The vacuolar iron transporter mediates iron detoxification in Toxoplasma gondii.

Iron is essential to cells as a cofactor in enzymes of respiration and replication, however without correct storage, iron leads to the formation of dangerous oxygen radicals. In yeast and plants, iron is transported into a membrane-bound vacuole by the vacuolar iron transporter (VIT). This transporter is conserved in the apicomplexan family of obligate intracellular parasites, including in Toxoplasma gondii. Here, we assess the role of VIT and iron storage in T. gondii. By deleting VIT, we find a slight growth defect in vitro, and iron hypersensitivity, confirming its essential role in parasite iron detoxification, which can be rescued by scavenging of oxygen radicals. We show VIT expression is regulated by iron at transcript and protein levels, and by altering VIT localization. In the absence of VIT, T. gondii responds by altering expression of iron metabolism genes and by increasing antioxidant protein catalase activity. We also show that iron detoxification has an important role both in parasite survival within macrophages and in virulence in a mouse model. Together, by demonstrating a critical role for VIT during iron detoxification in T. gondii, we reveal the importance of iron storage in the parasite and provide the first insight into the machinery involved.

Discovery and Optimization of Triazine Nitrile Inhibitors of Toxoplasma gondii Cathepsin L for the Potential Treatment of Chronic Toxoplasmosis in the CNS.

With roughly 2 billion people infected, the neurotropic protozoan Toxoplasma gondii remains one of the most pervasive and infectious parasites. Toxoplasma infection is the second leading cause of death due to foodborne illness in the United States, causes severe disease in immunocompromised patients, and is correlated with several cognitive and neurological disorders. Currently, no therapies exist that are capable of eliminating the persistent infection in the central nervous system (CNS). In this study we report the identification of triazine nitrile inhibitors of Toxoplasma cathepsin L (TgCPL) from a high throughput screen and their subsequent optimization. Through rational design, we improved inhibitor potency to as low as 5 nM, identified pharmacophore features that can be exploited for isoform selectivity (up to 7-fold for TgCPL versus human isoform), and improved metabolic stability (t1/2 > 60 min in mouse liver microsomes) guided by a metabolite ID study. We demonstrated that this class of compounds is capable of crossing the blood-brain barrier in mice (1:1 brain/plasma at 2 h). Importantly, we also show for the first time that treatment of T. gondii bradyzoite cysts in vitro with triazine nitrile inhibitors reduces parasite viability with efficacy equivalent to a TgCPL genetic knockout.

mSphere of Influence: Ushering in the CRISPR Revolution to Toxoplasma Biology.

Alfredo J. Guerra works in the field of molecular parasitology and structural biology. In this mSphere of Influence article, he reflects on how "Efficient Gene Disruption in Diverse Strains of Toxoplasma gondii Using CRISPR/CAS9" by Bang Shen et al. (mBio 5:e01114-14, 2014, https://doi.org/10.1128/mBio.01114-14) and "Efficient Genome Engineering of Toxoplasma gondii using CRISPR/CAS9" by Saima M. Sidik et al. (PLoS One 9:e100450, 2014, https://doi.org/10.1371/journal.pone.0100450) made an impact on him by successfully implementing strategies to genetically manipulate T. gondii using CRISPR/CAS9 gene editing technology.

Development of a conditional localization approach to control apicoplast protein trafficking in malaria parasites.

Secretory proteins are of particular importance to apicomplexan parasites and comprise over 15% of the genomes of the human pathogens that cause diseases like malaria, toxoplasmosis and babesiosis as well as other diseases of agricultural significance. Here, we developed an approach that allows us to control the trafficking destination of secretory proteins in the human malaria parasite Plasmodium falciparum. Based on the unique structural requirements of apicoplast transit peptides, we designed three conditional localization domains (CLD1, 2 and 3) that can be used to control protein trafficking via the addition of a cell permeant ligand. Studies comparing the trafficking dynamics of each CLD show that CLD2 has the most optimal trafficking efficiency. To validate this system, we tested whether CLD2 could conditionally localize a biotin ligase called holocarboxylase synthetase 1 (HCS1) without interfering with the function of the enzyme. In a parasite line expressing CLD2-HCS1, we were able to control protein biotinylation in the apicoplast in a ligand-dependent manner, demonstrating the full functionality of the CLD tool. We have developed and validated a novel molecular tool that may be used in future studies to help elucidate the function of secretory proteins in malaria parasites.

Structural basis of Toxoplasma gondii perforin-like protein 1 membrane interaction and activity during egress.

Intracellular pathogens must egress from the host cell to continue their infectious cycle. Apicomplexans are a phylum of intracellular protozoans that have evolved members of the membrane attack complex and perforin (MACPF) family of pore forming proteins to disrupt cellular membranes for traversing cells during tissue migration or egress from a replicative vacuole following intracellular reproduction. Previous work showed that the apicomplexan Toxoplasma gondii secretes a perforin-like protein (TgPLP1) that contains a C-terminal Domain (CTD) which is necessary for efficient parasite egress. However, the structural basis for CTD membrane binding and egress competency remained unknown. Here, we present evidence that TgPLP1 CTD prefers binding lipids that are abundant in the inner leaflet of the lipid bilayer. Additionally, solving the high-resolution crystal structure of the TgPLP1 APCß domain within the CTD reveals an unusual double-layered ß-prism fold that resembles only one other protein of known structure. Three direct repeat sequences comprise subdomains, with each constituting a wall of the ß-prism fold. One subdomain features a protruding hydrophobic loop with an exposed tryptophan at its tip. Spectrophotometric measurements of intrinsic tryptophan fluorescence are consistent with insertion of the hydrophobic loop into a target membrane. Using CRISPR/Cas9 gene editing we show that parasite strains bearing mutations in the hydrophobic loop, including alanine substitution of the tip tryptophan, are equally deficient in egress as a strain lacking TgPLP1 altogether. Taken together our findings suggest a crucial role for the hydrophobic loop in anchoring TgPLP1 to the membrane to support its cytolytic activity and egress function.

Optimization of dipeptidic inhibitors of cathepsin L for improved Toxoplasma gondii selectivity and CNS permeability.

The neurotropic protozoan Toxoplasma gondii is the second leading cause of death due to foodborne illness in the US, and has been designated as one of five neglected parasitic infections by the Center for Disease Control and Prevention. Currently, no treatment options exist for the chronic dormant-phase Toxoplasma infection in the central nervous system (CNS). T. gondii cathepsin L (TgCPL) has recently been implicated as a novel viable target for the treatment of chronic toxoplasmosis. In this study, we report the first body of SAR work aimed at developing potent inhibitors of TgCPL with selectivity vs the human cathepsin L. Starting from a known inhibitor of human cathepsin L, and guided by structure-based design, we were able to modulate the selectivity for Toxoplasma vs human CPL by nearly 50-fold while modifying physiochemical properties to be more favorable for metabolic stability and CNS penetrance. The overall potency of our inhibitors towards TgCPL was improved from 2 µM to as low as 110 nM and we successfully demonstrated that an optimized analog 18b is capable of crossing the BBB (0.5 brain/plasma). This work is an important first step toward development of a CNS-penetrant probe to validate TgCPL as a feasible target for the treatment of chronic toxoplasmosis.

Structural Features of Apicomplexan Pore-Forming Proteins and Their Roles in Parasite Cell Traversal and Egress.

Apicomplexan parasites cause diseases, including malaria and toxoplasmosis, in a range of hosts, including humans. These intracellular parasites utilize pore-forming proteins that disrupt host cell membranes to either traverse host cells while migrating through tissues or egress from the parasite-containing vacuole after replication. This review highlights recent insight gained from the newly available three-dimensional structures of several known or putative apicomplexan pore-forming proteins that contribute to cell traversal or egress. These new structural advances suggest that parasite pore-forming proteins use distinct mechanisms to disrupt host cell membranes at multiple steps in parasite life cycles. How proteolytic processing, secretion, environment, and the accessibility of lipid receptors regulate the membranolytic activities of such proteins is also discussed.

A novel lipoate attachment enzyme is shared by Plasmodium and Chlamydia species.

Lipoate is an essential cofactor for enzymes that are important for central metabolism and other processes. In malaria parasites, scavenged lipoate from the human host is required for survival. The Plasmodium falciparum mitochondrion contains two enzymes (PfLipL1 and PfLipL2) that are responsible for activating mitochondrial proteins through the covalent attachment of lipoate (lipoylation). Lipoylation occurs via a novel redox-gated mechanism that remains poorly understood. We show that PfLipL1 functions as a redox switch that determines which downstream proteins will be activated. Based on the lipoate redox state, PfLipL1 either functions as a canonical lipoate ligase or as a lipoate activating enzyme which works in conjunction with PfLipL2. We demonstrate that PfLipL2 is a lipoyltransferase and is a member of a novel clade of lipoate attachment enzymes. We show that a LipL2 enzyme from Chlamydia trachomatis has similar activity, demonstrating conservation between intracellular pathogens from different phylogenetic kingdoms and supporting the hypothesis that an early ancestor of malaria parasites once contained a chlamydial endosymbiont. Redox-dependent lipoylation may regulate processes such as central metabolism and oxidative defense pathways.

Crystal structure of lipoate-bound lipoate ligase 1, LipL1, from Plasmodium falciparum.

Plasmodium falciparum lipoate protein ligase 1 (PfLipL1) is an ATP-dependent ligase that belongs to the biotin/lipoate A/B protein ligase family (PFAM PF03099). PfLipL1 is the only known canonical lipoate ligase in Pf and functions as a redox switch between two lipoylation routes in the parasite mitochondrion. Here, we report the crystal structure of a deletion construct of PfLipL1 (PfLipL1Δ243-279 ) bound to lipoate, and validate the lipoylation activity of this construct in both an in vitro lipoylation assay and a cell-based lipoylation assay. This characterization represents the first step in understanding the redox dependence of the lipoylation mechanism in malaria parasites. Proteins 2017; 85:1777-1783. © 2017 Wiley Periodicals, Inc.

Backbone and sterospecific methyl side chain resonance assignments of the homodimeric zinc sensor AdcR (32 kDa) in the apo- and Zn(II)-bound states.

Streptococcus pneumoniae adhesin competence repressor (AdcR) is a Zn(II)-dependent 32 kDa homodimer that controls the transcription of a zinc-specific ABC uptake system (AdcABC), three pneumococcal histidine triad proteins (PhtA, PhtD and PhtE), and an AdcA homolog AdcAII. AdcR is the first metal-dependent member of the MarR family of prokaryotic transcriptional repressors. Two-dimensional NMR studies reveal large changes in the spectrum upon Zn(II) binding. Near complete backbone and stereospecific methyl group resonance assignments for apo- and Zn(II)-AdcR are presented here.

Solution NMR refinement of a metal ion bound protein using metal ion inclusive restrained molecular dynamics methods.

Correctly calculating the structure of metal coordination sites in a protein during the process of nuclear magnetic resonance (NMR) structure determination and refinement continues to be a challenging task. In this study, we present an accurate and convenient means by which to include metal ions in the NMR structure determination process using molecular dynamics (MD) simulations constrained by NMR-derived data to obtain a realistic and physically viable description of the metal binding site(s). This method provides the framework to accurately portray the metal ions and its binding residues in a pseudo-bond or dummy-cation like approach, and is validated by quantum mechanical/molecular mechanical (QM/MM) MD calculations constrained by NMR-derived data. To illustrate this approach, we refine the zinc coordination complex structure of the zinc sensing transcriptional repressor protein Staphylococcus aureus CzrA, generating over 130 ns of MD and QM/MM MD NMR-data compliant sampling. In addition to refining the first coordination shell structure of the Zn(II) ion, this protocol benefits from being performed in a periodically replicated solvation environment including long-range electrostatics. We determine that unrestrained (not based on NMR data) MD simulations correlated to the NMR data in a time-averaged ensemble. The accurate solution structure ensemble of the metal-bound protein accurately describes the role of conformational sampling in allosteric regulation of DNA binding by zinc and serves to validate our previous unrestrained MD simulations of CzrA. This methodology has potentially broad applicability in the structure determination of metal ion bound proteins, protein folding and metal template protein-design studies.

Allosteric inhibition of a zinc-sensing transcriptional repressor: insights into the arsenic repressor (ArsR) family.

The molecular basis of allosteric regulation remains a subject of intense interest. Staphylococcus aureus CzrA is a member of the ubiquitous arsenic repressor (ArsR) family of bacterial homodimeric metal-sensing proteins and has emerged as a model system for understanding allosteric regulation of operator DNA binding by transition metal ions. Using unnatural amino acid substitution and a standard linkage analysis, we show that a His97' NH(ε2)...O=C His67 quaternary structural hydrogen bond is an energetically significant contributor to the magnitude of the allosteric coupling free energy, ∆Gc. A "cavity" introduced just beneath this hydrogen bond in V66A/L68V CzrA results in a significant reduction in regulation by Zn(II) despite adopting a wild-type global structure and Zn(II) binding and DNA binding affinities only minimally affected from wild type. The energetics of Zn(II) binding and heterotropic coupling free energies (∆Hc, -T∆Sc) of the double mutant are also radically altered and suggest that increased internal dynamics leads to poorer allosteric negative regulation in V66A/L68V CzrA. A statistical coupling analysis of 3000 ArsR proteins reveals a sector that links the DNA-binding determinants and the α5 Zn(II)-sensing sites through V66/L68 in CzrA. We propose that distinct regulatory sites uniquely characteristic of individual ArsR proteins result from evolution of distinct connectivities to this sector, each capable of driving the same biological outcome, transcriptional derepression.

Energetics of zinc-mediated interactions in the allosteric pathways of metal sensor proteins.

A metal-mediated interprotomer hydrogen bond has been implicated in the allosteric mechanism of DNA operator binding in several metal-sensing proteins. Using computational methods, we investigate the energetics of such zinc-mediated interactions in members of the ArsR/SmtB family of proteins (CzrA, SmtB, CadC, and NmtR) and the MarR family zinc-uptake repressor AdcR, which feature similar interactions, but in sites that differ widely in their allosteric responsiveness. We provide novel structural insight into previously uncharacterized allosteric forms of these proteins using computational methodologies. We find this metal-mediated interaction to be significantly stronger (∼8 kcal/mol) at functional allosteric metal binding sites compared to a nonresponsive site (CadC) and the apo-proteins. Simulations of the apo-proteins further reveal that the high interaction energy works to overcome the considerable disorder at these hydrogen-bonding sites and functions as a "switch" to lock in a weak DNA-binding conformation once metal is bound. These findings suggest a conserved functional role of metal-mediated second coordination shell hydrogen bonds at allosterically responsive sites in zinc-sensing transcription regulators.

Metal site occupancy and allosteric switching in bacterial metal sensor proteins.

All prokaryotes encode a panel of metal sensor or metalloregulatory proteins that govern the expression of genes that allows an organism to quickly adapt to toxicity or deprivation of both biologically essential transition metal ions, e.g., Zn, Cu, Fe, and heavy metal pollutants. As such, metal sensor proteins can be considered arbiters of intracellular transition metal bioavailability and thus potentially control the metallation state of the metalloproteins in the cell. Metal sensor proteins are specialized allosteric proteins that regulate transcription as a result direct binding of one or two cognate metal ions, to the exclusion of all others. In most cases, the binding of the cognate metal ion induces a structural change in a protein oligomer that either activates or inhibits operator DNA binding. A quantitative measure of the degree to which a particular metal drives metalloregulation of operator DNA-binding is the allosteric coupling free energy, ΔGc. In this review, we summarize recent work directed toward understanding metal occupancy and metal selectivity of these allosteric switches in selected families of metal sensor proteins and examine the structural origins of ΔGc in the functional context a thermodynamic "set-point" model of intracellular metal homeostasis.

Crystal structure of the zinc-dependent MarR family transcriptional regulator AdcR in the Zn(II)-bound state.

Streptococcus pneumoniae adhesin competence regulator (AdcR), the first metal-dependent member of the multiple antibiotic resistance regulator (MarR) family of proteins, represses the transcription of a high-affinity zinc-specific uptake transporter, a group of surface antigen zinc-binding pneumococcal histidine triad proteins (PhtA, PhtB, PhtD, and PhtE), and an AdcA homologue (AdcAII). The 2.0 Å resolution structure of Zn(II)-bound AdcR reveals a highly helical two-fold-symmetric dimer with two distinct metal-binding sites per protomer. Zn(II) is tetrahedrally coordinated by E24, H42, H108, and H112 in what defines the primary sensing site in AdcR. Site 2 is a tetracoordinate site whose function is currently unknown. NMR methyl group perturbation experiments reveal that Zn(II) drives a global change in the structure of apo-AdcR that stabilizes a conformation that is compatible with DNA binding. This co-repression mechanism is unprecedented in MarR transcriptional regulators.

The metalloregulatory zinc site in Streptococcus pneumoniae AdcR, a zinc-activated MarR family repressor.

Streptococcus pneumoniae D39 AdcR (adhesin competence repressor) is the first metal-sensing member of the MarR (multiple antibiotic resistance repressor) family to be characterized. Expression profiling with a ΔadcR strain grown in liquid culture (brain-heart infusion) under microaerobic conditions revealed upregulation of 13 genes, including adcR and adcCBA, encoding a high-affinity ABC uptake system for zinc, and genes encoding cell-surface zinc-binding pneumococcal histidine triad (Pht) proteins and AdcAII (Lmb, laminin binding). The ΔadcR, H108Q and H112Q adcR mutant allelic strains grown in 0.2 mM Zn(II) exhibit a slow-growth phenotype and an approximately twofold increase in cell-associated Zn(II). Apo- and Zn(II)-bound AdcR are homodimers in solution and binding to a 28-mer DNA containing an adc operator is strongly stimulated by Zn(II) with K(DNA-Zn)=2.4 × 10(8) M(-1) (pH 6.0, 0.2 M NaCl, 25 °C). AdcR binds two Zn(II) per dimer, with stepwise Zn(II) affinities K(Zn1) and K(Zn2) of ≥10(9) M(-1) at pH 6.0 and ≥10(12) M(-1) at pH 8.0, and one to three lower affinity Zn(II) depending on the pH. X-ray absorption spectroscopy of the high-affinity site reveals a pentacoordinate N/O complex and no cysteine coordination, the latter finding corroborated by wild type-like functional properties of C30A AdcR. Alanine substitution of conserved residues His42 in the DNA-binding domain, and His108 and His112 in the C-terminal regulatory domain, abolish high-affinity Zn(II) binding and greatly reduce Zn(II)-activated binding to DNA. NMR studies reveal that these mutants adopt the same folded conformation as dimeric wild type apo-AdcR, but fail to conformationally switch upon Zn(II) binding. These studies implicate His42, His108 and H112 as metalloregulatory zinc ligands in S. pneumoniae AdcR.

Prevalencia de obesidad y sobrepesoen la población infantil del Centro de SaludOtero de Ceuta (EPOICE 2009) / Prevalence of obesity and overweight in the paediatric population in the Otero health care in Ceuta

Objetivo: determinar la prevalencia de la obesidad y sobrepesoen niños y niñas de entre 6 y 13 años adscritos alCentro de Salud Otero de Ceuta. También se pretendió analizarla obesidad y el sobrepeso en función del sexo, la edad,la realización del desayuno, hacer alguna actividad física y laadscripción cultural.Material y método: estudio descriptivo transversal en niñosy niñas de entre 6 y 13 años, adscritos al Centro de SaludOtero de Ceuta y que acudieron a la consulta de pediatríaentre el 1 de enero y el 30 de junio de 2009. Se planificóestudiar 255 sujetos (seguridad: 95%, proporción esperadade obesidad: 0,21, precisión absoluta: 5%). El instrumentode medida fue un cuestionario validado sobre hábitos dealimentación para la población infantil española (ProgramaPerseo). Se consideró obesidad un Índice de Masa Corporal(IMC) ≥ p 97; y sobrepeso un IMC p ≥ 85-p < 97. Para el análisisbivariante se utilizó el test de Chi cuadrado.Resultados: la prevalencia de obesidad fue del 22,8% (IC95%:17,6-28) y del 19,1% (IC 95%:14,1-24) para el sobrepeso,con una mayor incidencia en el sexo masculino. La obesidadapareció con mayor relevancia en el grupo etáreo 12-13años (45%). Los sujetos que no realizaban ningún tipo de actividaddeportiva presentaban mayor prevalencia de obesidad(29% frente al 17%). No se dio sobrepeso ni obesidad enaquellos niños/as que realizaban un desayuno completo.Conclusiones: los niveles de obesidad/sobrepeso presentadosponen de manifiesto el alarmante problema al que nosenfrentamos. Poner en práctica programas como el que selleva a cabo en la actualidad, a través del grupo PERSEO ola estrategia NAOS, orientados al fomento de hábitos devida saludable, pueden ser los pilares fundamentales dondese deba asentar nuestra política educacional para disminuirlas cifras de obesidad/sobrepeso (AU)

Objective: to determine the prevalence of obesity and overweightin boys and girls aged 6 to 13 years registered at theOtero health care centre in Ceuta. A second aim of the studywas to analyse both these conditions depending on the gender,age, eating or not eating breakfast, the level of physicalactivity and cultural affiliation.Material and method: cross-sectional descriptive study inchildren of both genders aged between 6 and 13 years registeredat the Otero health care centre in Ceuta who werevisited at the paediatric office between 1 January and 30June 2009. The study population included 255 subjects (safety:95%, expected obesity ratio: 0,21, absolute accuracy:5%). The measure instrument used was a validated questionnaireon the nutritional habits for the spanish paediatricpopulation (PERSEO programme). Obesity was consideredas a Body Mass Index (BMI) of p ≥ 85-p < 97. The chisquare test was used for multivariate analysis.Results: the prevalence of obesity was 22,8% (CI95%:17,6-28) and 19,1% (CI 95%: 14,1-24) for overweight, with agreater incidence in boys than girls. Obesity was identifiedin a greater proportion in the age group from 12-13 years(45%). Subjects who did not engage in any type of sportpresented a higher prevalence of obesity (29% vs 17%). Noobesity was found in children who had a full breakfast.Conclusions: the levels of obesity/overweight identifiedshow the very alarming problem we are facing. Programmessuch as the one being currently implemented throughthe PERSEO group or the NAOS strategy, aimed at promotinggood health and notional habits, should be put intoplace urgently, as they should be the fundamental cornerstone on which our educational policy should be based todecrease the observed obesity/overweight figures (AU)