Systemic Immune Modulation by Gastrointestinal Nematodes.

Gastrointestinal nematode (GIN) infection has applied significant evolutionary pressure to the mammalian immune system and remains a global economic and human health burden. Upon infection, type 2 immune sentinels activate a common antihelminth response that mobilizes and remodels the intestinal tissue for effector function; however, there is growing appreciation of the impact GIN infection also has on the distal tissue immune state. Indeed, this effect is observed even in tissues through which GINs never transit. This review highlights how GIN infection modulates systemic immunity through (a) induction of host resistance and tolerance responses, (b) secretion of immunomodulatory products, and (c) interaction with the intestinal microbiome. It also discusses the direct consequences that changes to distal tissue immunity can have for concurrent and subsequent infection, chronic noncommunicable diseases, and vaccination efficacy.

Structural Basis for Blocking Sugar Uptake into the Malaria Parasite Plasmodium falciparum.

Plasmodium species, the causative agent of malaria, rely on glucose for energy supply during blood stage. Inhibition of glucose uptake thus represents a potential strategy for the development of antimalarial drugs. Here, we present the crystal structures of PfHT1, the sole hexose transporter in the genome of Plasmodium species, at resolutions of 2.6 Å in complex with D-glucose and 3.7 Å with a moderately selective inhibitor, C3361. Although both structures exhibit occluded conformations, binding of C3361 induces marked rearrangements that result in an additional pocket. This inhibitor-binding-induced pocket presents an opportunity for the rational design of PfHT1-specific inhibitors. Among our designed C3361 derivatives, several exhibited improved inhibition of PfHT1 and cellular potency against P. falciparum, with excellent selectivity to human GLUT1. These findings serve as a proof of concept for the development of the next-generation antimalarial chemotherapeutics by simultaneously targeting the orthosteric and allosteric sites of PfHT1.

Oligopeptide Signaling through TbGPR89 Drives Trypanosome Quorum Sensing.

Trypanosome parasites control their virulence and spread by using quorum sensing (QS) to generate transmissible "stumpy forms" in their host bloodstream. However, the QS signal "stumpy induction factor" (SIF) and its reception mechanism are unknown. Although trypanosomes lack G protein-coupled receptor signaling, we have identified a surface GPR89-family protein that regulates stumpy formation. TbGPR89 is expressed on bloodstream "slender form" trypanosomes, which receive the SIF signal, and when ectopically expressed, TbGPR89 drives stumpy formation in a SIF-pathway-dependent process. Structural modeling of TbGPR89 predicts unexpected similarity to oligopeptide transporters (POT), and when expressed in bacteria, TbGPR89 transports oligopeptides. Conversely, expression of an E. coli POT in trypanosomes drives parasite differentiation, and oligopeptides promote stumpy formation in vitro. Furthermore, the expression of secreted trypanosome oligopeptidases generates a paracrine signal that accelerates stumpy formation in vivo. Peptidase-generated oligopeptide QS signals being received through TbGPR89 provides a mechanism for both trypanosome SIF production and reception.

Genetic crosses within and between species of Cryptosporidium.

Parasites and their hosts are engaged in reciprocal coevolution that balances competing mechanisms of virulence, resistance, and evasion. This often leads to host specificity, but genomic reassortment between different strains can enable parasites to jump host barriers and conquer new niches. In the apicomplexan parasite Cryptosporidium, genetic exchange has been hypothesized to play a prominent role in adaptation to humans. The sexual lifecycle of the parasite provides a potential mechanism for such exchange; however, the boundaries of Cryptosporidium sex are currently undefined. To explore this experimentally, we established a model for genetic crosses. Drug resistance was engineered using a mutated phenylalanyl tRNA synthetase gene and marking strains with this and the previously used Neo transgene enabled selection of recombinant progeny. This is highly efficient, and genomic recombination is evident and can be continuously monitored in real time by drug resistance, flow cytometry, and PCR mapping. Using this approach, multiple loci can now be modified with ease. We demonstrate that essential genes can be ablated by crossing a Cre recombinase driver strain with floxed strains. We further find that genetic crosses are also feasible between species. Crossing Cryptosporidium parvum, a parasite of cattle and humans, and Cryptosporidium tyzzeri a mouse parasite resulted in progeny with a recombinant genome derived from both species that continues to vigorously replicate sexually. These experiments have important fundamental and translational implications for the evolution of Cryptosporidium and open the door to reverse- and forward-genetic analysis of parasite biology and host specificity.

Duplication and neofunctionalization of a horizontally transferred xyloglucanase as a facet of the Red Queen coevolutionary dynamic.

Oomycete protists share phenotypic similarities with fungi, including the ability to cause plant diseases, but branch in a distant region of the tree of life. It has been suggested that multiple horizontal gene transfers (HGTs) from fungi-to-oomycetes contributed to the evolution of plant-pathogenic traits. These HGTs are predicted to include secreted proteins that degrade plant cell walls, a barrier to pathogen invasion and a rich source of carbohydrates. Using a combination of phylogenomics and functional assays, we investigate the diversification of a horizontally transferred xyloglucanase gene family in the model oomycete species Phytophthora sojae. Our analyses detect 11 xyloglucanase paralogs retained in P. sojae. Using heterologous expression in yeast, we show consistent evidence that eight of these paralogs have xyloglucanase function, including variants with distinct protein characteristics, such as a long-disordered C-terminal extension that can increase xyloglucanase activity. The functional variants analyzed subtend a phylogenetic node close to the fungi-to-oomycete transfer, suggesting the horizontally transferred gene was a bona fide xyloglucanase. Expression of three xyloglucanase paralogs in Nicotiana benthamiana triggers high-reactive oxygen species (ROS) generation, while others inhibit ROS responses to bacterial immunogens, demonstrating that the paralogs differentially stimulate pattern-triggered immunity. Mass spectrometry of detectable enzymatic products demonstrates that some paralogs catalyze the production of variant breakdown profiles, suggesting that secretion of variant xyloglucanases increases efficiency of xyloglucan breakdown as well as diversifying the damage-associated molecular patterns released. We suggest that this pattern of neofunctionalization and the variant host responses represent an aspect of the Red Queen host-pathogen coevolutionary dynamic.

Protective function and differentiation cues of brain-resident CD8+ T cells during surveillance of latent Toxoplasma gondii infection.

Chronic Toxoplasma gondii infection induces brain-resident CD8+ T cells (bTr), but the protective functions and differentiation cues of these cells remain undefined. Here, we used a mouse model of latent infection by T. gondii leading to effective CD8+ T cell-mediated parasite control. Thanks to antibody depletion approaches, we found that peripheral circulating CD8+ T cells are dispensable for brain parasite control during chronic stage, indicating that CD8+ bTr are able to prevent brain parasite reactivation. We observed that the retention markers CD69, CD49a, and CD103 are sequentially acquired by brain parasite-specific CD8+ T cells throughout infection and that a majority of CD69/CD49a/CD103 triple-positive (TP) CD8+ T cells also express Hobit, a transcription factor associated with tissue residency. This TP subset develops in a CD4+ T cell-dependent manner and is associated with effective parasite control during chronic stage. Conditional invalidation of Transporter associated with Antigen Processing (TAP)-mediated major histocompatibility complex (MHC) class I presentation showed that presentation of parasite antigens by glutamatergic neurons and microglia regulates the differentiation of CD8+ bTr into TP cells. Single-cell transcriptomic analyses revealed that resistance to encephalitis is associated with the expansion of stem-like subsets of CD8+ bTr. In summary, parasite-specific brain-resident CD8+ T cells are a functionally heterogeneous compartment which autonomously ensure parasite control during T. gondii latent infection and which differentiation is shaped by neuronal and microglial MHC I presentation. A more detailed understanding of local T cell-mediated immune surveillance of this common parasite is needed for harnessing brain-resident CD8+ T cells in order to enhance control of chronic brain infections.

Role of a novel uropod-like cell membrane protrusion in the pathogenesis of the parasite Trichomonas vaginalis.

Trichomonas vaginalis causes trichomoniasis, the most common non-viral sexually transmitted disease worldwide. As an extracellular parasite, adhesion to host cells is essential for the development of infection. During attachment, the parasite changes its tear ovoid shape to a flat ameboid form, expanding the contact surface and migrating through tissues. Here, we have identified a novel structure formed at the posterior pole of adherent parasite strains, resembling the previously described uropod, which appears to play a pivotal role as an anchor during the attachment process. Moreover, our research demonstrates that the overexpression of the tetraspanin T. vaginalis TSP5 protein (TvTSP5), which is localized on the cell surface of the parasite, notably enhances the formation of this posterior anchor structure in adherent strains. Finally, we demonstrate that parasites that overexpress TvTSP5 possess an increased ability to adhere to host cells, enhanced aggregation and reduced migration on agar plates. Overall, these findings unveil novel proteins and structures involved in the intricate mechanisms of T. vaginalis interactions with host cells.

HpARI Protein Secreted by a Helminth Parasite Suppresses Interleukin-33.

Infection by helminth parasites is associated with amelioration of allergic reactivity, but mechanistic insights into this association are lacking. Products secreted by the mouse parasite Heligmosomoides polygyrus suppress type 2 (allergic) immune responses through interference in the interleukin-33 (IL-33) pathway. Here, we identified H. polygyrus Alarmin Release Inhibitor (HpARI), an IL-33-suppressive 26-kDa protein, containing three predicted complement control protein (CCP) modules. In vivo, recombinant HpARI abrogated IL-33, group 2 innate lymphoid cell (ILC2) and eosinophilic responses to Alternaria allergen administration, and diminished eosinophilic responses to Nippostrongylus brasiliensis, increasing parasite burden. HpARI bound directly to both mouse and human IL-33 (in the cytokine's activated state) and also to nuclear DNA via its N-terminal CCP module pair (CCP1/2), tethering active IL-33 within necrotic cells, preventing its release, and forestalling initiation of type 2 allergic responses. Thus, HpARI employs a novel molecular strategy to suppress type 2 immunity in both infection and allergy.

Parasite effects on receivers in animal communication: Hidden impacts on behavior, ecology, and evolution.

Parasites exert a profound effect on biological processes. In animal communication, parasite effects on signalers are well-known drivers of the evolution of communication systems. Receiver behavior is also likely to be altered when they are parasitized or at risk of parasitism, but these effects have received much less attention. Here, we present a broad framework for understanding the consequences of parasitism on receivers for behavioral, ecological, and evolutionary processes. First, we outline the different kinds of effects parasites can have on receivers, including effects on signal processing from the many parasites that inhabit, occlude, or damage the sensory periphery and the central nervous system or that affect physiological processes that support these organs, and effects on receiver response strategies. We then demonstrate how understanding parasite effects on receivers could answer important questions about the mechanistic causes and functional consequences of variation in animal communication systems. Variation in parasitism levels is a likely source of among-individual differences in response to signals, which can affect receiver fitness and, through effects on signaler fitness, impact population levels of signal variability. The prevalence of parasitic effects on specific sensory organs may be an important selective force for the evolution of elaborate and multimodal signals. Finally, host-parasite coevolution across heterogeneous landscapes will generate geographic variation in communication systems, which could ultimately lead to evolutionary divergence. We discuss applications of experimental techniques to manipulate parasitism levels and point the way forward by calling for integrative research collaborations between parasitologists, neurobiologists, and behavioral and evolutionary ecologists.

The developmental hierarchy and scarcity of replicative slender trypanosomes in blood challenges their role in infection maintenance.

The development of Trypanosoma brucei in its mammalian host is marked by a distinct morphological change as replicative "slender" forms differentiate into cell cycle arrested "stumpy" forms in a quorum-sensing-dependent manner. Although stumpy forms dominate chronic infections at the population level, the proportion of replicative parasites at the individual cell level and the irreversibility of arrest in the bloodstream are unclear. Here, we experimentally demonstrate that developmental cell cycle arrest is definitively irreversible in acute and chronic infections in mice. Furthermore, analysis of replicative capacity and single-cell transcriptome profiling reveal a temporal hierarchy, whereby cell cycle arrest and appearance of a reversible stumpy-like transcriptome precede irreversible commitment and morphological change. Unexpectedly, we show that proliferating parasites are exceptionally scarce in the blood after infections are established. This challenges the ability of bloodstream trypanosomes to sustain infection by proliferation or antigenic variation, these parasites instead being overwhelmingly adapted for transmission.

Intracellular Plasmodium aquaporin 2 is important for sporozoite production in the mosquito vector and malaria transmission.

Malaria remains a devastating disease and, with current measures failing to control its transmission, there is a need for novel interventions. A family of proteins that have long been pursued as potential intervention targets are aquaporins, which are channels facilitating the movement of water and other solutes across membranes. We identify an aquaporin in malaria parasites and demonstrate that it is important for completion of Plasmodium development in the mosquito vector. Disruption of AQP2 in the human parasite Plasmodium falciparum and the rodent parasite Plasmodium berghei blocks sporozoite production inside oocysts established on mosquito midguts, greatly limiting parasite infection of salivary glands and transmission to a new host. In vivo epitope tagging of AQP2 in P. berghei, combined with immunofluorescence assays, reveals that the protein is localized in vesicle-like organelles found in the cytoplasm of gametocytes, ookinetes, and sporozoites. The number of these organelles varies between individual parasites and lifecycle stages suggesting that they are likely part of a dynamic endomembrane system. Phylogenetic analysis confirms that AQP2 is unique to malaria and closely related parasites and most closely resembles intracellular aquaporins. Structure prediction analyses identify several unusual features, including a large accessory extracellular loop and an arginine-to-phenylalanine substitution in the selectivity filter principally determining pore function, a unique feature among known aquaporins. This in conjunction with the importance of AQP2 for malaria transmission suggests that AQP2 may be a fruitful target of antimalarial interventions.

Temperature dependence of parasitoid infection and abundance of a diatom revealed by automated imaging and classification.

Diatoms are a group of phytoplankton that contribute disproportionately to global primary production. Traditional paradigms that suggest diatoms are consumed primarily by larger zooplankton are challenged by sporadic parasitic "epidemics" within diatom populations. However, our understanding of diatom parasitism is limited by difficulties in quantifying these interactions. Here, we observe the dynamics of Cryothecomonas aestivalis (a protist) infection of an important diatom on the Northeast U.S. Shelf (NES), Guinardia delicatula, with a combination of automated imaging-in-flow cytometry and a convolutional neural network image classifier. Application of the classifier to >1 billion images from a nearshore time series and >20 survey cruises across the broader NES reveals the spatiotemporal gradients and temperature dependence of G. delicatula abundance and infection dynamics. Suppression of parasitoid infection at temperatures <4 °C drives annual cycles in both G. delicatula infection and abundance, with an annual maximum in infection observed in the fall-winter preceding an annual maximum in host abundance in the winter-spring. This annual cycle likely varies spatially across the NES in response to variable annual cycles in water temperature. We show that infection remains suppressed for ~2 mo following cold periods, possibly due to temperature-induced local extinctions of the C. aestivalis strain(s) that infect G. delicatula. These findings have implications for predicting impacts of a warming NES surface ocean on G. delicatula abundance and infection dynamics and demonstrate the potential of automated plankton imaging and classification to quantify phytoplankton parasitism in nature across unprecedented spatiotemporal scales.

Engineered gut symbiont inhibits microsporidian parasite and improves honey bee survival.

Honey bees (Apis mellifera) are critical agricultural pollinators as well as model organisms for research on development, behavior, memory, and learning. The parasite Nosema ceranae, a common cause of honey bee colony collapse, has developed resistance to small-molecule therapeutics. An alternative long-term strategy to combat Nosema infection is therefore urgently needed, with synthetic biology offering a potential solution. Honey bees harbor specialized bacterial gut symbionts that are transmitted within hives. Previously, these have been engineered to inhibit ectoparasitic mites by expressing double-stranded RNA (dsRNA) targeting essential mite genes, via activation of the mite RNA interference (RNAi) pathway. In this study, we engineered a honey bee gut symbiont to express dsRNA targeting essential genes of N. ceranae via the parasite's own RNAi machinery. The engineered symbiont sharply reduced Nosema proliferation and improved bee survival following the parasite challenge. This protection was observed in both newly emerged and older forager bees. Furthermore, engineered symbionts were transmitted among cohoused bees, suggesting that introducing engineered symbionts to hives could result in colony-level protection.

Dual effector population modification gene-drive strains of the African malaria mosquitoes, Anopheles gambiae and Anopheles coluzzii.

Proposed genetic approaches for reducing human malaria include population modification, which introduces genes into vector mosquitoes to reduce or prevent parasite transmission. We demonstrate the potential of Cas9/guide RNA (gRNA)-based gene-drive systems linked to dual antiparasite effector genes to spread rapidly through mosquito populations. Two strains have an autonomous gene-drive system coupled to dual anti-Plasmodium falciparum effector genes comprising single-chain variable fragment monoclonal antibodies targeting parasite ookinetes and sporozoites in the African malaria mosquitoes Anopheles gambiae (AgTP13) and Anopheles coluzzii (AcTP13). The gene-drive systems achieved full introduction within 3 to 6 mo after release in small cage trials. Life-table analyses revealed no fitness loads affecting AcTP13 gene-drive dynamics but AgTP13 males were less competitive than wild types. The effector molecules reduced significantly both parasite prevalence and infection intensities. These data supported transmission modeling of conceptual field releases in an island setting that shows meaningful epidemiological impacts at different sporozoite threshold levels (2.5 to 10 k) for human infection by reducing malaria incidence in optimal simulations by 50 to 90% within as few as 1 to 2 mo after a series of releases, and by ≥90% within 3 mo. Modeling outcomes for low sporozoite thresholds are sensitive to gene-drive system fitness loads, gametocytemia infection intensities during parasite challenges, and the formation of potentially drive-resistant genome target sites, extending the predicted times to achieve reduced incidence. TP13-based strains could be effective for malaria control strategies following validation of sporozoite transmission threshold numbers and testing field-derived parasite strains. These or similar strains are viable candidates for future field trials in a malaria-endemic region.

Host-parasite coevolution and the stability of genetic kin recognition.

Crozier's paradox suggests that genetic kin recognition will not be evolutionarily stable. The problem is that more common tags (markers) are more likely to be recognized and helped. This causes common tags to increase in frequency, eliminating the genetic variability that is required for genetic kin recognition. Two potential solutions to this problem have been suggested: host-parasite coevolution and multiple social encounters. We show that the host-parasite coevolution hypothesis does not work as commonly assumed. Host-parasite coevolution only stabilizes kin recognition at a parasite resistance locus if parasites adapt rapidly to hosts and cause intermediate or high levels of damage (virulence). Additionally, when kin recognition is stabilized at a parasite resistance locus, this can have an additional cost of making hosts more susceptible to parasites. However, we show that if the genetic architecture is allowed to evolve, meaning natural selection can choose the recognition locus, genetic kin recognition is more likely to be stable. The reason for this is that host-parasite coevolution can maintain tag diversity at another (neutral) locus by genetic hitchhiking, allowing that other locus to be used for genetic kin recognition. These results suggest a way that host-parasite coevolution can resolve Crozier's paradox, without making hosts more susceptible to parasites. However, the opportunity for multiple social encounters may provide a more robust resolution of Crozier's paradox.

An immune memory-structured SIS epidemiological model for hyperdiverse pathogens.

A hyperdiverse class of pathogens of humans and wildlife, including the malaria parasite Plasmodium falciparum, relies on multigene families to encode antigenic variation. As a result, high (asymptomatic) prevalence is observed despite high immunity in local populations under high-transmission settings. The vast diversity of "strains" and genes encoding this variation challenges the application of established models for the population dynamics of such infectious diseases. Agent-based models have been formulated to address theory on strain coexistence and structure, but their complexity can limit application to gain insights into population dynamics. Motivated by P. falciparum malaria, we develop an alternative formulation in the form of a structured susceptible-infected-susceptible population model in continuous time, where individuals are classified not only by age, as is standard, but also by the diversity of parasites they have been exposed to and retain in their specific immune memory. We analyze the population dynamics and bifurcation structure of this system of partial-differential equations, showing the existence of alternative steady states and an associated tipping point with transmission intensity. We attribute the critical transition to the positive feedback between parasite genetic diversity and force of infection. Basins of attraction show that intervention must drastically reduce diversity to prevent a rebound to high infection levels. Results emphasize the importance of explicitly considering pathogen diversity and associated specific immune memory in the population dynamics of hyperdiverse epidemiological systems. This statement is discussed in a more general context for ecological competition systems with hyperdiverse trait spaces.

The parasite intraerythrocytic cycle and human circadian cycle are coupled during malaria infection.

During infections with the malaria parasites Plasmodium vivax, patients exhibit rhythmic fevers every 48 h. These fever cycles correspond with the time the parasites take to traverse the intraerythrocytic cycle (IEC). In other Plasmodium species that infect either humans or mice, the IEC is likely guided by a parasite-intrinsic clock [Rijo-Ferreiraet al., Science 368, 746-753 (2020); Smith et al., Science 368, 754-759 (2020)], suggesting that intrinsic clock mechanisms may be a fundamental feature of malaria parasites. Moreover, because Plasmodium cycle times are multiples of 24 h, the IECs may be coordinated with the host circadian clock(s). Such coordination could explain the synchronization of the parasite population in the host and enable alignment of IEC and circadian cycle phases. We utilized an ex vivo culture of whole blood from patients infected with P. vivax to examine the dynamics of the host circadian transcriptome and the parasite IEC transcriptome. Transcriptome dynamics revealed that the phases of the host circadian cycle and the parasite IEC are correlated across multiple patients, showing that the cycles are phase coupled. In mouse model systems, host-parasite cycle coupling appears to provide a selective advantage for the parasite. Thus, understanding how host and parasite cycles are coupled in humans could enable antimalarial therapies that disrupt this coupling.

Intestinal tuft cells: Sentinels, what else?

The intestinal epithelium plays crucial roles in maintaining gut homeostasis. A key function consists in constituting a physical and chemical barrier between self and non-self-compartments, and, based on its crosstalk with the luminal environment, in controlling activation of the host immune system. Tuft cells are a unique epithelial cell lineage, the function of which remained a mystery even 50 years after their initial discovery. The first function of intestinal tuft cells was recently described, with a central role in initiating type 2 immune responses following infection with helminth parasites. Since then, tuft cells have emerged as sentinel cells recognizing a variety of luminal cues, mediating the host-microorganisms crosstalk with additional pathogens, including viruses and bacteria. Although it can be anticipated that more functions will be discovered for tuft cells in the future, recent discoveries already propelled them at the forefront of gut mucosal homeostasis regulation, with important potential impact in gut physiopathology. This review focuses on intestinal tuft cells, from their initial description to the current understanding of their functions, and their potential impact in diseases.

The anthelmintic meclonazepam activates a schistosome transient receptor potential channel.

Parasitic flatworms cause various clinical and veterinary infections that impart a huge burden worldwide. The most clinically impactful infection is schistosomiasis, a neglected tropical disease caused by parasitic blood flukes. Schistosomiasis is treated with praziquantel (PZQ), an old drug introduced over 40 years ago. New drugs are urgently needed, as while PZQ is broadly effective it suffers from several limitations including poor efficacy against juvenile worms, which may prevent it from being completely curative. An old compound that retains efficacy against juvenile worms is the benzodiazepine meclonazepam (MCLZ). However, host side effects caused by benzodiazepines preclude development of MCLZ as a drug and MCLZ lacks an identified parasite target to catalyze rational drug design for engineering out human host activity. Here, we identify a transient receptor potential ion channel of the melastatin subfamily, named TRPMMCLZ, as a parasite target of MCLZ. MCLZ potently activates Schistosoma mansoni TRPMMCLZ through engagement of a binding pocket within the voltage-sensor-like domain of the ion channel to cause worm paralysis, tissue depolarization, and surface damage. TRPMMCLZ reproduces all known features of MCLZ action on schistosomes, including a lower activity versus Schistosoma japonicum, which is explained by a polymorphism within this voltage-sensor-like domain-binding pocket. TRPMMCLZ is distinct from the TRP channel targeted by PZQ (TRPMPZQ), with both anthelmintic chemotypes targeting unique parasite TRPM paralogs. This advances TRPMMCLZ as a novel druggable target that could circumvent any target-based resistance emerging in response to current mass drug administration campaigns centered on PZQ.

Leishmania profilin interacts with actin through an unusual structural mechanism to control cytoskeletal dynamics in parasites.

Diseases caused by Leishmania and Trypanosoma parasites are a major health problem in tropical countries. Because of their complex life cycle involving both vertebrate and insect hosts, and >1 billion years of evolutionarily distance, the cell biology of trypanosomatid parasites exhibits pronounced differences to animal cells. For example, the actin cytoskeleton of trypanosomatids is divergent when compared with other eukaryotes. To understand how actin dynamics are regulated in trypanosomatid parasites, we focused on a central actin-binding protein profilin. Co-crystal structure of Leishmania major actin in complex with L. major profilin revealed that, although the overall folds of actin and profilin are conserved in eukaryotes, Leishmania profilin contains a unique α-helical insertion, which interacts with the target binding cleft of actin monomer. This insertion is conserved across the Trypanosomatidae family and is similar to the structure of WASP homology-2 (WH2) domain, a small actin-binding motif found in many other cytoskeletal regulators. The WH2-like motif contributes to actin monomer binding and enhances the actin nucleotide exchange activity of Leishmania profilin. Moreover, Leishmania profilin inhibited formin-catalyzed actin filament assembly in a mechanism that is dependent on the presence of the WH2-like motif. By generating profilin knockout and knockin Leishmania mexicana strains, we show that profilin is important for efficient endocytic sorting in parasites, and that the ability to bind actin monomers and proline-rich proteins, and the presence of a functional WH2-like motif, are important for the in vivo function of Leishmania profilin. Collectively, this study uncovers molecular principles by which profilin regulates actin dynamics in trypanosomatids.