Plasmodium RON11 triggers biogenesis of the merozoite rhoptry pair and is essential for erythrocyte invasion.

Malaria is a global and deadly human disease caused by the apicomplexan parasites of the genus Plasmodium. Parasite proliferation within human red blood cells (RBC) is associated with the clinical manifestations of the disease. This asexual expansion within human RBCs, begins with the invasion of RBCs by P. falciparum, which is mediated by the secretion of effectors from two specialized club-shaped secretory organelles in merozoite-stage parasites known as rhoptries. We investigated the function of the Rhoptry Neck Protein 11 (RON11), which contains seven transmembrane domains and calcium-binding EF-hand domains. We generated conditional mutants of the P. falciparum RON11. Knockdown of RON11 inhibits parasite growth by preventing merozoite invasion. The loss of RON11 did not lead to any defects in processing of rhoptry proteins but instead led to a decrease in the amount of rhoptry proteins. We utilized ultrastructure expansion microscopy (U-ExM) to determine the effect of RON11 knockdown on rhoptry biogenesis. Surprisingly, in the absence of RON11, fully developed merozoites had only one rhoptry each. The single rhoptry in RON11 deficient merozoites were morphologically typical with a bulb and a neck oriented into the apical polar ring. Moreover, rhoptry proteins are trafficked accurately to the single rhoptry in RON11 deficient parasites. These data show that in the absence of RON11, the first rhoptry is generated during schizogony but upon the start of cytokinesis, the second rhoptry never forms. Interestingly, these single-rhoptry merozoites were able to attach to host RBCs but are unable to invade RBCs. Instead, RON11 deficient merozoites continue to engage with RBC for prolonged periods eventually resulting in echinocytosis, a result of secreting the contents from the single rhoptry into the RBC. Together, our data show that RON11 triggers the de novo biogenesis of the second rhoptry and functions in RBC invasion.

Structure guided mimicry of an essential P. falciparum receptor-ligand complex enhances cross neutralizing antibodies.

Invasion of human erythrocytes by Plasmodium falciparum (Pf) merozoites relies on the interaction between two parasite proteins: apical membrane antigen 1 (AMA1) and rhoptry neck protein 2 (RON2). While antibodies to AMA1 provide limited protection against Pf in non-human primate malaria models, clinical trials using recombinant AMA1 alone (apoAMA1) yielded no protection due to insufficient functional antibodies. Immunization with AMA1 bound to RON2L, a 49-amino acid peptide from its ligand RON2, has shown superior protection by increasing the proportion of neutralizing antibodies. However, this approach relies on the formation of a complex in solution between the two vaccine components. To advance vaccine development, here we engineered chimeric antigens by replacing the AMA1 DII loop, displaced upon ligand binding, with RON2L. Structural analysis confirmed that the fusion chimera (Fusion-FD12) closely mimics the binary AMA1-RON2L complex. Immunization studies in female rats demonstrated that Fusion-FD12 immune sera, but not purified IgG, neutralized vaccine-type parasites more efficiently compared to apoAMA1, despite lower overall anti-AMA1 titers. Interestingly, Fusion-FD12 immunization enhanced antibodies targeting conserved epitopes on AMA1, leading to increased neutralization of non-vaccine type parasites. Identifying these cross-neutralizing antibody epitopes holds promise for developing an effective, strain-transcending malaria vaccine.

Structure guided mimicry of an essential P. falciparum receptor-ligand complex enhances cross neutralizing antibodies.

Invasion of human red blood cells (RBCs) by Plasmodium falciparum (Pf) merozoites relies on the interaction between two parasite proteins, apical membrane antigen 1 (AMA1) and rhoptry neck protein 2 (RON2) 1,2 . Antibodies to AMA1 confer limited protection against P. falciparum in non-human primate malaria models 3,4 . However, clinical trials with recombinant AMA1 alone (apoAMA1) saw no protection, likely due to inadequate levels of functional antibodies 5-8 . Notably, immunization with AMA1 in its ligand bound conformation using RON2L, a 49 amino acid peptide from RON2, confers superior protection against P. falciparum malaria by enhancing the proportion of neutralizing antibodies 9,10 . A limitation of this approach, however, is that it requires the two vaccine components to form a complex in solution. To facilitate vaccine development, we engineered chimeric antigens by strategically replacing the AMA1 DII loop that is displaced upon ligand binding with RON2L. Structural characterization of the fusion chimera, Fusion-F D12 to 1.55 Å resolution showed that it closely mimics the binary receptor-ligand complex. Immunization studies showed that Fusion-F D12 immune sera neutralized parasites more efficiently than apoAMA1 immune sera despite having an overall lower anti-AMA1 titer, suggesting improvement in antibody quality. Furthermore, immunization with Fusion-F D12 enhanced antibodies targeting conserved epitopes on AMA1 resulting in greater neutralization of non-vaccine type parasites. Identifying epitopes of such cross-neutralizing antibodies will help in the development of an effective, strain-transcending malaria vaccine. Our fusion protein design is a robust vaccine platform that can be enhanced by incorporating polymorphisms in AMA1 to effectively neutralize all P. falciparum parasites.

Characterization of AMA1-RON2L complex with native gel electrophoresis and capillary isoelectric focusing.

Rhoptry neck protein 2 (RON2) binds to the hydrophobic groove of apical membrane antigen 1 (AMA1), an interaction essential for invasion of red blood cells (RBCs) by Plasmodium falciparum (Pf) parasites. Vaccination with AMA1 alone has been shown to be immunogenic, but unprotective even against homologous challenge in human trials. However, the AMA1-RON2L (L is referred to as the loop region of RON2 peptide) complex is a promising candidate, as preclinical studies with Freund's adjuvant have indicated complete protection against lethal challenge in mice and superior protection against virulent infection in Aotus monkeys. To prepare for clinical trials of the AMA1-RON2L complex, identity and integrity of the candidate vaccine must be assessed, and characterization methods must be carefully designed to not dissociate the delicate complex during evaluation. In this study, we developed a native Tris-glycine gel method to separate and identify the AMA1-RON2L complex, which was further identified and confirmed by Western blotting using anti-AMA1 monoclonal antibodies (mAbs 4G2 and 2C2) and anti-RON2L polyclonal Ab coupled with mass spectrometry. The formation of complex was also confirmed by Capillary Isoelectric Focusing (cIEF). A short-term (48 h and 72 h at 4°C) stability study of AMA1-RON2L complex was also performed. The results indicate that the complex was stable for 72 h at 4°C. Our research demonstrates that the native Tris-glycine gel separation/Western blotting coupled with mass spectrometry and cIEF can fully characterize the identity and integrity of the AMA1-RON2L complex and provide useful quality control data for the subsequent clinical trials.

Malaria parasites use a soluble RhopH complex for erythrocyte invasion and an integral form for nutrient uptake.

Malaria parasites use the RhopH complex for erythrocyte invasion and channel-mediated nutrient uptake. As the member proteins are unique to Plasmodium spp., how they interact and traffic through subcellular sites to serve these essential functions is unknown. We show that RhopH is synthesized as a soluble complex of CLAG3, RhopH2, and RhopH3 with 1:1:1 stoichiometry. After transfer to a new host cell, the complex crosses a vacuolar membrane surrounding the intracellular parasite and becomes integral to the erythrocyte membrane through a PTEX translocon-dependent process. We present a 2.9 Å single-particle cryo-electron microscopy structure of the trafficking complex, revealing that CLAG3 interacts with the other subunits over large surface areas. This soluble complex is tightly assembled with extensive disulfide bonding and predicted transmembrane helices shielded. We propose a large protein complex stabilized for trafficking but poised for host membrane insertion through large-scale rearrangements, paralleling smaller two-state pore-forming proteins in other organisms.

Malaria is an infectious disease caused by the family of Plasmodium parasites, which pass between mosquitoes and animals to complete their life cycle. With one bite, mosquitoes can deposit up to one hundred malaria parasites into the human skin, from where they enter the bloodstream. After increasing their numbers in liver cells, the parasites hijack, invade and remodel red blood cells to create a safe space to grow and mature. This includes inserting holes in the membrane of red blood cells to take up nutrients from the bloodstream. A complex of three tightly bound RhopH proteins plays an important role in these processes. These proteins are unique to malaria parasites, and so far, it has been unclear how they collaborate to perform these specialist roles. Here, Schureck et al. have purified the RhopH complex from Plasmodium-infected human blood to determine its structure and reveal how it moves within an infected red blood cell. Using cryo-electron microscopy to visualise the assembly in fine detail, Schureck et al. showed that the three proteins bind tightly to each other over large areas using multiple anchor points. As the three proteins are produced, they assemble into a complex that remains dissolved and free of parasite membranes until the proteins have been delivered to their target red blood cells. Some hours after delivery, specific sections of the RhopH complex are inserted into the red blood cell membrane to produce pores that allow them to take up nutrients and to grow. The study of Schureck et al. provides important new insights into how the RhopH complex serves multiple roles during Plasmodium infection of human red blood cells. The findings provide a framework for the development of effective antimalarial treatments that target RhopH proteins to block red blood cell invasion and nutrient uptake.

Multiple genetic loci define Ca++ utilization by bloodstream malaria parasites.

BACKGROUND: Bloodstream malaria parasites require Ca++ for their development, but the sites and mechanisms of Ca++ utilization are not well understood. We hypothesized that there may be differences in Ca++ uptake or utilization by genetically distinct lines of P. falciparum. These differences, if identified, may provide insights into molecular mechanisms. RESULTS: Dose response studies with the Ca++ chelator EGTA (ethylene glycol-bis(ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid) revealed stable differences in Ca++ requirement for six geographically divergent parasite lines used in previous genetic crosses, with the largest difference seen between the parents of the HB3 x Dd2 cross. Genetic mapping of Ca++ requirement yielded complex inheritance in 34 progeny clones with a single significant locus on chromosome 7 and possible contributions from other loci. Although encoded by a gene in the significant locus and a proposed Ca++ target, PfCRT (P. falciparum chloroquine resistance transporter), the primary determinant of clinical resistance to the antimalarial drug chloroquine, does not appear to contribute to this quantitative trait. Stage-specific application of extracellular EGTA also excluded determinants associated with merozoite egress and erythrocyte reinvasion. CONCLUSIONS: We have identified differences in Ca++ utilization amongst P. falciparum lines. These differences are under genetic regulation, segregating as a complex trait in genetic cross progeny. Ca++ uptake and utilization throughout the bloodstream asexual cycle of malaria parasites represents an unexplored target for therapeutic intervention.

Malaria parasite detection and cell counting for human and mouse using thin blood smear microscopy.

Despite the remarkable progress that has been made to reduce global malaria mortality by 29% in the past 5 years, malaria is still a serious global health problem. Inadequate diagnostics is one of the major obstacles in fighting the disease. An automated system for malaria diagnosis can help to make malaria screening faster and more reliable. We present an automated system to detect and segment red blood cells (RBCs) and identify infected cells in Wright-Giemsa stained thin blood smears. Specifically, using image analysis and machine learning techniques, we process digital images of thin blood smears to determine the parasitemia in each smear. We use a cell extraction method to segment RBCs, in particular overlapping cells. We show that a combination of RGB color and texture features outperforms other features. We evaluate our method on microscopic blood smear images from human and mouse and show that it outperforms other techniques. For human cells, we measure an absolute error of 1.18% between the true and the automatic parasite counts. For mouse cells, our automatic counts correlate well with expert and flow cytometry counts. This makes our system the first one to work for both human and mouse.

Profiling invasive Plasmodium falciparum merozoites using an integrated omics approach.

The symptoms of malaria are brought about by blood-stage parasites, which are established when merozoites invade human erythrocytes. Our understanding of the molecular events that underpin erythrocyte invasion remains hampered by the short-period of time that merozoites are invasive. To address this challenge, a Plasmodium falciparum gamma-irradiated long-lived merozoite (LLM) line was developed and investigated. Purified LLMs invaded erythrocytes by an increase of 10-300 fold compared to wild-type (WT) merozoites. Using an integrated omics approach, we investigated the basis for the phenotypic difference. Only a few single nucleotide polymorphisms within the P. falciparum genome were identified and only marginal differences were observed in the merozoite transcriptomes. By contrast, using label-free quantitative mass-spectrometry, a significant change in protein abundance was noted, of which 200 were proteins of unknown function. We determined the relative molar abundance of over 1100 proteins in LLMs and further characterized the major merozoite surface protein complex. A unique processed MSP1 intermediate was identified in LLM but not observed in WT suggesting that delayed processing may be important for the observed phenotype. This integrated approach has demonstrated the significant role of the merozoite proteome during erythrocyte invasion, while identifying numerous unknown proteins likely to be involved in invasion.

A malaria vaccine protects Aotus monkeys against virulent Plasmodium falciparum infection.

The Plasmodium falciparum protein, apical membrane antigen 1 forms a complex with another parasite protein, rhoptry neck protein 2, to initiate junction formation with the erythrocyte and is essential for merozoite invasion during the blood stage of infection. Consequently, apical membrane antigen 1 has been a target of vaccine development but vaccination with apical membrane antigen 1 alone in controlled human malaria infections failed to protect and showed limited efficacy in field trials. Here we show that vaccination with AMA1-RON2L complex in Freund's adjuvant protects Aotus monkeys against a virulent Plasmodium falciparum infection. Vaccination with AMA1 alone gave only partial protection, delaying infection in one of eight animals. However, the AMA1-RON2L complex vaccine completely protected four of eight monkeys and substantially delayed infection (>25 days) in three of the other four animals. Interestingly, antibodies from monkeys vaccinated with the AMA1-RON2L complex had significantly higher neutralizing activity than antibodies from monkeys vaccinated with AMA1 alone. Importantly, we show that antibodies from animals vaccinated with the complex have significantly higher neutralization activity against non-vaccine type parasites. We suggest that vaccination with the AMA1-RON2L complex induces functional antibodies that better recognize AMA1 as it appears complexed with RON2 during merozoite invasion. These data justify progression of this next generation AMA1 vaccine towards human trials.

Recent advances in recombinant protein-based malaria vaccines.

Plasmodium parasites are the causative agent of human malaria, and the development of a highly effective vaccine against infection, disease and transmission remains a key priority. It is widely established that multiple stages of the parasite's complex lifecycle within the human host and mosquito vector are susceptible to vaccine-induced antibodies. The mainstay approach to antibody induction by subunit vaccination has been the delivery of protein antigen formulated in adjuvant. Extensive efforts have been made in this endeavor with respect to malaria vaccine development, especially with regard to target antigen discovery, protein expression platforms, adjuvant testing, and development of soluble and virus-like particle (VLP) delivery platforms. The breadth of approaches to protein-based vaccines is continuing to expand as innovative new concepts in next-generation subunit design are explored, with the prospects for the development of a highly effective multi-component/multi-stage/multi-antigen formulation seeming ever more likely. This review will focus on recent progress in protein vaccine design, development and/or clinical testing for a number of leading malaria antigens from the sporozoite-, merozoite- and sexual-stages of the parasite's lifecycle-including PfCelTOS, PfMSP1, PfAMA1, PfRH5, PfSERA5, PfGLURP, PfMSP3, Pfs48/45 and Pfs25. Future prospects and challenges for the development, production, human delivery and assessment of protein-based malaria vaccines are discussed.

CD68 acts as a major gateway for malaria sporozoite liver infection.

After being delivered by the bite from an infected mosquito, Plasmodium sporozoites enter the blood circulation and infect the liver. Previous evidence suggests that Kupffer cells, a macrophage-like component of the liver blood vessel lining, are traversed by sporozoites to initiate liver invasion. However, the molecular determinants of sporozoite-Kupffer cell interactions are unknown. Understanding the molecular basis for this specific recognition may lead to novel therapeutic strategies to control malaria. Using a phage display library screen, we identified a peptide, P39, that strongly binds to the Kupffer cell surface and, importantly, inhibits sporozoite Kupffer cell entry. Furthermore, we determined that P39 binds to CD68, a putative receptor for sporozoite invasion of Kupffer cells that acts as a gateway for malaria infection of the liver.

Reversible Conformational Change in the Plasmodium falciparum Circumsporozoite Protein Masks Its Adhesion Domains.

The extended rod-like Plasmodium falciparum circumsporozoite protein (CSP) is comprised of three primary domains: a charged N terminus that binds heparan sulfate proteoglycans, a central NANP repeat domain, and a C terminus containing a thrombospondin-like type I repeat (TSR) domain. Only the last two domains are incorporated in RTS,S, the leading malaria vaccine in phase 3 trials that, to date, protects about 50% of vaccinated children against clinical disease. A seroepidemiological study indicated that the N-terminal domain might improve the efficacy of a new CSP vaccine. Using a panel of CSP-specific monoclonal antibodies, well-characterized recombinant CSPs, label-free quantitative proteomics, and in vitro inhibition of sporozoite invasion, we show that native CSP is N-terminally processed in the mosquito host and undergoes a reversible conformational change to mask some epitopes in the N- and C-terminal domains until the sporozoite interacts with the liver hepatocyte. Our findings show the importance of understanding processing and the biophysical change in conformation, possibly due to a mechanical or molecular signal, and may aid in the development of a new CSP vaccine.

A feasibility study of agricultural and sewage biomass as biochar, bioenergy and biocomposite feedstock: production, characterization and potential applications.

In this study, we pyrolysed six waste derived biomass: pine sawdust (PSD), paunch grass (PG), broiler litter (BL), sewage sludge (SS), dewatered pond sludge (DWP), and dissolved air-floatation sludge (DAF) into biochar. Biochars were characterized using scanning electron microscopy, energy dispersive X-ray spectrometry, X-ray diffraction, Fourier transform infrared spectroscopy, inductively-coupled plasma mass spectrometry, (13)C-solid-state nuclear magnetic resonance spectroscopy, and X-ray photoelectron spectroscopy to evaluate their feasibility for potential agronomic and environmental applications. Syngas produced during the pyrolysis process was also analyzed to determine the energy values. Results show that PSD biochar has the utmost potential for carbon sequestration and contaminant remediation due to its high surface area, aromaticity and carbon content. Additionally given its low ash content, PSD biochar could also potentially be used as filler in wood plastic biocomposites. Low levels of heavy metals (Cr, Cu, Zn, As, Cd, Hg, and Pb) in all biochars suggest that biochars are also applicable for land application according to the United States Environmental Protection Agency regulation 40 CFR part 503. The composition of syngas evolved during the pyrolysis of feedstocks showed little difference in the calorific values, ranging from 12-16 MJ/dsm with PSD having the maximum calorific value of 16 MJ/dsm.

Characterisation of agricultural waste-derived biochars and their sorption potential for sulfamethoxazole in pasture soil: a spectroscopic investigation.

We investigated the effects of feedstock type and pyrolysis temperatures on the sorptive potential of a model pastoral soil amended with biochars for sulfamethoxazole (SMO), using laboratory batch sorption studies. The results indicated that high temperature chars exhibited enhanced adsorptive potential, compared to low temperature chars. Pine sawdust (PSD) biochar produced at 700°C using the steam gasification process exhibited the highest sorptive capacity (2-fold greater than the control treatment) for SMO among the three biochars used. Soils amended with green waste (GW) biochars produced at three different pyrolysis temperatures showed a small increase in SMO sorption with the increases in temperature. The NMR spectra, the elemental molar ratios (H/C, O/C) and polarity index (O+N)/C of the biochars revealed that PSD biochar possessed the highest degree of aromatic condensation compared to CC and GW chars. These results correlated well with the sorption affinity of each biochar, with effective distribution coefficient (Kd(eff)) being highest for PSD and lowest for GW biochars. X-ray photoelectron spectroscopy results for the biochars showed a relatively large difference in oxygen containing surface functional groups amongst the GW biochars. However, they exhibited nearly identical sorption affinity to SMO, indicating negligible role of oxygen containing surface functional groups on SMO sorption. These observations provide important information on the use of biochars as engineered sorbents for environmental applications, such as reducing the bioavailability of antibiotics and/or predicting the fate of sulfonamides in biochar-amended soils.

Immunization with a functional protein complex required for erythrocyte invasion protects against lethal malaria.

An essential step in the invasion of red blood cells (RBCs) by Plasmodium falciparum (Pf) merozoites is the binding of rhoptry neck protein 2 (RON2) to the hydrophobic groove of apical membrane antigen 1 (AMA1), triggering junction formation between the apical end of the merozoite and the RBC surface to initiate invasion. Vaccination with AMA1 provided protection against homologous parasites in one of two phase 2 clinical trials; however, despite its ability to induce high-titer invasion-blocking antibodies in a controlled human challenge trial, the vaccine conferred little protection even against the homologous parasite. Here we provide evidence that immunization with an AMA1-RON2 peptide complex, but not with AMA1 alone, provided complete protection against a lethal Plasmodium yoelii challenge in mice. Significantly, IgG from mice immunized with the complex transferred protection. Furthermore, IgG from PfAMA1-RON2-immunized animals showed enhanced invasion inhibition compared with IgG elicited by AMA1 alone. Interestingly, this qualitative increase in inhibitory activity appears to be related, at least in part, to a switch in the proportion of IgG specific for certain loop regions in AMA1 surrounding the binding site of RON2. Antibodies induced by the complex were not sufficient to block the FVO strain heterologous parasite, however, reinforcing the need to include multiallele AMA1 to cover polymorphisms. Our results suggest that AMA1 subunit vaccines may be highly effective when presented to the immune system as an invasion complex with RON2.

Assessing the sorption and leaching behaviour of three sulfonamides in pasture soils through batch and column studies.

We investigated the sorption potential and transport behaviour of three sulfonamides, namely, sulfamethoxazole (SMO), sulfachloropyridazine (SCP) and sulfamethazine (SM), and a conservative bromide tracer (Br(-)) in two undisturbed soil columns collected from the dairy farming regions in the North Island of New Zealand. Based on the low log Koc values obtained from the sorption study, all three sulfonamides are likely to have high mobility, making them a potential threat to surface and ground water. Soil column studies also showed that the mobility of the sulfonamides varied among soils and antibiotic type. Sulfonamides exhibited a mobility pattern similar to that of conservative Br(-) tracer. Considerable retardation was observed for the Hamilton soil, and the delayed peak arrival time (or maxima) was due to the role of sorption-related retention processes under saturated flow conditions. Residual antibiotic concentrations for SMO and SCP were detected in all soil sections including at 18 cm depth, while no resident concentration of SM was detected at any depth in the entire length of the core for both soils. The deterministic, physical equilibrium model (CXTFIT) described the peak arrival time as well as the maximum concentration of the antibiotic breakthrough curves reasonably, but showed some underestimation at the advanced stages of the leaching process. There was a significant difference in the model estimated retardation factors obtained from column study and the experimental retardation factors obtained from the conventional batch sorption experiments.

Deriving sulfamethoxazole dissipation endpoints in pasture soils using first order and biphasic kinetic models.

Single first-order (SFO) kinetic model is often used to derive the dissipation endpoints of an organic chemical in soil. This model is used due to its simplicity and requirement by regulatory agencies. However, using the SFO model for all types of decay pattern could lead to under- or overestimation of dissipation endpoints when the deviation from first-order is significant. In this study the performance of three biphasic kinetic models - bi-exponential decay (BEXP), first-order double exponential decay (FODED), and first-order two-compartment (FOTC) models was evaluated using dissipation datasets of sulfamethoxazole (SMO) antibiotic in three different soils under varying concentration, depth, temperature, and sterile conditions. Corresponding 50% (DT50) and 90% (DT90) dissipation times for the antibiotics were numerically obtained and compared against those obtained using the SFO model. The fit of each model to the measured values was evaluated based on an array of statistical measures such as coefficient of determination (R(2)adj), root mean square error (RMSE), chi-square (χ(2)) test at 1% significance, Bayesian Information Criteria (BIC) and % model error. Box-whisker residual plots were also used to compare the performance of each model to the measured datasets. The antibiotic dissipation was successfully predicted by all four models. However, the nonlinear biphasic models improved the goodness-of-fit parameters for all datasets. Deviations from datasets were also often less evident with the biphasic models. The fits of FOTC and FODED models for SMO dissipation datasets were identical in most cases, and were found to be superior to the BEXP model. Among the biphasic models, the FOTC model was found to be the most suitable for obtaining the endpoints and could provide a mechanistic explanation for SMO dissipation in the soils.

Dissipation of sulfamethoxazole in pasture soils as affected by soil and environmental factors.

The dissipation of sulfamethoxazole (SMO) antibiotic in three different soils was investigated through laboratory incubation studies. The experiments were conducted under different incubation conditions such as initial chemical concentration, soil depth, temperature, and with sterilisation. The results indicate that SMO dissipated rapidly in New Zealand pasture soils, and the 50% dissipation times (DT50) in Hamilton, Te Kowhai and Horotiu soils under non-sterile conditions were 9.24, 4.3 and 13.33 days respectively. During the incubation period for each sampling event the soil dehydrogenase activity (DHA) and the variation in microbial community were monitored thorough phospholipid fatty acid extraction analysis (PLFA). The DHA data correlated well with the dissipation rate constants of SMO antibiotic, an increase in the DHA activity resulted in faster antibiotic dissipation. The PLFA analysis was indicative of higher bacterial presence as compared to fungal community, highlighting the type of microbial community responsible for dissipation. The results indicate that with increasing soil depth, SMO dissipation in soil was slower (except for Horotiu) while with increase in temperature the antibiotic loss was faster, and was noticeable in all the soils. Both the degree of biological activity and the temperature of the soil influenced overall SMO dissipation. SMO is not likely to persist more than 5-6 months in all three soils suggesting that natural biodegradation may be sufficient for the removal of these contaminants from the soil. Its dissipation in sterile soils indicated abiotic factors such as strong sorption onto soil components to play a role in the dissipation of SMO.

Sorption of selected veterinary antibiotics onto dairy farming soils of contrasting nature.

The sorption potential for three sulfonamides (SAs), sulfamethoxazole (SMO), sulfachloropyridazine (SCP) and sulfamethazine (SM) and a macrolide, tylosin tartrate (TT) was assessed on six New Zealand dairy farming soils of contrasting physico-chemical properties. Kinetics studies showed that the sorption was rapid in the first few hours of the contact time (0-2h for SA and 0-4h for TT) and thereafter apparent equilibrium was achieved. Batch sorption isotherm data revealed that the degree of isotherm linearity (N) for SCP and SM varied between 0.50 and 1.08 in the six soils. Isotherms of both TT and SMO were mostly non-linear with the degree of non-linearity for TT (N=0.38-0.71) being greater than for SMO (0.42-0.75) in all soils except Manawatu (TT) and Te Kowhai (SMO) where a linear pattern was observed. Concentration-dependent effective distribution coefficient (Kd(eff)) values for the SMO, SCP and SM antibiotics in the soils ranged from 0.85 to 16.35 L kg(-1), while that for TT was 1.6 to 1,042 L kg(-1). The sorption affinity for all soils followed an order: TT>SCP>SM>SMO. Remarkable high sorption for tylosin in Matawhero soil as compared to other soils was attributed to the presence of oxygen containing acidic polar functional groups as evident in the FT-IR spectra of the soil. Furthermore, it was hypothesised that sorption of TT onto soils was mostly driven by metal oxide-surface mediated transformations whereas for sulfonamides it was primarily due to hydrophobic interactions.

Disrupting malaria parasite AMA1-RON2 interaction with a small molecule prevents erythrocyte invasion.

Plasmodium falciparum resistance to artemisinin derivatives, the first-line antimalarial drug, drives the search for new classes of chemotherapeutic agents. Current discovery is primarily directed against the intracellular forms of the parasite. However, late schizont-infected red blood cells (RBCs) may still rupture and cause disease by sequestration; consequently targeting invasion may reduce disease severity. Merozoite invasion of RBCs requires interaction between two parasite proteins AMA1 and RON2. Here we identify the first inhibitor of this interaction that also blocks merozoite invasion in genetically distinct parasites by screening a library of over 21,000 compounds. We demonstrate that this inhibition is mediated by the small molecule binding to AMA1 and blocking the formation of AMA1-RON complex. Electron microscopy confirms that the inhibitor prevents junction formation, a critical step in invasion that results from AMA1-RON2 binding. This study uncovers a strategy that will allow for highly effective combination therapies alongside existing antimalarial drugs.