Furio Spano

Large sequence variation at two microsatellite loci among zoonotic (genotype C) isolates of Cryptosporidium parvum

The genetic polymorphism among 57 Cryptosporidium parvum isolates belonging to genotype C was studied by PCR amplification and the sequencing of two microsatellite loci (ML1 and ML2). A comparative analysis of DNA sequences showed the presence of three (ML1-238, ML1-226, and ML1-220) and seven (ML2-231, ML2-229, ML2-227, ML2-213, ML2-193, ML2-191, and ML2-187) different alleles at these two loci. Alleles differed by expansions/contractions of the microsatellite repeats that generated length polymorphisms. Some alleles were found to be associated with infections of all examined hosts (calf, kid, lamb, and human), whereas others were either associated with a single host, or were geographically restricted. When considering the information from both loci, some preferential associations between alleles are apparent. These data confirm the utility of microsatellite markers for the molecular identification of C. parvum, which is of particular relevance in the investigation of the source of infection of outbreaks and single cases, as well as for genetic studies.

Molecular characterisation of a novel family of cysteine-rich proteins of Toxoplasma gondii and ultrastructural evidence of oocyst wall localisation.

Among apicomplexan parasites, the coccidia and Cryptosporidium spp. are important pathogens of livestock and humans, and the environmentally resistant stage (oocyst) is essential for their transmission. Little is known of the chemical and molecular composition of the oocyst wall. Currently, the only parasite molecules shown to be involved in oocyst wall formation are the tyrosine-rich proteins gam56, gam82 and gam230 of Eimeria spp. and the cysteine-rich proteins COWP1 and COWP8 of Cryptosporidium parvum. In the present study, we searched the ToxoDB database for the presence of putative Toxoplasma gondii oocyst wall proteins (OWPs) and identified seven candidates, herein named TgOWP1 through TgOWP7, showing homology to the Cryptosporidium COWPs. We analysed a cDNA library from partially sporulated oocysts of T. gondii and cloned the full-length cDNAs encoding TgOWP1, TgOWP2 and TgOWP3, which consist of 499, 462 and 640 amino acids, respectively. The three proteins share 24% sequence identity with each other and a markedly similar overall structure, based on the presence of an N-terminal leader peptide followed by tandem duplications of a six-cysteine amino acid motif closely related to the Type I repeat of COWPs. Using antisera to recombinant TgOWP1, TgOWP2 and TgOWP3, we showed by Western blot that these molecules are expressed in T. gondii oocysts but are not detectable in tachyzoites. The solubilisation of TgOWP1-3 strictly depended on the presence of reducing agents, consistent with a likely involvement of these proteins in multimeric complexes mediated by disulphide bridges. Immunofluorescence analysis allowed the localisation of TgOWP1, TgOWP2 and TgOWP3 to the oocyst wall. Additionally, using immunoelectron microscopy and the 1G12 monoclonal antibody, TgOWP3 was specifically detected in the outer layer of the oocyst wall, thus representing the first validated molecular marker of this structure in T. gondii.

Global proteomic analysis of the oocyst/ sporozoite of Toxoplasma gondii reveals commitment to a host-independent lifestyle

BackgroundToxoplasmosis is caused by the apicomplexan parasite Toxoplasma gondii and can be acquired either congenitally or via the oral route. In the latter case, transmission is mediated by two distinct invasive stages, i.e., bradyzoites residing in tissue cysts or sporozoites contained in environmentally resistant oocysts shed by felids in their feces. The oocyst plays a central epidemiological role, yet this stage has been scarcely investigated at the molecular level and the knowledge of its expressed proteome is very limited.ResultsUsing one-dimensional gel electrophoresis coupled to liquid chromatography-linked tandem mass spectrometry, we analysed total or fractionated protein extracts of partially sporulated T. gondii oocysts, producing a dataset of 1304 non reduntant proteins (~18% of the total predicted proteome), ~59% of which were classified according to the MIPS functional catalogue database. Notably, the comparison of the oocyst dataset with the extensively covered proteome of T. gondii tachyzoite, the invasive stage responsible for the clinical signs of toxoplasmosis, identified 154 putative oocyst/sporozoite-specific proteins, some of which were validated by Western blot. The analysis of this protein subset showed that, compared to tachyzoites, oocysts have a greater capability of de novo amino acid biosynthesis and are well equipped to fuel the Krebs cycle with the acetyl-CoA generated through fatty acid β-oxidation and the degradation of branched amino acids.ConclusionsThe study reported herein significantly expanded our knowledge of the proteome expressed by the oocyst/sporozoite of T. gondii, shedding light on a stage-specifc subset of proteins whose functional profile is consistent with the adaptation of T. gondii oocysts to the nutrient-poor and stressing extracellular environment.

The SAG5 locus of Toxoplasma gondii encodes three novel proteins belonging to the SAG1 family of surface antigens

We have identified three novel Toxoplasma gondii proteins showing close structural similarity to molecules of the SAG1 family, a group of glycosylphosphatidylinositol-anchored surface antigens expressed by the invasive stages of T. gondii. The novel proteins, denominated SAG5A, SAG5B and SAG5C, are encoded by tandemly arrayed and tightly clustered genes containing no introns. The 367 amino acid-long SAG5B and SAG5C are 97.5% identical to each other, whereas SAG5A (362 amino acids) consists of a C-terminal domain sharing 98% identity with SAG5B and SAG5C, and an N-terminal domain whose identity to the other SAG5 polypeptides is only 42%. Expression analysis of the T. gondii strains RH (virulent) and 76 K (avirulent) showed that all members of the SAG5 cluster are transcribed in T. gondii tachyzoites and bradyzoites. However, immunoblot studies on the RH strain revealed that the synthesis of SAG5A does not occur in tachyzoites and is possibly controlled at the post-transcriptional level. On the contrary, SAG5B and SAG5C were detected by immunoblot in tachyzoite lysates and found to migrate in the 40-45 kDa range under reducing conditions or at approximately 34 kDa under unreduced conditions. Triton X-114 partitioning of tachyzoite protein lysates treated with phosphatidylinositol-specific phospholipase C indicated that SAG5B and SAG5C are glycosylphosphatidylinositol-anchored membrane-associated molecules. Consistently, immunofluorescence analysis of transformed tachyzoites over-expressing SAG5B or SAG5C showed that these molecules are targeted to the parasite surface. The characterisation of the SAG5 locus sheds further light on the complex repertoire of SAG1-related genes in T. gondii, that now comprises 14 highly homologous members and five distantly related genes belonging to the SAG2 family.

Dissecting the interface between apicomplexan parasite and host cell: Insights from a divergent AMA–RON2 pair

Significance Parasites of phylum Apicomplexa cause significant morbidity and mortality on a global scale. Central to the pathogenesis of these parasites is their ability to invade host cells through a junction formed by members of the apical membrane antigen (AMA) and rhoptry neck protein 2 (RON2) families localized to the parasite surface and host outer membrane, respectively. Here we structurally and functionally characterize Toxoplasma gondii AMA4 (TgAMA4), a highly divergent AMA protein. Structural analyses of TgAMA4 in the apo and RON2L1 bound forms reveal a previously underappreciated level of molecular diversity at the parasite–host-cell interface that offers important insight into stage-dependent invasion strategies and yields a more comprehensive model of apicomplexan invasion. Plasmodium falciparum and Toxoplasma gondii are widely studied parasites in phylum Apicomplexa and the etiological agents of severe human malaria and toxoplasmosis, respectively. These intracellular pathogens have evolved a sophisticated invasion strategy that relies on delivery of proteins into the host cell, where parasite-derived rhoptry neck protein 2 (RON2) family members localize to the host outer membrane and serve as ligands for apical membrane antigen (AMA) family surface proteins displayed on the parasite. Recently, we showed that T. gondii harbors a novel AMA designated as TgAMA4 that shows extreme sequence divergence from all characterized AMA family members. Here we show that sporozoite-expressed TgAMA4 clusters in a distinct phylogenetic clade with Plasmodium merozoite apical erythrocyte-binding ligand (MAEBL) proteins and forms a high-affinity, functional complex with its coevolved partner, TgRON2L1. High-resolution crystal structures of TgAMA4 in the apo and TgRON2L1-bound forms complemented with alanine scanning mutagenesis data reveal an unexpected architecture and assembly mechanism relative to previously characterized AMA–RON2 complexes. Principally, TgAMA4 lacks both a deep surface groove and a key surface loop that have been established to govern RON2 ligand binding selectivity in other AMAs. Our study reveals a previously underappreciated level of molecular diversity at the parasite–host-cell interface and offers intriguing insight into the adaptation strategies underlying sporozoite invasion. Moreover, our data offer the potential for improved design of neutralizing therapeutics targeting a broad range of AMA–RON2 pairs and apicomplexan invasive stages.

Evidence of tRNA cleavage in apicomplexan parasites: Half-tRNAs as new potential regulatory molecules of Toxoplasma gondii and Plasmodium berghei

Several lines of evidence demonstrated that organisms ranging from bacteria to higher animals possess a regulated endonucleolytic cleavage pathway producing half-tRNA fragments. In the present study, we investigated the occurrence of this phenomenon in two distantly related apicomplexan parasites, Toxoplasma gondii, the agent of toxoplasmosis, and the rodent malaria parasite Plasmodium berghei. A low-scale molecular characterization of the small RNA fraction of T. gondii revealed the endonucleolytic processing of 10 distinct tRNA species, with cleavage in the anticodon loop and upstream of the 3-terminal CCA sequence yielding 5- or 3-end half-tRNAs. T. gondii and P. berghei exhibited variable rates of tRNA cleavage upon egress from host cells and in response to stage differentiation, amino acid starvation and heat-shock. Moreover, avirulent isolates of T. gondii and attenuated P. berghei parasites showed a higher rate of tRNA cleavage than virulent strains. Interestingly, half-tRNA production was significantly higher in the metabolically quiescent bradyzoite and sporozoite stages of T. gondii, compared to the fast-growing tachyzoite. Collectively, our findings shed light for the first time on the occurrence of tRNA cleavage in apicomplexan parasites and suggest a relationship between half-tRNA production and growth rate in this important group of organisms.

Analysis of the SAG5 locus reveals a distinct genomic organisation in virulent and avirulent strains of Toxoplasma gondii

We have recently characterised, in the virulent strain RH of Toxoplasma gondii, three glycosylphosphatidylinositol-anchored surface antigens related to SAG1 (p30) and encoded by highly homologous, tandemly arrayed genes named SAG5A, SAG5B and SAG5C. In the present study, we compared the genomic organisation of the SAG5 locus in strains belonging to the three major genotypes of T. gondii. Southern blot analysis using a SAG5-specific probe produced two related but distinct hybridisation patterns, one exclusive of genotype I virulent strains, the other shared by avirulent strains of either genotype II or genotype III. To understand the molecular bases of this intergenotypic heterogeneity, we cloned and sequenced the SAG5 locus in the genotype II strain Me49. We found that in this isolate the SAG5B gene is missing, with SAG5A and SAG5C laying contiguously. This genomic arrangement explains the hybridisation profiles observed for all the avirulent strains examined and indicates that the presence of SAG5B is a distinctive trait of genotype I. Furthermore, we identified two novel SAG1-related genes, SAG5D and SAG5E, mapping respectively 1.8 and 4.0 kb upstream of SAG5A. SAG5D is transcribed in tachyzoites and encodes a polypeptide of 362 amino acids sharing 50% identity with SAG5A-C, whereas SAG5E is a transcribed pseudogene. We also evaluated polymorphisms at the SAG5 locus by comparing the coding regions of SAG5A-E from strains representative of the three archetypal genotypes. In agreement with the strict allelic dimorphism of T. gondii, we identified two alleles for SAG5D, whereas SAG5A, SAG5C and SAG5E were found to be three distinct nucleotide variants. The higher intergenotypic polymorphism of SAG5A, SAG5C and SAG5E suggests that these genes underwent a more rapid genetic drift than the other members of the SAG1 family. Finally, we developed a new PCR-restriction fragment length polymorphism method based on the SAG5C gene that is able to discriminate between strains of genotype I, II and III by a single endonuclease digestion.

The initiation translation factor eIF-4A of Cryptosporidium parvum is encoded by two distinct mRNA forms and shows DNA sequence polymorphism distinguishing genotype 1 and 2 isolates

The eukaryotic translation initiation factor eIF-4A is an ATP-dependent RNA helicase involved in ribosome attachment to the 5′ end of mRNAs. Employing as a probe a Cryptosporidium parvum genomic amplicon encoding a partial polypeptide related to eIF-4A, we screened a C. parvum sporozoite cDNA library to clone the full length of the gene. Two complete cDNAs were characterized, Cp.F6 and Cp.F10, which consisted of 1,900 and 1,418 bp, respectively. The overlapping portions of the sequences shared 100% identity and encoded a polypeptide of 405 amino acids whose identity to known eIF-4A molecules ranged between 77 and 39%. The 2 cDNAs differed in the length of their respective 3′ untranslated regions, of 577 bp in Cp.F6 and 72 bp in Cp.F10, in both of which a putative polyadenylation signal was identified. The structure of the cloned cDNAs, along with genomic Southern blot data indicating that eIF-4A is encoded by a single copy gene, strongly suggested that Cp.F6 and Cp.F10 reflect a differential 3′ end processing of mRNA precursors, not observed so far in C. parvum. Northern blot analysis confirmed that the sporozoites express 2 eIF-4A mRNAs and showed that the lower molecular weight transcript is 10- to 20-fold more abundant. We also investigated the polymorphism of the eIF-4A gene and defined a novel polymerase chain reaction–restriction fragment length polymorphism marker discriminating between C. parvum isolates of genotypes 1 and 2.

Alternative splicing mechanisms orchestrating post-transcriptional gene expression: intron retention and the intron-rich genome of apicomplexan parasites

Apicomplexan parasites including Toxoplasma gondii and Plasmodium species have complex life cycles that include multiple hosts and differentiation through several morphologically distinct stages requiring marked changes in gene expression. This review highlights emerging evidence implicating regulation of mRNA splicing as a mechanism to prime these parasites for rapid gene expression upon differentiation. We summarize the most important insights in alternative splicing including its role in regulating gene expression by decreasing mRNA abundance via ‘Regulated Unproductive Splicing and Translation’. As a related but less well-understood mechanism, we discuss also our recent work suggesting a role for intron retention for precluding translation of stage specific isoforms of T. gondii glycolytic enzymes. We additionally provide new evidence that intron retention might be a widespread mechanism during parasite differentiation. Supporting this notion, recent genome-wide analysis of Toxoplasma and Plasmodium suggests intron retention is more pervasive than heretofore thought. These findings parallel recent emergence of intron retention being more prevalent in mammals than previously believed, thereby adding to the established roles in plants, fungi and unicellular eukaryotes. Deeper mechanistic studies of intron retention will provide important insight into its role in regulating gene expression in apicomplexan parasites and more general in eukaryotic organisms.

CCp5A Protein from Toxoplasma gondii as a Serological Marker of Oocyst-driven Infections in Humans and Domestic Animals.

Considering that the current immunoassays are not able to distinguish the infective forms that cause Toxoplasma gondii infection, the present study was carried out to evaluate the reactivity of two recombinant proteins (CCp5A and OWP1) from oocyst/sporozoite, in order to differentiate infections occurring by ingestion of oocysts or tissue cysts. The reactivity of the recombinant proteins was assessed against panels of serum samples from animals (chickens, pigs, and mice) that were naturally or experimentally infected by different infective stages of the parasite. Also, we tested sera from humans who have been infected by oocysts during a well-characterized toxoplasmosis outbreak, as well as sera from pregnant women tested IgM+/IgG+ for T. gondii, which source of infection was unknown. Only the sporozoite-specific CCp5A protein was able to differentiate the parasite stage that infected chickens, pigs and mice, with specific reactivity for oocyst-infected animals. Furthermore, the CCp5A showed preferential reactivity for recent infection by oocyst/sporozoite in pigs and mice. In humans, CCp5A showed higher reactivity with serum samples from the outbreak, compared with serum from pregnant women. Altogether, these findings demonstrate the usefulness of the CCp5A protein as a new tool to identify the parasite stage of T. gondii infection, allowing its application for diagnosis and epidemiological investigations in animals and humans. The identification of parasite infective stage can help to design effective strategies to minimize severe complications in immunocompromised people and, particularly, in pregnant women to prevent congenital infection.