Role of Pfs47 in the dispersal of ancestral Plasmodium falciparum malaria through adaptation to different anopheline vectors.

Plasmodium falciparum malaria originated when Plasmodium praefalciparum, a gorilla malaria parasite transmitted by African sylvan anopheline mosquitoes, adapted to humans. Pfs47, a protein on the parasite surface mediates P. falciparum evasion of the mosquito immune system by interacting with a midgut receptor and is critical for Plasmodium adaptation to different anopheline species. Genetic analysis of 4,971 Pfs47 gene sequences from different continents revealed that Asia and Papua New Guinea harbor Pfs47 haplotypes more similar to its ortholog in P. praefalciparum at sites that determine vector compatibility, suggesting that ancestral P. falciparum readily adapted to Asian vectors. Consistent with this observation, Pfs47-receptor gene sequences from African sylvan malaria vectors, such as Anopheles moucheti and An. marshallii, were found to share greater similarity with those of Asian vectors than those of vectors of the African An. gambiae complex. Furthermore, experimental infections provide direct evidence that transformed P. falciparum parasites carrying Pfs47 orthologs of P. praefalciparum or P. reichenowi were more effective at evading the immune system of the Asian malaria vector An. dirus than An. gambiae. We propose that high compatibility of ancestral P. falciparum Pfs47 with the receptors of Asian vectors facilitated the early dispersal of human malaria to the Asian continent, without having to first adapt to sub-Saharan vectors of the An. gambiae complex.

Double peroxidase and histone acetyltransferase AgTip60 maintain innate immune memory in primed mosquitoes.

Immune priming in Anopheles gambiae is mediated by the systemic release of a hemocyte differentiation factor (HDF), a complex of lipoxin A4 bound to Evokin, a lipid carrier. HDF increases the proportion of circulating granulocytes and enhances mosquito cellular immunity. Here, we show that Evokin is present in hemocytes and fat-body cells, and messenger RNA (mRNA) expression increases significantly after immune priming. The double peroxidase (DBLOX) enzyme, present in insects but not in vertebrates, is essential for HDF synthesis. DBLOX is highly expressed in oenocytes in the fat-body tissue, and these cells increase in number in primed mosquitoes. We provide direct evidence that the histone acetyltransferase AgTip60 (AGAP001539) is also essential for a sustained increase in oenocyte numbers, HDF synthesis, and immune priming. We propose that oenocytes may function as a population of cells that are reprogrammed, and orchestrate and maintain a broad, systemic, and long-lasting state of enhanced immune surveillance in primed mosquitoes.

Plasmodium falciparum evades immunity of anopheline mosquitoes by interacting with a Pfs47 midgut receptor.

The surface protein Pfs47 allows Plasmodium falciparum parasites to survive and be transmitted by making them "undetectable" to the mosquito immune system. P. falciparum parasites express Pfs47 haplotypes compatible with their sympatric vectors, while those with incompatible haplotypes are eliminated by the mosquito. We proposed that Pfs47 serves as a "key" that mediates immune evasion by interacting with a mosquito receptor "the lock," which differs in evolutionarily divergent anopheline mosquitoes. Recombinant Pfs47 (rPfs47) was used to identify the mosquito Pfs47 receptor protein (P47Rec) using far-Western analysis. rPfs47 bound to a single 31-kDa band and the identity of this protein was determined by mass spectrometry. The mosquito P47Rec has two natterin-like domains and binds to Pfs47 with high affinity (17 to 32 nM). P47Rec is a highly conserved protein with submicrovillar localization in midgut cells. It has structural homology to a cytoskeleton-interacting protein and accumulates at the site of ookinete invasion. Silencing P47Rec expression reduced P. falciparum infection, indicating that the interaction of Pfs47 with the receptor is critical for parasite survival. The binding specificity of P47Rec from distant anophelines (Anopheles gambiae, Anopheles dirus, and Anopheles albimanus) with Pfs47-Africa (GB4) and Pfs47-South America (7G8) haplotypes was evaluated, and it is in agreement with the previously documented compatibility between P. falciparum parasites expressing different Pfs47 haplotypes and these three anopheline species. Our findings give further support to the role of Pfs47 in the adaptation of P. falciparum to different vectors.

Effect of naturally occurring Wolbachia in Anopheles gambiae s.l. mosquitoes from Mali on Plasmodium falciparum malaria transmission.

A naturally occurring Wolbachia strain (wAnga-Mali) was identified in mosquitoes of the Anopheles gambiae complex collected in the Malian villages of Dangassa and Kenieroba. Phylogenetic analysis of the nucleotide sequence of two 16S rRNA regions showed that wAnga-Mali clusters with Wolbachia strains from supergroup A and has the highest homology to a Wolbachia strain isolated from cat fleas (Ctenocephalides). wAnga-Mali is different from two Wolbachia strains previously reported in A. gambiae from Burkina Faso (wAnga_VK5_STP and wAnga_VK5_3.1a). Quantitative analysis of Wolbachia and Plasmodium sporozoite infection in field-collected mosquitoes indicates that the prevalence and intensity of Plasmodium falciparum sporozoite infection is significantly lower in Wolbachia-infected females. The presence of Wolbachia in females from a laboratory Anopheles coluzzii (A. gambiae, M form) colony experimentally infected with P. falciparum (NF54 strain) gametocyte cultures slightly enhanced oocyst infection. However, Wolbachia infection significantly reduced the prevalence and intensity of sporozoite infection, as observed in the field. This indicates that wAnga-Mali infection does not limit early stages of Plasmodium infection in the mosquito, but it has a strong deleterious effect on sporozoites and reduces malaria transmission.

Plasmodium falciparum evades mosquito immunity by disrupting JNK-mediated apoptosis of invaded midgut cells.

The malaria parasite, Plasmodium, must survive and develop in the mosquito vector to be successfully transmitted to a new host. The Plasmodium falciparum Pfs47 gene is critical for malaria transmission. Parasites that express Pfs47 (NF54 WT) evade mosquito immunity and survive, whereas Pfs47 knockouts (KO) are efficiently eliminated by the complement-like system. Two alternative approaches were used to investigate the mechanism of action of Pfs47 on immune evasion. First, we examined whether Pfs47 affected signal transduction pathways mediating mosquito immune responses, and show that the Jun-N-terminal kinase (JNK) pathway is a key mediator of Anopheles gambiae antiplasmodial responses to P. falciparum infection and that Pfs47 disrupts JNK signaling. Second, we used microarrays to compare the global transcriptional responses of A. gambiae midguts to infection with WT and KO parasites. The presence of Pfs47 results in broad and profound changes in gene expression in response to infection that are already evident 12 h postfeeding, but become most prominent at 26 h postfeeding, the time when ookinetes invade the mosquito midgut. Silencing of 15 differentially expressed candidate genes identified caspase-S2 as a key effector of Plasmodium elimination in parasites lacking Pfs47. We provide experimental evidence that JNK pathway regulates activation of caspases in Plasmodium-invaded midgut cells, and that caspase activation is required to trigger midgut epithelial nitration. Pfs47 alters the cell death pathway of invaded midgut cells by disrupting JNK signaling and prevents the activation of several caspases, resulting in an ineffective nitration response that makes the parasite undetectable by the mosquito complement-like system.

Plasmodium evasion of mosquito immunity and global malaria transmission: The lock-and-key theory.

Plasmodium falciparum malaria originated in Africa and became global as humans migrated to other continents. During this journey, parasites encountered new mosquito species, some of them evolutionarily distant from African vectors. We have previously shown that the Pfs47 protein allows the parasite to evade the mosquito immune system of Anopheles gambiae mosquitoes. Here, we investigated the role of Pfs47-mediated immune evasion in the adaptation of P. falciparum to evolutionarily distant mosquito species. We found that P. falciparum isolates from Africa, Asia, or the Americas have low compatibility to malaria vectors from a different continent, an effect that is mediated by the mosquito immune system. We identified 42 different haplotypes of Pfs47 that have a strong geographic population structure and much lower haplotype diversity outside Africa. Replacement of the Pfs47 haplotypes in a P. falciparum isolate is sufficient to make it compatible to a different mosquito species. Those parasites that express a Pfs47 haplotype compatible with a given vector evade antiplasmodial immunity and survive. We propose that Pfs47-mediated immune evasion has been critical for the globalization of P. falciparum malaria as parasites adapted to new vector species. Our findings predict that this ongoing selective force by the mosquito immune system could influence the dispersal of Plasmodium genetic traits and point to Pfs47 as a potential target to block malaria transmission. A new model, the "lock-and-key theory" of P. falciparum globalization, is proposed, and its implications are discussed.

Multiple treatment interruptions and protecting HIV-specific CD4 T cells enable durable CD8 T cell response and viral control.

Human Immunodeficiency Virus (HIV) remains a global health challenge, and novel approaches to improve HIV control are significantly important. The cell and gene therapy product AGT103-T was previously evaluated (NCT04561258) for safety, immunogenicity, and persistence in seven patients for up to 180 days post infusion. In this study, we sought to investigate the impact of AGT103-T treatment upon analytical treatment interruptions (ATIs). Six patients previously infused with AGT103-T were enrolled into an ATI study (NCT05540964), wherein they suspended their antiretroviral therapy (ART) until their viral load reached 100,000 copies/mL in two successive visits, or their CD4 count was reduced to below 300 cells/µL. During the ATI, all patients experienced viral rebound followed by a notable expansion in HIV specific immune responses. The participants demonstrated up to a five-fold increase in total CD8 counts over baseline approximately 1-2 weeks followed by the peak viremia. This coincided with a rise in HIV-specific CD8 T cells, which was attributed to the increase in antigen availability and memory recall. Thus, the protocol was amended to include a second ATI with the first ATI serving as an "auto-vaccination." Four patients participated in a second ATI. During the second ATI, the Gag-specific CD8 T cells were either maintained or rose in response to viral rebound and the peak viremia was substantially decreased. The patients reached a viral set point ranging from 7,000 copies/mL to 25,000 copies/mL. Upon resuming ART, all participants achieved viral control more rapidly than during the first ATI, with CD4 counts remaining within 10% of baseline measurements and without any serious adverse events or evidence of drug resistance. In summary, the rise in CD8 counts and the viral suppression observed in 100% of the study participants are novel observations demonstrating that AGT103-T gene therapy when combined with multiple ATIs, is a safe and effective approach for achieving viral control, with viral setpoints consistently below 25,000 copies/mL and relatively stable CD4 T cell counts. We conclude that HIV cure-oriented cell and gene therapy trials should include ATI and may benefit from designs that include multiple ATIs when induction of CD8 T cells is required to establish viral control.

Structural features affecting trafficking, processing, and secretion of Trypanosoma cruzi mucins.

Trypanosoma cruzi is wrapped by a dense coat of mucin-type molecules encoded by complex gene families termed TcSMUG and TcMUC, which are expressed in the insect- and mammal-dwelling forms of the parasite, respectively. Here, we dissect the contribution of distinct post-translational modifications on the trafficking of these glycoconjugates. In vivo tracing and characterization of tagged-variants expressed by transfected epimastigotes indicate that although the N-terminal signal peptide is responsible for targeting TcSMUG products to the endoplasmic reticulum (ER), the glycosyl phosphatidylinositol (GPI)-anchor likely functions as a forward transport signal for their timely progression along the secretory pathway. GPI-minus variants accumulate in the ER, with only a minor fraction being ultimately released to the medium as anchorless products. Secreted products, but not ER-accumulated ones, display several diagnostic features of mature mucin-type molecules including extensive O-type glycosylation, Galf-based epitopes recognized by monoclonal antibodies, and terminal Galp residues that become readily sialylated upon addition of parasite trans-sialidases. Processing of N-glycosylation site(s) is dispensable for the overall TcSMUG mucin-type maturation and secretion. Despite undergoing different O-glycosylation elaboration, TcMUC reporters yielded quite similar results, thus indicating that (i) molecular trafficking signals are structurally and functionally conserved between mucin families, and (ii) TcMUC and TcSMUG products are recognized and processed by a distinct repertoire of stage-specific glycosyltransferases. Thus, using the fidelity of a homologous expression system, we have defined some biosynthetic aspects of T. cruzi mucins, key molecules involved in parasite protection and virulence.

Involvement of TSSA (trypomastigote small surface antigen) in Trypanosoma cruzi invasion of mammalian cells.

TSSA (trypomastigote small surface antigen) is a polymorphic mucin-like molecule displayed on the surface of Trypanosoma cruzi trypomastigote forms. To evaluate its functional properties, we undertook comparative biochemical and genetic approaches on isoforms present in parasite stocks from extant evolutionary lineages (CL Brener and Sylvio X-10). We show that CL Brener TSSA, but not the Sylvio X-10 counterpart, exhibits dose-dependent and saturable binding towards non-macrophagic cell lines. This binding triggers Ca(2+)-based signalling responses in the target cell while providing an anchor for the invading parasite. Accordingly, exogenous addition of either TSSA-derived peptides or specific antibodies significantly inhibits invasion of CL Brener, but not Sylvio X-10, trypomastigotes. Non-infective epimastigote forms, which do not express detectable levels of TSSA, were stably transfected with TSSA cDNA from either parasite stock. Although both transfectants produced a surface-associated mucin-like TSSA product, epimastigotes expressing CL Brener TSSA showed a ~2-fold increase in their attachment to mammalian cells. Overall, these findings indicate that CL Brener TSSA functions as a parasite adhesin, engaging surface receptor(s) and inducing signalling pathways on the host cell as a prerequisite for parasite internalization. More importantly, the contrasting functional features of TSSA isoforms provide one appealing mechanism underlying the differential infectivity of T. cruzi stocks.

The heat shock protein Hsc70-3 mediates an anti-apoptotic response critical for Plasmodium evasion of Anopheles gambiae immunity.

IMPORTANCE: Malaria transmission by Anopheles gambiae mosquitoes is very effective, in part because the parasite expresses a surface protein called Pfs47 that allows it to evade the mosquito immune system. Here we investigate how this protein changes the response of mosquito midgut epithelial cells to invasion by the parasite. Pfs47 is known to interact with P47Rec, a mosquito midgut receptor. We found that Pf47Rec inhibits caspase-mediated apoptosis by interacting with the Hsc70-3. This disrupts nitration of midgut epithelial cells invaded by the parasite and the release of hemocyte-derived microvesicles, which are critical for effective activation of the mosquito complement system that eliminates the parasite.

Lysine transporters in human trypanosomatid pathogens.

In previous studies we characterized arginine transporter genes from Trypanosoma cruzi and Leishmania donovani, the etiological agents of chagas disease and kala azar, respectively, both fatal diseases in humans. Unlike arginine transporters in higher eukaryotes that transport also lysine, these parasite transporters translocate only arginine. This phenomenon prompted us to identify and characterize parasite lysine transporters. Here we demonstrate that LdAAP7 and TcAAP7 encode lysine-specific permeases in L. donovani and T. cruzi, respectively. These two lysine permeases are both members of the large amino acid/auxin permease family and share certain biochemical properties, such as specificity and Km. However, we evidence that LdAAP7 and TcAAP7 differ in their regulation and localization, such differences are likely a reflection of the dissimilar L. donovani and T. cruzi life cycles. Failed attempts to delete both alleles of LdAAP7 support the premise that this is an essential gene that encodes the only lysine permeases expressed in L. donovani promastigotes and T. cruzi epimastigotes, respectively.

[The mammalian TOR pathway is present in Trypanosoma cruzi. In silico reconstruction and possible functions]. / La vía de transducción de señales TOR de mamíferos está presente en Trypanosoma cruzi. Reconstrucción in silico y posibles funciones.

The mammalian TOR pathway ("Target Of Rapamycin") is a regulatory protein network involved in a wide range of processes including cell growth and differentiation, providing a functional switch between anabolic and catabolic cell metabolism. Trypanosoma cruzi, the etiologic agent of Chagas disease, has a complex life cycle with different morphological stages in various hosts. This life cycle implies that parasites have to deal with fluctuations in the extracellular medium that should be detected and counteracted adapting their metabolism. A candidate to be the mediator between the receptors / sensors of the environment and cellular adaptive response is the TOR pathway. In this paper we integrate the bibliographic data of the TOR pathway in trypanosomatids by in silico analysis (computer simulation of biological structures and processes) of the parasite's genome. Possible effectors and processes regulated by this metabolic pathway are also proposed. Given that the information on the mechanisms of signal transduction in trypanosomatids is scarce, we consider the model presented in this work may be a reference for future experimental work.

In vivo Characterization of Plasmodium berghei P47 (Pbs47) as a Malaria Transmission-Blocking Vaccine Target.

An effective vaccine to reduce malaria transmission is central to control and ultimately achieve disease eradication. Recently, we demonstrated that antibodies targeting the Plasmodium falciparum surface protein P47 (Pfs47) reduce parasite transmission to Anopheles gambiae mosquitoes. Here, Plasmodium berghei (Pb) was used as a model to assess the in vivo efficacy of a P47-targeted transmission blocking vaccine (Pbs47). Mice were immunized following a prime/boost regimen and infected with P. berghei. The effect of immunization on infectivity to mosquitoes was evaluated by direct feeding on P. berghei-infected mice. The key region in Pbs47 where antibody binding confers protection was mapped, and the immunogenicity of this protective antigen was enhanced by conjugation to a virus-like particle. Passive immunization with 100 and 50 µg/mL of anti-Pbs47 IgG reduced oocyst density by 77 and 67%, respectively. Furthermore, affinity purified Pbs47-specific IgG significantly reduced oocyst density by 88 and 77%, respectively at doses as low as 10 and 1 µg/mL. These studies suggest that P47 is a promising transmission blocking target and show that antibodies to the same specific region in Pfs47 and Pbs47 confer protection.

Antibody Therapy Goes to Insects: Monoclonal Antibodies Can Block Plasmodium Transmission to Mosquitoes.

Malaria eradication is a global priority but requires innovative strategies. Humoral immune responses attack different parasite stages, and antibody-based therapy may prevent malaria infection or transmission. Here, we discuss targets of monoclonal antibodies in mosquito sexual stages of Plasmodium.

Subcellular localization of Trypanosoma cruzi arginine kinase.

Phosphoarginine is a cell energy buffer molecule synthesized by the enzyme arginine kinase. In Trypanosoma cruzi, the aetiological agent of Chagas' disease, 2 different isoforms were identified by data mining, but only 1 was expressed during the parasite life cycle. The digitonin extraction pattern of arginine kinase differed from those obtained for reservosomes, glycosomes and mitochondrial markers, and similar to the cytosolic marker. Immunofluorescence analysis revealed that although arginine kinase is localized mainly in unknown punctuated structures and also in the cytosol, it did not co-localize with any of the subcelular markers. This punctuated pattern has previously been observed in many cytosolic proteins of trypanosomatids. The knowledge of the subcellular localization of phosphagen kinases is a crucial issue to understand their physiological role in protozoan parasites.

Integrating Scientific English into Biological Sciences PhD Programs in Developing Countries: Strategies from Trainees and Mentor.

Successful researchers in the biological sciences communicate their work to a global audience and must do so in English to be widely recognized and cited. This applies equally to scientific talks, posters, and published articles; thus, scientific English must be prioritized in nonnative English-speaking (NNES) academic institutions to prepare their trainees for successful careers. Here, we propose strategies for integrating scientific English into PhD programs operating in NNES countries. Many graduate students from NNES countries strive for an international career and encounter English as an important barrier. Based on our own experiences as NNES postdoctoral fellows at a US institution, or as a US mentor of these trainees, we contend that conventional learning processes at home institutions do not sufficiently prioritize scientific English as the medium for regular discussions of laboratory-generated data. Principal investigators, mentors, and supervisors are key in promoting English language usage as a structured component of PhD training. If these stakeholders routinely integrate English training and education within the research laboratory program, graduates will be equipped to pursue international academic careers. The ideas presented here are intended for NNES PhD students (and their mentors) who seek an international scientific career in the biological sciences.

Engineering a Virus-Like Particle as an Antigenic Platform for a Pfs47-Targeted Malaria Transmission-Blocking Vaccine.

We recently characterized Pfs47, a protein expressed on the surface of sexual stages and ookinetes of Plasmodium falciparum, as a malaria transmission-blocking vaccine (TBV) target. Mice immunization induced antibodies that conferred strong transmission-reducing activity (TRA) at a concentration of 200 µg/mL. Here, we sought to optimize the Pfs47 vaccine to elicit higher titers of high-affinity antibodies, capable of inducing strong TRA at a lower concentration. We report the development and evaluation of a Pfs47-based virus-like particle (VLP) vaccine generated by conjugating our 58 amino acid Pfs47 antigen to Acinetobacter phage AP205-VLP using the SpyCatcher:SpyTag adaptor system. AP205-Pfs47 complexes (VLP-P47) formed particles of ~22 nm diameter that reacted with polyclonal anti-Pfs47 antibodies, indicating that the antigen was accessible on the surface of the particle. Mice immunized with VLP-P47 followed by a boost with Pfs47 monomer induced significantly higher antibody titers, with higher binding affinity to Pfs47, than mice that received two immunizations with either VLP-P47 (VLP-P47/VLP-P47) or the Pfs47 monomer (P47/P47). Purified IgG from VLP-P47/P47 mice had strong TRA (83-98%) at concentrations as low as 5 µg/mL. These results indicate that conjugating the Pfs47 antigen to AP205-VLP significantly enhanced antigenicity and confirm the potential of Pfs47 as a TBV candidate.

Homologous Expression of Glycosylphosphatidylinositol-Anchored Glycoproteins in Trypanosoma cruzi.

The surface coat of Trypanosoma cruzi is covered with glycosylphosphatidylinositol (GPI)-anchored glycoproteins (GAGPs) that contribute to parasite protection and to the establishment of a persistent infection in both the insect vector and the mammalian host. Multiple GAGPs that vary by amino acid sequence and/or posttranslational modifications are co-expressed on the parasite surface coat, hence curtailing structural/functional analyses on these molecules. Studies in our lab have indicated that GAGP-tagged variants expressed by transfected parasites undergo analogous posttranslational processing than endogenous ones and therefore constitute suitable tools to overcome these limitations. In this chapter, we detail the entire methodological pipeline for the efficient homologous expression of GAGPs in T. cruzi: from a simple strategy for the simultaneously cloning and tagging of the gene of interest to the biochemical validation of the parasite-expressed product.

Metabolic Labeling of Surface Neo-sialylglyconjugates Catalyzed by Trypanosoma cruzi trans-Sialidase.

Trypanosoma cruzi, the protozoan agent of Chagas disease, has evolved an innovative metabolic pathway by which protective sialic acid (SA) residues are scavenged from host sialylglycoconjugates and transferred onto parasite surface mucin-like molecules (or surface glycoconjugates from host target cells) by means of a unique trans-sialidase (TS) enzyme. TS-induced changes in the glycoprotein sialylation profile of both parasite and host cells are crucial for the establishment of a persistent T. cruzi infection and for the development of Chagas disease-associated pathogenesis. In this chapter, we describe a novel metabolic labeling method developed in our labs that enables straightforward identification and molecular characterization of SA acceptors of the TS-catalyzed reaction.

Molecular and antigenic characterization of Trypanosoma cruzi TolT proteins.

BACKGROUND: TolT was originally described as a Trypanosoma cruzi molecule that accumulated on the trypomastigote flagellum bearing similarity to bacterial TolA colicins receptors. Preliminary biochemical studies indicated that TolT resolved in SDS-PAGE as ~3-5 different bands with sizes between 34 and 45 kDa, and that this heterogeneity could be ascribed to differences in polypeptide glycosylation. However, the recurrent identification of TolT-deduced peptides, and variations thereof, in trypomastigote proteomic surveys suggested an intrinsic TolT complexity, and prompted us to undertake a thorough reassessment of this antigen. METHODS/PRINCIPLE FINDINGS: Genome mining exercises showed that TolT constitutes a larger-than-expected family of genes, with at least 12 polymorphic members in the T. cruzi CL Brener reference strain and homologs in different trypanosomes. According to structural features, TolT deduced proteins could be split into three robust groups, termed TolT-A, TolT-B, and TolT-C, all of them showing marginal sequence similarity to bacterial TolA proteins and canonical signatures of surface localization/membrane association, most of which were herein experimentally validated. Further biochemical and microscopy-based characterizations indicated that this grouping may have a functional correlate, as TolT-A, TolT-B and TolT-C molecules showed differences in their expression profile, sub-cellular distribution, post-translational modification(s) and antigenic structure. We finally used a recently developed fluorescence magnetic beads immunoassay to validate a recombinant protein spanning the central and mature region of a TolT-B deduced molecule for Chagas disease serodiagnosis. CONCLUSION/SIGNIFICANCE: This study unveiled an unexpected genetic and biochemical complexity within the TolT family, which could be exploited for the development of novel T. cruzi biomarkers with diagnostic/therapeutic applications.