce gametocytes.

Conclusions: PfGEXP5 represents the earliest post-invasion sexual stage marker described to date. This provides a tool that can be used to identify sexually committed ring stage parasites in natural infections. This early gametocyte marker would enable the identification and mapping of malaria transmission reservoirs in human populations and the study of gametocyte sequestration dynamics in infected individuals. The fact that regulation of PfGEXP5 does not depend on the AP2-G master regulator of parasite sexual development suggests that, after sexual commitment, differentiation progresses through multiple checkpoints in the early phase of gametocytogenesis.

No MeSH data available.


Related in: MedlinePlus