Michael W. White

Toxoplasma co-opts host gene expression by injection of a polymorphic kinase homologue.

Toxoplasma gondii, an obligate intracellular parasite of the phylum Apicomplexa, can cause severe disease in humans with an immature or suppressed immune system. The outcome of Toxoplasma infection is highly dependent on the strain type, as are many of its in vitro growth properties. Here we use genetic crosses between type II and III lines to show that strain-specific differences in the modulation of host cell transcription are mediated by a putative protein kinase, ROP16. Upon invasion by the parasite, this polymorphic protein is released from the apical organelles known as rhoptries and injected into the host cell, where it ultimately affects the activation of signal transducer and activator of transcription (STAT) signalling pathways and consequent downstream effects on a key host cytokine, interleukin (IL)-12. Our findings provide a new mechanism for how an intracellular eukaryotic pathogen can interact with its host and reveal important differences in how different Toxoplasma lineages have evolved to exploit this interaction.

A Secreted Serine-Threonine Kinase Determines Virulence in the Eukaryotic Pathogen Toxoplasma gondii

Toxoplasma gondii strains differ dramatically in virulence despite being genetically very similar. Genetic mapping revealed two closely adjacent quantitative trait loci on parasite chromosome VIIa that control the extreme virulence of the type I lineage. Positional cloning identified the candidate virulence gene ROP18, a highly polymorphic serine-threonine kinase that was secreted into the host cell during parasite invasion. Transfection of the virulent ROP18 allele into a nonpathogenic type III strain increased growth and enhanced mortality by 4 to 5 logs. These attributes of ROP18 required kinase activity, which revealed that secretion of effectors is a major component of parasite virulence.

Coordinated progression through two subtranscriptomes underlies the tachyzoite cycle of Toxoplasma gondii

Background Apicomplexan parasites replicate by varied and unusual processes where the typically eukaryotic expansion of cellular components and chromosome cycle are coordinated with the biosynthesis of parasite-specific structures essential for transmission. Methodology/Principal Findings Here we describe the global cell cycle transcriptome of the tachyzoite stage of Toxoplasma gondii. In dividing tachyzoites, more than a third of the mRNAs exhibit significant cyclical profiles whose timing correlates with biosynthetic events that unfold during daughter parasite formation. These 2,833 mRNAs have a bimodal organization with peak expression occurring in one of two transcriptional waves that are bounded by the transition into S phase and cell cycle exit following cytokinesis. The G1-subtranscriptome is enriched for genes required for basal biosynthetic and metabolic functions, similar to most eukaryotes, while the S/M-subtranscriptome is characterized by the uniquely apicomplexan requirements of parasite maturation, development of specialized organelles, and egress of infectious daughter cells. Two dozen AP2 transcription factors form a series through the tachyzoite cycle with successive sharp peaks of protein expression in the same timeframes as their mRNA patterns, indicating that the mechanisms responsible for the timing of protein delivery might be mediated by AP2 domains with different promoter recognition specificities. Conclusion/Significance Underlying each of the major events in apicomplexan cell cycles, and many more subordinate actions, are dynamic changes in parasite gene expression. The mechanisms responsible for cyclical gene expression timing are likely crucial to the efficiency of parasite replication and may provide new avenues for interfering with parasite growth.

Recovery of the coral Montastrea annularis in the Florida Keys after the 1987 Caribbean “bleaching event”

Many reef-building corals and other cnidarians lost photosynthetic pigments and symbiotic algae (zooxanthellae) during the coral bleaching event in the Caribbean in 1987. The Florida Reef Tract included some of the first documented cases, with widespread bleaching of the massive coral Montastrea annularis beginning in late August. Phototransects at Carysfort Reef showed discoloration of >90% of colonies of this species in March 1988 compared to 0% in July 1986; however no mortality was observed between 1986 and 1988. Samples of corals collected in February and June 1988 had zooxanthellae densities ranging from 0.1 in the most lightly colored corals, to 1.6x106 cells/cm2 in the darker corals. Minimum densities increased to 0.5x106 cells/cm2 by August 1989. Chlorophyll-a content of zooxanthellae and zooxanthellar mitotic indices were significantly higher in corals with lower densities of zooxanthellae, suggesting that zooxanthellar at low densities may be more nutrientsufficient than those in unbleached corals. Ash-free dry weight of coral tissue was positively correlated with zooxanthellae density at all sample times and was significantly lower in June 1988 compared to August 1989. Proteins and lipids per cm2 were significantly higher in August 1989 than in February or June, 1988. Although recovery of zooxanthellae density and coral pigmentation to normal levels may occur in less than one year, regrowth of tissue biomass and energy stores lost during the period of low symbiont densities may take significantly longer.

Defining the cell cycle for the tachyzoite stage of Toxoplasma gondii.

Tachyzoite endodyogeny is characterized by a three phase cell cycle comprised of major G1 and S phases with mitosis following immediately upon the conclusion of DNA replication. Cytokinesis, which begins with the formation of daughter apical complexes, initiates in late S phase and overlaps mitosis. There is no evidence to support an extended G2 period in these parasites. In all strains, parasites with a 2 N DNA content are a relatively small subpopulation and when tachyzoites expressing a fluorescent nuclear marker (green-fluorescent-protein fused to proliferating-cell-nuclear-antigen) were observed by time-lapse microscopy, there appeared to be little delay between S phase and mitosis. Measurements of the DNA content of RH parasites by flow cytometry demonstrated that the G1 and S periods were approximately 60 and approximately 30% of a single division cycle, although these phases were longer in strains that display a slower growth rate. The overall length of S phase was determined by [3H]-thymidine autoradiography using transgenic parasites expressing herpes simplex thymidine kinase and validated by Northern analysis of S phase specific genes during synchronous growth. The fraction of S phase parasites by flow cytometry paralleled autoradiography, however, within S phase, the distribution of parasites was bimodal in all strains examined. Parasites containing a 1-1.7 N DNA complement were a small fraction when compared to the major S phase population which contained a near-diploid ( approximately 1.8 N) complement, suggesting parasites in late S phase have a slower rate of DNA replication. In lieu of a short or missing G2, where checkpoints are thought to operate in other eukaryotes, the bimodal replication of tachyzoite chromosomes may represent a distinct premitotic checkpoint associated with endodyogeny.

The transcriptome of Toxoplasma gondii

BackgroundToxoplasma gondii gives rise to toxoplasmosis, among the most prevalent parasitic diseases of animals and man. Transformation of the tachzyoite stage into the latent bradyzoite-cyst form underlies chronic disease and leads to a lifetime risk of recrudescence in individuals whose immune system becomes compromised. Given the importance of tissue cyst formation, there has been intensive focus on the development of methods to study bradyzoite differentiation, although the molecular basis for the developmental switch is still largely unknown.ResultsWe have used serial analysis of gene expression (SAGE) to define the Toxoplasma gondii transcriptome of the intermediate-host life cycle that leads to the formation of the bradyzoite/tissue cyst. A broad view of gene expression is provided by >4-fold coverage from nine distinct libraries (~300,000 SAGE tags) representing key developmental transitions in primary parasite populations and in laboratory strains representing the three canonical genotypes. SAGE tags, and their corresponding mRNAs, were analyzed with respect to abundance, uniqueness, and antisense/sense polarity and chromosome distribution and developmental specificity.ConclusionThis study demonstrates that phenotypic transitions during parasite development were marked by unique stage-specific mRNAs that accounted for 18% of the total SAGE tags and varied from 1–5% of the tags in each developmental stage. We have also found that Toxoplasma mRNA pools have a unique parasite-specific composition with 1 in 5 transcripts encoding Apicomplexa-specific genes functioning in parasite invasion and transmission. Developmentally co-regulated genes were dispersed across all Toxoplasma chromosomes, as were tags representing each abundance class, and a variety of biochemical pathways indicating that trans-acting mechanisms likely control gene expression in this parasite. We observed distinct similarities in the specificity and expression levels of mRNAs in primary populations (Day-6 post-sporozoite infection) that occur prior to the onset of bradyzoite development that were uniquely shared with the virulent Type I-RH laboratory strain suggesting that development of RH may be arrested. By contrast, strains from Type II-Me49B7 and Type III-VEGmsj contain SAGE tags corresponding to bradyzoite genes, which suggests that priming of developmental expression likely plays a role in the greater capacity of these strains to complete bradyzoite development.

Genetic complementation in apicomplexan parasites

A robust forward genetic model for Apicomplexa could greatly enhance functional analysis of genes in these important protozoan pathogens. We have developed and successfully tested a genetic complementation strategy based on genomic insertion in Toxoplasma gondii. Adapting recombination cloning to genomic DNA, we show that complementing sequences can be shuttled between parasite genome and bacterial plasmid, providing an efficient tool for the recovery and functional assessment of candidate genes. We show complementation, gene cloning, and biological verification with a mutant parasite lacking hypoxanthine-xanthine-guanine phosphoribosyltransferase and a T. gondii cDNA library. We also explored the utility of this approach to clone genes based on function from other apicomplexan parasites using Toxoplasma as a surrogate. A heterologous library containing Cryptosporidium parvum genomic DNA was generated, and we identified a C. parvum gene coding for inosine 5-monophosphate-dehydrogenase (IMPDH). Interestingly, phylogenetic analysis demonstrates a clear eubacterial origin of this gene and strongly suggests its lateral transfer from ɛ-proteobacteria. The prokaryotic origin of this enzyme might make it a promising target for therapeutics directed against Cryptosporidium.

Just one cross appears capable of dramatically altering the population biology of a eukaryotic pathogen like Toxoplasma gondii

Toxoplasma gondii, an obligate intracellular protozoan of the phylum Apicomplexa, is estimated to infect over a billion people worldwide as well as a great many other mammalian and avian hosts. Despite this ubiquity, the vast majority of human infections in Europe and North America are thought to be due to only three genotypes. Using a genome-wide analysis of single-nucleotide polymorphisms, we have constructed a genealogy for these three lines. The data indicate that types I and III are second- and first-generation offspring, respectively, of a cross between a type II strain and one of two ancestral strains. An extant T. gondii strain (P89) appears to be the modern descendant of the non-type II parent of type III, making the full genealogy of the type III clonotype known. The simplicity of this family tree demonstrates that even a single cross can lead to the emergence and dominance of a new clonal genotype that completely alters the population biology of a sexual pathogen.

A Systematic Screen to Discover and Analyze Apicoplast Proteins Identifies a Conserved and Essential Protein Import Factor

Parasites of the phylum Apicomplexa cause diseases that impact global health and economy. These unicellular eukaryotes possess a relict plastid, the apicoplast, which is an essential organelle and a validated drug target. However, much of its biology remains poorly understood, in particular its elaborate compartmentalization: four membranes defining four different spaces. Only a small number of organellar proteins have been identified in particular few proteins are known for non-luminal apicoplast compartments. We hypothesized that enlarging the catalogue of apicoplast proteins will contribute toward identifying new organellar functions and expand the realm of targets beyond a limited set of characterized pathways. We developed a bioinformatic screen based on mRNA abundance over the cell cycle and on phyletic distribution. We experimentally assessed 57 genes, and of 30 successful epitope tagged candidates eleven novel apicoplast proteins were identified. Of those, seven appear to target to the lumen of the organelle, and four localize to peripheral compartments. To address their function we then developed a robust system for the construction of conditional mutants via a promoter replacement strategy. We confirm the feasibility of this system by establishing conditional mutants for two selected genes – a luminal and a peripheral apicoplast protein. The latter is particularly intriguing as it encodes a hypothetical protein that is conserved in and unique to Apicomplexan parasites and other related organisms that maintain a red algal endosymbiont. Our studies suggest that this peripheral plastid protein, PPP1, is likely localized to the periplastid compartment. Conditional disruption of PPP1 demonstrated that it is essential for parasite survival. Phenotypic analysis of this mutant is consistent with a role of the PPP1 protein in apicoplast biogenesis, specifically in import of nuclear-encoded proteins into the organelle.

Serial Analysis of Gene Expression in Circulating γδ T Cell Subsets Defines Distinct Immunoregulatory Phenotypes and Unexpected Gene Expression Profiles

Gene expression profiles were compared in circulating bovine GD3.5+ (CD8−) and GD3.5− (predominantly CD8+) γδ T cells using serial analysis of gene expression (SAGE). Approximately 20,000 SAGE tags were generated from each library. A comparison of the two libraries demonstrated 297 and 173 tags representing genes with 5-fold differential expression in GD3.5+ and GD3.5− γδ T cells, respectively. Consistent with their localization into sites of inflammation, GD3.5+ γδ T cells appeared transcriptionally and translationally more active than GD3.5− γδ cells. GD3.5− γδ T cells demonstrated higher expression of the cell proliferation inhibitor BAP 37, which was associated with their less activated gene expression phenotype. The immune regulatory and apoptosis-inducing molecule, galectin-1, was identified as a highly abundant molecule and was higher in GD3.5+γδ T cells. Surface molecules attributed to myeloid cells, such as CD14, CD68, and scavenger receptor-1, were identified in both populations. Furthermore, expression of B lymphocyte-induced maturation protein, a master regulator of B cell and myeloid cell differentiation, was identified by SAGE analysis and was confirmed at the RNA level to be selectively expressed in γδ T cells vs αβ T cells. These results provide new insights into the inherent differences between circulating γδ T cell subsets.