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Type of in vitro cultivation influences cytoadhesion, knob structure, protein localization and transcriptome profile of Plasmodium falciparum.

Tilly AK, Thiede J, Metwally N, Lubiana P, Bachmann A, Roeder T, Rockliffe N, Lorenzen S, Tannich E, Gutsmann T, Bruchhaus I - Sci Rep (2015)

Bottom Line: In addition, there was a greater reduction in the cytoadhesion of IEs to various endothelial receptors in the presence of AlbuMAX than in the presence of human serum.Surprisingly, cytoadhesion did not correlate with the presence or absence of knobs.Greater numbers of the variant surface antigen families RIFIN, STEVOR, and PfMC-2TM were found at the IE membrane when cultivated in the presence of AlbuMAX.

View Article: PubMed Central - PubMed

Affiliation: Bernhard Nocht Institute for Tropical Medicine, Bernhard-Nocht-Str. 74, 20359 Hamburg, Germany.

ABSTRACT
In vitro cultivation of Plasmodium falciparum is critical for studying the biology of this parasite. However, it is likely that different in vitro cultivation conditions influence various aspects of the parasite's life cycle. In the present study two P. falciparum isolates were cultivated using the two most common methods, in which AlbuMAX or human serum as additives are used, and the results were compared. The type of cultivation influenced the knob structure of P. falciparum-infected erythrocytes (IEs). IEs cultivated with AlbuMAX had fewer knobs than those cultivated with human serum. Furthermore, knob size varied between isolates and is also depended on the culture medium. In addition, there was a greater reduction in the cytoadhesion of IEs to various endothelial receptors in the presence of AlbuMAX than in the presence of human serum. Surprisingly, cytoadhesion did not correlate with the presence or absence of knobs. Greater numbers of the variant surface antigen families RIFIN, STEVOR, and PfMC-2TM were found at the IE membrane when cultivated in the presence of AlbuMAX. Moreover, the type of cultivation had a marked influence on the transcriptome profile. Compared with cultivation with human serum, cultivation with AlbuMAX increased the expression of approximately 500-870 genes.

No MeSH data available.


Related in: MedlinePlus

Localization of small VSA within infected erythrocytes.The localization of RIFIN, STEVOR, and PfMC-2TM proteins was quantified in trophozoite-stage parasites (28-32 hpi). Shown are percentages of proteins inside the parasitic boundaries (parasite, dark grey), associated within the Maurer’s clefts (MC, light grey), and at the erythrocyte membrane (E, black) as well as without specific fluorescence signal (negative, white). One hundred IEs were counted for each staining; however, the overall percentage is greater than 100 because some proteins localized to multiple sites within a single cell. A. Immunofluorescence microscopy of 3D7 IEs. B. Immunofluorescence microscopy of FCR3 IEs. K-, cultivation in the presence of AlbuMAX or human serum; K+, cultivation in the presence of AlbuMAX or human serum plus gelatin sedimentation to enrich knobby IEs. HS, human serum.
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f6: Localization of small VSA within infected erythrocytes.The localization of RIFIN, STEVOR, and PfMC-2TM proteins was quantified in trophozoite-stage parasites (28-32 hpi). Shown are percentages of proteins inside the parasitic boundaries (parasite, dark grey), associated within the Maurer’s clefts (MC, light grey), and at the erythrocyte membrane (E, black) as well as without specific fluorescence signal (negative, white). One hundred IEs were counted for each staining; however, the overall percentage is greater than 100 because some proteins localized to multiple sites within a single cell. A. Immunofluorescence microscopy of 3D7 IEs. B. Immunofluorescence microscopy of FCR3 IEs. K-, cultivation in the presence of AlbuMAX or human serum; K+, cultivation in the presence of AlbuMAX or human serum plus gelatin sedimentation to enrich knobby IEs. HS, human serum.

Mentions: We next investigated whether cultivation in the presence of AlbuMAX or human serum, as well as the presence or absence of knobs, influenced the localization of the small variant surface antigen families RIFIN, STEVOR, and PfMC-2TM (Fig. 1). The localization studies were based on immunofluorescence microscopy using three different antisera (a-RIF40 (RIFIN), a-PFC0025c (STEVOR), and a-PfMC2TM-CT (PfMC-2TM)). The three antisera were selected, because it is known that they are cross-reactive recognizing semi-conserved portions of RIFIN, STEVOR or PfMC-2TM respectively in the isolates 3D7 and FCR327. For RIFIN, between 75% and 100% of the 3D7 and FCR3 parasites within the IEs were stained. Occasionally RIFIN was detected in the Maurer’s clefts. Staining of the erythrocyte membrane was detected more frequently when the parasites were cultivated in the presence of AlbuMAX (17%–37%) rather than in human serum (1%–5%). The presence or absence of knobs had no influence on localization (Fig. 6A,B, Table 1). The localization pattern of STEVOR was not as uniform as that of RIFIN. Between 75% and 89% of 3D7 parasites within IEs were stained, with little staining of Maurer’s clefts observed. In addition, 40% of knobless IEs cultivated in the presence of AlbuMAX showed staining of the erythrocyte membrane. This increased 94% if the IEs were enriched for the presence of knobs. Cultivation with human serum yielded contrasting results. Knobless IEs showed 34% erythrocyte membrane localization with anti-STEVOR serum, whereas only 10% of knobby IEs exported STEVOR to the host cell membrane. When FCR3 were cultivated in the presence of AlbuMAX, STEVOR antiserum stained the parasites in 48% of knobless and 14% of knobby IEs; however, 84% of knobless IEs and 98% of knobby IEs showed staining of the erythrocyte membrane. Host cell membrane staining was markedly reduced in the presence of human serum (5% of knobless and 8% of knobby IEs), whereas approximately 85% of the parasites within knobless and knobby IEs were stained (Fig. 6A,B, Table 1).


Type of in vitro cultivation influences cytoadhesion, knob structure, protein localization and transcriptome profile of Plasmodium falciparum.

Tilly AK, Thiede J, Metwally N, Lubiana P, Bachmann A, Roeder T, Rockliffe N, Lorenzen S, Tannich E, Gutsmann T, Bruchhaus I - Sci Rep (2015)

Localization of small VSA within infected erythrocytes.The localization of RIFIN, STEVOR, and PfMC-2TM proteins was quantified in trophozoite-stage parasites (28-32 hpi). Shown are percentages of proteins inside the parasitic boundaries (parasite, dark grey), associated within the Maurer’s clefts (MC, light grey), and at the erythrocyte membrane (E, black) as well as without specific fluorescence signal (negative, white). One hundred IEs were counted for each staining; however, the overall percentage is greater than 100 because some proteins localized to multiple sites within a single cell. A. Immunofluorescence microscopy of 3D7 IEs. B. Immunofluorescence microscopy of FCR3 IEs. K-, cultivation in the presence of AlbuMAX or human serum; K+, cultivation in the presence of AlbuMAX or human serum plus gelatin sedimentation to enrich knobby IEs. HS, human serum.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4645185&req=5

f6: Localization of small VSA within infected erythrocytes.The localization of RIFIN, STEVOR, and PfMC-2TM proteins was quantified in trophozoite-stage parasites (28-32 hpi). Shown are percentages of proteins inside the parasitic boundaries (parasite, dark grey), associated within the Maurer’s clefts (MC, light grey), and at the erythrocyte membrane (E, black) as well as without specific fluorescence signal (negative, white). One hundred IEs were counted for each staining; however, the overall percentage is greater than 100 because some proteins localized to multiple sites within a single cell. A. Immunofluorescence microscopy of 3D7 IEs. B. Immunofluorescence microscopy of FCR3 IEs. K-, cultivation in the presence of AlbuMAX or human serum; K+, cultivation in the presence of AlbuMAX or human serum plus gelatin sedimentation to enrich knobby IEs. HS, human serum.
Mentions: We next investigated whether cultivation in the presence of AlbuMAX or human serum, as well as the presence or absence of knobs, influenced the localization of the small variant surface antigen families RIFIN, STEVOR, and PfMC-2TM (Fig. 1). The localization studies were based on immunofluorescence microscopy using three different antisera (a-RIF40 (RIFIN), a-PFC0025c (STEVOR), and a-PfMC2TM-CT (PfMC-2TM)). The three antisera were selected, because it is known that they are cross-reactive recognizing semi-conserved portions of RIFIN, STEVOR or PfMC-2TM respectively in the isolates 3D7 and FCR327. For RIFIN, between 75% and 100% of the 3D7 and FCR3 parasites within the IEs were stained. Occasionally RIFIN was detected in the Maurer’s clefts. Staining of the erythrocyte membrane was detected more frequently when the parasites were cultivated in the presence of AlbuMAX (17%–37%) rather than in human serum (1%–5%). The presence or absence of knobs had no influence on localization (Fig. 6A,B, Table 1). The localization pattern of STEVOR was not as uniform as that of RIFIN. Between 75% and 89% of 3D7 parasites within IEs were stained, with little staining of Maurer’s clefts observed. In addition, 40% of knobless IEs cultivated in the presence of AlbuMAX showed staining of the erythrocyte membrane. This increased 94% if the IEs were enriched for the presence of knobs. Cultivation with human serum yielded contrasting results. Knobless IEs showed 34% erythrocyte membrane localization with anti-STEVOR serum, whereas only 10% of knobby IEs exported STEVOR to the host cell membrane. When FCR3 were cultivated in the presence of AlbuMAX, STEVOR antiserum stained the parasites in 48% of knobless and 14% of knobby IEs; however, 84% of knobless IEs and 98% of knobby IEs showed staining of the erythrocyte membrane. Host cell membrane staining was markedly reduced in the presence of human serum (5% of knobless and 8% of knobby IEs), whereas approximately 85% of the parasites within knobless and knobby IEs were stained (Fig. 6A,B, Table 1).

Bottom Line: In addition, there was a greater reduction in the cytoadhesion of IEs to various endothelial receptors in the presence of AlbuMAX than in the presence of human serum.Surprisingly, cytoadhesion did not correlate with the presence or absence of knobs.Greater numbers of the variant surface antigen families RIFIN, STEVOR, and PfMC-2TM were found at the IE membrane when cultivated in the presence of AlbuMAX.

View Article: PubMed Central - PubMed

Affiliation: Bernhard Nocht Institute for Tropical Medicine, Bernhard-Nocht-Str. 74, 20359 Hamburg, Germany.

ABSTRACT
In vitro cultivation of Plasmodium falciparum is critical for studying the biology of this parasite. However, it is likely that different in vitro cultivation conditions influence various aspects of the parasite's life cycle. In the present study two P. falciparum isolates were cultivated using the two most common methods, in which AlbuMAX or human serum as additives are used, and the results were compared. The type of cultivation influenced the knob structure of P. falciparum-infected erythrocytes (IEs). IEs cultivated with AlbuMAX had fewer knobs than those cultivated with human serum. Furthermore, knob size varied between isolates and is also depended on the culture medium. In addition, there was a greater reduction in the cytoadhesion of IEs to various endothelial receptors in the presence of AlbuMAX than in the presence of human serum. Surprisingly, cytoadhesion did not correlate with the presence or absence of knobs. Greater numbers of the variant surface antigen families RIFIN, STEVOR, and PfMC-2TM were found at the IE membrane when cultivated in the presence of AlbuMAX. Moreover, the type of cultivation had a marked influence on the transcriptome profile. Compared with cultivation with human serum, cultivation with AlbuMAX increased the expression of approximately 500-870 genes.

No MeSH data available.


Related in: MedlinePlus