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Thomas J Templeton

Publications and source records attributed to Thomas J Templeton.

13 recordsLinked to original sources
PubMed2006

Hypervariability within the Rifin, Stevor and Pfmc-2TM superfamilies in Plasmodium falciparum.

The human malaria parasite, Plasmodium falciparum, possesses a broad repertoire of proteins that are proposed to be trafficked to the erythrocyte cytoplasm or surface, based upon the presence within these proteins of a Pexel/VTS erythrocyte-trafficking motif. This catalog includes large families of predicted 2 transmembrane (2TM) proteins, including the Rifin, Stevor and Pfmc-2TM superfamilies, of which each possesses a region of extensive sequence diversity across paralogs and between isolates that is confined to a proposed surface-exposed loop on the infected erythrocyte. Here we express epitope-tagged versions of the 2TM proteins in transgenic NF54 parasites and present evidence that the Stevor and Pfmc-2TM families are exported to the erythrocyte membrane, thus supporting the hypothesis that host immune pressure drives antigenic diversity within the loop. An examination of multiple P.falciparum isolates demonstrates that the hypervariable loop within Stevor and Pfmc-2TM proteins possesses sequence diversity across isolate boundaries. The Pfmc-2TM genes are encoded within large amplified loci that share profound nucleotide identity, which in turn highlight the divergences observed within the hypervariable loop. The majority of Pexel/VTS proteins are organized together within sub-telomeric genome neighborhoods, and a mechanism must therefore exist to differentially generate sequence diversity within select genes, as well as within highly defined regions within these genes.

Amino Acid Sequence↗
PubMed2006

Variant antigen gene expression in malaria.

Pathogens of the genus Plasmodium are unicellular parasites that infect a variety of animals, including reptiles, birds and mammals. All Plasmodium species target host erythrocytes and replicate asexually within this niche. In humans, proliferation within erythrocytes causes disease symptoms ranging from asymtomatic infection to severe disease, including mild to severe febrile and respiratory symptoms, profound anaemia and obstruction of blood flow. The most serious form of human malaria is caused by Plasmodium falciparum, a pathogen that is responsible for several million deaths annually throughout the developing world. Malaria parasites succeed in evading the host immune response to establish long-term, persistent infections, thus increasing the efficiency by which they are transmitted to the mosquito vector. The ability to evade the host immune system, in particular the avoidance of antibody-mediated immunity against parasite-encoded surface proteins, is the result of amplification of extensive repertoires of multicopy, hypervariable gene families that encode infected erythrocyte or merozoite surface proteins. Via switching between antigenically diverse genes within these large families, populations of parasites have the capacity for rapid variation in antigenicity and virulence over the course of an infection. Here we review the amplification and generation of antigenic diversity within the Plasmodium variant gene families, as well as discuss the mechanisms underlying their tightly controlled gene expression and antigenic switching.

Animals↗
PubMed2006

The Plasmodium vivax homolog of the ookinete adhesive micronemal protein, CTRP.

The Plasmodium circumsporozoite protein/thrombospondin-related anonymous protein-related protein (CTRP) is expressed at the mosquito midgut ookinete stage and is considered to be a transmission-blocking vaccine candidate. CTRP is composed of multiple von Willebrand factor A (vWA) and thrombospondin type 1 domains in the extracellular portion of the molecule, and a short acidic cytoplasmic domain that interacts with the actomyosin machinery. As a means to predict functionally relevant domains within CTRP we determined the nucleotide sequences of CTRP from the Plasmodium vivax Sall and the Plasmodium yoelii 17XL strains and characterized the conservation of domain architectures and motifs across Plasmodium genera. Sequence alignments indicate that the CTRP 1st to 4th vWA domains exhibit greater conservation, and thereby are perhaps functionally more important than the 5th and 6th domains. This point should be considered for the development of a transmission-blocking vaccine that includes CTRP recombinant subunit. To complement previous cellular studies on CTRP, we further determined the expression and cellular localization of CTRP protein in P. vivax and P. yoelii.

Amino Acid Motifs↗
PubMed2006

Plasmodium falciparum: Co-dependent expression and co-localization of the PfCCp multi-adhesion domain proteins.

In Plasmodium falciparum, a family of six secreted proteins having a conserved architecture of multiple adhesive domains was recently identified by genome annotation. Three of the proteins, termed PfCCp1, PfCCp2, and PfCCp3 due to a common LCCL domain, are expressed inside the gametocyte parasitophorous vacuole and released during gamete emergence, where they relocate extracellularly surrounding exflagellation complexes. In this study we show that the three PfCCp proteins are co-expressed at the gametocyte surface. Abrogation of PfCCp3 in gene disruptant parasites leads to the loss of PfCCp1 and PfCCp2 protein but not transcript, indicating a co-dependent protein expression. In view of these data we propose that the three PfCCp proteins interact during gametocytogenesis by formation of a protein complex.

Animals↗
PubMed2006

Programmed transcription of the var gene family, but not of stevor, in Plasmodium falciparum gametocytes.

The var genes encode Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) proteins, a set of highly diverse surface-expressed proteins that mediate adhesion of erythrocytes infected with asexual blood-stage parasites to host endothelium. Switching among expressed PfEMP1 variants in the course of a blood-stage infection is a key component of antigenic variation, and thus immune evasion, by the parasite. The majority of var loci are found in the subtelomeric regions of P. falciparum chromosomes associated with members of other multigene families, including stevor. Both PfEMP1 and STEVOR are expressed in gametocytes, the transmissible parasite stage, but the role of these proteins in the biology of sexual-stage parasites remains unknown. PfEMP1 may continue to mediate antigenic variation in gametocytes, which need to persist in the host for many days before reaching maturity. Using quantitative reverse transcription-PCR and Northern hybridization, we demonstrate that transcription of a defined subset of type C var loci occurs during gametocyte development in vitro. This transcriptional program occurs in gametocytes regardless of the var expression phenotype of their asexual progenitors and therefore is subject to regulatory processes distinct from those that manage antigenic variation in the asexual parasite. In contrast, the same stevor variants are transcribed in both gametocytes and their asexual progenitors. We also provide evidence that for both asexual parasites and gametocytes, var and stevor transcription patterns are not linked to each other.

Animals↗
PubMed2005

Targeting malaria parasite proteins to the erythrocyte.

The intraerythrocytic stages of the protozoan parasite Plasmodium falciparum reside within a parasitophorous vacuole (PV) and set up unique "extraparasite, intraerythrocyte" protein-trafficking pathways that target parasite-encoded proteins to the erythrocyte cytoplasm and cell surface. Two recent articles report the identification of trafficking motifs that regulate the transport of parasite-encoded proteins across the PV. These articles greatly aid the annotation of the parasite "secretome" catalog of proteins that are targeted to the erythrocyte cytoplasm or cell membrane.

Amino Acid Sequence↗
PubMed2004

A multidomain adhesion protein family expressed in Plasmodium falciparum is essential for transmission to the mosquito.

The recent sequencing of several apicomplexan genomes has provided the opportunity to characterize novel antigens essential for the parasite life cycle that might lead to the development of new diagnostic and therapeutic markers. Here we have screened the Plasmodium falciparum genome sequence for genes encoding extracellular multidomain putative adhesive proteins. Three of these identified genes, named PfCCp1, PfCCp2, and PfCCp3, have multiple adhesive modules including a common Limulus coagulation factor C domain also found in two additional Plasmodium genes. Orthologues were identified in the Cryptosporidium parvum genome sequence, indicating an evolutionary conserved function. Transcript and protein expression analysis shows sexual stage-specific expression of PfCCp1, PfCCp2, and PfCCp3, and cellular localization studies revealed plasma membrane-associated expression in mature gametocytes. During gametogenesis, PfCCps are released and localize surrounding complexes of newly emerged microgametes and macrogametes. PfCCp expression markedly decreased after formation of zygotes. To begin to address PfCCp function, the PfCCp2 and PfCCp3 gene loci were disrupted by homologous recombination, resulting in parasites capable of forming oocyst sporozoites but blocked in the salivary gland transition. Our results describe members of a conserved apicomplexan protein family expressed in sexual stage Plasmodium parasites that may represent candidates for subunits of a transmission-blocking vaccine.

Amino Acid Sequence↗
PubMed2004

Plasmodium ookinete-secreted proteins secreted through a common micronemal pathway are targets of blocking malaria transmission.

The mosquito midgut ookinete stage of the malaria parasite, Plasmodium, possesses microneme secretory organelles that mediate locomotion and midgut wall egress to establish sporogonic stages and subsequent transmission. The purpose of this study was 2-fold: 1) to determine whether there exists a single micronemal population with respect to soluble and membrane-associated secreted proteins; and 2) to evaluate the ookinete micronemal proteins chitinase (PgCHT1), circumsporozoite and TRAP-related protein (CTRP), and von Willebrand factor A domain-related protein (WARP) as immunological targets eliciting sera-blocking malaria parasite infectivity to mosquitoes. Indirect immunofluorescence localization studies in Plasmodium gallinaceum using specific antisera showed that all three proteins are distributed intracellularly with a similar granular cytoplasmic appearance and with focal concentration of PgCHT1 and PgCTRP, but not PgWARP, at the ookinete apical end. Immunogold double-labeling electron microscopy, using antisera against the membrane-associated protein CTRP and the soluble WARP, showed that these two proteins co-localized to the same micronemal population. Within the microneme CTRP was associated peripherally at the microneme membrane, whereas PgCHT1 and WARP were diffuse within the micronemal lumen. Sera produced against Plasmodium falciparum WARP significantly reduced the infectivity of P. gallinaceum to Aedes aegypti and P. falciparum to Anopheles mosquitoes. Antisera against PgCTRP and PgCHT1 also significantly reduced the infectivity of P. gallinaceum for A. aegypti. These results support the concept that ookinete micronemal proteins may constitute a general class of malaria transmission-blocking vaccine candidates.

Amino Acid Sequence↗
PubMed2004

Complete genome sequence of the apicomplexan, Cryptosporidium parvum.

The apicomplexan Cryptosporidium parvum is an intestinal parasite that affects healthy humans and animals, and causes an unrelenting infection in immunocompromised individuals such as AIDS patients. We report the complete genome sequence of C. parvum, type II isolate. Genome analysis identifies extremely streamlined metabolic pathways and a reliance on the host for nutrients. In contrast to Plasmodium and Toxoplasma, the parasite lacks an apicoplast and its genome, and possesses a degenerate mitochondrion that has lost its genome. Several novel classes of cell-surface and secreted proteins with a potential role in host interactions and pathogenesis were also detected. Elucidation of the core metabolism, including enzymes with high similarities to bacterial and plant counterparts, opens new avenues for drug development.

Animals↗
PubMed2004

Comparative analysis of apicomplexa and genomic diversity in eukaryotes.

The apicomplexans Plasmodium and Cryptosporidium have developed distinctive adaptations via lineage-specific gene loss and gene innovation in the process of diverging from a common parasitic ancestor. The two lineages have acquired distinct but overlapping sets of surface protein adhesion domains typical of animal proteins, but in no case do they share multidomain architectures identical to animals. Cryptosporidium, but not Plasmodium, possesses an animal-type O-linked glycosylation pathway, along with >30 predicted surface proteins having mucin-like segments. The two parasites have notable qualitative differences in conserved protein architectures associated with chromatin dynamics and transcription. Cryptosporidium shows considerable reduction in the number of introns and a concomitant loss of spliceosomal machinery components. We also describe additional molecular characteristics distinguishing Apicomplexa from other eukaryotes for which complete genome sequences are available.

Animals↗
PubMed2004

The Cryptosporidium oocyst wall protein is a member of a multigene family and has a homolog in Toxoplasma.

Coccidian parasites are transmitted via a fecal oocyst stage that is exceptionally resistant to environmental stress and harsh chemical treatments, which allows parasites to stably persist outside a host. Because of its oocyst durability Cryptosporidium parvum is a significant water- and food-borne pathogen of humans, as well as animals of agricultural importance. To date, only one apicomplexan oocyst membrane protein has been identified, Cryptosporidium oocyst wall protein 1 (COWP1). COWP1 has a highly cysteine-rich periodicity due to arrays of two apicomplexan-specific motifs, designated the type I and type II domains. In this study, exhaustive BLAST screening of a complete C. parvum genome sequence database resulted in identification of eight additional genes encoding similar arrays of cysteine-rich type I and/or type II domains. Transcript expression analysis revealed that all COWP genes are abundantly expressed at a time when developing oocysts are observed, roughly 48 to 72 h after inoculation of in vitro cultures. A monoclonal antibody recognizing COWP8 specifically localized to the C. parvum oocyst wall, supporting the hypothesis that multiple COWPs play a role in the oocyst wall structure. BLAST screening of the Toxoplasma gondii genome sequence database resulted in identification of a gene encoding at least one COWP homolog (TgOWP1), and this multiexon sequence information was used to isolate a full-length cDNA. Exhaustive screening of Plasmodium sp. genome sequence databases by using COWP genes as BLAST queries failed to detect similar proteins in PLASMODIUM: We therefore propose that the COWP family of proteins have a structural role in apicomplexan species that produce durable shed cysts capable of surviving environmental stress.

Amino Acid Sequence↗
PubMed2002

Cryptosporidium parvum genes containing thrombospondin type 1 domains.

Cryptosporidium parvum is recognized as an enteropathogen of great worldwide medical and veterinary importance, yet understanding of its pathogenesis has been hampered in part by limited knowledge of the invasion machinery of this parasite. Recently, genes containing thrombospondin type 1 (TSP1) domains have been identified in several genera of apicomplexans, including thrombospondin-related adhesive proteins (TRAPs) that have been implicated as key molecules for parasite motility and adhesion onto host cell surfaces. Previously, a large-scale random survey of the C. parvum genome conducted in our laboratory revealed the presence of multiple genomic DNA sequences with a high degree of similarity to known apicomplexan TRAP genes. In the present study, TBLASTN screening of available C. parvum genomic sequences by using TSP1 domains as queries identified a total of 12 genes possessing TSP1-like domains. All genes have putative signal peptide sequences, one or more TSP1-like domains, plus additional extracellular protein modules such as Kringle, epidermal growth factor, and Apple domains. Two genes, putative paralogs CpTSP8 and CpTSP9, contain predicted introns near their amino termini, which were verified by comparing PCR products from cDNA versus genomic DNA templates. Reverse transcription-PCR analysis of transcript levels reveals that C. parvum TSP genes were developmentally regulated with distinct patterns of expression during in vitro infection. TRAPC1, CpTSP3, and CpTSP11 were expressed at high levels during both early and late stages of infection, whereas CpTSP2, CpTSP5, CpTSP6, CpTSP8, and CpTSP9 were maximally expressed during the late stages of infection. Only CpTSP4 was highly expressed solely at an early stage of infection.

Amino Acid Sequence↗