The influence of oviposition status on measures of transmission potential in malaria-infected mosquitoes depends on sugar availability.

BACKGROUND: Like other oviparous organisms, the gonotrophic cycle of mosquitoes is not complete until they have selected a suitable habitat to oviposit. In addition to the evolutionary constraints associated with selective oviposition behavior, the physiological demands relative to an organism's oviposition status also influence their nutrient requirement from the environment. Yet, studies that measure transmission potential (vectorial capacity or competence) of mosquito-borne parasites rarely consider whether the rates of parasite replication and development could be influenced by these constraints resulting from whether mosquitoes have completed their gonotrophic cycle. METHODS: Anopheles stephensi mosquitoes were infected with Plasmodium berghei, the rodent analog of human malaria, and maintained on 1% or 10% dextrose and either provided oviposition sites ('oviposited' herein) to complete their gonotrophic cycle or forced to retain eggs ('non-oviposited'). Transmission potential in the four groups was measured up to 27 days post-infection as the rates of (i) sporozoite appearance in the salivary glands ('extrinsic incubation period' or EIP), (ii) vector survival and (iii) sporozoite densities. RESULTS: In the two groups of oviposited mosquitoes, rates of sporozoite appearance and densities in the salivary glands were clearly dependent on sugar availability, with shorter EIP and higher sporozoite densities in mosquitoes fed 10% dextrose. In contrast, rates of appearance and densities in the salivary glands were independent of sugar concentrations in non-oviposited mosquitoes, although both measures were slightly lower than in oviposited mosquitoes fed 10% dextrose. Vector survival was higher in non-oviposited mosquitoes. CONCLUSIONS: Costs to parasite fitness and vector survival were buffered against changes in nutritional availability from the environment in non-oviposited but not oviposited mosquitoes. Taken together, these results suggest vectorial capacity for malaria parasites may be dependent on nutrient availability and oviposition/gonotrophic status and, as such, argue for more careful consideration of this interaction when estimating transmission potential. More broadly, the complex patterns resulting from physiological (nutrition) and evolutionary (egg-retention) trade-offs described here, combined with the ubiquity of selective oviposition behavior, implies the fitness of vector-borne pathogens could be shaped by selection for these traits, with implications for disease transmission and management. For instance, while reducing availability of oviposition sites and environmental sources of nutrition are key components of integrated vector management strategies, their abundance and distribution are under strong selection pressure from the patterns associated with climate change.

Extended blood stage sensitivity profiles of Plasmodium cynomolgi to doxycycline and tafenoquine, as a model for Plasmodium vivax.

Testing Plasmodium vivax antimicrobial sensitivity is limited to ex vivo schizont maturation assays, which preclude determining the IC50s of delayed action antimalarials such as doxycycline. Using Plasmodium cynomolgi as a model for P. vivax, we determined the physiologically significant delayed death effect induced by doxycycline [IC50(96 h), 1,401 ± 607 nM]. As expected, IC50(96 h) to chloroquine (20.4 nM), piperaquine (12.6 µM), and tafenoquine (1,424 nM) were not affected by extended exposure.

In Vitro Antimalarial Activity of Trichothecenes against Liver and Blood Stages of Plasmodium Species.

Trichothecenes (TCNs) are a large group of tricyclic sesquiterpenoid mycotoxins that have intriguing structural features and remarkable biological activities. Herein, we focused on three TCNs (anguidine, verrucarin A, and verrucarol) and their ability to target both the blood and liver stages of Plasmodium species, the parasite responsible for malaria. Anguidine and verrucarin A were found to be highly effective against the blood and liver stages of malaria, while verrucarol had no effect at the highest concentration tested. However, these compounds were also found to be cytotoxic and, thus, not selective, making them unsuitable for drug development. Nonetheless, they could be useful as chemical probes for protein synthesis inhibitors due to their direct impact on parasite synthesis processes.

Corrigendum: Identification of novel anti-amoebic pharmacophores from kinase inhibitor chemotypes.

[This corrects the article DOI: 10.3389/fmicb.2023.1149145.].

Blood meals from 'dead-end' vertebrate hosts enhance transmission potential of malaria-infected mosquitoes.

Ingestion of an additional blood meal(s) by a hematophagic insect can accelerate development of several vector-borne parasites and pathogens. Most studies, however, offer blood from the same vertebrate host species as the original challenge (for e.g., human for primary and additional blood meals). Here, we show a second blood meal from bovine and canine hosts can also enhance sporozoite migration in Anopheles stephensi mosquitoes infected with the human- and rodent-restricted Plasmodium falciparum and P. berghei, respectively. The extrinsic incubation period (time to sporozoite appearance in salivary glands) showed more consistent reductions with blood from human and bovine donors than canine blood, although the latter's effect may be confounded by the toxicity, albeit non-specific, associated with the anticoagulant used to collect whole blood from donors. The complex patterns of enhancement highlight the limitations of a laboratory system but are nonetheless reminiscent of parasite host-specificity and mosquito adaptations, and the genetic predisposition of An. stephensi for bovine blood. We suggest that in natural settings, a blood meal from any vertebrate host could accentuate the risk of human infections by P. falciparum: targeting vectors that also feed on animals, via endectocides for instance, may reduce the number of malaria-infected mosquitoes and thus directly lower residual transmission. Since endectocides also benefit animal health, our results underscore the utility of the One Health framework, which postulates that human health and well-being is interconnected with that of animals. We posit this framework will be further validated if our observations also apply to other vector-borne diseases which together are responsible for some of the highest rates of morbidity and mortality in socio-economically disadvantaged populations.

Shotgun Kinetic Target-Guided Synthesis Approach Enables the Discovery of Small-Molecule Inhibitors against Pathogenic Free-Living Amoeba Glucokinases.

Pathogenic free-living amoebae (pFLA) can cause life-threatening central nervous system (CNS) infections and warrant the investigation of new chemical agents to combat the rise of infection from these pathogens. Naegleria fowleri glucokinase (NfGlck), a key metabolic enzyme involved in generating glucose-6-phosphate, was previously identified as a potential target due to its limited sequence similarity with human Glck (HsGlck). Herein, we used our previously demonstrated multifragment kinetic target-guided synthesis (KTGS) screening strategy to identify inhibitors against pFLA glucokinases. Unlike the majority of previous KTGS reports, our current study implements a "shotgun" approach, where fragments were not biased by predetermined binding potentials. The study resulted in the identification of 12 inhibitors against 3 pFLA glucokinase enzymes─NfGlck, Balamuthia mandrillaris Glck (BmGlck), and Acanthamoeba castellanii Glck (AcGlck). This work demonstrates the utility of KTGS to identify small-molecule binders for biological targets where resolved X-ray crystal structures are not readily accessible.

Characterization of the extracellular vesicles, ultrastructural morphology, and intercellular interactions of multiple clinical isolates of the brain-eating amoeba, Naegleria fowleri.

Introduction: As global temperatures rise to unprecedented historic levels, so too do the latitudes of habitable niches for the pathogenic free-living amoeba, Naegleria fowleri. This opportunistic parasite causes a rare, but >97% fatal, neurological infection called primary amoebic meningoencephalitis. Despite its lethality, this parasite remains one of the most neglected and understudied parasitic protozoans. Methods: To better understand amoeboid intercellular communication, we elucidate the structure, proteome, and potential secretion mechanisms of amoeba-derived extracellular vesicles (EVs), which are membrane-bound communication apparatuses that relay messages and can be used as biomarkers for diagnostics in various diseases. Results and Discussion: Herein we propose that N. fowleri secretes EVs in clusters from the plasma membrane, from multivesicular bodies, and via beading of thin filaments extruding from the membrane. Uptake assays demonstrate that EVs are taken up by other amoebae and mammalian cells, and we observed a real-time increase in metabolic activity for mammalian cells exposed to EVs from amoebae. Proteomic analysis revealed >2,000 proteins within the N. fowleri-secreted EVs, providing targets for the development of diagnostics or therapeutics. Our work expands the knowledge of intercellular interactions among these amoebae and subsequently deepens the understanding of the mechanistic basis of PAM.

Sheptide A: an antimalarial cyclic pentapeptide from a fungal strain in the Herpotrichiellaceae.

As part of ongoing efforts to isolate biologically active fungal metabolites, a cyclic pentapeptide, sheptide A (1), was discovered from strain MSX53339 (Herpotrichiellaceae). The structure and sequence of 1 were determined primarily by analysis of 2D NMR and HRMS/MS data, while the absolute configuration was assigned using a modified version of Marfey's method. In an in vitro assay for antimalarial potency, 1 displayed a pEC50 value of 5.75 ± 0.49 against malaria-causing Plasmodium falciparum. Compound 1 was also tested in a counter screen for general cytotoxicity against human hepatocellular carcinoma (HepG2), yielding a pCC50 value of 5.01 ± 0.45 and indicating a selectivity factor of ~6. This makes 1 the third known cyclic pentapeptide biosynthesized by fungi with antimalarial activity.

Identification of novel anti-amoebic pharmacophores from kinase inhibitor chemotypes.

Acanthamoeba species, Naegleria fowleri, and Balamuthia mandrillaris are opportunistic pathogens that cause a range of brain, skin, eye, and disseminated diseases in humans and animals. These pathogenic free-living amoebae (pFLA) are commonly misdiagnosed and have sub-optimal treatment regimens which contribute to the extremely high mortality rates (>90%) when they infect the central nervous system. To address the unmet medical need for effective therapeutics, we screened kinase inhibitor chemotypes against three pFLA using phenotypic drug assays involving CellTiter-Glo 2.0. Herein, we report the activity of the compounds against the trophozoite stage of each of the three amoebae, ranging from nanomolar to low micromolar potency. The most potent compounds that were identified from this screening effort were: 2d (A. castellanii EC50: 0.92 ± 0.3 µM; and N. fowleri EC50: 0.43 ± 0.13 µM), 1c and 2b (N. fowleri EC50s: <0.63 µM, and 0.3 ± 0.21 µM), and 4b and 7b (B. mandrillaris EC50s: 1.0 ± 0.12 µM, and 1.4 ± 0.17 µM, respectively). With several of these pharmacophores already possessing blood-brain barrier (BBB) permeability properties, or are predicted to penetrate the BBB, these hits present novel starting points for optimization as future treatments for pFLA-caused diseases.

AIM2 sensors mediate immunity to Plasmodium infection in hepatocytes.

Malaria, caused by Plasmodium parasites is a severe disease affecting millions of people around the world. Plasmodium undergoes obligatory development and replication in the hepatocytes, before initiating the life-threatening blood-stage of malaria. Although the natural immune responses impeding Plasmodium infection and development in the liver are key to controlling clinical malaria and transmission, those remain relatively unknown. Here we demonstrate that the DNA of Plasmodium parasites is sensed by cytosolic AIM2 (absent in melanoma 2) receptors in the infected hepatocytes, resulting in Caspase-1 activation. Remarkably, Caspase-1 was observed to undergo unconventional proteolytic processing in hepatocytes, resulting in the activation of the membrane pore-forming protein, Gasdermin D, but not inflammasome-associated proinflammatory cytokines. Nevertheless, this resulted in the elimination of Plasmodium-infected hepatocytes and the control of malaria infection in the liver. Our study uncovers a pathway of natural immunity critical for the control of malaria in the liver.

Integrative Genetic Manipulation of Plasmodium cynomolgi Reveals Multidrug Resistance-1 Y976F Associated With Increased In Vitro Susceptibility to Mefloquine.

The lack of a long-term in vitro culture method has severely restricted the study of Plasmodium vivax, in part because it limits genetic manipulation and reverse genetics. We used the recently optimized Plasmodium cynomolgi Berok in vitro culture model to investigate the putative P. vivax drug resistance marker MDR1 Y976F. Introduction of this mutation using clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9 (CRISPR-Cas9) increased sensitivity to mefloquine, but had no significant effect on sensitivity to chloroquine, amodiaquine, piperaquine, and artesunate. To our knowledge, this is the first reported use of CRISPR-Cas9 in P. cynomolgi, and the first reported integrative genetic manipulation of this species.

Cryopreservation of Plasmodium Sporozoites.

Malaria is a deadly disease caused by the parasite, Plasmodium, and impacts the lives of millions of people around the world. Following inoculation into mammalian hosts by infected mosquitoes, the sporozoite stage of Plasmodium undergoes obligate development in the liver before infecting erythrocytes and causing clinical malaria. The most promising vaccine candidates for malaria rely on the use of attenuated live sporozoites to induce protective immune responses. The scope of widespread testing or clinical use of such vaccines is limited by the absence of efficient, reliable, or transparent strategies for the long-term preservation of live sporozoites. Here we outline a method to cryopreserve the sporozoites of various human and murine Plasmodium species. We found that the structural integrity, viability, and in vivo or in vitro infectiousness were conserved in the recovered cryopreserved sporozoites. Cryopreservation using our approach also retained the transgenic properties of sporozoites and immunization with cryopreserved radiation attenuated sporozoites (RAS) elicited strong immune responses. Our work offers a reliable protocol for the long-term storage and recovery of human and murine Plasmodium sporozoites and lays the groundwork for the widespread use of live sporozoites for research and clinical applications.

Liver-stage fate determination in Plasmodium vivax parasites: Characterization of schizont growth and hypnozoite fating from patient isolates.

Plasmodium vivax, one species of parasite causing human malaria, forms a dormant liver stage, termed the hypnozoite, which activate weeks, months or years after the primary infection, causing relapse episodes. Relapses significantly contribute to the vivax malaria burden and are only killed with drugs of the 8-aminoquinoline class, which are contraindicated in many vulnerable populations. Development of new therapies targeting hypnozoites is hindered, in part, by the lack of robust methods to continuously culture and characterize this parasite. As a result, the determinants of relapse periodicity and the molecular processes that drive hypnozoite formation, persistence, and activation are largely unknown. While previous reports have described vastly different liver-stage growth metrics attributable to which hepatocyte donor lot is used to initiate culture, a comprehensive assessment of how different P. vivax patient isolates behave in the same lots at the same time is logistically challenging. Using our primary human hepatocyte-based P. vivax liver-stage culture platform, we aimed to simultaneously test the effects of how hepatocyte donor lot and P. vivax patient isolate influence the fate of sporozoites and growth of liver schizonts. We found that, while environmental factors such as hepatocyte donor lot can modulate hypnozoite formation rate, the P. vivax case is also an important determinant of the proportion of hypnozoites observed in culture. In addition, we found schizont growth to be mostly influenced by hepatocyte donor lot. These results suggest that, while host hepatocytes harbor characteristics making them more- or less-supportive of a quiescent versus growing intracellular parasite, sporozoite fating toward hypnozoites is isolate-specific. Future studies involving these host-parasite interactions, including characterization of individual P. vivax strains, should consider the impact of culture conditions on hypnozoite formation, in order to better understand this important part of the parasite's lifecycle.

Streamlining sporozoite isolation from mosquitoes by leveraging the dynamics of migration to the salivary glands.

BACKGROUND: Sporozoites isolated from the salivary glands of Plasmodium-infected mosquitoes are a prerequisite for several basic and pre-clinical applications. Although salivary glands are pooled to maximize sporozoite recovery, insufficient yields pose logistical and analytical hurdles; thus, predicting yields prior to isolation would be valuable. Preceding oocyst densities in the midgut is an obvious candidate. However, it is unclear whether current understanding of its relationship with sporozoite densities can be used to maximize yields, or whether it can capture the potential density-dependence in rates of sporozoite invasion of the salivary glands. METHODS: This study presents a retrospective analysis of Anopheles stephensi mosquitoes infected with two strains of the rodent-specific Plasmodium berghei. Mean oocyst densities were estimated in the midguts earlier in the infection (11-15 days post-blood meal), with sporozoites pooled from the salivary glands later in the infection (17-29 days). Generalized linear mixed effects models were used to determine if (1) mean oocyst densities can predict sporozoite yields from pooled salivary glands, (2) whether these densities can capture differences in rates of sporozoite invasion of salivary glands, and (3), if the interaction between oocyst densities and time could be leveraged to boost overall yields. RESULTS: The non-linear effect of mean oocyst densities confirmed the role of density-dependent constraints in limiting yields beyond certain oocyst densities. Irrespective of oocyst densities however, the continued invasion of salivary glands by the sporozoites boosted recoveries over time (17-29 days post-blood meal) for either parasite strain. CONCLUSIONS: Sporozoite invasion of the salivary glands over time can be leveraged to maximize yields for P. berghei. In general, however, invasion of the salivary glands over time is a critical fitness determinant for all Plasmodium species (extrinsic incubation period, EIP). Thus, delaying sporozoite collection could, in principle, substantially reduce dissection effort for any parasite within the genus, with the results also alluding to the potential for changes in sporozoites densities over time to modify infectivity for the next host.

Single-cell RNA profiling of Plasmodium vivax-infected hepatocytes reveals parasite- and host- specific transcriptomic signatures and therapeutic targets.

The resilience of Plasmodium vivax, the most widely-distributed malaria-causing parasite in humans, is attributed to its ability to produce dormant liver forms known as hypnozoites, which can activate weeks, months, or even years after an initial mosquito bite. The factors underlying hypnozoite formation and activation are poorly understood, as is the parasite's influence on the host hepatocyte. Here, we shed light on transcriptome-wide signatures of both the parasite and the infected host cell by sequencing over 1,000 P. vivax-infected hepatocytes at single-cell resolution. We distinguish between replicating schizonts and hypnozoites at the transcriptional level, identifying key differences in transcripts encoding for RNA-binding proteins associated with cell fate. In infected hepatocytes, we show that genes associated with energy metabolism and antioxidant stress response are upregulated, and those involved in the host immune response downregulated, suggesting both schizonts and hypnozoites alter the host intracellular environment. The transcriptional markers in schizonts, hypnozoites, and infected hepatocytes revealed here pinpoint potential factors underlying dormancy and can inform therapeutic targets against P. vivax liver-stage infection.

Chemistry and Bioactivity of the Deep-Water Antarctic Octocoral Alcyonium sp.

Chemical investigation of an Antarctic deep-water octocoral has led to the isolation of four new compounds, including three illudalane sesquiterpenoids (1-3) related to the alcyopterosins, a highly oxidized steroid, alcyosterone (5), and five known alcyopterosins (4, 6-9). The structures were established by extensive 1D and 2D NMR analyses, while 9 was verified by XRD. Alcyopterosins are unusual for their nitrate ester functionalization and have been characterized with cytotoxicity related to their DNA binding properties. Alcyopterosins V (3) and E (4) demonstrated single-digit micromolar activity against Clostridium difficile, an intestinal bacterium capable of causing severe diarrhea that is increasingly associated with drug resistance. Alcyosterone (5) and several alcyopterosins were similarly potent against the protist Leishmania donovani, the causative agent of leishmaniasis, a disfiguring disease that can be fatal if not treated. While the alcyopterosin family of sesquiterpenes is known for mild cytotoxicity, the observed activity against C. difficile and L. donovani is selective for the infectious agents.

Direct type I interferon signaling in hepatocytes controls malaria.

Malaria is a devastating disease impacting over half of the world's population. Plasmodium parasites that cause malaria undergo obligatory development and replication in hepatocytes before infecting red blood cells and initiating clinical disease. While type I interferons (IFNs) are known to facilitate innate immune control to Plasmodium in the liver, how they do so has remained unresolved, precluding the manipulation of such responses to combat malaria. Utilizing transcriptomics, infection studies, and a transgenic Plasmodium strain that exports and traffics Cre recombinase, we show that direct type I IFN signaling in Plasmodium-infected hepatocytes is necessary to control malaria. We also show that the majority of infected hepatocytes naturally eliminate Plasmodium infection, revealing the potential existence of anti-malarial cell-autonomous immune responses in such hepatocytes. These discoveries challenge the existing paradigms in Plasmodium immunobiology and are expected to inspire anti-malarial drugs and vaccine strategies.

Improving in vitro continuous cultivation of Plasmodium cynomolgi, a model for P. vivax.

The absence of a routine continuous in vitro cultivation method for Plasmodium vivax, an important globally distributed parasite species causing malaria in humans, has restricted investigations to field and clinical sampling. Such a method has recently been developed for the Berok strain of P. cynomolgi, a parasite of macaques that has long been used as a model for P. vivax, as these two parasites are nearly indistinguishable biologically and are genetically closely related. The availability of the P. cynomolgi Berok in routine continuous culture provides for the first time an opportunity to conduct a plethora of functional studies. However, the initial cultivation protocol proved unsuited for investigations requiring extended cultivation times, such as reverse genetics and drug resistance. Here we have addressed some of the critical obstacles to this, and we propose a set of modifications that help overcome them.

Alkyne modified purines for assessment of activation of Plasmodium vivax hypnozoites and growth of pre-erythrocytic and erythrocytic stages in Plasmodium spp.

Malaria is a major global health problem which predominantly afflicts developing countries. Although many antimalarial therapies are currently available, the protozoan parasite causing this disease, Plasmodium spp., continues to evade eradication efforts. One biological phenomenon hampering eradication efforts is the parasite's ability to arrest development, transform into a drug-insensitive form, and then resume growth post-therapy. Currently, the mechanisms by which the parasite enters arrested development, or dormancy, and later recrudesces or reactivates to continue development, are unknown and the malaria field lacks techniques to study these elusive mechanisms. Since Plasmodium spp. salvage purines for DNA synthesis, we hypothesised that alkyne-containing purine nucleosides could be used to develop a DNA synthesis marker which could be used to investigate mechanisms behind dormancy. Using copper-catalysed click chemistry methods, we observe incorporation of alkyne modified adenosine, inosine, and hypoxanthine in actively replicating asexual blood stages of Plasmodium falciparum and incorporation of modified adenosine in actively replicating liver stage schizonts of Plasmodium vivax. Notably, these modified purines were not incorporated in dormant liver stage hypnozoites, suggesting this marker could be used as a tool to differentiate replicating and non-replicating liver forms and, more broadly, as a tool for advancing our understanding of Plasmodium dormancy mechanisms.

Metabolic, Pharmacokinetic, and Activity Profile of the Liver Stage Antimalarial (RC-12).

The catechol derivative RC-12 (WR 27653) (1) is one of the few non-8-aminoquinolines with good activity against hypnozoites in the gold-standard Plasmodium cynomolgi-rhesus monkey (Macaca mulatta) model, but in a small clinical trial, it had no efficacy against Plasmodium vivax hypnozoites. In an attempt to better understand the pharmacokinetic and pharmacodynamic profile of 1 and to identify potential active metabolites, we now describe the phase I metabolism, rat pharmacokinetics, and in vitro liver-stage activity of 1 and its metabolites. Compound 1 had a distinct metabolic profile in human vs monkey liver microsomes, and the data suggested that the O-desmethyl, combined O-desmethyl/N-desethyl, and N,N-didesethyl metabolites (or a combination thereof) could potentially account for the superior liver stage antimalarial efficacy of 1 in rhesus monkeys vs that seen in humans. Indeed, the rate of metabolism was considerably lower in human liver microsomes in comparison to rhesus monkey microsomes, as was the formation of the combined O-desmethyl/N-desethyl metabolite, which was the only metabolite tested that had any activity against liver-stage P. vivax; however, it was not consistently active against liver-stage P. cynomolgi. As 1 and all but one of its identified Phase I metabolites had no in vitro activity against P. vivax or P. cynomolgi liver-stage malaria parasites, we suggest that there may be additional unidentified active metabolites of 1 or that the exposure of 1 achieved in the reported unsuccessful clinical trial of this drug candidate was insufficient to kill the P. vivax hypnozoites.