The Myosin Family of Mechanoenzymes: From Mechanisms to Therapeutic Approaches.

Myosins are among the most fascinating enzymes in biology. As extremely allosteric chemomechanical molecular machines, myosins are involved in myriad pivotal cellular functions and are frequently sites of mutations leading to disease phenotypes. Human ß-cardiac myosin has proved to be an excellent target for small-molecule therapeutics for heart muscle diseases, and, as we describe here, other myosin family members are likely to be potentially unique targets for treating other diseases as well. The first part of this review focuses on how myosins convert the chemical energy of ATP hydrolysis into mechanical movement, followed by a description of existing therapeutic approaches to target human ß-cardiac myosin. The next section focuses on the possibility of targeting nonmuscle members of the human myosin family for several diseases. We end the review by describing the roles of myosin in parasites and the therapeutic potential of targeting them to block parasitic invasion of their hosts.

Tuft cell acetylcholine is released into the gut lumen to promote anti-helminth immunity.

Upon parasitic helminth infection, activated intestinal tuft cells secrete interleukin-25 (IL-25), which initiates a type 2 immune response during which lamina propria type 2 innate lymphoid cells (ILC2s) produce IL-13. This causes epithelial remodeling, including tuft cell hyperplasia, the function of which is unknown. We identified a cholinergic effector function of tuft cells, which are the only epithelial cells that expressed choline acetyltransferase (ChAT). During parasite infection, mice with epithelial-specific deletion of ChAT had increased worm burden, fitness, and fecal egg counts, even though type 2 immune responses were comparable. Mechanistically, IL-13-amplified tuft cells release acetylcholine (ACh) into the gut lumen. Finally, we demonstrated a direct effect of ACh on worms, which reduced their fecundity via helminth-expressed muscarinic ACh receptors. Thus, tuft cells are sentinels in naive mice, and their amplification upon helminth infection provides an additional type 2 immune response effector function.

Differential plasticity and fate of brain-resident and recruited macrophages during the onset and resolution of neuroinflammation.

Microglia and border-associated macrophages (BAMs) are brain-resident self-renewing cells. Here, we examined the fate of microglia, BAMs, and recruited macrophages upon neuroinflammation and through resolution. Upon infection, Trypanosoma brucei parasites invaded the brain via its border regions, triggering brain barrier disruption and monocyte infiltration. Fate mapping combined with single-cell sequencing revealed microglia accumulation around the ventricles and expansion of epiplexus cells. Depletion experiments using genetic targeting revealed that resident macrophages promoted initial parasite defense and subsequently facilitated monocyte infiltration across brain barriers. These recruited monocyte-derived macrophages outnumbered resident macrophages and exhibited more transcriptional plasticity, adopting antimicrobial gene expression profiles. Recruited macrophages were rapidly removed upon disease resolution, leaving no engrafted monocyte-derived cells in the parenchyma, while resident macrophages progressively reverted toward a homeostatic state. Long-term transcriptional alterations were limited for microglia but more pronounced in BAMs. Thus, brain-resident and recruited macrophages exhibit diverging responses and dynamics during infection and resolution.

A Genome-wide CRISPR Screen in Toxoplasma Identifies Essential Apicomplexan Genes.

Apicomplexan parasites are leading causes of human and livestock diseases such as malaria and toxoplasmosis, yet most of their genes remain uncharacterized. Here, we present the first genome-wide genetic screen of an apicomplexan. We adapted CRISPR/Cas9 to assess the contribution of each gene from the parasite Toxoplasma gondii during infection of human fibroblasts. Our analysis defines ∼200 previously uncharacterized, fitness-conferring genes unique to the phylum, from which 16 were investigated, revealing essential functions during infection of human cells. Secondary screens identify as an invasion factor the claudin-like apicomplexan microneme protein (CLAMP), which resembles mammalian tight-junction proteins and localizes to secretory organelles, making it critical to the initiation of infection. CLAMP is present throughout sequenced apicomplexan genomes and is essential during the asexual stages of the malaria parasite Plasmodium falciparum. These results provide broad-based functional information on T. gondii genes and will facilitate future approaches to expand the horizon of antiparasitic interventions.

Defensive-lipid droplets: Cellular organelles designed for antimicrobial immunity.

Microbes have developed many strategies to subvert host organisms, which, in turn, evolved several innate immune responses. As major lipid storage organelles of eukaryotes, lipid droplets (LDs) are an attractive source of nutrients for invaders. Intracellular viruses, bacteria, and protozoan parasites induce and physically interact with LDs, and the current view is that they "hijack" LDs to draw on substrates for host colonization. This dogma has been challenged by the recent demonstration that LDs are endowed with a protein-mediated antibiotic activity, which is upregulated in response to danger signals and sepsis. Dependence on host nutrients could be a generic "Achilles' heel" of intracellular pathogens and LDs a suitable chokepoint harnessed by innate immunity to organize a front-line defense. Here, we will provide a brief overview of the state of the conflict and discuss potential mechanisms driving the formation of the 'defensive-LDs' functioning as hubs of innate immunity.

The direct binding of Plasmodium vivax AMA1 to erythrocytes defines a RON2-independent invasion pathway.

We used a transgenic parasite in which Plasmodium falciparum parasites were genetically modified to express Plasmodium vivax apical membrane antigen 1 (PvAMA1) protein in place of PfAMA1 to study PvAMA1-mediated invasion. In P. falciparum, AMA1 interaction with rhoptry neck protein 2 (RON2) is known to be crucial for invasion, and PfRON2 peptides (PfRON2p) blocked the invasion of PfAMA1 wild-type parasites. However, PfRON2p has no effect on the invasion of transgenic parasites expressing PvAMA1 indicating that PfRON2 had no role in the invasion of PvAMA1 transgenic parasites. Interestingly, PvRON2p blocked the invasion of PvAMA1 transgenic parasites in a dose-dependent manner. We found that recombinant PvAMA1 domains 1 and 2 (rPvAMA1) bound to reticulocytes and normocytes indicating that PvAMA1 directly interacts with erythrocytes during the invasion, and invasion blocking of PvRON2p may result from it interfering with PvAMA1 binding to erythrocytes. It was previously shown that the peptide containing Loop1a of PvAMA1 (PvAMA1 Loop1a) is also bound to reticulocytes. We found that the Loop1a peptide blocked the binding of PvAMA1 to erythrocytes. PvAMA1 Loop1a has no polymorphisms in contrast to other PvAMA1 loops and may be an attractive vaccine target. We thus present the evidence that PvAMA1 binds to erythrocytes in addition to interacting with PvRON2 suggesting that the P. vivax merozoites may exploit complex pathways during the invasion process.

The mitochondrion of Plasmodium falciparum is required for cellular acetyl-CoA metabolism and protein acetylation.

Coenzyme A (CoA) biosynthesis is an excellent target for antimalarial intervention. While most studies have focused on the use of CoA to produce acetyl-CoA in the apicoplast and the cytosol of malaria parasites, mitochondrial acetyl-CoA production is less well understood. In the current study, we performed metabolite-labeling experiments to measure endogenous metabolites in Plasmodium falciparum lines with genetic deletions affecting mitochondrial dehydrogenase activity. Our results show that the mitochondrion is required for cellular acetyl-CoA biosynthesis and identify a synthetic lethal relationship between the two main ketoacid dehydrogenase enzymes. The activity of these enzymes is dependent on the lipoate attachment enzyme LipL2, which is essential for parasite survival solely based on its role in supporting acetyl-CoA metabolism. We also find that acetyl-CoA produced in the mitochondrion is essential for the acetylation of histones and other proteins outside of the mitochondrion. Taken together, our results demonstrate that the mitochondrion is required for cellular acetyl-CoA metabolism and protein acetylation essential for parasite survival.

Where is the EXIT? Phenotypic screens for new egress factors in apicomplexan parasites.

Apicomplexans, such as Plasmodium and Toxoplasma are obligate intracellular parasites that invade, replicate and finally EXIT their host cell. During replication within a parasitophorous vacuole (PV), the parasites establish an extensive F-actin-containing network that connects individual parasites and is required for material exchange, recycling and the final steps of daughter cell assembly. After multiple rounds of replication, the parasites exit the host cell involving multiple signalling cascades, disassembly of the network, secretion of microneme proteins and activation of the acto-myosin motor. Blocking the host cell EXIT process leads to the formation of large PVs, making the screening for genes involved in exiting the cell relatively straightforward. Given that apicomplexans are highly diverse from other eukaryotes, approximately 30% of all genes are annotated as hypothetical, some apicomplexan-specific factors are likely to be critical during EXIT. This motivated several labs to design and perform forward genetic and phenotypic screens using various approaches, such as random insertion mutagenesis, temperature-sensitive mutants and, more recently, CRISPR/Cas9-mediated targeted editing and conditional mutagenesis. Here we will provide an overview of the technological developments over recent years and the most successful stories that led to the identification of new critical factors in Toxoplasma gondii.

Glycan Complexity and Heterogeneity of Glycoproteins in Somatic Extracts and Secretome of the Infective Stage of the Helminth Fasciola hepatica.

Fasciola hepatica is a global helminth parasite of humans and their livestock. The invasive stage of the parasite, the newly excysted juvenile (NEJs), relies on glycosylated excreted-secreted (ES) products and surface/somatic molecules to interact with host cells and tissues and to evade the host's immune responses, such as disarming complement and shedding bound antibody. While -omics technologies have generated extensive databases of NEJs' proteins and their expression, detailed knowledge of the glycosylation of proteins is still lacking. Here, we employed glycan, glycopeptide, and proteomic analyses to determine the glycan profile of proteins within the NEJs' somatic (Som) and ES extracts. These analyses characterized 123 NEJ glycoproteins, 71 of which are secreted proteins, and allowed us to map 356 glycopeptides and their associated 1690 N-glycan and 37 O-glycan forms to their respective proteins. We discovered abundant micro-heterogeneity in the glycosylation of individual glycosites and between different sites of multi-glycosylated proteins. The global heterogeneity across NEJs' glycoproteome was refined to 53 N-glycan and 16 O-glycan structures, ranging from highly truncated paucimannosidic structures to complex glycans carrying multiple phosphorylcholine (PC) residues, and included various unassigned structures due to unique linkages, particularly in pentosylated O-glycans. Such exclusive glycans decorate some well-known secreted molecules involved in host invasion, including cathepsin B and L peptidases, and a variety of membrane-bound glycoproteins, suggesting that they participate in host interactions. Our findings show that F. hepatica NEJs generate exceptional protein variability via glycosylation, suggesting that their molecular portfolio that communicates with the host is far more complex than previously anticipated by transcriptomic and proteomic analyses. This study opens many avenues to understand the glycan biology of F. hepatica throughout its life-stages, as well as other helminth parasites, and allows us to probe the glycosylation of individual NEJs proteins in the search for innovative diagnostics and vaccines against fascioliasis.

Human Passage of Schistosoma incognitum, Tamil Nadu, India, and Review of Autochthonous Schistosomiasis, South Asia.

A fecal survey in Tamil Nadu, India, revealed 2 persons passed schistosome eggs, later identified as Schistosoma incognitum, a parasite of pigs, dogs, and rats. We investigated those cases and reviewed autochthonous schistosomiasis cases from India and Nepal. Whether the 2 new cases represent true infection or spurious passage is undetermined.

Taenia martis Neurocysticercosis-Like Lesion in Child, Associated with Local Source, the Netherlands.

A neurocysticercosis-like lesion in an 11-year-old boy in the Netherlands was determined to be caused by the zoonotic Taenia martis tapeworm. Subsequent testing revealed that 15% of wild martens tested in that region were infected with T. martis tapeworms with 100% genetic similarity; thus, the infection source was most likely local.

Source Tracing of Leishmania donovani in Emerging Foci of Visceral Leishmaniasis, Western Nepal.

We sequenced Leishmania donovani genomes in blood samples collected in emerging foci of visceral leishmaniasis in western Nepal. We detected lineages very different from the preelimination main parasite population, including a new lineage and a rare one previously reported in eastern Nepal. Our findings underscore the need for genomic surveillance.

Successful Treatment of Confirmed Naegleria fowleri Primary Amebic Meningoencephalitis.

Primary amebic meningoencephalitis caused by Naegleria fowleri is a rare but nearly always fatal parasitic infection of the brain. Globally, few survivors have been reported, and the disease has no specific treatment. We report a confirmed case in Pakistan in a 22-year-old man who survived after aggressive therapy.

Confirmed Autochthonous Case of Human Alveolar Echinococcosis, Italy, 2023.

In September 2023, a patient in Italy who had never traveled abroad was referred for testing for suspected hepatic cystic echinococcosis. Lesions were incompatible with cystic echinococcosis; instead, autochthonous alveolar echinococcosis was confirmed. Alveolar echinococcosis can be fatal, and awareness must be raised of the infection's expanding distribution.

Wuchereria bancrofti Lymphatic Filariasis, Barrancabermeja, Colombia, 2023.

We describe a recent case of lymphatic filariasis in Colombia caused by Wuchereria bancrofti nematodes. Our study combines clinical-epidemiologic findings with phylogenetic data. Resurgence of lymphatic filariasis may be linked to increasing urbanization trends and migration from previously endemic regions. Fieldwork can be a beneficial tool for screening and containing transmission.

Effect of Rodent Control Program on Incidence of Zoonotic Cutaneous Leishmaniasis, Iran.

We report the effect of a rodent control program on the incidence of zoonotic cutaneous leishmaniasis in an endemic region of Iran. A 1-year interruption in rodent control led to 2 years of increased incidence of zoonotic cutaneous leishmaniasis. Restarting rodent control led to a decline of zoonotic cutaneous leishmaniasis.

Molecular Confirmation of Anopheles stephensi Mosquitoes in the Al Hudaydah Governorate, Yemen, 2021 and 2022.

We detected malaria vector Anopheles stephensi mosquitoes in the Al Hudaydah governorate in Yemen by using DNA sequencing. We report 2 cytochrome c oxidase subunit I haplotypes, 1 previously found in Ethiopia, Somalia, Djibouti, and Yemen. These findings provide insight into invasive An. stephensi mosquitoes in Yemen and their connection to East Africa.

Disseminated Leishmaniasis, a Severe Form of Leishmania braziliensis Infection.

Disseminated leishmaniasis (DL) is an emergent severe disease manifesting with multiple lesions. To determine the relationship between immune response and clinical and therapeutic outcomes, we studied 101 DL and 101 cutaneous leishmaniasis (CL) cases and determined cytokines and chemokines in supernatants of mononuclear cells stimulated with leishmania antigen. Patients were treated with meglumine antimoniate (20 mg/kg) for 20 days (CL) or 30 days (DL); 19 DL patients were instead treated with amphotericin B, miltefosine, or miltefosine and meglumine antimoniate. High levels of chemokine ligand 9 were associated with more severe DL. The cure rate for meglumine antimoniate was low for both DL (44%) and CL (60%), but healing time was longer in DL (p = 0.003). The lowest cure rate (22%) was found in DL patients with >100 lesions. However, meglumine antimoniate/miltefosine treatment cured all DL patients who received it; therefore, that combination should be considered as first choice therapy.

Enterocytozoon bieneusi Infection after Hematopoietic Stem Cell Transplant in Child, Argentina.

We report a case of Enterocytozoon bieneusi infection in a pediatric hematopoietic stem cell transplant recipient in Argentina. Spores were visualized in feces using Calcofluor White and modified trichrome stainings. PCR and sequencing identified E. bieneusi genotype D in fecal samples and liver samples, confirming extraintestinal dissemination of the parasite.

Emergence of Thelaziosis Caused by Thelazia callipaeda in Dogs and Cats, United States.

We report 2 autochthonous feline thelaziosis cases caused by the eyeworm Thelazia callipaeda and discuss the spread among dogs in the northeastern United States. Phylogenetic analysis suggests the parasite was introduced from Europe. Adopting a One Health approach is needed to limit further spread of T. callipaeda eyeworms in North America.