Michael S. Behnke

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.

Compensatory dendritic cell development mediated by BATF-IRF interactions

The AP1 transcription factor Batf3 is required for homeostatic development of CD8α+ classical dendritic cells that prime CD8 T-cell responses against intracellular pathogens. Here we identify an alternative, Batf3-independent pathway in mice for CD8α+ dendritic cell development operating during infection with intracellular pathogens and mediated by the cytokines interleukin (IL)-12 and interferon-γ. This alternative pathway results from molecular compensation for Batf3 provided by the related AP1 factors Batf, which also functions in T and B cells, and Batf2 induced by cytokines in response to infection. Reciprocally, physiological compensation between Batf and Batf3 also occurs in T cells for expression of IL-10 and CTLA4. Compensation among BATF factors is based on the shared capacity of their leucine zipper domains to interact with non-AP1 factors such as IRF4 and IRF8 to mediate cooperative gene activation. Conceivably, manipulating this alternative pathway of dendritic cell development could be of value in augmenting immune responses to vaccines.

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.

Virulence differences in Toxoplasma mediated by amplification of a family of polymorphic pseudokinases.

The population structure of Toxoplasma gondii includes three highly prevalent clonal lineages referred to as types I, II, and III, which differ greatly in virulence in the mouse model. Previous studies have implicated a family of serine/threonine protein kinases found in rhoptries (ROPs) as important in mediating virulence differences between strain types. Here, we explored the genetic basis of differences in virulence between the highly virulent type I lineage and moderately virulent type II based on successful genetic cross between these lineages. Genome-wide association revealed that a single quantitative trait locus controls the dramatic difference in lethality between these strain types. Neither ROP16 nor ROP18, previously implicated in virulence of T. gondii, was found to contribute to differences between types I and II. Instead, the major virulence locus contained a tandem cluster of polymorphic alleles of ROP5, which showed similar protein expression between strains. ROP5 contains a conserved serine/threonine protein kinase domain that includes only part of the catalytic triad, and hence, all members are considered to be pseudokinases. Genetic disruption of the entire ROP5 locus in the type I lineage led to complete attenuation of acute virulence, and complementation with ROP5 restored lethality to WT levels. These findings reveal that a locus of polymorphic pseudokinases plays an important role in pathogenesis of toxoplasmosis in the mouse model.

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.

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.

The Polymorphic Pseudokinase ROP5 Controls Virulence in Toxoplasma gondii by Regulating the Active Kinase ROP18

Secretory polymorphic serine/threonine kinases control pathogenesis of Toxoplasma gondii in the mouse. Genetic studies show that the pseudokinase ROP5 is essential for acute virulence, but do not reveal its mechanism of action. Here we demonstrate that ROP5 controls virulence by blocking IFN-γ mediated clearance in activated macrophages. ROP5 was required for the catalytic activity of the active S/T kinase ROP18, which phosphorylates host immunity related GTPases (IRGs) and protects the parasite from clearance. ROP5 directly regulated activity of ROP18 in vitro, and both proteins were necessary to avoid IRG recruitment and clearance in macrophages. Clearance of both the Δrop5 and Δrop18 mutants was reversed in macrophages lacking Irgm3, which is required for IRG function, and the virulence defect was fully restored in Irgm3−/− mice. Our findings establish that the pseudokinase ROP5 controls the activity of ROP18, thereby blocking IRG mediated clearance in macrophages. Additionally, ROP5 has other functions that are also Irgm3 and IFN-γ dependent, indicting it plays a general role in governing virulence factors that block immunity.

The transcription of bradyzoite genes in Toxoplasma gondii is controlled by autonomous promoter elements

Experimental evidence suggests that apicomplexan parasites possess bipartite promoters with basal and regulated cis‐elements similar to other eukaryotes. Using a dual luciferase model adapted for recombinational cloning and use in Toxoplasma gondii, we show that genomic regions flanking 16 parasite genes, which encompass examples of constitutive and tachyzoite‐ and bradyzoite‐specific genes, are able to reproduce the appropriate developmental stage expression in a transient luciferase assay. Mapping of cis‐acting elements in several bradyzoite promoters led to the identification of short sequence spans that are involved in control of bradyzoite gene expression in multiple strains and under different bradyzoite induction conditions. Promoters that regulate the heat shock protein BAG1 and a novel bradyzoite‐specific NTPase during bradyzoite development were fine mapped to a 6–8 bp resolution and these minimal cis‐elements were capable of converting a constitutive promoter to one that is induced by bradyzoite conditions. Gel‐shift experiments show that mapped cis‐elements are bound by parasite protein factors with the appropriate functional sequence specificity. These studies are the first to identify the minimal sequence elements that are required and sufficient for bradyzoite gene expression and to show that bradyzoite promoters are maintained in a ‘poised’ chromatin state throughout the intermediate host life cycle in low passage strains. Together, these data demonstrate that conventional eukaryotic promoter mechanisms work with epigenetic processes to regulate developmental gene expression during tissue cyst formation.

Changes in the expression of human cell division autoantigen-1 influence Toxoplasma gondii growth and development.

Toxoplasma is a significant opportunistic pathogen in AIDS, and bradyzoite differentiation is the critical step in the pathogenesis of chronic infection. Bradyzoite development has an apparent tropism for cells and tissues of the central nervous system, suggesting the need for a specific molecular environment in the host cell, but it is unknown whether this environment is parasite directed or the result of molecular features specific to the host cell itself. We have determined that a trisubstituted pyrrole acts directly on human and murine host cells to slow tachyzoite replication and induce bradyzoite-specific gene expression in type II and III strain parasites but not type I strains. New mRNA synthesis in the host cell was required and indicates that novel host transcripts encode signals that were able to induce parasite development. We have applied multivariate microarray analyses to identify and correlate host gene expression with specific parasite phenotypes. Human cell division autoantigen-1 (CDA1) was identified in this analysis, and small interfering RNA knockdown of this gene demonstrated that CDA1 expression causes the inhibition of parasite replication that leads subsequently to the induction of bradyzoite differentiation. Overexpression of CDA1 alone was able to slow parasite growth and induce the expression of bradyzoite-specific proteins, and thus these results demonstrate that changes in host cell transcription can directly influence the molecular environment to enable bradyzoite development. Investigation of host biochemical pathways with respect to variation in strain type response will help provide an understanding of the link(s) between the molecular environment in the host cell and parasite development.