Johannes Dessens - Academia.edu (original) (raw)
Papers by Johannes Dessens
Parasitol Today, 2000
Plasmodium, the causative agent of malaria, has many morphologically and functionally distinct de... more Plasmodium, the causative agent of malaria, has many morphologically and functionally distinct developmental stages. In the mosquito host alone, there are five transitions during the development of a gametocyte into a sporozoite. Determining which genes are expressed at the different developmental stages is vital to our understanding of the parasite. There are a growing number of techniques designed to study gene expression, including microarray. Here, Johannes Dessens, Gabrielle Margos, Maria del Carmen Rodriguez and Robert Sinden describe a novel method: suppression subtractive hybridization (SSH) and its successful application in obtaining mosquito midgut stage-specific genes of Plasmodium.
Full-length cDNAs of barley mild mosaic bymovirus RNA1 and RNA2 were cloned downstream of a modif... more Full-length cDNAs of barley mild mosaic bymovirus RNA1 and RNA2 were cloned downstream of a modified cauliflower mosaic virus 35S promoter with double enhancer. Mechanical inoculation of barley seedlings with a mixture of both cDNAs resulted in systemic mosaic symptoms, typical of barley mild mosaic virus infection. The presence of both RNA species and their gene products in the systemically
Trends in Parasitology
Malaria parasites possess many unique subcellular structures and organelles that are essential fo... more Malaria parasites possess many unique subcellular structures and organelles that are essential for the successful completion of the complex life cycle of Plasmodium in the vertebrate host and mosquito vector. Among these are the crystalloids: transient structures whose presence is restricted to the mosquito-specific ookinete and young oocyst stages of the parasite. Nearly five decades after they were first described, the crystalloids are back in the spotlight, with recent discoveries pointing to an important role in protein trafficking and sporozoite transmission that could be exploited as new targets for control of malaria transmission.
Journal of Virology
The tick-borne Thogoto virus (THOV) is the type species of a new genus in the family Orthomyxovir... more The tick-borne Thogoto virus (THOV) is the type species of a new genus in the family Orthomyxoviridae. Its genome comprises six segments of single-stranded, negative-sense RNA. Each segment possesses conserved regions of semicomplementary nucleotides at the 3' and 5' termini which strongly resemble those of influenza virus. An in vitro polymerase assay based on reconstituted THOV viral cores was developed, and activity was shown to rely on an interaction between the conserved 3'- and 5'-terminal sequences and to be primer dependent. Addition of globin mRNA primed transcription, catalyzing the addition of an extra nucleotide to the transcripts, corresponding to the 5'-terminal m7G cap residue. Priming with various cap analogs suggested that THOV transcription is initiated preferentially with m7GpppAm and involves base pairing. This is the first experimental evidence of endonuclease activity in THOV as part of a unique cap-snatching mechanism.
Journal of Virology
In the accompanying report, we describe an in vitro polymerase assay based on reconstituted Thogo... more In the accompanying report, we describe an in vitro polymerase assay based on reconstituted Thogoto virus (THOV) cores which provided evidence of a double-stranded vRNA promoter consisting of both the 3' and 5' sequences of vRNA (M. B. Leahy, J. T. Dessens, and P. A. Nuttall, J. Virol. 71:8347-8351, 1997). This system was used to investigate further the THOV vRNA promoter structure by using short, synthetic vRNA promoters. The results obtained show that interstrand base pairing between residues 10 and 11 of the 3' promoter arm with residues 11 and 12 of the 5' promoter arm, respectively, is important for promoter activity. In addition, intrastrand base pairing between residues 2 and 3 with residues 9 and 8 of the 5' promoter arm, respectively, was shown to be involved in promoter activity, while no evidence of intrastrand base pairing between residues 2 and 9 of the 3' promoter arm was obtained. These observations are consistent with a hook-like structure in ...
Journal of Virology
An in vitro assay was developed to investigate endonuclease activity of Thogoto virus, a tick-bor... more An in vitro assay was developed to investigate endonuclease activity of Thogoto virus, a tick-borne orthomyxovirus. Endonuclease activity relied on an interaction between the 3' and 5' termini of virion RNA (vRNA) and not those of cRNA. Evidence was obtained that cap structures are cleaved directly from cap donors and that cleavage does not occur after pyrimidines. A 5' hook structure, present in the vRNA promoter but not the cRNA promoter, was introduced into cRNA promoter mutants. These mutants stimulated endonuclease activity, although at levels slightly lower than that of vRNA. The ability of the cRNA promoter to stimulate endonuclease activity when mutated to contain a 5' hook structure indicates that this structure constitutes a switching mechanism for endonuclease activity between the vRNA and cRNA promoters.
Journal of Virology
The interferon-induced mouse Mx1 protein has intrinsic antiviral activity against orthomyxoviruse... more The interferon-induced mouse Mx1 protein has intrinsic antiviral activity against orthomyxoviruses, including Thogoto virus. Thus, Mx1(+) A2G mice are apparently resistant to infection following needle- or tick-borne virus challenge. However, tick-borne challenge and, to a lesser degree, injection of virus mixed with tick salivary gland extract resulted in virus transmission to uninfected ticks feeding on the A2G mice. The data indicate that immunomodulatory components in tick saliva can overcome a natural antiviral mechanism.
The Journal of general virology, 1998
The cRNA promoter of Thogoto virus, a tick-borne orthomyxovirus, was investigated using an in vit... more The cRNA promoter of Thogoto virus, a tick-borne orthomyxovirus, was investigated using an in vitro polymerase assay based on purified viral cores and synthetic oligoribonucleotides corresponding to the 3' and 5' ends of cRNA. In vitro polymerase activity relied on an interaction between the 3' and 5' ends of cRNA and was ApG primer-dependent. Mutational analysis of the promoter showed that interstrand base-pairing of residues 11 and 12 of the 3' promoter arm with residues 10 and 11 of the 5' promoter arm, respectively, was essential for polymerase activity. These data provide the first clear evidence for a cRNA panhandle in an orthomyxovirus. No evidence was obtained for the presence of a 5' or 3' hook structure in the cRNA promoter, and transcription could not be primed with rabbit globin mRNA or synthetic cap analogues. This demonstrates that cap snatching activity relies on the presence of the vRNA terminal sequences.
The Journal of general virology, 1997
Full-length cDNAs of barley mild mosaic bymovirus RNA1 and RNA2 were cloned downstream of a modif... more Full-length cDNAs of barley mild mosaic bymovirus RNA1 and RNA2 were cloned downstream of a modified cauliflower mosaic virus 35S promoter with double enhancer. Mechanical inoculation of barley seedlings with a mixture of both cDNAs resulted in systemic mosaic symptoms, typical of barley mild mosaic virus infection. The presence of both RNA species and their gene products in the systemically infected leaves was demonstrated by RT-PCR and Western blot analyses, respectively. Virions were detected by immunogold labelling, demonstrating that the RNAs are encapsidated. This is the first report of the 35S promoter used in successfully infecting a monocot plant host with cDNA from a strictly monocot plant RNA virus.
Journal of virology, 1997
In the accompanying report, we describe an in vitro polymerase assay based on reconstituted Thogo... more In the accompanying report, we describe an in vitro polymerase assay based on reconstituted Thogoto virus (THOV) cores which provided evidence of a double-stranded vRNA promoter consisting of both the 3' and 5' sequences of vRNA (M. B. Leahy, J. T. Dessens, and P. A. Nuttall, J. Virol. 71:8347-8351, 1997). This system was used to investigate further the THOV vRNA promoter structure by using short, synthetic vRNA promoters. The results obtained show that interstrand base pairing between residues 10 and 11 of the 3' promoter arm with residues 11 and 12 of the 5' promoter arm, respectively, is important for promoter activity. In addition, intrastrand base pairing between residues 2 and 3 with residues 9 and 8 of the 5' promoter arm, respectively, was shown to be involved in promoter activity, while no evidence of intrastrand base pairing between residues 2 and 9 of the 3' promoter arm was obtained. These observations are consistent with a hook-like structure in ...
Virology, 1990
Potato virus C (PVC), a non-aphid transmissible strain of potato virus Y (PVY), was found to code... more Potato virus C (PVC), a non-aphid transmissible strain of potato virus Y (PVY), was found to code for a protein (PVC-HC) which is similar in molecular weight and immunological reactivity to the helper component protein of PVY (PVY-HC). PVC-HC, however, was inactive with respect to its ability to effect aphid transmission of either PVC or PVY. The 5'-terminal 2.7-kb regions of PVC and PVY were sequenced. Within the HC region there was 92% nucleotide homology between the two strains; comparison of the derived amino acid sequences revealed 24 amino acid differences. Comparison of the PVC-HC sequence with that of five potyviruses revealed 2 amino acid changes which were specific to PVC-HC. These amino acids are prime targets for mutational analysis of HC activity.
Molecular and Biochemical Parasitology, 2012
Malaria parasites express a conserved family of LCCL-lectin adhesive-like domain proteins (LAPs) ... more Malaria parasites express a conserved family of LCCL-lectin adhesive-like domain proteins (LAPs) that have essential functions in sporozoite transmission. In Plasmodium falciparum all six family members are expressed in gametocytes and form a multi-protein complex. Intriguingly, knockout of P. falciparum LCCL proteins adversely affects expression of other family members at protein, but not at mRNA level, a phenomenon termed co-dependent expression. Here, we investigate this in Plasmodium berghei by crossing a PbLAP1 null mutant parasite with a parasite line expressing GFP-tagged PbLAP3 that displays strong fluorescence in gametocytes. Selected and validated double mutants show normal synthesis and subcellular localization of PbLAP3::GFP. However, GFP-based fluorescence is dramatically reduced without PbLAP1 present, indicating that PbLAP1 and PbLAP3 interact. Moreover, absence of PbLAP1 markedly reduces the half-life of PbLAP3, consistent with a scenario of misfolding. These findings unveil a potential mechanism of conformational interdependence that facilitates assembly and stability of the functional LCCL protein complex.
Molecular microbiology, 2008
Crystalloids are transient organelles that form in developing malaria ookinetes and disappear aft... more Crystalloids are transient organelles that form in developing malaria ookinetes and disappear after ookinete-to-oocyst transition. Their origins and functions remain poorly understood. The Plasmodium berghei scavenger receptor-like protein PbSR is essential for mosquito-to-host transmission of the parasite: PbSR knockout parasites produce normal numbers of oocysts that fail to form sporozoites, pointing to a role for PbSR in the oocyst during sporogony. Here, using fluorescent protein tagging and targeted gene disruption, we show that PbSR is synthesized in macrogametocytes, gets targeted to the crystalloids of developing ookinetes and is involved in crystalloid formation. While oocyst sporulation rates of PbSR knockout parasites are highly reduced in parasite-infected mosquitoes, sporulation rates in vitro are not adversely affected, supporting the view that mosquito factors could be involved in the PbSR loss-of-function phenotype. These findings are the first to identify a parasit...
Parasitology Research, 2014
The invasive and motile life stages of malaria parasites (merozoite, ookinete and sporozoite) pos... more The invasive and motile life stages of malaria parasites (merozoite, ookinete and sporozoite) possess a distinctive cortical structure termed the pellicle. The pellicle is characterised by a double-layered 'inner membrane complex' (IMC) located underneath the plasma membrane, which is supported by a cytoskeletal structure termed the subpellicular network (SPN). The SPN consists of intermediate filaments, whose major constituents include a family of proteins called alveolins. Here, we re-appraise the alveolins in the genus Plasmodium with respect to their repertoire, structure and interrelatedness. Amongst 13 family members identified, we distinguish two domain types that, albeit distinct at the primary structure level, are structurally related and contain tandem repeats with a consensus 12-amino acid periodicity. Analysis in Plasmodium berghei of the most divergent alveolin, PbIMC1d, reveals a zoite-specific expression in ookinetes and a subcellular localisation in the pellicle, consistent with its predicted role as a SPN component. Knockout of PbIMC1d gives rise to a wild-type phenotype with respect to ookinete morphogenesis, tensile strength, gliding motility and infectivity, presenting the first example of apparent functional redundancy amongst alveolin family members.
Parasitology Research, 2014
The zoite stages of malaria parasites (merozoite, ookinete and sporozoite) possess a distinctive ... more The zoite stages of malaria parasites (merozoite, ookinete and sporozoite) possess a distinctive cortical structure termed the pellicle, which is defined by a double membrane layer named the inner membrane complex (IMC). The IMC is supported by a cytoskeleton of intermediate filaments, termed the subpellicular network (SPN). Plasmodium IMC1 proteins, or alveolins, make up a conserved family of structurally related proteins that comprise building blocks of the SPN. Here, using green fluorescent protein (GFP) tagging in P. berghei, we show that the alveolins PbIMC1c and PbIMC1e are expressed in all three zoite stages. Our data reveal that PbIMC1e is assembled into the SPN concurrent with pellicle development, while PbIMC1c is assembled after pellicle formation. In the sexual stages, these processes are accompanied by different gene expressions from maternal and paternal alleles: PbIMC1e is expressed uniquely from the maternal allele, while PbIMC1c is expressed from the maternal allele in gametocytes, but from both parental alleles during ookinete development. These findings establish biogenesis of the cortical cytoskeleton in Plasmodium to be a complex and dynamic process, involving distinct parental gene expression and chronological recruitment of its protein constituents. While allelic replacement of the pbimc1c and pbimc1e genes with GFP-tagged versions was readily achieved using double crossover homologous recombination, attempts to disrupt these genes by this strategy only resulted in the integration of the selectable marker and GFP reporter into non-specific genomic locations. The recurrent inability to disrupt these genes provides the first genetic evidence that alveolins are necessary for asexual blood-stage parasite development in Plasmodium.
Virus Research, 1997
The tick-borne Thogoto virus (THOV) is the type species of a newly recognized fourth genus, Thogo... more The tick-borne Thogoto virus (THOV) is the type species of a newly recognized fourth genus, Thogotovirus, in the family Orthomyxo6iridae. Because of the distant relationship of THOV with the influenza viruses, determination of its genomic information can potentially be used to identify important domains in influenza virus proteins. We have determined the complete nucleotide sequence of the second longest RNA segment of THOV. The molecule comprises 2212 nucleotides with a single large open reading frame encoding a protein of 710 amino acids, estimated M r 81 284. The protein shares 77% amino acid similarity with the PB1-like protein of Dhori virus, a related tick-borne virus, and 50-53% with the PB1 polymerase proteins of influenza virus A, B and C. All the motifs characteristic of RNA-dependent polymerases were identified including the SSDD motif common to all RNA-dependant RNA polymerases, indicating that the THOV protein is functionally analogous to the influenza virus PB1 proteins and involved in chain elongation. We also report the corrected sequence of the third longest RNA segment of THOV, encoding a protein which shares 44-47% amino acid similarity with the PA-like polymerase proteins of influenza virus A, B and C. The biological significance of conserved domains in these orthomyxovirid proteins is discussed.
Parasitol Today, 2000
Plasmodium, the causative agent of malaria, has many morphologically and functionally distinct de... more Plasmodium, the causative agent of malaria, has many morphologically and functionally distinct developmental stages. In the mosquito host alone, there are five transitions during the development of a gametocyte into a sporozoite. Determining which genes are expressed at the different developmental stages is vital to our understanding of the parasite. There are a growing number of techniques designed to study gene expression, including microarray. Here, Johannes Dessens, Gabrielle Margos, Maria del Carmen Rodriguez and Robert Sinden describe a novel method: suppression subtractive hybridization (SSH) and its successful application in obtaining mosquito midgut stage-specific genes of Plasmodium.
Full-length cDNAs of barley mild mosaic bymovirus RNA1 and RNA2 were cloned downstream of a modif... more Full-length cDNAs of barley mild mosaic bymovirus RNA1 and RNA2 were cloned downstream of a modified cauliflower mosaic virus 35S promoter with double enhancer. Mechanical inoculation of barley seedlings with a mixture of both cDNAs resulted in systemic mosaic symptoms, typical of barley mild mosaic virus infection. The presence of both RNA species and their gene products in the systemically
Trends in Parasitology
Malaria parasites possess many unique subcellular structures and organelles that are essential fo... more Malaria parasites possess many unique subcellular structures and organelles that are essential for the successful completion of the complex life cycle of Plasmodium in the vertebrate host and mosquito vector. Among these are the crystalloids: transient structures whose presence is restricted to the mosquito-specific ookinete and young oocyst stages of the parasite. Nearly five decades after they were first described, the crystalloids are back in the spotlight, with recent discoveries pointing to an important role in protein trafficking and sporozoite transmission that could be exploited as new targets for control of malaria transmission.
Journal of Virology
The tick-borne Thogoto virus (THOV) is the type species of a new genus in the family Orthomyxovir... more The tick-borne Thogoto virus (THOV) is the type species of a new genus in the family Orthomyxoviridae. Its genome comprises six segments of single-stranded, negative-sense RNA. Each segment possesses conserved regions of semicomplementary nucleotides at the 3' and 5' termini which strongly resemble those of influenza virus. An in vitro polymerase assay based on reconstituted THOV viral cores was developed, and activity was shown to rely on an interaction between the conserved 3'- and 5'-terminal sequences and to be primer dependent. Addition of globin mRNA primed transcription, catalyzing the addition of an extra nucleotide to the transcripts, corresponding to the 5'-terminal m7G cap residue. Priming with various cap analogs suggested that THOV transcription is initiated preferentially with m7GpppAm and involves base pairing. This is the first experimental evidence of endonuclease activity in THOV as part of a unique cap-snatching mechanism.
Journal of Virology
In the accompanying report, we describe an in vitro polymerase assay based on reconstituted Thogo... more In the accompanying report, we describe an in vitro polymerase assay based on reconstituted Thogoto virus (THOV) cores which provided evidence of a double-stranded vRNA promoter consisting of both the 3' and 5' sequences of vRNA (M. B. Leahy, J. T. Dessens, and P. A. Nuttall, J. Virol. 71:8347-8351, 1997). This system was used to investigate further the THOV vRNA promoter structure by using short, synthetic vRNA promoters. The results obtained show that interstrand base pairing between residues 10 and 11 of the 3' promoter arm with residues 11 and 12 of the 5' promoter arm, respectively, is important for promoter activity. In addition, intrastrand base pairing between residues 2 and 3 with residues 9 and 8 of the 5' promoter arm, respectively, was shown to be involved in promoter activity, while no evidence of intrastrand base pairing between residues 2 and 9 of the 3' promoter arm was obtained. These observations are consistent with a hook-like structure in ...
Journal of Virology
An in vitro assay was developed to investigate endonuclease activity of Thogoto virus, a tick-bor... more An in vitro assay was developed to investigate endonuclease activity of Thogoto virus, a tick-borne orthomyxovirus. Endonuclease activity relied on an interaction between the 3' and 5' termini of virion RNA (vRNA) and not those of cRNA. Evidence was obtained that cap structures are cleaved directly from cap donors and that cleavage does not occur after pyrimidines. A 5' hook structure, present in the vRNA promoter but not the cRNA promoter, was introduced into cRNA promoter mutants. These mutants stimulated endonuclease activity, although at levels slightly lower than that of vRNA. The ability of the cRNA promoter to stimulate endonuclease activity when mutated to contain a 5' hook structure indicates that this structure constitutes a switching mechanism for endonuclease activity between the vRNA and cRNA promoters.
Journal of Virology
The interferon-induced mouse Mx1 protein has intrinsic antiviral activity against orthomyxoviruse... more The interferon-induced mouse Mx1 protein has intrinsic antiviral activity against orthomyxoviruses, including Thogoto virus. Thus, Mx1(+) A2G mice are apparently resistant to infection following needle- or tick-borne virus challenge. However, tick-borne challenge and, to a lesser degree, injection of virus mixed with tick salivary gland extract resulted in virus transmission to uninfected ticks feeding on the A2G mice. The data indicate that immunomodulatory components in tick saliva can overcome a natural antiviral mechanism.
The Journal of general virology, 1998
The cRNA promoter of Thogoto virus, a tick-borne orthomyxovirus, was investigated using an in vit... more The cRNA promoter of Thogoto virus, a tick-borne orthomyxovirus, was investigated using an in vitro polymerase assay based on purified viral cores and synthetic oligoribonucleotides corresponding to the 3' and 5' ends of cRNA. In vitro polymerase activity relied on an interaction between the 3' and 5' ends of cRNA and was ApG primer-dependent. Mutational analysis of the promoter showed that interstrand base-pairing of residues 11 and 12 of the 3' promoter arm with residues 10 and 11 of the 5' promoter arm, respectively, was essential for polymerase activity. These data provide the first clear evidence for a cRNA panhandle in an orthomyxovirus. No evidence was obtained for the presence of a 5' or 3' hook structure in the cRNA promoter, and transcription could not be primed with rabbit globin mRNA or synthetic cap analogues. This demonstrates that cap snatching activity relies on the presence of the vRNA terminal sequences.
The Journal of general virology, 1997
Full-length cDNAs of barley mild mosaic bymovirus RNA1 and RNA2 were cloned downstream of a modif... more Full-length cDNAs of barley mild mosaic bymovirus RNA1 and RNA2 were cloned downstream of a modified cauliflower mosaic virus 35S promoter with double enhancer. Mechanical inoculation of barley seedlings with a mixture of both cDNAs resulted in systemic mosaic symptoms, typical of barley mild mosaic virus infection. The presence of both RNA species and their gene products in the systemically infected leaves was demonstrated by RT-PCR and Western blot analyses, respectively. Virions were detected by immunogold labelling, demonstrating that the RNAs are encapsidated. This is the first report of the 35S promoter used in successfully infecting a monocot plant host with cDNA from a strictly monocot plant RNA virus.
Journal of virology, 1997
In the accompanying report, we describe an in vitro polymerase assay based on reconstituted Thogo... more In the accompanying report, we describe an in vitro polymerase assay based on reconstituted Thogoto virus (THOV) cores which provided evidence of a double-stranded vRNA promoter consisting of both the 3' and 5' sequences of vRNA (M. B. Leahy, J. T. Dessens, and P. A. Nuttall, J. Virol. 71:8347-8351, 1997). This system was used to investigate further the THOV vRNA promoter structure by using short, synthetic vRNA promoters. The results obtained show that interstrand base pairing between residues 10 and 11 of the 3' promoter arm with residues 11 and 12 of the 5' promoter arm, respectively, is important for promoter activity. In addition, intrastrand base pairing between residues 2 and 3 with residues 9 and 8 of the 5' promoter arm, respectively, was shown to be involved in promoter activity, while no evidence of intrastrand base pairing between residues 2 and 9 of the 3' promoter arm was obtained. These observations are consistent with a hook-like structure in ...
Virology, 1990
Potato virus C (PVC), a non-aphid transmissible strain of potato virus Y (PVY), was found to code... more Potato virus C (PVC), a non-aphid transmissible strain of potato virus Y (PVY), was found to code for a protein (PVC-HC) which is similar in molecular weight and immunological reactivity to the helper component protein of PVY (PVY-HC). PVC-HC, however, was inactive with respect to its ability to effect aphid transmission of either PVC or PVY. The 5'-terminal 2.7-kb regions of PVC and PVY were sequenced. Within the HC region there was 92% nucleotide homology between the two strains; comparison of the derived amino acid sequences revealed 24 amino acid differences. Comparison of the PVC-HC sequence with that of five potyviruses revealed 2 amino acid changes which were specific to PVC-HC. These amino acids are prime targets for mutational analysis of HC activity.
Molecular and Biochemical Parasitology, 2012
Malaria parasites express a conserved family of LCCL-lectin adhesive-like domain proteins (LAPs) ... more Malaria parasites express a conserved family of LCCL-lectin adhesive-like domain proteins (LAPs) that have essential functions in sporozoite transmission. In Plasmodium falciparum all six family members are expressed in gametocytes and form a multi-protein complex. Intriguingly, knockout of P. falciparum LCCL proteins adversely affects expression of other family members at protein, but not at mRNA level, a phenomenon termed co-dependent expression. Here, we investigate this in Plasmodium berghei by crossing a PbLAP1 null mutant parasite with a parasite line expressing GFP-tagged PbLAP3 that displays strong fluorescence in gametocytes. Selected and validated double mutants show normal synthesis and subcellular localization of PbLAP3::GFP. However, GFP-based fluorescence is dramatically reduced without PbLAP1 present, indicating that PbLAP1 and PbLAP3 interact. Moreover, absence of PbLAP1 markedly reduces the half-life of PbLAP3, consistent with a scenario of misfolding. These findings unveil a potential mechanism of conformational interdependence that facilitates assembly and stability of the functional LCCL protein complex.
Molecular microbiology, 2008
Crystalloids are transient organelles that form in developing malaria ookinetes and disappear aft... more Crystalloids are transient organelles that form in developing malaria ookinetes and disappear after ookinete-to-oocyst transition. Their origins and functions remain poorly understood. The Plasmodium berghei scavenger receptor-like protein PbSR is essential for mosquito-to-host transmission of the parasite: PbSR knockout parasites produce normal numbers of oocysts that fail to form sporozoites, pointing to a role for PbSR in the oocyst during sporogony. Here, using fluorescent protein tagging and targeted gene disruption, we show that PbSR is synthesized in macrogametocytes, gets targeted to the crystalloids of developing ookinetes and is involved in crystalloid formation. While oocyst sporulation rates of PbSR knockout parasites are highly reduced in parasite-infected mosquitoes, sporulation rates in vitro are not adversely affected, supporting the view that mosquito factors could be involved in the PbSR loss-of-function phenotype. These findings are the first to identify a parasit...
Parasitology Research, 2014
The invasive and motile life stages of malaria parasites (merozoite, ookinete and sporozoite) pos... more The invasive and motile life stages of malaria parasites (merozoite, ookinete and sporozoite) possess a distinctive cortical structure termed the pellicle. The pellicle is characterised by a double-layered 'inner membrane complex' (IMC) located underneath the plasma membrane, which is supported by a cytoskeletal structure termed the subpellicular network (SPN). The SPN consists of intermediate filaments, whose major constituents include a family of proteins called alveolins. Here, we re-appraise the alveolins in the genus Plasmodium with respect to their repertoire, structure and interrelatedness. Amongst 13 family members identified, we distinguish two domain types that, albeit distinct at the primary structure level, are structurally related and contain tandem repeats with a consensus 12-amino acid periodicity. Analysis in Plasmodium berghei of the most divergent alveolin, PbIMC1d, reveals a zoite-specific expression in ookinetes and a subcellular localisation in the pellicle, consistent with its predicted role as a SPN component. Knockout of PbIMC1d gives rise to a wild-type phenotype with respect to ookinete morphogenesis, tensile strength, gliding motility and infectivity, presenting the first example of apparent functional redundancy amongst alveolin family members.
Parasitology Research, 2014
The zoite stages of malaria parasites (merozoite, ookinete and sporozoite) possess a distinctive ... more The zoite stages of malaria parasites (merozoite, ookinete and sporozoite) possess a distinctive cortical structure termed the pellicle, which is defined by a double membrane layer named the inner membrane complex (IMC). The IMC is supported by a cytoskeleton of intermediate filaments, termed the subpellicular network (SPN). Plasmodium IMC1 proteins, or alveolins, make up a conserved family of structurally related proteins that comprise building blocks of the SPN. Here, using green fluorescent protein (GFP) tagging in P. berghei, we show that the alveolins PbIMC1c and PbIMC1e are expressed in all three zoite stages. Our data reveal that PbIMC1e is assembled into the SPN concurrent with pellicle development, while PbIMC1c is assembled after pellicle formation. In the sexual stages, these processes are accompanied by different gene expressions from maternal and paternal alleles: PbIMC1e is expressed uniquely from the maternal allele, while PbIMC1c is expressed from the maternal allele in gametocytes, but from both parental alleles during ookinete development. These findings establish biogenesis of the cortical cytoskeleton in Plasmodium to be a complex and dynamic process, involving distinct parental gene expression and chronological recruitment of its protein constituents. While allelic replacement of the pbimc1c and pbimc1e genes with GFP-tagged versions was readily achieved using double crossover homologous recombination, attempts to disrupt these genes by this strategy only resulted in the integration of the selectable marker and GFP reporter into non-specific genomic locations. The recurrent inability to disrupt these genes provides the first genetic evidence that alveolins are necessary for asexual blood-stage parasite development in Plasmodium.
Virus Research, 1997
The tick-borne Thogoto virus (THOV) is the type species of a newly recognized fourth genus, Thogo... more The tick-borne Thogoto virus (THOV) is the type species of a newly recognized fourth genus, Thogotovirus, in the family Orthomyxo6iridae. Because of the distant relationship of THOV with the influenza viruses, determination of its genomic information can potentially be used to identify important domains in influenza virus proteins. We have determined the complete nucleotide sequence of the second longest RNA segment of THOV. The molecule comprises 2212 nucleotides with a single large open reading frame encoding a protein of 710 amino acids, estimated M r 81 284. The protein shares 77% amino acid similarity with the PB1-like protein of Dhori virus, a related tick-borne virus, and 50-53% with the PB1 polymerase proteins of influenza virus A, B and C. All the motifs characteristic of RNA-dependent polymerases were identified including the SSDD motif common to all RNA-dependant RNA polymerases, indicating that the THOV protein is functionally analogous to the influenza virus PB1 proteins and involved in chain elongation. We also report the corrected sequence of the third longest RNA segment of THOV, encoding a protein which shares 44-47% amino acid similarity with the PA-like polymerase proteins of influenza virus A, B and C. The biological significance of conserved domains in these orthomyxovirid proteins is discussed.