Parasite Multiplication Research Articles

The Description of Microbial Pattern and Antibiotic Sensitivity Among Pediatric Patients at RSUP M Djamil Padang

Background Infection alludes to the invasion and multiplication of either microorganisms or parasites inside bodily tissues. The management of such infections is closely interrelated to the use of antibiotics. Ineffective use of antibiotics is contributed to the antibiotic resistance. Severe disease complications and lengthy hospital stays are among essential reasons why physicians may prescribe myriad types of antibiotics for longer durations. In effort to diminish resistance, the selection of antibiotics ought to be based on information about the bacterial spectrum causing the infection and the patterns of sensitivity to antibiotics. Annual reporting of microbial patterns and sensitivity testing is essential, particularly for pediatric ward patients, as it provides a guide for administering relevant antibiotics and preventing further spread of infections. Objectives This study evaluated microbial patterns, sensitivity test results, and antibiotic resistance among patients in the pediatric ward of RSUP M Djamil Padang from January-December 2020, with a total of 138 samples. Results The proportion of gram-negative and gram-positive bacteria are 83.3% and 16.7%, respectively. The most prevalent gram-negative bacteria are Klebsiella pneumoniae (28.9%), Escherichia coli (26.1%). Pseudomonas aeruginosa (7.9%), Staphylococcus epidermidis (7.2%) and Acinetobacter baumannii (3.7%) also present. These bacteria are predominantly found in samples of feces, urine, and blood. Gram-positive bacteria show notable resistance to the antibiotics such as erythromycin (22.2%), trimethoprim-sulfamethoxazole (19%), and ciprofloxacin (15.8%), while being sensitive to chloramphenicol (17.4%), vancomycin (15.1%), and tetracycline (12%). In contrast, gram-negative bacteria exhibit resistance to ampicillin (16.5%), ceftriaxone (12.1%), and ceftazidime (11.3%), but are sensitive to amikacin (19.8%), meropenem (19.4%), and gentamicin (10.9%). Keywords: microbial patterns, antibiotic sensitivity, pediatric ward

Pyrimidine Azepine Targets the Plasmodium bc 1 Complex and Displays Multistage Antimalarial Activity.

Malaria control and elimination efforts would benefit from the identification and validation of new malaria chemotherapeutics. Recently, a transgenic Plasmodium berghei line was used to perform a series of high-throughput in vitro screens for new antimalarials acting against the parasite sexual stages. The screens identified pyrimidine azepine chemotypes with potent activity. Here, we validate the activity of PyAz90, the most potent pyrimidine azepine chemotype identified, against P. falciparum and P. vivax in the asexual and sexual stages. PyAz90 blocked parasite transmission to the mosquito vector at nanomolar concentrations and inhibited in vitro asexual parasite multiplication with a fast-action profile. Through the generation of P. falciparum PyAz90-resistant parasites and in vitro assays of mitochondrial activity, we identified cytochrome b as a molecular target of PyAz90. This work characterizes a promising chemotype that can be explored for the future development of new antimalarials targeting the Plasmodium cytochrome bc 1 complex.

Effects of Amphotericin B-Conjugated Functionalized Carbon Nanoparticles in the Treatment of Cutaneous Leishmaniasis.

Leishmaniasis is a parasitic disease spread by the bite of an infected sandfly and caused by protozoan parasites of the genus Leishmania. Currently, there is no vaccine available for leishmaniasis in humans, and the existing chemotherapy methods face various clinical challenges. The majority of drugs are limited to a few toxic compounds, with some parasite strains developing resistance. Therefore, the discovery and development of a new anti-leishmanial compound is crucial. One promising strategy involves the use of nanoparticle delivery systems to accelerate the effectiveness of existing treatments. In this study, Amphotericin B (AmB) was incorporated into functionalized carbon nanotube (f-CNT) and evaluated for its efficacy against Leishmania major in vitro and in a BALB/c mice model. The increase in footpad thickness was measured, and real-time PCR was used to quantify the parasite load post-infection. Levels of nitric oxide and cytokines IL-4 and IFN-γ were also determined. We found that f-CNT-AmB significantly reduced the levels of promastigotes and amastigotes of the Leishmania parasite. The nanoparticle showed strong anti-leishmanial activity with an IC50 of 0.00494 ± 0.00095 mg/mL for promastigotes and EC50 of 0.00294 ± 0.00065 mg/mL for amastigotes at 72 h post-infection, without causing harm to mice macrophages. Treatment of infected BALB/c mice with f-CNT-AmB resulted in a significant decrease in cutaneous leishmania (CL) lesion size in the foot pad, as well as reduced Leishmania burden in both lymph nodes and spleen. The levels of nitric oxide and IFN-γ significantly increased in the f-CNT-AmB treated groups. Also, our results showed that the level of IL-4 significantly decreased after f-CNT-AmB treatment in comparison to other groups. In conclusion, our results demonstrate that AmB loaded into f-CNT is significantly more effective than AmB alone in inhibiting parasite propagation and promoting a shift towards a Th1 response.

Application of optical tweezer technology reveals that PfEBA and PfRH ligands, not PfMSP1, play a central role in Plasmodium falciparum merozoite-erythrocyte attachment.

Malaria pathogenesis and parasite multiplication depend on the ability of Plasmodium merozoites to invade human erythrocytes. Invasion is a complex multi-step process involving multiple parasite proteins which can differ between species and has been most extensively studied in P. falciparum. However, dissecting the precise role of individual proteins has to date been limited by the availability of quantifiable phenotypic assays. In this study, we apply a new approach to assigning function to invasion proteins by using optical tweezers to directly manipulate recently egressed P. falciparum merozoites and erythrocytes and quantify the strength of attachment between them, as well as the frequency with which such attachments occur. Using a range of inhibitors, antibodies, and genetically modified strains including some generated specifically for this work, we quantitated the contribution of individual P. falciparum proteins to these merozoite-erythrocyte attachment interactions. Conditional deletion of the major P. falciparum merozoite surface protein PfMSP1, long thought to play a central role in initial attachment, had no impact on the force needed to pull merozoites and erythrocytes apart, whereas interventions that disrupted the function of several members of the EBA-175 like Antigen (PfEBA) family and Reticulocyte Binding Protein Homologue (PfRH) invasion ligand families did have a significant negative impact on attachment. Deletion of individual PfEBA and PfRH ligands reinforced the known redundancy within these families, with the deletion of some ligands impacting detachment force while others did not. By comparing over 4000 individual merozoite-erythrocyte interactions in a range of conditions and strains, we establish that the PfEBA/PfRH families play a central role in P. falciparum merozoite attachment, not the major merozoite surface protein PfMSP1.

Molecular, structural, and functional characterization of delta subunit of T-complex protein-1 from Leishmania donovani.

Chaperonins/Heat shock protein 60 are ubiquitous multimeric protein complexes that assist in the folding of partially and/or misfolded proteins using metabolic energy into their native stage. The eukaryotic group II chaperonin, also referred as T-complex protein-1 ring complex (TRiC)/T-complex protein-1 (TCP1)/chaperonin containing T-complex protein (CCT), contains 8-9 paralogous subunits, arranged in each of the two rings of hetero-oligomeric complex. In Leishmania, till date, only one subunit, LdTCP1γ, has been well studied. Here, we report the molecular, structural, and functional characterization of TCP1δ subunit of Leishmania donovani (LdTCP1δ), the causative agent of Indian kala-azar. LdTCP1δ gene exhibited only 27.9% identity with LdTCP1γ and clustered in a separate branch in the phylogenic tree of LdTCP1 subunits. The purified recombinant protein formed a high molecular weight complex (0.75 MDa), arranged into 16-mer assembly, and performed in vitro chaperonin activity as assayed by ATP-dependent luciferase folding. LdTCP1δ exhibits 1.8-fold upregulated expression in metabolically active, rapidly dividing log phase promastigotes. Over-expression of LdTCP1δ in promastigotes results in increased infectivity and rate of multiplication of intracellular amastigotes. The study thus establishes the existence of an individual functionally active homo-oligomeric complex of LdTCP1δ chaperonin with its role in parasite infectivity and multiplication.

Toxoplasma acyl-CoA synthetase TgACS3 is crucial to channel host fatty acids in lipid droplets and for parasite propagation

Apicomplexa comprise important pathogenic parasitic protists that heavily depend on lipid acquisition to survive within their human host cells. Lipid synthesis relies on the incorporation of an essential combination of fatty acids (FAs) either generated by a metabolically adaptable de novo synthesis in the parasite or by scavenging from the host cell. The incorporation of FAs into membrane lipids depends on their obligate metabolic activation by specific enzyme groups, acyl-CoA synthetases (ACSs). Each ACS has its own specificity, so it can fulfill specific metabolic functions. Whilst such functionalities have been well studied in other eukaryotic models, their roles and importance in Apicomplexa are currently very limited, especially for Toxoplasma gondii. Here, we report the identification of seven putative ACSs encoded by the genome of T. gondii (TgACS), which localize to different sub-cellular compartments of the parasite, suggesting exclusive functions. We show that the perinuclear/cytoplasmic TgACS3 regulates the replication and growth of Toxoplasma tachyzoites. Conditional disruption of TgACS3 shows that the enzyme is required for parasite propagation and survival, especially under high host nutrient content. Lipidomic analysis of parasites lacking TgACS3 reveals its role in the activation of host-derived FAs that are used for i) parasite membrane phospholipid and ii) storage triacylglycerol (TAG) syntheses, allowing proper membrane biogenesis of parasite progenies. Altogether, our results reveal the role of TgACS3 as the bulk FA activator for membrane biogenesis allowing intracellular division and survival in T. gondii tachyzoites, further pointing to the importance of ACS and FA metabolism for the parasite.

NDV targets the invasion pathway in malaria parasite through cell surface sialic acid interaction.

Merozoites utilize sialic acids on the red blood cell (RBC) cell surface to rapidly adhere to and invade the RBCs. Newcastle disease virus (NDV) displays a strong affinity toward membrane-bound sialic acids. Incubation of NDV with the malaria parasites dose-dependently reduces its cellular viability. The antiplasmodial activity of NDV is specific, as incubation with Japanese encephalitis virus, duck enteritis virus, infectious bronchitis virus, and influenza virus did not affect the parasite propagation. Interestingly, NDV is reducing more than 80% invasion when RBCs are pretreated with the virus. Removal of the RBC surface proteins or the NDV coat proteins results in disruption of the virus binding to RBC. It suggests the involvement of specific protein: ligand interaction in virus binding. We established that the virus engages with the parasitized RBCs (PRBCs) through its hemagglutinin neuraminidase (HN) protein by recognizing sialic acid-containing glycoproteins on the cell surface. Blocking of the HN protein with free sialic acid or anti-HN antibodies abolished the virus binding as well as its ability to reduce parasite growth. Interestingly, the purified HN from the virus alone could inhibit the parasite's growth in a dose-dependent manner. NDV binds strongly to knobless murine parasite strain Plasmodium yoelii and restricted the parasite growth in mice. Furthermore, the virus was found to preferentially target the PRBCs compared to normal erythrocytes. Immunolocalization studies reveal that NDV is localized on the plasma membrane as well as weakly inside the PRBC. NDV causes neither any infection nor aggregation of the human RBCs. Our findings suggest that NDV is a potential candidate for developing targeted drug delivery platforms for the Plasmodium-infected RBCs.

Mixed malaria: the paradox of visual diagnosis in the tropics.

Malaria is the parasitic disease with the greatest impact on humans. It is an infectious disease caused by protozoa of the genus Plasmodium spp. and transmitted to humans by the bite of the mosquito Anopheles spp. Its incidence has increased in the tropical regions of Africa and Southeast Asia, areas already defined by the World Health Organization (WHO) as malarial zones [1]. In these places, malaria generally disappears at altitudes above 2.000 meters above sea level. The most frequently circulating parasites are P. vivax and P. falciparum; P. malarie is also widely distributed, but less frequent. In West Africa, P. ovale replaces P. vivax. The most widespread species in the world is P. vivax, which has been found from sea level to 2.770 meters above sea level. The endemic areas, however, are generally in the tropics. By definition, the number of parasites found in the peripheral blood depends on the species: the highest number corresponds to P. falciparum which infects 10 to 40% of all red blood cells; it is worth mentioning that when parasitaemia of this species reaches more than 25%, it is usually fatal [2]. Multiple invasion of red blood cells is frequent with P. falciparum, rare with P. vivax (which has a predominance of reticulocytes) and very rare with P. malariae [1- 3]. Without treatment, maximum parasite multiplication is reached in 2 weeks in P. vivax, and in 10 days in the case of P. falciparum. The simultaneous presence of erythrocytic parasitic forms (asexual and/or sexual) of two or more species of Plasmodium spp. is called mixed plasmodial infection. If, in addition, there are symptoms such as fever, chills, headache, sweating, among others, it corresponds to the disease called mixed malaria. In the world, the prevalence of this last entity is uncertain, depending on the diagnostic method used: 2% by light microscopy (e.g., thick blood smear [TBS] or peripheral blood smear) and up to 65% by polymerase chain reaction (PCR) [4]. In Latin America, prevalence is estimated at 0.46% by TBS and 12.8% by PCR. In terms of performance, the thick smear is 20-30 times more sensitive than the thin smear (peripheral blood smear), although less specific for the identification of the erythrocytic asexual (young rings or trophozoites, mature trophozoites, schizonts) and sexual (gametocytes) forms of the five species of Plasmodium spp. that parasitize humans (P. falciparum, P. vivax, P. ovale, P. malariae and P. knowlesi), which explains an underdiagnosis of both clinical scenarios (both plasmodial infection and mixed malaria) by light microscopy, with serious implications for diagnostic and therapeutic guidance. Nevertheless, some methods have been developed to improve the diagnostic capability of TBS [2,3]: • Fluorescent stains (acridine orange): used to increase sensitivity of point-of-care diagnosis, without improving specificity of species identification; additionally, some stains may be toxic. • Fluorescence staining + microcentrifuge: increases diagnostic speed and sensitivity for P. falciparum; reduces sensitivity for other species. • Magnetic deposition: takes advantage of the magnetic characteristics of hemozoin to precipitate the parasites to the plate with a magnet. It is an inexpensive method. It increases sensitivity; however, it is not species specific, without good representation of ring stage parasites.

Breadth of Fc-mediated effector function correlates with clinical immunity following human malaria challenge

Malaria is a life-threatening disease of global health importance, particularly in sub-Saharan Africa. The growth inhibition assay (GIA) is routinely used to evaluate, prioritize, and quantify the efficacy of malaria blood-stage vaccine candidates but does not reliably predict either naturally acquired or vaccine-induced protection. Controlled human malaria challenge studies in semi-immune volunteers provide an unparalleled opportunity to robustly identify mechanistic correlates of protection. We leveraged this platform to undertake a head-to-head comparison of seven functional antibody assays that are relevant to immunity against the erythrocytic merozoite stage of Plasmodium falciparum. Fc-mediated effector functions were strongly associated with protection from clinical symptoms of malaria and exponential parasite multiplication, while the gold standard GIA was not. The breadth of Fc-mediated effector function discriminated clinical immunity following the challenge. These findings present a shift in the understanding of the mechanisms that underpin immunity to malaria and have important implications for vaccine development.

A longer-chain acylated derivative of Dictyostelium differentiation-inducing factor-1 enhances the antimalarial activity against Plasmodium parasites

The spread of malarial parasites resistant to first-line treatments such as artemisinin combination therapies is a global health concern. Differentiation-inducing factor 1 (DIF-1) is a chlorinated alkylphenone (1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl) hexan-1-one) originally found in the cellular slime mould Dictyostelium discoideum. We previously showed that some derivatives of DIF-1, particularly DIF-1(+2) (1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl) octan-1-one), exert potent antimalarial activities. In this study, we synthesised DIF-1(+3) (1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl) nonan-1-one). We then evaluated the effects of DIF-1(+3) in vitro on Plasmodium falciparum and in vivo over 7 days (50–100 mg/kg/day) in a mouse model of Plasmodium berghei. DIF-1(+3) exhibited a half-maximal inhibitory concentration of approximately 20–30 % of DIF-1(+2) in three laboratory strains with a selectivity index > 263, including in strains resistant to chloroquine and artemisinin. Parasite growth and multiplication were almost completely suppressed by treatment with 100 mg/kg DIF-1(+3). The survival time of infected mice was significantly increased (P = 0.006) with no apparent adverse effects. In summary, addition of an acyl group to DIF-1(+2) to prepare DIF-1(+3) substantially enhanced antimalarial activity, even in drug-resistant malaria, indicating the potential of applying DIF-1(+3) for malaria treatment.

The essential malaria protein PfCyRPA targets glycans to invade erythrocytes

Plasmodium falciparum is a human-adapted apicomplexan parasite that causes the most dangerous form of malaria. P.falciparum cysteine-rich protective antigen (PfCyRPA) is an invasion complex protein essential for erythrocyte invasion. The precise role of PfCyRPA in this process has not been resolved. Here, we show that PfCyRPA is a lectin targeting glycans terminating with α2-6-linked N-acetylneuraminic acid (Neu5Ac). PfCyRPA has a >50-fold binding preference for human, α2-6-linked Neu5Ac over non-human, α2-6-linked N-glycolylneuraminic acid. PfCyRPA lectin sites were predicted by molecular modeling and validated by mutagenesis studies. Transgenic parasite lines expressing endogenous PfCyRPA with single amino acid exchange mutants indicated that the lectin activity of PfCyRPA has an important role in parasite invasion. Blocking PfCyRPA lectin activity with small molecules or with lectin-site-specific monoclonal antibodies can inhibit blood-stage parasite multiplication. Therefore, targeting PfCyRPA lectin activity with drugs, immunotherapy, or a vaccine-primed immune response is a promising strategy to prevent and treat malaria.

The acyl-CoA synthetase TgACS1 allows neutral lipid metabolism and extracellular motility in Toxoplasma gondii through relocation via its peroxisomal targeting sequence (PTS) under low nutrient conditions.

Apicomplexa parasites cause major diseases such as toxoplasmosis and malaria that have major health and economic burdens. These unicellular pathogens are obligate intracellular parasites that heavily depend on lipid metabolism for the survival within their hosts. Their lipid synthesis relies on an essential combination of fatty acids (FAs) obtained from both de novo synthesis and scavenging from the host. The constant flux of scavenged FA needs to be channeled toward parasite lipid storage, and these FA storages are timely mobilized during parasite division. In eukaryotes, the utilization of FA relies on their obligate metabolic activation mediated by acyl-co-enzyme A (CoA) synthases (ACSs), which catalyze the thioesterification of FA to a CoA. Besides the essential functions of FA for parasite survival, the presence and roles of ACS are yet to be determined in Apicomplexa. Here, we identified TgACS1 as a Toxoplasma gondii cytosolic ACS that is involved in FA mobilization in the parasite specifically during low host nutrient conditions, especially in extracellular stages where it adopts a different localization. Heterologous complementation of yeast ACS mutants confirmed TgACS1 as being an Acyl-CoA synthetase of the bubble gum family that is most likely involved in β-oxidation processes. We further demonstrate that TgACS1 is critical for gliding motility of extracellular parasite facing low nutrient conditions, by relocating to peroxisomal-like area.IMPORTANCEToxoplasma gondii, causing human toxoplasmosis, is an Apicomplexa parasite and model within this phylum that hosts major infectious agents, such as Plasmodium spp., responsible for malaria. The diseases caused by apicomplexans are responsible for major social and economic burdens affecting hundreds of millions of people, like toxoplasmosis chronically present in about one-third of the world's population. Lack of efficient vaccines, rapid emergence of resistance to existing treatments, and toxic side effects of current treatments all argue for the urgent need to develop new therapeutic tools to combat these diseases. Understanding the key metabolic pathways sustaining host-intracellular parasite interactions is pivotal to develop new efficient ways to kill these parasites. Current consensus supports parasite lipid synthesis and trafficking as pertinent target for novel treatments. Many processes of this essential lipid metabolism in the parasite are not fully understood. The capacity for the parasites to sense and metabolically adapt to the host physiological conditions has only recently been unraveled. Our results clearly indicate the role of acyl-co-enzyme A (CoA) synthetases for the essential metabolic activation of fatty acid (FA) used to maintain parasite propagation and survival. The significance of our research is (i) the identification of seven of these enzymes that localize at different cellular areas in T. gondii parasites; (ii) using lipidomic approaches, we show that TgACS1 mobilizes FA under low host nutrient content; (iii) yeast complementation showed that acyl-CoA synthase 1 (ACS1) is an ACS that is likely involved in peroxisomal β-oxidation; (iv) the importance of the peroxisomal targeting sequence for correct localization of TgACS1 to a peroxisomal-like compartment in extracellular parasites; and lastly, (v) that TgACS1 has a crucial role in energy production and extracellular parasite motility.

In vitro assay to determine inactivation of Toxoplasma gondii in meat samples

Consumption of raw and undercooked meat is considered as an important source of Toxoplasma gondii infections. However, most non-heated meat products contain salt and additives, which affect T. gondii viability. It was our aim to develop an in vitro method to substitute the mouse bioassay for determining the effect of salting on T. gondii viability. Two sheep were experimentally infected by oral inoculation with 6.5 × 104 oocysts. Grinded meat samples of 50 g were prepared from heart, diaphragm, and four meat cuts. Also, pooled meat samples were either kept untreated (positive control), frozen (negative control) or supplemented with 0.6 %, 0.9 %, 1.2 % or 2.7 % NaCl. All samples were digested in pepsin-HCl solution, and digests were inoculated in duplicate onto monolayers of RK13 (a rabbit kidney cell line). Cells were maintained for up to four weeks and parasite growth was monitored by assessing Cq-values using the T. gondii qPCR on cell culture supernatant in intervals of one week and ΔCq-values determined. Additionally, 500 μL of each digest from the individual meat cuts, heart and diaphragm were inoculated in duplicate in IFNγ KO mice. Both sheep developed an antibody response and tissue samples contained similar concentrations of T. gondii DNA. From all untreated meat samples positive ΔCq-values were obtained in the in vitro assay, indicating presence and multiplication of viable parasites. This was in line with the mouse bioassay, with the exception of a negative mouse bioassay on one heart sample. Samples supplemented with 0.6 %–1.2 % NaCl showed positive ΔCq-values over time. The frozen sample and the sample supplemented with 2.7 % NaCl remained qPCR positive but with high Cq-values, which indicated no growth. In conclusion, the in vitro method has successfully been used to detect viable T. gondii in tissues of experimentally infected sheep, and a clear difference in T. gondii viability was observed between the samples supplemented with 2.7 % NaCl and those with 1.2 % NaCl or less.

Stearoyl-CoA desaturase regulates organelle biogenesis and hepatic merozoite formation in Plasmodium berghei.

Plasmodium is an obligate intracellular parasite that requires intense lipid synthesis for membrane biogenesis and survival. One of the principal membrane components is oleic acid, which is needed to maintain the membrane's biophysical properties and fluidity. The malaria parasite can modify fatty acids, and stearoyl-CoA Δ9-desaturase (Scd) is an enzyme that catalyzes the synthesis of oleic acid by desaturation of stearic acid. Scd is dispensable in P. falciparum blood stages; however, its role in mosquito and liver stages remains unknown. We show that P. berghei Scd localizes to the ER in the blood and liver stages. Disruption of Scd in the rodent malaria parasite P. berghei did not affect parasite blood stage propagation, mosquito stage development, or early liver-stage development. However, when Scd KO sporozoites were inoculated intravenously or by mosquito bite into mice, they failed to initiate blood-stage infection. Immunofluorescence analysis revealed that organelle biogenesis was impaired and merozoite formation was abolished, which initiates blood-stage infections. Genetic complementation of the KO parasites restored merozoite formation to a level similar to that of WT parasites. Mice immunized with Scd KO sporozoites confer long-lasting sterile protection against infectious sporozoite challenge. Thus, the Scd KO parasite is an appealing candidate for inducing protective pre-erythrocytic immunity and hence its utility as a GAP.

Leishmania highjack host lipid body for its proliferation in macrophages by overexpressing host Rab18 and TRAPPC9 by downregulating miR-1914-3p expression.

Lipids stored in lipid-bodies (LBs) in host cells are potential sources of fatty acids for pathogens. However, the mechanism of recruitment of LBs from the host cells by pathogens to acquire fatty acids is not known. Here, we have found that Leishmania specifically upregulates the expression of host Rab18 and its GEF, TRAPPC9 by downregulating the expression of miR-1914-3p by reducing the level of Dicer in macrophages via their metalloprotease gp63. Our results also show that miR-1914-3p negatively regulates the expression of Rab18 and its GEF in cells. Subsequently, Leishmania containing parasitophorous vacuoles (Ld-PVs) recruit and retain host Rab18 and TRAPPC9. Leishmania infection also induces LB biogenesis in host cells and recruits LBs on Ld-PVs and acquires FLC12-labeled fatty acids from LBs. Moreover, overexpression of miR-1914-3p in macrophages significantly inhibits the recruitment of LBs and thereby suppresses the multiplication of parasites in macrophages as parasites are unable to acquire fatty acids. These results demonstrate a novel mechanism how Leishmania acquire fatty acids from LBs for their growth in macrophages.

Prodomain-driven enzyme dimerization: a pH-dependent autoinhibition mechanism that controls Plasmodium Sub1 activity before merozoite egress.

Malaria symptoms are associated with the asexual multiplication of Plasmodium falciparum within human red blood cells (RBCs) and fever peaks coincide with the egress of daughter merozoites following the rupture of the parasitophorous vacuole (PV) and the RBC membranes. Over the last two decades, it has emerged that the release of competent merozoites is tightly regulated by a complex cascade of events, including the unusual multi-step activation mechanism of the pivotal subtilisin-like protease 1 (Sub1) that takes place in three different cellular compartments and remains poorly understood. Following an initial auto-maturation in the endoplasmic reticulum (ER) between its pro- and catalytic domains, the Sub1 prodomain (PD) undergoes further cleavages by the parasite aspartic protease plasmepsin X (PmX) within acidic secretory organelles that ultimately lead to full Sub1 activation upon discharge into the PV. Here, we report the crystal structure of full-length P. falciparum Sub1 (PfS1FL) and demonstrate, through structural, biochemical, and biophysical studies, that the atypical Plasmodium-specific Sub1 PD directly promotes the assembly of inactive enzyme homodimers at acidic pH, whereas Sub1 is primarily monomeric at neutral pH. Our results shed new light into the finely tuned Sub1 spatiotemporal activation during secretion, explaining how PmX processing and full activation of Sub1 can occur in different cellular compartments, and uncover a robust mechanism of pH-dependent subtilisin autoinhibition that plays a key role in P. falciparum merozoites egress from infected host cells.IMPORTANCEMalaria fever spikes are due to the rupture of infected erythrocytes, allowing the egress of Plasmodium sp. merozoites and further parasite propagation. This fleeting tightly regulated event involves a cascade of enzymes, culminating with the complex activation of the subtilisin-like protease 1, Sub1. Differently than other subtilisins, Sub1 activation strictly depends upon the processing by a parasite aspartic protease within acidic merozoite secretory organelles. However, Sub1 biological activity is required in the pH neutral parasitophorous vacuole, to prime effectors involved in the rupture of the vacuole and erythrocytic membranes. Here, we show that the unusual, parasite-specific Sub1 prodomain is directly responsible for its acidic-dependent dimerization and autoinhibition, required for protein secretion, before its full activation at neutral pH in a monomeric form. pH-dependent Sub1 dimerization defines a novel, essential regulatory element involved in the finely tuned spatiotemporal activation of the egress of competent Plasmodium merozoites.

PvDBPII elicits multiple antibody-mediated mechanisms that reduce growth in a Plasmodium vivax challenge trial

The receptor-binding domain, region II, of the Plasmodium vivax Duffy binding protein (PvDBPII) binds the Duffy antigen on the reticulocyte surface to mediate invasion. A heterologous vaccine challenge trial recently showed that a delayed dosing regimen with recombinant PvDBPII SalI variant formulated with adjuvant Matrix-MTM reduced the in vivo parasite multiplication rate (PMR) in immunized volunteers challenged with the Thai P. vivax isolate PvW1. Here, we describe extensive analysis of the polyfunctional antibody responses elicited by PvDBPII immunization and identify immune correlates for PMR reduction. A classification algorithm identified antibody features that significantly contribute to PMR reduction. These included antibody titre, receptor-binding inhibitory titre, dissociation constant of the PvDBPII-antibody interaction, complement C1q and Fc gamma receptor binding and specific IgG subclasses. These data suggest that multiple immune mechanisms elicited by PvDBPII immunization are likely to be associated with protection and the immune correlates identified could guide the development of an effective vaccine for P. vivax malaria. Importantly, all the polyfunctional antibody features that correlated with protection cross-reacted with both PvDBPII SalI and PvW1 variants, suggesting that immunization with PvDBPII should protect against diverse P. vivax isolates.

In vitro anti-parasitic effect of the alkaloids harmaline and piperine on Toxoplasma gondii.

Toxoplasma gondii is a coccidian protozoan of zoonotic importance that causes toxoplasmosis. Although the current treatments for toxoplasmosis may be associated with adverse effects and limited efficacy for different biological forms of the parasite, evidence suggests that alkaloid molecules such as harmaline and piperine exhibit antiparasitic effects against protozoa parasites. This investigation aimed to evaluate the in vitro effect of harmaline and piperine against T. gondii tachyzoites in infected Vero cell cultures. After 24 hours of host cell infection, the cultures were treated with harmaline or piperine (0.49 to 15.63 µg/mL). Negative and positive controls were RPMI/DMSO (0.1%) and sulfadiazine (200 µg/mL). Harmaline significantly reduced parasite multiplication by 20% compared to the negative control, while piperine decreased between 55.56% and 88.89% in a dose-dependent manner. According to an intracellular parasite proportion scale, it was observed that the Vero cells with low or moderate parasitic proliferation were more prevalent after the alkaloid treatment. The study demonstrated that the alkaloids had antiparasitic effects on T. gondii, with piperine being the most effective. Additional studies must be carried out to clarify other aspects of the action of the alkaloids on parasites.

Effects of IDO1 and AhR Inhibition on Trypanosoma musculi co-cultured with macrophages in vitro

During coevolution with their hosts, trypanosomes have developed mechanisms to modulate immune responses. Some of these mechanisms involve deregulating amino acid metabolism, including L-arginine and potentially L-tryptophan metabolism. Then, we wanted to decipher tryptophan (Trp) metabolism via the IDO1 pathway, involving the AhR (Aryl Hydrocarbon Receptor) receptor. The inhibitors 1-M-LT for IDO1 and CH-223191 for AhR were used to study the involvement of IDO1. The method consisted of carrying out a series of trypanosome/macrophage co-cultures with the various inhibitors, assessing the parasite load, and then characterizing the effects of these inhibitors in vivo. Our preliminary results demonstrated that in vitro inhibitors of IDO1 and AhR distinctly reduced parasite multiplication in co-culture with macrophages. These results show that in vitro, IDO1 and AhR are involved in T. musculi-induced immunomodulation.

Does the leishmaniasis protects against SARS-CoV-2

The relationship between leishmaniasis and COVID-19 remains uncertain, with no evidence suggesting that leishmaniasis provides protection against COVID-19. Leishmaniasis, transmitted by sandflies, and COVID-19, caused by the SARS-CoV-2 virus, are unrelated diseases with distinct transmission modes. It is essential to rely on established preventive measures for COVID-19, such as vaccination and hygiene practices, as leishmaniasis requires separate medical attention. Some studies suggest that visceral Leishmania-COVID-19 co-infection may reactivate asymptomatic leishmaniasis. Severe COVID-19 cases may exacerbate parasitic multiplication and organ involvement. The immunological response to visceral leishmaniasis may increase susceptibility to subsequent SARS-CoV-2 infection, emphasizing the importance of an effective immune response. Cutaneous leishmaniasis may confer cross-protection against COVID-19, particularly in tropical regions. IFN-γ plays a critical role in parasite control and antiviral defense, highlighting its importance in both diseases. Further research is needed to understand the mechanisms underlying the interaction between SARS-CoV-2 and leishmaniasis.