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Longitudinal analysis of Plasmodium sporozoite motility in the dermis reveals component of blood vessel recognition.

Hopp CS, Chiou K, Ragheb DR, Salman A, Khan SM, Liu AJ, Sinnis P - Elife (2015)

Bottom Line: How sporozoites locate and enter a blood vessel is a critical, but poorly understood process.Our data suggest that sporozoites exhibit two types of motility: in regions far from blood vessels, they exhibit 'avascular motility', defined by high speed and less confinement, while in the vicinity of blood vessels their motility is more constrained.Imaging of sporozoites with mutations in key adhesive proteins highlight the importance of the sporozoite's gliding speed and its ability to modulate adhesive properties for successful exit from the inoculation site.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, United States.

ABSTRACT
Malaria infection starts with injection of Plasmodium sporozoites by an Anopheles mosquito into the skin of the mammalian host. How sporozoites locate and enter a blood vessel is a critical, but poorly understood process. In this study, we examine sporozoite motility and their interaction with dermal blood vessels, using intravital microscopy in mice. Our data suggest that sporozoites exhibit two types of motility: in regions far from blood vessels, they exhibit 'avascular motility', defined by high speed and less confinement, while in the vicinity of blood vessels their motility is more constrained. We find that curvature of sporozoite tracks engaging with vasculature optimizes contact with dermal capillaries. Imaging of sporozoites with mutations in key adhesive proteins highlight the importance of the sporozoite's gliding speed and its ability to modulate adhesive properties for successful exit from the inoculation site.

No MeSH data available.


Related in: MedlinePlus

Generation and verification of a marker-free Plasmodium berghei line expressing mCherry under control of the uis4 promoter, using GIMO transfection.(A) Schematic representation of the introduction of an mCherry-expression cassette under the control of uis4-promoter into the GIMOPbANKA mother line 1596cl1. Transfection construct pL1937 containing the uis4-mCherry-3ʹpbdhfr cassette is integrated into the modified P. berghei 230p locus containing the hdhfr::yfcu selectable marker cassette (black box) by double cross-over homologous recombination at the target regions (hatched boxes). Negative selection with 5-FC selects for parasites that have the mCherry reporter introduced into the genome and the hdhfr::yfcu marker removed (PbANKAmCherry line 2204). Location of primers used for PCR analysis and sizes of PCR products are shown. (B) Diagnostic PCRs confirm integration as expected in PbANKAmCherry line 2204 clone 5, shown by the absence of the hdhfr::yfcu marker (amplification of hdhfr::yfcu with primers 4698/4699) and correct 5′- and 3′-integration PCR product sizes (primer pairs 5510/4958 and 5515/5511, respectively). See Supplementary file 1 for all primer sequences.DOI:http://dx.doi.org/10.7554/eLife.07789.007
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fig1s3: Generation and verification of a marker-free Plasmodium berghei line expressing mCherry under control of the uis4 promoter, using GIMO transfection.(A) Schematic representation of the introduction of an mCherry-expression cassette under the control of uis4-promoter into the GIMOPbANKA mother line 1596cl1. Transfection construct pL1937 containing the uis4-mCherry-3ʹpbdhfr cassette is integrated into the modified P. berghei 230p locus containing the hdhfr::yfcu selectable marker cassette (black box) by double cross-over homologous recombination at the target regions (hatched boxes). Negative selection with 5-FC selects for parasites that have the mCherry reporter introduced into the genome and the hdhfr::yfcu marker removed (PbANKAmCherry line 2204). Location of primers used for PCR analysis and sizes of PCR products are shown. (B) Diagnostic PCRs confirm integration as expected in PbANKAmCherry line 2204 clone 5, shown by the absence of the hdhfr::yfcu marker (amplification of hdhfr::yfcu with primers 4698/4699) and correct 5′- and 3′-integration PCR product sizes (primer pairs 5510/4958 and 5515/5511, respectively). See Supplementary file 1 for all primer sequences.DOI:http://dx.doi.org/10.7554/eLife.07789.007

Mentions: To quantitatively assess sporozoite motility over the first 120 min after inoculation into the skin of a mouse, we generated P. berghei sporozoites expressing the fluorescent protein mCherry under the control of a strong sporozoite-stage promoter (Figure 1—figure supplement 3) and visualized them in the ear pinna. 4-min time-lapse stacks were acquired 5 min, 10 min, 20 min, 30 min, 60 min, and 120 min after intradermal inoculation (see Video 1) and the paths of migrating sporozoites were manually tracked using Imaris software. Reconstructed tracks were re-centered by plotting to a common origin (Figure 1A), which revealed a gradual decrease in parasite dispersal over the first 120 min after inoculation. The mean square displacement (MSD) reflects the dissemination of a motile population from their origin (Beltman et al., 2009). To characterize changes in sporozoite dissemination over the first 120 min after inoculation, the MSD of sporozoites was plotted over time. Highest dispersals were seen within the first 15 min after intradermal inoculation (Figure 1B). Over the following 110 min, parasite dispersal gradually decreases, which is reflected in a gradual reduction of the slope obtained from linear regression fitting of the MSD plot (Figure 1B, inset).Video 1.Time-lapse microscopy of wild-type control P. berghei sporozoites from 5 min to 120 min after intradermal inoculation.


Longitudinal analysis of Plasmodium sporozoite motility in the dermis reveals component of blood vessel recognition.

Hopp CS, Chiou K, Ragheb DR, Salman A, Khan SM, Liu AJ, Sinnis P - Elife (2015)

Generation and verification of a marker-free Plasmodium berghei line expressing mCherry under control of the uis4 promoter, using GIMO transfection.(A) Schematic representation of the introduction of an mCherry-expression cassette under the control of uis4-promoter into the GIMOPbANKA mother line 1596cl1. Transfection construct pL1937 containing the uis4-mCherry-3ʹpbdhfr cassette is integrated into the modified P. berghei 230p locus containing the hdhfr::yfcu selectable marker cassette (black box) by double cross-over homologous recombination at the target regions (hatched boxes). Negative selection with 5-FC selects for parasites that have the mCherry reporter introduced into the genome and the hdhfr::yfcu marker removed (PbANKAmCherry line 2204). Location of primers used for PCR analysis and sizes of PCR products are shown. (B) Diagnostic PCRs confirm integration as expected in PbANKAmCherry line 2204 clone 5, shown by the absence of the hdhfr::yfcu marker (amplification of hdhfr::yfcu with primers 4698/4699) and correct 5′- and 3′-integration PCR product sizes (primer pairs 5510/4958 and 5515/5511, respectively). See Supplementary file 1 for all primer sequences.DOI:http://dx.doi.org/10.7554/eLife.07789.007
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Related In: Results  -  Collection

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fig1s3: Generation and verification of a marker-free Plasmodium berghei line expressing mCherry under control of the uis4 promoter, using GIMO transfection.(A) Schematic representation of the introduction of an mCherry-expression cassette under the control of uis4-promoter into the GIMOPbANKA mother line 1596cl1. Transfection construct pL1937 containing the uis4-mCherry-3ʹpbdhfr cassette is integrated into the modified P. berghei 230p locus containing the hdhfr::yfcu selectable marker cassette (black box) by double cross-over homologous recombination at the target regions (hatched boxes). Negative selection with 5-FC selects for parasites that have the mCherry reporter introduced into the genome and the hdhfr::yfcu marker removed (PbANKAmCherry line 2204). Location of primers used for PCR analysis and sizes of PCR products are shown. (B) Diagnostic PCRs confirm integration as expected in PbANKAmCherry line 2204 clone 5, shown by the absence of the hdhfr::yfcu marker (amplification of hdhfr::yfcu with primers 4698/4699) and correct 5′- and 3′-integration PCR product sizes (primer pairs 5510/4958 and 5515/5511, respectively). See Supplementary file 1 for all primer sequences.DOI:http://dx.doi.org/10.7554/eLife.07789.007
Mentions: To quantitatively assess sporozoite motility over the first 120 min after inoculation into the skin of a mouse, we generated P. berghei sporozoites expressing the fluorescent protein mCherry under the control of a strong sporozoite-stage promoter (Figure 1—figure supplement 3) and visualized them in the ear pinna. 4-min time-lapse stacks were acquired 5 min, 10 min, 20 min, 30 min, 60 min, and 120 min after intradermal inoculation (see Video 1) and the paths of migrating sporozoites were manually tracked using Imaris software. Reconstructed tracks were re-centered by plotting to a common origin (Figure 1A), which revealed a gradual decrease in parasite dispersal over the first 120 min after inoculation. The mean square displacement (MSD) reflects the dissemination of a motile population from their origin (Beltman et al., 2009). To characterize changes in sporozoite dissemination over the first 120 min after inoculation, the MSD of sporozoites was plotted over time. Highest dispersals were seen within the first 15 min after intradermal inoculation (Figure 1B). Over the following 110 min, parasite dispersal gradually decreases, which is reflected in a gradual reduction of the slope obtained from linear regression fitting of the MSD plot (Figure 1B, inset).Video 1.Time-lapse microscopy of wild-type control P. berghei sporozoites from 5 min to 120 min after intradermal inoculation.

Bottom Line: How sporozoites locate and enter a blood vessel is a critical, but poorly understood process.Our data suggest that sporozoites exhibit two types of motility: in regions far from blood vessels, they exhibit 'avascular motility', defined by high speed and less confinement, while in the vicinity of blood vessels their motility is more constrained.Imaging of sporozoites with mutations in key adhesive proteins highlight the importance of the sporozoite's gliding speed and its ability to modulate adhesive properties for successful exit from the inoculation site.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, United States.

ABSTRACT
Malaria infection starts with injection of Plasmodium sporozoites by an Anopheles mosquito into the skin of the mammalian host. How sporozoites locate and enter a blood vessel is a critical, but poorly understood process. In this study, we examine sporozoite motility and their interaction with dermal blood vessels, using intravital microscopy in mice. Our data suggest that sporozoites exhibit two types of motility: in regions far from blood vessels, they exhibit 'avascular motility', defined by high speed and less confinement, while in the vicinity of blood vessels their motility is more constrained. We find that curvature of sporozoite tracks engaging with vasculature optimizes contact with dermal capillaries. Imaging of sporozoites with mutations in key adhesive proteins highlight the importance of the sporozoite's gliding speed and its ability to modulate adhesive properties for successful exit from the inoculation site.

No MeSH data available.


Related in: MedlinePlus