Synchronous versus asynchronous oscillations for antigenically varying Plasmodium falciparum with host immune response

We consider a deterministic intra-host model for Plasmodium falciparum (Pf) malaria infection, which accounts for antigenic variation between n clonal variants of PfEMP1 and the corresponding host immune response (IR). Specifically, the model separates the IR into two components, specific and cross-reactive, respectively, in order to demonstrate that the latter can be a mechanism for the sequential appearance of variants observed in actual Pf infections. We show that a strong variant-specific IR relative to the cross-reactive IR favours the asynchronous oscillations (sequential dominance) over the synchronous oscillations in a number of ways. The decay rate of asynchronous oscillations is smaller than that for the synchronous oscillations, allowing for the parasite to survive longer. With the introduction of a delay in the stimulation of the IR, we show that only a small delay is necessary to cause persistent asynchronous oscillations and that a strong variant-specific IR increases the amplitude of the asynchronous oscillations.     

Conflicting immune responses can prolong the length of infection in Plasmodium falciparum malaria

We have recently proposed a new model for antigenic variation in Plasmodium falciparum that relies on a network of partially cross-protective immune responses to orchestrate this complex immune evasion process. In addition to exhibiting prolonged oscillations of single variants that resemble the sequential dominance of immunologically distinct antigenic types, the model implies that a higher efficacy of cross-reactive immunity actually increases the length of infection while reducing severity of disease. Here, we analyse the behaviour of a reduced system under conditions of perfect synchrony between variants to demonstrate that these features of this system can be attributed to the antagonism between cross-reactive and variant-specific responses.     

Detailed functional characterization of glycosylated and nonglycosylated variants of malaria vaccine candidate PfAMA1 produced in Nicotiana benthamiana and analysis of growth inhibitory responses in rabbits

One of the most promising malaria vaccine candidate antigens is the Plasmodium falciparum apical membrane antigen 1 (PfAMA1). Several studies have shown that this blood‐stage antigen can induce strong parasite growth inhibitory antibody responses. PfAMA1 contains up to six recognition sites for N‐linked glycosylation, a post‐translational modification that is absent in P. falciparum. To prevent any potential negative impact of N‐glycosylation, the recognition sites have been knocked out in most PfAMA1 variants expressed in eukaryotic hosts. However, N‐linked glycosylation may increase efficacy by improving immunogenicity and/or focusing the response towards relevant epitopes by glycan masking. We describe the production of glycosylated and nonglycosylated PfAMA1 in Nicotiana benthamiana and its detailed characterization in terms of yield, integrity and protective efficacy. Both PfAMA1 variants accumulated to high levels (>510 μg/g fresh leaf weight) after transient expression, and high‐mannose‐type N‐glycans were confirmed for the glycosylated variant. No significant differences between the N. benthamiana and Pichia pastoris PfAMA1 variants were detected in conformation‐sensitive ligand‐binding studies. Specific titres of >2 × 10⁶ were induced in rabbits, and strong reactivity with P. falciparum schizonts was observed in immunofluorescence assays, as well as up to 100% parasite growth inhibition for both variants, with IC₅₀ values of ~35 μg/mL. Competition assays indicated that a number of epitopes were shielded from immune recognition by N‐glycans, warranting further studies to determine how glycosylation can be used for the directed targeting of immune responses. These results highlight the potential of plant transient expression systems as a production platform for vaccine candidates.     

Humoral Immune Response to Mixed PfAMA1 Alleles; Multivalent PfAMA1 Vaccines Induce Broad Specificity

Apical Membrane Antigen 1 (AMA1), a merozoite protein essential for red cell invasion, is a candidate malaria vaccine component. Immune responses to AMA1 can protect in experimental animal models and antibodies isolated from AMA1-vaccinated or malaria-exposed humans can inhibit parasite multiplication in vitro. The parasite is haploid in the vertebrate host and the genome contains a single copy of AMA1, yet on a population basis a number of AMA1 molecular surface residues are polymorphic, a property thought to be primarily as a result of selective immune pressure. After immunisation with AMA1, antibodies more effectively inhibit strains carrying homologous AMA1 genes, suggesting that polymorphism may compromise vaccine efficacy. Here, we analyse induction of broad strain inhibitory antibodies with a multi-allele Plasmodium falciparum AMA1 (PfAMA1) vaccine, and determine the relative importance of cross-reactive and strain-specific IgG fractions by competition ELISA and in vitro parasite growth inhibition assays. Immunisation of rabbits with a PfAMA1 allele mixture yielded an increased proportion of antibodies to epitopes common to all vaccine alleles, compared to single allele immunisation. Competition ELISA with the anti-PfAMA1 antibody fraction that is cross-reactive between FVO and 3D7 AMA1 alleles showed that over 80% of these common antibodies were shared with other PfAMA1 alleles. Furthermore, growth inhibition assays revealed that for any PfAMA1 allele (FVO or 3D7), the cross-reactive fraction alone, on basis of weight, had the same functional capacity on homologous parasites as the total affinity-purified IgGs (cross-reactive+strain-specific). By contrast, the strain-specific IgG fraction of either PfAMA1 allele showed slightly less inhibition of red cell invasion by homologous strains. Thus multi-allele immunisation relatively increases the levels of antibodies to common allele epitopes. This explains the broadened cross inhibition of diverse malaria parasites, and suggests multi-allele approaches warrant further clinical investigation.     

Trypanosoma congolense: Surface glycoproteins of two early bloodstream variants: II. Purification and partial chemical characterization

Two sequential variant-specific glycoproteins have been purified from two variants of Trypanosoma congolense expressed during a relapsing infection. Isolation of the two glycoproteins, termed VSG-1 and VSG-2, respectively, employed glycerol lysis followed by purification on concanavalin A, Sephadex G-25, and gradient-eluted DE-52 columns. Partially purified VSG proteins were immunologically cross-reactive, but highly purified VSGs showed no cross-reactivity under the conditions employed. Both VSG-1 and VSG-2 consisted of a triplet of polypeptides. Although each member of a triplet subset could be distinguished by isoelectric focusing, all three gave identical N-terminal amino acid sequences and nearly identical tryptic peptide maps. The members of the VSG-1 polypeptide subset differed from those of the VSG-2 subset both with regard to N-terminal amino acid sequence and in tryptic peptide map patterns. Comparison of N-terminal sequences of VSG-1 and VSG-2 did, however, show that the sequences could be aligned to give a modest degree of amino acid homology (27%). This alignment also produced a minimum in the number of two-base changes, suggesting that the observed homology is not a coincidence and that these two proteins may well have arisen by gene duplication followed by retention of multiple point mutations.     

<Emphasis Type="Italic">In Silico</Emphasis> sequence analysis and molecular modeling of the three-dimensional structure of DAHP synthase from <Emphasis Type="Italic">Pseudomonas fragi</Emphasis>

The shikimate pathway is involved in production of aromatic amino acids in microorganisms and plants. The enzymes of this biosynthetic pathway are a potential target for the design of antimicrobial compounds and herbicides. 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase (DAHPS) catalyzes the first step of the pathway. The gene encoding DAHPS was cloned and sequenced from Pseudomonas fragi, the bacterium responsible for spoilage of milk, dairy products and meat. Amino acid sequence deduced from the nucleotide sequence revealed that P. fragi DAHPS (Pf-DAHPS) consists of 448 amino acids with calculated molecular weight of ∼50 kDa and isoelectric point of 5.81. Primary sequence analysis of Pf-DAHPS shows that it has more than 84% identity with DAHPS of other Pseudomonas species, 46% identity with Mycobacterium tuberculosis DAHPS (Mt-DAHPS), the type II DAHPS and less than 11% sequence identity with the type I DAHPS. The three-dimensional structure of Pf-DAHPS was predicted by homology modeling based on the crystal structure of Mt-DAHPS. Pf-DAHPS model contains a (β/α)₈ TIM barrel structure. Sequence alignment, phylogenetic analysis and 3D structure model classifies Pf-DAHPS as a type II DAHPS. Sequence analysis revealed the presence of DAHPS signature motif DxxHxN in Pf-DAHPS. Highly conserved sequence motif RxxxxxxKPRT(S/T) and xGxR present in type II DAHPS were also identified in Pf-DAHPS sequence. High sequence homology of DAHPS within Pseudomonas species points to the option of designing a broad spectrum drug for the genus. Pf-DAHPS 3D model provides molecular insights that may be beneficial in rationale inhibitor design for developing effective food preservative against P. fragi.     

Slow oscillations in neural networks with facilitating synapses

The synchronous oscillatory activity characterizing many neurons in a network is often considered to be a mechanism for representing, binding, conveying, and organizing information. A number of models have been proposed to explain high-frequency oscillations, but the mechanisms that underlie slow oscillations are still unclear. Here, we show by means of analytical solutions and simulations that facilitating excitatory (E _f) synapses onto interneurons in a neural network play a fundamental role, not only in shaping the frequency of slow oscillations, but also in determining the form of the up and down states observed in electrophysiological measurements. Short time constants and strong E _f synapse-connectivity were found to induce rapid alternations between up and down states, whereas long time constants and weak E _f synapse connectivity prolonged the time between up states and increased the up state duration. These results suggest a novel role for facilitating excitatory synapses onto interneurons in controlling the form and frequency of slow oscillations in neuronal circuits.     

Biological Control of Rice Blast byPseudomonas fluorescensStrainPf7–14: Evaluation of a Marker Gene and Formulations

Pseudomonas fluorescensstrainPf7–14 was evaluated for biological control of rice blast in field experiments. StrainPf7–14 was formulated in methylcellulose:talc (1:4) and applied to IR50 rice (Oryza sativa) seeds as a seed treatment and as foliar sprays in seedbed and field experiments. When applied as a seed treatment followed by three foliar applications, the strain provided a 68.5% suppression of rice blast in the seedbed experiment and a 59.6% suppression in the field experiment. The persistence and migration ofPf7–14 on the rice plant was studied with the aid oflacZYgenes inserted into the bacterium. In greenhouse experiments,Pf7–14gal was detected on rice roots at 10⁶to 10⁵cfu/g of root tissue for 110 days, the duration of the rice crop. Migration of the strain from the seeds to the leaves occurred only until the seedlings were 16 days old. WhenPf7–14 was applied to the rice plants by foliar sprays, 10⁴cfu of the bacterium per gram of leaf tissue was detected for the next 40 days. The limited migration of the bacterial biocontrol agent emphasizes the need for multiple foliar applications of the bacterium to sustain the bacterial population for effective suppression of rice blast.     

Multicomplex-based pharmacophore modeling in conjunction with multi-target docking and molecular dynamics simulations for the identification of PfDHFR inhibitors

Abstract

Plasmodium falciparum dihydrofolate reductase enzyme (PfDHFR) is counted as one of the attractive and validated antimalarial drug targets. However, the point mutations in the active site of wild-type PfDHFR have developed resistance against the well-known antifolates. Therefore, there is a dire need for the development of inhibitors that can inhibit both wild-type and mutant-type DHFR enzyme. In the present contribution, we have constructed the common feature pharmacophore models from the available PfDHFR. A representative hypothesis was prioritized and then employed for the screening of natural product library to search for the molecules with complementary features responsible for the inhibition. The screened candidates were processed via drug-likeness filters and molecular docking studies. The docking was carried out on the wild-type PfDHFR (3QGT); double-mutant PfDHFR (3UM5 and 1J3J) and quadruple-mutant PfDHFR (1J3K) enzymes. A total of eight common hits were obtained from the docking calculations that could be the potential inhibitors for both wild and mutant type DHFR enzymes. Eventually, the stability of these candidates with the selected proteins was evaluated via molecular dynamics simulations. Except for SPECS14, all the prioritized candidates were found to be stable throughout the simulation run. Overall, the strategy employed in the present work resulted in the retrieval of seven candidates that may show inhibitory activity against PfDHFR and could be further exploited as a scaffold to develop novel antimalarials.

Communicated by Ramaswamy H. Sarma     

The effects of host heterogeneity on pathogen population structure.

We have shown that among pathogens, populations may self-organize into strains with non-overlapping repertoires of antigenic variants as a consequence of strong immune selection operating on polymorphic antigens. Recently, we have also demonstrated that over a wide range of intermediate levels of immune selection, pathogens may still be structured into discrete strains, but different sets of non-overlapping pathogen types will replace each other in a cyclical or chaotic manner. These models assume that the ranking of antigens in terms of the strength of the induced immune response is the same for every host. However, host immune responses may be restricted by the genotype of the individual. To explore this issue, a mathematical model was constructed under the assumption that a proportion of the host population responds principally to a variable antigen while the remainder of the population responds principally to a conserved antigen. The results of this analysis indicate that discrete strain structure (DSS) will be maintained even with a high frequency of hosts that do not respond in a variant-specific manner. Furthermore, the range of the immune selection pressure over which DSS prevails is increased (and the region of cyclical or chaotic behaviour reduced) by the inclusion of hosts that respond in a cross-reactive rather than a variant-specific manner.     

Two-patch dispersal-linked compensatory-overcompensatory spatially discrete population models

We study the role of asynchronous and synchronous dispersals on discrete-time two-patch dispersal-linked population models, where the pre-dispersal local patch dynamics are of mixed compensatory and overcompensatory types. Single-species dispersal-linked models behave as single-species single-patch models whenever all pre-dispersal local patch dynamics are compensatory and dispersal is synchronous. However, the dynamics of the corresponding two-patch population model connected by asynchronous dispersal depends on the dispersal rates. The species goes extinct on at least one patch when the asynchronous dispersal rates are high, while it persists when the rates are low. We use numerical simulations to show that in both synchronous and asynchronous mixed compensatory and overcompensatory systems, symmetric and asymmetric dispersals can control and impede the onset of cyclic population oscillations via period-doubling reversal bifurcations. Also, we show that in mixed systems both asynchronous and synchronous dispersals are capable of altering the pre-dispersal local patch dynamics from overcompensatory to compensatory dynamics. Dispersal-linked population models with ‘unstructured’ overcompensatory pre-dispersal local dynamics connected by synchronous dispersal can generate multiple attractors with fractal basin boundaries. However, mixed compensatory and overcompensatory systems appear to exhibit single attractors and not coexisting (multiple) attractors.     

Two-patch dispersal-linked compensatory-overcompensatory spatially discrete population models

We study the role of asynchronous and synchronous dispersals on discrete-time two-patch dispersal-linked population models, where the pre-dispersal local patch dynamics are of mixed compensatory and overcompensatory types. Single-species dispersal-linked models behave as single-species single-patch models whenever all pre-dispersal local patch dynamics are compensatory and dispersal is synchronous. However, the dynamics of the corresponding two-patch population model connected by asynchronous dispersal depends on the dispersal rates. The species goes extinct on at least one patch when the asynchronous dispersal rates are high, while it persists when the rates are low. We use numerical simulations to show that in both synchronous and asynchronous mixed compensatory and overcompensatory systems, symmetric and asymmetric dispersals can control and impede the onset of cyclic population oscillations via period-doubling reversal bifurcations. Also, we show that in mixed systems both asynchronous and synchronous dispersals are capable of altering the pre-dispersal local patch dynamics from overcompensatory to compensatory dynamics. Dispersal-linked population models with 'unstructured' overcompensatory pre-dispersal local dynamics connected by synchronous dispersal can generate multiple attractors with fractal basin boundaries. However, mixed compensatory and overcompensatory systems appear to exhibit single attractors and not coexisting (multiple) attractors.     

A redox-active crosslinker reveals an essential and inhibitable oxidative folding network in the endoplasmic reticulum of malaria parasites

Malaria remains a major global health problem, creating a constant need for research to identify druggable weaknesses in P. falciparum biology. As important components of cellular redox biology, members of the Thioredoxin (Trx) superfamily of proteins have received interest as potential drug targets in Apicomplexans. However, the function and essentiality of endoplasmic reticulum (ER)-localized Trx-domain proteins within P. falciparum has not been investigated. We generated conditional mutants of the protein PfJ2—an ER chaperone and member of the Trx superfamily—and show that it is essential for asexual parasite survival. Using a crosslinker specific for redox-active cysteines, we identified PfJ2 substrates as PfPDI8 and PfPDI11, both members of the Trx superfamily as well, which suggests a redox-regulatory role for PfJ2. Knockdown of these PDIs in PfJ2 conditional mutants show that PfPDI11 may not be essential. However, PfPDI8 is required for asexual growth and our data suggest it may work in a complex with PfJ2 and other ER chaperones. Finally, we show that the redox interactions between these Trx-domain proteins in the parasite ER and their substrates are sensitive to small molecule inhibition. Together these data build a model for how Trx-domain proteins in the P. falciparum ER work together to assist protein folding and demonstrate the suitability of ER-localized Trx-domain proteins for antimalarial drug development.     

The distance between the anticodon loops of two tRNAs bound to the 70 S Escherichia coli ribosome.

Using singlet-singlet energy transfer, we have measured the distance between the anticodons of two transfer RNAs simultaneously bound to a messengerprogramed Escherichia coli 70 S ribosome. The fluorescent Y base adjacent to the anticodon of yeast tRNA_Y^Phe serves as a donor. A proflavine (Pf) chemically substituted for the Y base in tRNA_Pf^Phe serves as an acceptor. By exploiting the sequential binding properties of 70 S ribosomes for two deacylated tRNAs, we can fill the strong site with either tRNA_Y^Phe or tRNA_Pf^Phe and then the weak site with the other tRNA. In both cases donor quenching and sensitized emission of the acceptor are observed. Analysis of these results leads to an estimate for the Y-proflavine distance of 18 ± 2 Å. This distance is very short and suggests strongly that the two tRNAs are simultaneously in contact with adjacent codons of the message. Separate experiments show that binding of a tRNA to the weak site does not perturb the environment of the hypermodified base of a tRNA bound to the strong site. This supports the assignment of the strong site as the peptidyl site. It also indicates that binding of the second tRNA proceeds without a change in the anticodon structure of a pre-existing tRNA at the peptidyl site.     

Origins of the specificity of inhibitor P218 toward wild-type and mutant PfDHFR: a molecular dynamics analysis

Molecular dynamics simulations were performed to evaluate the origin of the antimalarial effect of the lead compound P218. The simulations of the ligand in the cavities of wild-type, mutant Plasmodium falciparum Dihydrofolate Reductase (PfDHFR) and the human DHFR revealed the differences in the atomic-level interactions and also provided explanation for the specificity of this ligand toward PfDHFR. The binding free energy estimation using Molecular Mechanics Poisson-Boltzmann Surface Area method revealed that P218 has higher binding affinity (~ ?30 to ?35 kcal/mol) toward PfDHFR (both in wild-type and mutant forms) than human DHFR (~ ?22 kcal/mol), corroborating the experimental observations. Intermolecular hydrogen bonding analysis of the trajectories showed that P218 formed two stable hydrogen bonds with human DHFR (Ile7 and Glu30), wild-type and double-mutant PfDHFR’s (Asp54 and Arg122), while it formed three stable hydrogen bonds with quadruple-mutant PfDHFR (Asp54, Arg59, and Arg122). Additionally, P218 binding in PfDHFR is stabilized by hydrogen bonds with residues Ile14 and Ile164. It was found that mutant residues do not reduce the binding affinity of P218 to PfDHFR, in contrast, Cys59Arg mutation strongly favors inhibitor binding to quadruple-mutant PfDHFR. The atomistic-level details explored in this work will be highly useful for the design of non-resistant novel PfDHFR inhibitors as antimalarial agents.     

X‐ray crystal structures of an orally available aminopeptidase inhibitor,Tosedostat, bound to anti‐malarial drug targets PfA‐M1 and PfA‐M17

New anti‐malarial treatments are desperately required to face the spread of drug resistant parasites. Inhibition of metalloaminopeptidases, PfA‐M1 and PfA‐M17, is a validated therapeutic strategy for treatment of Plasmodium falciparum malaria. Here, we describe the crystal structures of PfA‐M1 and PfA‐M17 bound to chemotherapeutic agent Tosedostat. The inhibitor occupies the enzymes' putative product egress channels in addition to the substrate binding pockets; however, adopts different binding poses when bound to PfA‐M1 and PfA‐M17. These findings will be valuable for the continued development of selective inhibitors of PfA‐M1 and PfA‐M17. Proteins 2015; 83:789–795. © 2015 Wiley Periodicals, Inc.     

Glutathione mediated regulation of oligomeric structure and functional activity of <Emphasis Type="Italic">Plasmodium falciparum</Emphasis> glutathione S-transferase

Background  

In contrast to many other organisms, the malarial parasite Plasmodium falciparum possesses only one typical glutathione S-transferase. This enzyme, PfGST, cannot be assigned to any of the known GST classes and represents a most interesting target for antimalarial drug development. The PfGST under native conditions forms non-covalently linked higher aggregates with major population (~98%) being tetramer. However, in the presence of 2 mM GSH, a dimer of PfGST is observed. Recently reported study on binding and catalytic properties of PfGST indicated a GSH dependent low-high affinity transition with simultaneous binding of two GSH molecules to PfGST dimer suggesting that GSH binds to low affinity inactive enzyme dimer converting it to high affinity functionally active dimer. In order to understand the role of GSH in tetramer-dimer transition of PfGST as well as in modulation of functional activity of the enzyme, detailed structural, functional and stability studies on recombinant PfGST in the presence and absence of GSH were carried out.     

Solution NMR characterization of apical membrane antigen 1 and small molecule interactions as a basis for designing new antimalarials

Plasmodium falciparum apical membrane antigen 1 (PfAMA1) plays an important role in the invasion by merozoites of human red blood cells during a malaria infection. A key region of PfAMA1 is a conserved hydrophobic cleft formed by 12 hydrophobic residues. As anti‐apical membrane antigen 1 antibodies and other inhibitory molecules that target this hydrophobic cleft are able to block the invasion process, PfAMA1 is an attractive target for the development of strain‐transcending antimalarial agents. As solution nuclear magnetic resonance spectroscopy is a valuable technique for the rapid characterization of protein–ligand interactions, we have determined the sequence‐specific backbone assignments for PfAMA1 from two P. falciparum strains, FVO and 3D7. Both selective labelling and unlabelling strategies were used to complement triple‐resonance experiments in order to facilitate the assignment process. We have then used these assignments for mapping the binding sites for small molecules, including benzimidazoles, pyrazoles and 2‐aminothiazoles, which were selected on the basis of their affinities measured from surface plasmon resonance binding experiments. Among the compounds tested, benzimidazoles showed binding to a similar region on both FVO and 3D7 PfAMA1, suggesting that these compounds are promising scaffolds for the development of novel PfAMA1 inhibitors. Copyright © 2016 John Wiley & Sons, Ltd.