Viral and Host Factors Are Associated With Mortality in Hospitalized Patients With COVID-19.

BACKGROUND: Persistent mortality in adults hospitalized due to acute COVID-19 justifies pursuit of disease mechanisms and potential therapies. The aim was to evaluate which virus and host response factors were associated with mortality risk among participants in Therapeutics for Inpatients with COVID-19 (TICO/ACTIV-3) trials. METHODS: A secondary analysis of 2625 adults hospitalized for acute SARS-CoV-2 infection randomized to 1 of 5 antiviral products or matched placebo in 114 centers on 4 continents. Uniform, site-level collection of participant baseline clinical variables was performed. Research laboratories assayed baseline upper respiratory swabs for SARS-CoV-2 viral RNA and plasma for anti-SARS-CoV-2 antibodies, SARS-CoV-2 nucleocapsid antigen (viral Ag), and interleukin-6 (IL-6). Associations between factors and time to mortality by 90 days were assessed using univariate and multivariable Cox proportional hazards models. RESULTS: Viral Ag ≥4500 ng/L (vs <200 ng/L; adjusted hazard ratio [aHR], 2.07; 1.29-3.34), viral RNA (<35 000 copies/mL [aHR, 2.42; 1.09-5.34], ≥35 000 copies/mL [aHR, 2.84; 1.29-6.28], vs below detection), respiratory support (<4 L O2 [aHR, 1.84; 1.06-3.22]; ≥4 L O2 [aHR, 4.41; 2.63-7.39], or noninvasive ventilation/high-flow nasal cannula [aHR, 11.30; 6.46-19.75] vs no oxygen), renal impairment (aHR, 1.77; 1.29-2.42), and IL-6 >5.8 ng/L (aHR, 2.54 [1.74-3.70] vs ≤5.8 ng/L) were significantly associated with mortality risk in final adjusted analyses. Viral Ag, viral RNA, and IL-6 were not measured in real-time. CONCLUSIONS: Baseline virus-specific, clinical, and biological variables are strongly associated with mortality risk within 90 days, revealing potential pathogen and host-response therapeutic targets for acute COVID-19 disease.

Real-world effectiveness of early remdesivir and sotrovimab in the highest-risk COVID-19 outpatients during the Omicron surge.

BACKGROUND: Remdesivir and sotrovimab both have clinical trial data in the outpatient setting demonstrating reduction in the risk of hospitalizations and emergency department (ED) visits related to COVID-19. OBJECTIVES: To evaluate the effectiveness of remdesivir in comparison with sotrovimab and matched high-risk control patients in preventing COVID-19-related hospitalizations and ED visits during the Omicron B.1.1.529 surge. PATIENTS AND METHODS: This retrospective cohort study included outpatients positive for SARS-CoV-2, with non-severe symptoms for ≤7 days and deemed high-risk for severe COVID-19 by an internal scoring matrix. Patients who received remdesivir or sotrovimab from 27/12/2021 to 04/02/2022 were included (n = 82 and n = 88, respectively). These were compared with a control cohort of high-risk COVID-19 outpatients who did not receive therapy (n = 90). The primary outcome was a composite of 29 day COVID-19-related hospitalizations and/or ED visits. Pre-specified secondary outcomes included components of the primary endpoint, 29 day all-cause mortality and serious adverse drug events. RESULTS: Patients treated with remdesivir were significantly less likely to be hospitalized or visit the ED within 29 days from symptom onset (11% versus 23.3%; OR = 0.41, 95% CI = 0.17-0.95). Patients receiving sotrovimab were also less likely to be hospitalized or visit the ED (8% versus 23.3%; OR = 0.28, 95% CI = 0.11-0.71). There was no difference in the incidence of hospitalizations/ED visits between sotrovimab and remdesivir. CONCLUSIONS: Our highest-risk outpatients with Omicron-related COVID-19 who received early sotrovimab or remdesivir had significantly lower likelihoods of a hospitalization and/or ED visit.

Restriction Checkpoint Controls Bradyzoite Development in Toxoplasma gondii.

Human toxoplasmosis is a life-threatening disease caused by the apicomplexan parasite Toxoplasma gondii. Rapid replication of the tachyzoite is associated with symptomatic disease, while suppressed division of the bradyzoite is responsible for chronic disease. Here, we identified the T. gondii cell cycle mechanism, the G1 restriction checkpoint (R-point), that operates the switch between parasite growth and differentiation. Apicomplexans lack conventional R-point regulators, suggesting adaptation of alternative factors. We showed that Cdk-related G1 kinase TgCrk2 forms alternative complexes with atypical cyclins (TgCycP1, TgCycP2, and TgCyc5) in the rapidly dividing developmentally incompetent RH and slower dividing developmentally competent ME49 tachyzoites and bradyzoites. Examination of cyclins verified the correlation of cyclin expression with growth dependence and development capacity of RH and ME49 strains. We demonstrated that rapidly dividing RH tachyzoites were dependent on TgCycP1 expression, which interfered with bradyzoite differentiation. Using the conditional knockdown model, we established that TgCycP2 regulated G1 duration in the developmentally competent ME49 tachyzoites but not in the developmentally incompetent RH tachyzoites. We tested the functions of TgCycP2 and TgCyc5 in alkaline induced and spontaneous bradyzoite differentiation (rat embryonic brain cells) models. Based on functional and global gene expression analyses, we determined that TgCycP2 also regulated bradyzoite replication, while signal-induced TgCyc5 was critical for efficient tissue cyst maturation. In conclusion, we identified the central machinery of the T. gondii restriction checkpoint comprised of TgCrk2 kinase and three atypical T. gondii cyclins and demonstrated the independent roles of TgCycP1, TgCycP2, and TgCyc5 in parasite growth and development. IMPORTANCE Toxoplasma gondii is a virulent and abundant human pathogen that puts millions of silently infected people at risk of reactivation of the chronic disease. Encysted bradyzoites formed during the chronic stage are resistant to current therapies. Therefore, insights into the mechanism of tissue cyst formation and reactivation are major areas of investigation. The fact that rapidly dividing parasites differentiate poorly strongly suggests that there is a threshold of replication rate that must be crossed to be considered for differentiation. We discovered a cell cycle mechanism that controls the T. gondii growth-rest switch involved in the conversion of dividing tachyzoites into largely quiescent bradyzoites. This switch operates the T. gondii restriction checkpoint using a set of atypical and parasite-specific regulators. Importantly, the novel T. gondii R-point network was not present in the parasite's human and animal hosts, offering a wealth of new and parasite-specific drug targets to explore in the future.

A latent ability to persist: differentiation in Toxoplasma gondii.

A critical factor in the transmission and pathogenesis of Toxoplasma gondii is the ability to convert from an acute disease-causing, proliferative stage (tachyzoite), to a chronic, dormant stage (bradyzoite). The conversion of the tachyzoite-containing parasitophorous vacuole membrane into the less permeable bradyzoite cyst wall allows the parasite to persist for years within the host to maximize transmissibility to both primary (felids) and secondary (virtually all other warm-blooded vertebrates) hosts. This review presents our current understanding of the latent stage, including the factors that are important in bradyzoite induction and maintenance. Also discussed are the recent studies that have begun to unravel the mechanisms behind stage switching.

Linking EPCR-Binding PfEMP1 to Brain Swelling in Pediatric Cerebral Malaria.

Brain swelling is a major predictor of mortality in pediatric cerebral malaria (CM). However, the mechanisms leading to swelling remain poorly defined. Here, we combined neuroimaging, parasite transcript profiling, and laboratory blood profiles to develop machine-learning models of malarial retinopathy and brain swelling. We found that parasite var transcripts encoding endothelial protein C receptor (EPCR)-binding domains, in combination with high parasite biomass and low platelet levels, are strong indicators of CM cases with malarial retinopathy. Swelling cases presented low platelet levels and increased transcript abundance of parasite PfEMP1 DC8 and group A EPCR-binding domains. Remarkably, the dominant transcript in 50% of swelling cases encoded PfEMP1 group A CIDRα1.7 domains. Furthermore, a recombinant CIDRα1.7 domain from a pediatric CM brain autopsy inhibited the barrier-protective properties of EPCR in human brain endothelial cells in vitro. Together, these findings suggest a detrimental role for EPCR-binding CIDRα1 domains in brain swelling.

Toxoplasma gondii Cyclic AMP-Dependent Protein Kinase Subunit 3 Is Involved in the Switch from Tachyzoite to Bradyzoite Development.

UNLABELLED: Toxoplasma gondii is an obligate intracellular apicomplexan parasite that infects warm-blooded vertebrates, including humans. Asexual reproduction in T. gondii allows it to switch between the rapidly replicating tachyzoite and quiescent bradyzoite life cycle stages. A transient cyclic AMP (cAMP) pulse promotes bradyzoite differentiation, whereas a prolonged elevation of cAMP inhibits this process. We investigated the mechanism(s) by which differential modulation of cAMP exerts a bidirectional effect on parasite differentiation. There are three protein kinase A (PKA) catalytic subunits (TgPKAc1 to -3) expressed in T. gondii Unlike TgPKAc1 and TgPKAc2, which are conserved in the phylum Apicomplexa, TgPKAc3 appears evolutionarily divergent and specific to coccidian parasites. TgPKAc1 and TgPKAc2 are distributed in the cytomembranes, whereas TgPKAc3 resides in the cytosol. TgPKAc3 was genetically ablated in a type II cyst-forming strain of T. gondii (PruΔku80Δhxgprt) and in a type I strain (RHΔku80Δhxgprt), which typically does not form cysts. The Δpkac3 mutant exhibited slower growth than the parental and complemented strains, which correlated with a higher basal rate of tachyzoite-to-bradyzoite differentiation. 3-Isobutyl-1-methylxanthine (IBMX) treatment, which elevates cAMP levels, maintained wild-type parasites as tachyzoites under bradyzoite induction culture conditions (pH 8.2/low CO2), whereas the Δpkac3 mutant failed to respond to the treatment. This suggests that TgPKAc3 is the factor responsible for the cAMP-dependent tachyzoite maintenance. In addition, the Δpkac3 mutant had a defect in the production of brain cysts in vivo, suggesting that a substrate of TgPKAc3 is probably involved in the persistence of this parasite in the intermediate host animals. IMPORTANCE: Toxoplasma gondii is one of the most prevalent eukaryotic parasites in mammals, including humans. Parasites can switch from rapidly replicating tachyzoites responsible for acute infection to slowly replicating bradyzoites that persist as a latent infection. Previous studies have demonstrated that T. gondii cAMP signaling can induce or suppress bradyzoite differentiation, depending on the strength and duration of cAMP signal. Here, we report that TgPKAc3 is responsible for cAMP-dependent tachyzoite maintenance while suppressing differentiation into bradyzoites, revealing one mechanism underlying how this parasite transduces cAMP signals during differentiation.

A Bradyzoite is a Bradyzoite is a Bradyzoite?

Bradyzoite forms of Toxoplasma gondii persist in tissue cysts for the lifetime of an infected host and can reactivate to cause clinical disease. It was thought that in vivo bradyzoites within tissue cysts are biologically inactive dormant forms that rarely replicate. Apparently, consensus was wrong.

A nucleolar AAA-NTPase is required for parasite division.

Apicomplexa division involves several distinct phases shared with other eukaryote cell cycles including a gap period (G1) prior to chromosome synthesis, although how progression through the parasite cell cycle is controlled is not understood. Here we describe a cell cycle mutant that reversibly arrests in the G1 phase. The defect in this mutant was mapped by genetic complementation to a gene encoding a novel AAA-ATPase/CDC48 family member called TgNoAP1. TgNoAP1 is tightly regulated and expressed in the nucleolus during the G1/S phases. A tyrosine to a cysteine change upstream of the second AAA+ domain in the temperature sensitive TgNoAP1 allele leads to conditional protein instability, which is responsible for rapid cell cycle arrest and a primary defect in 28S rRNA processing as confirmed by knock-in of the mutation back into the parent genome. The interaction of TgNoAP1 with factors of the snoRNP and R2TP complexes indicates this protein has a role in pre-rRNA processing. This is a novel role for a cdc48-related chaperone protein and indicates that TgNoAP1 may be part of a dynamic mechanism that senses the health of the parasite protein machinery at the initial steps of ribosome biogenesis and conveys that information to the parasite cell cycle checkpoint controls.

The Toxoplasma nuclear factor TgAP2XI-4 controls bradyzoite gene expression and cyst formation.

Toxoplasma gondii undergoes many phenotypic changes during its life cycle. The recent identification of AP2 transcription factors in T. gondii has provided a platform for studying the mechanisms controlling gene expression. In the present study, we report that a recombinant protein encompassing the TgAP2XI-4 AP2 domain was able to specifically bind to a DNA motif using gel retardation assays. TgAP2XI-4 protein is localized in the parasite nucleus throughout the tachyzoite life cycle in vitro, with peak expression occurring after cytokinesis. We found that the TgAP2XI-4 transcript level was higher in bradyzoite cysts isolated from brains of chronically infected mice than in the rapidly replicating tachyzoites. A knockout of the TgAP2XI-4 gene in both T. gondii virulent type I and avirulent type II strains reveals its role in modulating expression and promoter activity of genes involved in stage conversion of the rapidly replicating tachyzoites to the dormant cyst forming bradyzoites. Furthermore, mice infected with the type II KO mutants show a drastically reduced brain cyst burden. Thus, our results validate TgAP2XI-4 as a novel nuclear factor that regulates bradyzoite gene expression during parasite differentiation and cyst formation.

The Toxoplasma gondii cyst wall protein CST1 is critical for cyst wall integrity and promotes bradyzoite persistence.

Toxoplasma gondii infects up to one third of the world's population. A key to the success of T. gondii as a parasite is its ability to persist for the life of its host as bradyzoites within tissue cysts. The glycosylated cyst wall is the key structural feature that facilitates persistence and oral transmission of this parasite. Because most of the antibodies and reagents that recognize the cyst wall recognize carbohydrates, identification of the components of the cyst wall has been technically challenging. We have identified CST1 (TGME49_064660) as a 250 kDa SRS (SAG1 related sequence) domain protein with a large mucin-like domain. CST1 is responsible for the Dolichos biflorus Agglutinin (DBA) lectin binding characteristic of T. gondii cysts. Deletion of CST1 results in reduced cyst number and a fragile brain cyst phenotype characterized by a thinning and disruption of the underlying region of the cyst wall. These defects are reversed by complementation of CST1. Additional complementation experiments demonstrate that the CST1-mucin domain is necessary for the formation of a normal cyst wall structure, the ability of the cyst to resist mechanical stress, and binding of DBA to the cyst wall. RNA-seq transcriptome analysis demonstrated dysregulation of bradyzoite genes within the various cst1 mutants. These results indicate that CST1 functions as a key structural component that confers essential sturdiness to the T. gondii tissue cyst critical for persistence of bradyzoite forms.

Compton scattering by internal shields based on melanin-containing mushrooms provides protection of gastrointestinal tract from ionizing radiation.

There is a need for radioprotectors that protect normal tissues from ionizing radiation in patients receiving high doses of radiation and during nuclear emergencies. We investigated the possibility of creating an efficient oral radioprotector based on the natural pigment melanin that would act as an internal shield and protect the tissues via Compton scattering followed by free radical scavenging. CD-1 mice were fed melanin-containing black edible mushrooms Auricularia auricila-judae before 9 Gy total body irradiation. The location of the mushrooms in the body before irradiation was determined by in vivo fluorescent imaging. Black mushrooms protected 80% of mice from the lethal dose, while control mice or those given melanin-devoid mushrooms died from gastrointestinal syndrome. The crypts of mice given black mushrooms showed less apoptosis and more cell division than those in control mice, and their white blood cell and platelet counts were restored at 45 days to preradiation levels. The role of melanin in radioprotection was proven by the fact that mice given white mushrooms supplemented with melanin survived at the same rate as mice given black mushrooms. The ability of melanin-containing mushrooms to provide remarkable protection against radiation suggests that they could be developed into oral radioprotectors.

Bright and stable near-infrared fluorescent protein for in vivo imaging.

Imaging biological processes in mammalian tissues will be facilitated by fluorescent probes with excitation and emission bands within the near-infrared optical window of high transparency. Here we report a phytochrome-based near-infrared fluorescent protein (iRFP) with excitation and emission maxima at 690 nm and 713 nm, respectively. iRFP does not require an exogenous supply of the chromophore biliverdin and has higher effective brightness, intracellular stability and photostability than earlier phytochrome-derived fluorescent probes. Compared with far-red GFP-like proteins, iRFP has a substantially higher signal-to-background ratio in a mouse model due to its infrared-shifted spectra.

Imaging devices for use in small animals.

Imaging devices for small animals have emerged in the past 10 years as extraordinarily useful tools in translational research and drug development. The Food and Drug Administration requires animal testing after in vitro drug discovery but before human application. Many small animal instruments have been developed in analogy to human scale devices, including positron emission tomography, single-photon emission computed tomography, computed tomography, magnetic resonance imaging, and ultrasound. Conversely, optical imaging with fluorescent and bioluminescent tracer technology, originating in single-cell in vitro studies, has been scaled up to whole-body animal imaging. Imaging that uses multiple devices permits a comparison of different aspects of function, anatomy, gene expression, and phenotype by the use of software algorithms or more recently with hybrid instruments. Animal imaging facilitates "bench-to-bedside" drug development in 2 ways. Longitudinal imaging improves the science of animal research through the benefit of paired statistics with the use of animals as their own controls while it simultaneously reduces animal sacrifice. In addition, imaging makes explicit the development of diagnostic and therapeutic agents on nearly identical molecular synthesis platforms, therefore linking drug discovery to the development of imaging tracers. This powerful paradigm, now known as diagnostic/therapeutic pairing or theranostics, is already familiar from the use of (123)I used for thyroid diagnosis and (131)I for therapy of benign and malignant thyroid conditions. Many newer examples exist, such as "cold" or "hot" octreotide and meta-iodobenzylguanidine in neuroendocrine tumors; and rituximab in pharmaceutical doses, or with beta emitter tags, for therapy of indolent non-Hodgkin's lymphoma. Theranostic agents are also rapidly emerging that use nanoparticles, aptamers, peptides, and antibodies for magnetic resonance imaging/positron emission tomography/single-photo emission computed tomography/computed tomography imaging devices in animals with subsequent therapeutic drug development for translation to human use.

Comprehensive proteomic analysis of membrane proteins in Toxoplasma gondii.

Toxoplasma gondii (T. gondii) is an obligate intracellular protozoan parasite that is an important human and animal pathogen. Experimental information on T. gondii membrane proteins is limited, and the majority of gene predictions with predicted transmembrane motifs are of unknown function. A systematic analysis of the membrane proteome of T. gondii is important not only for understanding this parasite's invasion mechanism(s), but also for the discovery of potential drug targets and new preventative and therapeutic strategies. Here we report a comprehensive analysis of the membrane proteome of T. gondii, employing three proteomics strategies: one-dimensional gel liquid chromatography-tandem MS analysis (one-dimensional gel electrophoresis LC-MS/MS), biotin labeling in conjunction with one-dimensional gel LC-MS/MS analysis, and a novel strategy that combines three-layer "sandwich" gel electrophoresis with multidimensional protein identification technology. A total of 2241 T. gondii proteins with at least one predicted transmembrane segment were identified and grouped into 841 sequentially nonredundant protein clusters, which account for 21.8% of the predicted transmembrane protein clusters in the T. gondii genome. A large portion (42%) of the identified T. gondii membrane proteins are hypothetical proteins. Furthermore, many of the membrane proteins validated by mass spectrometry are unique to T. gondii or to the Apicomplexa, providing a set of gene predictions ripe for experimental investigation, and potentially suitable targets for the development of therapeutic strategies.

Post-translational modifications to Toxoplasma gondii alpha- and beta-tubulins include novel C-terminal methylation.

Toxoplasma gondii is an apicomplexan of both medical and veterinary importance which is classified as an NIH Category B priority pathogen. It is best known for its ability to cause congenital infection in immune competent hosts and encephalitis in immune compromised hosts. The highly stable and specialized microtubule-based cytoskeleton participates in the invasion process. The genome encodes three isoforms of both alpha- and beta-tubulin and we show that the tubulin is extensively altered by specific post-translational modifications (PTMs) in this paper. T. gondii tubulin PTMs were analyzed by mass spectrometry and immunolabeling using specific antibodies. The PTMs identified on alpha-tubulin included acetylation of Lys40, removal of the last C-terminal amino acid residue Tyr453 (detyrosinated tubulin) and truncation of the last five amino acid residues. Polyglutamylation was detected on both alpha- and beta-tubulins. An antibody directed against mammalian alpha-tubulin lacking the last two C-terminal residues (Delta2-tubulin) labeled the apical region of this parasite. Detyrosinated tubulin was diffusely present in subpellicular microtubules and displayed an apparent accumulation at the basal end. Methylation, a PTM not previously described on tubulin, was also detected. Methylated tubulins were not detected in the host cells, human foreskin fibroblasts, suggesting that this may be a modification specific to the Apicomplexa.

Structural and metabolic specificity of methylthiocoformycin for malarial adenosine deaminases.

Plasmodium falciparum is a purine auxotroph requiring hypoxanthine as a key metabolic precursor. Erythrocyte adenine nucleotides are the source of the purine precursors, making adenosine deaminase (ADA) a key enzyme in the pathway of hypoxanthine formation. Methylthioadenosine (MTA) is a substrate for most malarial ADAs, but not for human ADA. The catalytic site specificity of malarial ADAs permits methylthiocoformycin (MT-coformycin) to act as a Plasmodium-specific transition state analogue with low affinity for human ADA [Tyler, P. C., Taylor, E. A., Frohlich, R. G. G., and Schramm, V. L. (2007) J. Am. Chem. Soc. 129, 6872-6879]. The structural basis for MTA and MT-coformycin specificity in malarial ADAs is the subject of speculation [Larson, E. T., et al. (2008) J. Mol. Biol. 381, 975-988]. Here, the crystal structure of ADA from Plasmodium vivax (PvADA) in a complex with MT-coformycin reveals an unprecedented binding geometry for 5'-methylthioribosyl groups in the malarial ADAs. Compared to malarial ADA complexes with adenosine or deoxycoformycin, 5'-methylthioribosyl groups are rotated 130 degrees . A hydrogen bonding network between Asp172 and the 3'-hydroxyl of MT-coformycin is essential for recognition of the 5'-methylthioribosyl group. Water occupies the 5'-hydroxyl binding site when MT-coformycin is bound. Mutagenesis of Asp172 destroys the substrate specificity for MTA and MT-coformycin. Kinetic, mutagenic, and structural analyses of PvADA and kinetic analysis of five other Plasmodium ADAs establish the unique structural basis for its specificity for MTA and MT-coformycin. Plasmodium gallinaceum ADA does not use MTA as a substrate, is not inhibited by MT-coformycin, and is missing Asp172. Treatment of P. falciparum cultures with coformycin or MT-coformycin in the presence of MTA is effective in inhibiting parasite growth.

Role of CpSUB1, a subtilisin-like protease, in Cryptosporidium parvum infection in vitro.

The apicomplexan parasite Cryptosporidium is a significant cause of diarrheal disease worldwide. Previously, we reported that a Cryptosporidium parvum subtilisin-like serine protease activity with furin-type specificity cleaves gp40/15, a glycoprotein that is proteolytically processed into gp40 and gp15, which are implicated in mediating infection of host cells. Neither the enzyme(s) responsible for the protease activity in C. parvum lysates nor those that process gp40/15 are known. There are no furin or other proprotein convertase genes in the C. parvum genome. However, a gene encoding CpSUB1, a subtilisin-like serine protease, is present. In this study, we cloned the CpSUB1 genomic sequence and expressed and purified the recombinant prodomain. Reverse transcriptase PCR analysis of RNA from C. parvum-infected HCT-8 cells revealed that CpSUB1 is expressed throughout infection in vitro. In immunoblots, antiserum to the recombinant CpSUB1 prodomain revealed two major bands, of approximately 64 kDa and approximately 48 kDa, for C. parvum lysates and proteins "shed" during excystation. In immunofluorescence assays, the antiserum reacted with the apical region of sporozoites and merozoites. The recombinant prodomain inhibited protease activity and processing of recombinant gp40/15 by C. parvum lysates but not by furin. Since prodomains are often selective inhibitors of their cognate enzymes, these results suggest that CpSUB1 may be a likely candidate for the protease activity in C. parvum and for processing of gp40/15. Importantly, the recombinant prodomain inhibited C. parvum infection of HCT-8 cells. These studies indicate that CpSUB1 plays a significant role in infection of host cells by the parasite and suggest that this enzyme may serve as a target for intervention.

Computational analysis and experimental validation of gene predictions in Toxoplasma gondii.

BACKGROUND: Toxoplasma gondii is an obligate intracellular protozoan that infects 20 to 90% of the population. It can cause both acute and chronic infections, many of which are asymptomatic, and, in immunocompromised hosts, can cause fatal infection due to reactivation from an asymptomatic chronic infection. An essential step towards understanding molecular mechanisms controlling transitions between the various life stages and identifying candidate drug targets is to accurately characterize the T. gondii proteome. METHODOLOGY/PRINCIPAL FINDINGS: We have explored the proteome of T. gondii tachyzoites with high throughput proteomics experiments and by comparison to publicly available cDNA sequence data. Mass spectrometry analysis validated 2,477 gene coding regions with 6,438 possible alternative gene predictions; approximately one third of the T. gondii proteome. The proteomics survey identified 609 proteins that are unique to Toxoplasma as compared to any known species including other Apicomplexan. Computational analysis identified 787 cases of possible gene duplication events and located at least 6,089 gene coding regions. Commonly used gene prediction algorithms produce very disparate sets of protein sequences, with pairwise overlaps ranging from 1.4% to 12%. Through this experimental and computational exercise we benchmarked gene prediction methods and observed false negative rates of 31 to 43%. CONCLUSIONS/SIGNIFICANCE: This study not only provides the largest proteomics exploration of the T. gondii proteome, but illustrates how high throughput proteomics experiments can elucidate correct gene structures in genomes.

Plasmodium falciparum purine nucleoside phosphorylase is critical for viability of malaria parasites.

Human malaria infections resulting from Plasmodium falciparum have become increasingly difficult to treat due to the emergence of drug-resistant parasites. The P. falciparum purine salvage enzyme purine nucleoside phosphorylase (PfPNP) is a potential drug target. Previous studies, in which PfPNP was targeted by transition state analogue inhibitors, found that those inhibiting human PNP and PfPNPs killed P. falciparum in vitro. However, many drugs have off-target interactions, and genetic evidence is required to demonstrate single target action for this class of potential drugs. We used targeted gene disruption in P. falciparum strain 3D7 to ablate PNP expression, yielding transgenic 3D7 parasites (Deltapfpnp). Lysates of the Deltapfpnp parasites showed no PNP activity, but activity of another purine salvage enzyme, adenosine deaminase (PfADA), was normal. When compared with wild-type 3D7, the Deltapfpnp parasites showed a greater requirement for exogenous purines and a severe growth defect at physiological concentrations of hypoxanthine. Drug assays using immucillins, specific transition state inhibitors of PNP, were performed on wild-type and Deltapfpnp parasites. The Deltapfpnp parasites were more sensitive to PNP inhibitors that bound hPNP tighter and less sensitive to MT-ImmH, an inhibitor with 100-fold preference for PfPNP over hPNP. The results demonstrate the importance of purine salvage in P. falciparum and validate PfPNP as the target of immucillins.

Toxoplasma gondii purine nucleoside phosphorylase biochemical characterization, inhibitor profiles, and comparison with the Plasmodium falciparum ortholog.

Purine nucleoside phosphorylase (PNP) is an important component of the nucleotide salvage pathway in apicomplexan parasites and a potential target for drug development. The intracellular pathogen Toxoplasma gondii was therefore tested for sensitivity to immucillins, transition state analogs that exhibit high potency against PNP in the malaria parasite Plasmodium falciparum. Growth of wild-type T. gondii is unaffected by up to 10 microm immucillin-H (ImmH), but mutants lacking the (redundant) purine salvage pathway enzyme adenosine kinase are susceptible to the drug, with an IC50 of 23 nm. This effect is rescued by the reaction product hypoxanthine, but not the substrate inosine, indicating that ImmH acts via inhibition of T. gondii PNP. The primary amino acid sequence of TgPNP is >40% identical to PfPNP, and recombinant enzymes exhibit similar kinetic parameters for most substrates. Unlike the Plasmodium enzyme, however, TgPNP cannot utilize 5'-methylthio-inosine (MTI). Moreover, TgPNP is insensitive to methylthio-immucillin-H (MT-ImmH), which inhibits PfPNP with a Ki* of 2.7 nm. MTI arises through the deamination of methylthio-adenosine, a product of the polyamine biosynthetic pathway, and its further metabolism to hypoxanthine involves PfPNP in purine recycling (in addition to salvage). Remarkably, analysis of the recently completed T. gondii genome indicates that polyamine biosynthetic machinery is completely lacking in this species, obviating the need for TgPNP to metabolize MTI. Differences in purine and polyamine metabolic pathways among members of the phylum Apicomplexa and these parasites and their human hosts are likely to influence drug target selection strategies. Targeting T. gondii PNP alone is unlikely to be efficacious for treatment of toxoplasmosis.