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Proteomes of plasmodium knowlesi early and late ring-stage parasites and infected host erythrocytes.Journal of Proteomics Jun 2024The emerging malaria parasite Plasmodium knowlesi threatens the goal of worldwide malaria elimination due to its zoonotic spread in Southeast Asia. After brief ex-vivo...
The emerging malaria parasite Plasmodium knowlesi threatens the goal of worldwide malaria elimination due to its zoonotic spread in Southeast Asia. After brief ex-vivo culture we used 2D LC/MS/MS to examine the early and late ring stages of infected Macaca mulatta red blood cells harboring P. knowlesi. The M. mulatta clathrin heavy chain and T-cell and macrophage inhibitor ERMAP were overexpressed in the early ring stage; glutaredoxin 3 was overexpressed in the late ring stage; GO term differential enrichments included response to oxidative stress and the cortical cytoskeleton in the early ring stage. P. knowlesi clathrin heavy chain and 60S acidic ribosomal protein P2 were overexpressed in the late ring stage; GO term differential enrichments included vacuoles in the early ring stage, ribosomes and translation in the late ring stage, and Golgi- and COPI-coated vesicles, proteasomes, nucleosomes, vacuoles, ion-, peptide-, protein-, nucleocytoplasmic- and RNA-transport, antioxidant activity and glycolysis in both stages. SIGNIFICANCE: Due to its zoonotic spread, cases of the emerging human pathogen Plasmodium knowlesi in southeast Asia, and particularly in Malaysia, threaten regional and worldwide goals for malaria elimination. Infection by this parasite can be fatal to humans, and can be associated with significant morbidity. Due to zoonotic transmission from large macaque reservoirs that are untreatable by drugs, and outdoor biting mosquito vectors that negate use of preventive measures such as bed nets, its containment remains a challenge. Its biology remains incompletely understood. Thus we examine the expressed proteome of the early and late ex-vivo cultured ring stages, the first intraerythrocyte developmental stages after infection of host rhesus macaque erythrocytes. We used GO term enrichment strategies and differential protein expression to compare early and late ring stages. The early ring stage is characterized by the enrichment of P. knowlesi vacuoles, and overexpression of the M. mulatta clathrin heavy chain, important for clathrin-coated pits and vesicles, and clathrin-mediated endocytosis. The M. mulatta protein ERMAP was also overexpressed in the early ring stage, suggesting a potential role in early ring stage inhibition of T-cells and macrophages responding to P. knowlesi infection of reticulocytes. This could allow expansion of the host P. knowlesi cellular niche, allowing parasite adaptation to invasion of a wider age range of RBCs than the preferred young RBCs or reticulocytes, resulting in proliferation and increased pathogenesis in infected humans. Other GO terms differentially enriched in the early ring stage include the M. mulatta cortical cytoskeleton and response to oxidative stress. The late ring stage is characterized by overexpression of the P. knowlesi clathrin heavy chain. Combined with late ring stage GO term enrichment of Golgi-associated and coated vesicles, and enrichment of COPI-coated vesicles in both stages, this suggests the importance to P. knowlesi biology of clathrin-mediated endocytosis. P. knowlesi ribosomes and translation were also differentially enriched in the late ring stage. With expression of a variety of heat shock proteins, these results suggest production of folded parasite proteins is increasing by the late ring stage. M. mulatta endocytosis was differentially enriched in the late ring stage, as were clathrin-coated vesicles and endocytic vesicles. This suggests that M. mulatta clathrin-based endocytosis, perhaps in infected reticulocytes rather than mature RBC, may be an important process in the late ring stage. Additional ring stage biology from enriched GO terms includes M. mulatta proteasomes, protein folding and the chaperonin-containing T complex, actin and cortical actin cytoskeletons. P knowlesi biology also includes proteasomes, as well as nucleosomes, antioxidant activity, a variety of transport processes, glycolysis, vacuoles and protein folding. Mature RBCs have lost internal organelles, suggesting infection here may involve immature reticulocytes still retaining organelles. P. knowlesi parasite proteasomes and translational machinery may be ring stage drug targets for known selective inhibitors of these processes in other Plasmodium species. To our knowledge this is the first examination of more than one timepoint within the ring stage. Our results expand knowledge of both host and parasite proteins, pathways and organelles underlying P. knowlesi ring stage biology.
Topics: Plasmodium knowlesi; Macaca mulatta; Animals; Erythrocytes; Proteome; Protozoan Proteins; Malaria; Humans; Host-Parasite Interactions
PubMed: 38759952
DOI: 10.1016/j.jprot.2024.105197 -
Infection, Genetics and Evolution :... Oct 2023Plasmodium knowlesi is the leading cause of malaria in Malaysia. Serine Repeat Antigens (SERAs) have an essential role in the parasite life cycle. However, genetic...
Plasmodium knowlesi is the leading cause of malaria in Malaysia. Serine Repeat Antigens (SERAs) have an essential role in the parasite life cycle. However, genetic characterization on P. knowlesi SERA3 Ag2 (PkSERA3 Ag2) is lacking. In the present study, nucleotide diversity, natural selection, and haplotypes of PkSERA3 Ag2 in clinical samples from Peninsular Malaysia and Malaysian Borneo were investigated. A total of 50 P. knowlesi clinical samples were collected from Peninsular Malaysia and Malaysian Borneo. The PkSERA3 Ag2 gene was amplified using PCR, and subsequently cloned and sequenced. Genetic diversity, haplotype, natural selection as well as genetic structure and differentiation of PkSERA3 Ag2 were analysed. In addition, in silico analyses were performed to identify repeat motifs, B-cell epitopes, and antigenicity indices of the protein. Analysis of 114 PkSERA3 Ag2 sequences revealed high nucleotide diversity of the gene in Malaysia. A codon-based Z-test indicated that the gene underwent purifying selection. Haplotype and population structure analyses identified two distinct PkSERA3 Ag2 clusters (K = 2, ΔK = 721.14) but no clear genetic distinction between PkSERA3 Ag2 from Peninsular Malaysia and Malaysian Borneo. F index indicated moderate differentiation of the gene. In silico analyses revealed unique repeat motifs among PkSERA3 Ag2 isolates. Moreover, the amino acid sequence of PkSERA3 Ag2 exhibited potential B-cell epitopes and possessed high antigenicity indices. These findings enhance the understanding of PkSERA3 Ag2 gene as well as its antigenic properties. Further validation is necessary to ascertain the utility of PkSERA3 Ag2 as a serological marker for P. knowlesi infection.
Topics: Genetic Variation; Protozoan Proteins; Plasmodium knowlesi; Malaysia; Epitopes, B-Lymphocyte; Nucleotides
PubMed: 37595939
DOI: 10.1016/j.meegid.2023.105490 -
Parasitology Nov 2023Of the 5 human malarial parasites, and are the most prevalent species globally, while and are less prevalent and typically occur as mixed-infections. , previously... (Review)
Review
Of the 5 human malarial parasites, and are the most prevalent species globally, while and are less prevalent and typically occur as mixed-infections. , previously considered a non-human primate (NHP) infecting species, is now a cause of human malaria in Malaysia. The other NHP species, , , , , and cause malaria in primates, which are mainly reported in southeast Asia and South America. The non- NHP species also emerged and were found to cross-transmit from their natural hosts (NHP) – to human hosts in natural settings. Here we have reviewed and collated data from the literature on the NHPs-to-human-transmitting species. It was observed that the natural transmission of these NHP parasites to humans had been reported from 2010 onwards. This study shows that: (1) the majority of the non- NHP mixed species infecting human cases were from Yala province of Thailand; (2) mono/mixed infections with other human-infecting species were prevalent in Malaysia and Thailand and (3) and were found in Central and South America.
Topics: Animals; Humans; Malaria; Plasmodium knowlesi; Primates; Asia, Southeastern; Plasmodium vivax
PubMed: 37929579
DOI: 10.1017/S003118202300077X -
Genome Medicine Nov 2023Malaria continues to be a major threat to global public health. Whole genome sequencing (WGS) of the underlying Plasmodium parasites has provided insights into the...
BACKGROUND
Malaria continues to be a major threat to global public health. Whole genome sequencing (WGS) of the underlying Plasmodium parasites has provided insights into the genomic epidemiology of malaria. Genome sequencing is rapidly gaining traction as a diagnostic and surveillance tool for clinical settings, where the profiling of co-infections, identification of imported malaria parasites, and detection of drug resistance are crucial for infection control and disease elimination. To support this informatically, we have developed the Malaria-Profiler tool, which rapidly (within minutes) predicts Plasmodium species, geographical source, and resistance to antimalarial drugs directly from WGS data.
RESULTS
The online and command line versions of Malaria-Profiler detect ~ 250 markers from genome sequences covering Plasmodium speciation, likely geographical source, and resistance to chloroquine, sulfadoxine-pyrimethamine (SP), and other anti-malarial drugs for P. falciparum, but also providing mutations for orthologous resistance genes in other species. The predictive performance of the mutation library was assessed using 9321 clinical isolates with WGS and geographical data, with most being single-species infections (P. falciparum 7152/7462, P. vivax 1502/1661, P. knowlesi 143/151, P. malariae 18/18, P. ovale ssp. 5/5), but co-infections were identified (456/9321; 4.8%). The accuracy of the predicted geographical profiles was high to both continental (96.1%) and regional levels (94.6%). For P. falciparum, markers were identified for resistance to chloroquine (49.2%; regional range: 24.5% to 100%), sulfadoxine (83.3%; 35.4- 90.5%), pyrimethamine (85.4%; 80.0-100%) and combined SP (77.4%). Markers associated with the partial resistance of artemisinin were found in WGS from isolates sourced from Southeast Asia (30.6%).
CONCLUSIONS
Malaria-Profiler is a user-friendly tool that can rapidly and accurately predict the geographical regional source and anti-malarial drug resistance profiles across large numbers of samples with WGS data. The software is flexible with modifiable bioinformatic pipelines. For example, it is possible to select the sequencing platform, display specific variants, and customise the format of outputs. With the increasing application of next-generation sequencing platforms on Plasmodium DNA, Malaria-Profiler has the potential to be integrated into point-of-care and surveillance settings, thereby assisting malaria control. Malaria-Profiler is available online (bioinformatics.lshtm.ac.uk/malaria-profiler) and as standalone software ( https://github.com/jodyphelan/malaria-profiler ).
Topics: Humans; Animals; Antimalarials; Parasites; Coinfection; Malaria; Plasmodium; Malaria, Falciparum; Chloroquine; Malaria, Vivax; Drug Resistance; Plasmodium falciparum
PubMed: 37950308
DOI: 10.1186/s13073-023-01247-7 -
RSC Advances Dec 2023Development and discovery of new antimalarial drugs are needed to overcome the multi-resistance of parasites to commercially available drugs. Modifying the...
Development and discovery of new antimalarial drugs are needed to overcome the multi-resistance of parasites to commercially available drugs. Modifying the substitutions on the amine groups has been shown to increase antimalarial activities and decrease cross-resistance with chloroquine. In this study, we have synthesized several chalcone derivatives the substitution of aminoalkyl groups into the aromatic chalcone ring using the Mannich-type reaction. The chalcone derivatives were evaluated for their antimalarial properties against A1H1 and 3D7, as well as their molecular docking on dihydrofolate reductases-thymidylate synthase (PfDHFR-TS). Data from evaluation showed that chalcone Mannich-type base derivatives 2a, 2e, and 2h displayed potential antimalarial activities against with EC of 2.64, 2.98, and 0.10 μM, respectively, and 3D7 with EC of 0.08, 2.69, and 0.15 μM, respectively. The synthesized compounds 2a, 2e, and 2h exerted high selectivity index (SI > 10) values on the A1H1 and 3D7 strains. The molecular docking analysis on PfDHFR-TS supported the assay of 2a, 2e, and 2h by displaying CDOCKER energy of -48.224, -43.292, and -45.851 kcal mol. Therefore, the evidence obtained here supports that PfDHFR-TS is a putative molecular target for the synthesized compound.
PubMed: 38090066
DOI: 10.1039/d3ra05361j -
MedRxiv : the Preprint Server For... Apr 2024Malaria remains a major public health concern with substantial morbidity and mortality worldwide. In Malaysia, the emergence of has led to a surge in zoonotic malaria...
INTRODUCTION
Malaria remains a major public health concern with substantial morbidity and mortality worldwide. In Malaysia, the emergence of has led to a surge in zoonotic malaria cases and deaths in recent years. Signs of cerebral involvement have been observed in a non-comatose, fatal case of severe knowlesi infection, but the potential impact of this malaria species on the brain remains underexplored. To address this gap, we investigated circulating levels of brain injury, inflammation, and vascular biomarkers in a cohort of knowlesi-infected patients and controls.
METHODS
Archived plasma samples from 19 patients with confirmed symptomatic knowlesi infection and 19 healthy, age-matched controls from Peninsular Malaysia were analysed. A total of 52 plasma biomarkers of brain injury, inflammation, and vascular activation were measured using Luminex and SIMOA assays. Wilcoxon tests were used to examine group differences, and biomarker profiles were explored through hierarchical clustering heatmap analysis.
RESULTS
Bonferroni-corrected analyses revealed significantly elevated brain injury biomarker levels in knowlesi-infected patients, including S100B (p<0.0001), Tau (p=0.0007), UCH-L1 (p<0.0001), αSyn (p<0.0001), Park7 (p=0.0006), NRGN (p=0.0022), and TDP-43 (p=0.005). Compared to controls, levels were lower in the infected group for BDNF (p<0.0001), CaBD (p<0.0001), CNTN1 (p<0.0001), NCAM-1 (p<0.0001), GFAP (p=0.0013), and KLK6 (p=0.0126). Hierarchical clustering revealed distinct group profiles for circulating levels of brain injury and vascular activation biomarkers.
CONCLUSIONS
Our findings highlight for the first time the impact of infection on the brain, with distinct alterations in cerebral injury and endothelial activation biomarker profiles compared to healthy controls. Further studies are warranted to investigate the pathophysiology and clinical significance of these altered surrogate markers, through both neuroimaging and long-term neurocognitive assessments.
PubMed: 38712121
DOI: 10.1101/2024.04.25.24306382 -
Tropical Medicine and Infectious Disease Oct 2023() causes zoonotic malaria and is known as the "fifth human malaria parasite". malaria is an emerging threat because infections are increasing and can be fatal. While... (Review)
Review
() causes zoonotic malaria and is known as the "fifth human malaria parasite". malaria is an emerging threat because infections are increasing and can be fatal. While most infections are in Southeast Asia (SEA), especially Malaysia, travelers frequently visit this region and can present with malaria around the world. So, clinicians need to know (1) patients who present with fever after recent travel to SEA might be infected with and (2) is often misdiagnosed as (which typically causes less severe malaria). Here we review the history, pathophysiology, clinical features, diagnosis, and treatment of malaria. Severe disease is most common in adults. Signs and symptoms can include fever, abdominal pain, jaundice, acute kidney injury, acute respiratory distress syndrome, hyponatremia, hyperparasitemia, and thrombocytopenia. Dengue is one of the diseases to be considered in the differential. Regarding pathophysiologic mechanisms, when parasites invade mature red blood cells (RBCs, i.e., normocytes) and reticulocytes, changes in the red blood cell (RBC) surface can result in life-threatening cytoadherence, sequestration, and reduced RBC deformability. Since molecular mechanisms involving the erythrocytic stage are responsible for onset of severe disease and lethal outcomes, it is biologically plausible that manual exchange transfusion (ET) or automated RBC exchange (RBCX) could be highly beneficial by replacing "sticky" parasitized RBCs with uninfected, deformable, healthy donor RBCs. Here we suggest use of special -resistant donor RBCs to optimize adjunctive manual ET/RBCX for malaria. "Therapeutically-rational exchange transfusion" (T-REX) is proposed in which -resistant RBCs are transfused (instead of disease-promoting RBCs). Because expression of the Duffy antigen on the surface of human RBCs is essential for parasite invasion, T-REX of Duffy-negative RBCs-also known as Fy(a-b-) RBCs-could replace the majority of the patient's circulating normocytes with invasion-resistant RBCs (in a single procedure lasting about 2 h). When sequestered or non-sequestered iRBCs rupture-in a 24 h asexual life cycle-the released merozoites cannot invade Fy(a-b-) RBCs. When Fy(a-b-) RBC units are scarce (e.g., in Malaysia), clinicians can consider the risks and benefits of transfusing plausibly -resistant RBCs, such as glucose-6-phosphate dehydrogenase deficient (G6PDd) RBCs and Southeast Asian ovalocytes (SAO). Patients typically require a very short recovery time (<1 h) after the procedure. Fy(a-b-) RBCs should have a normal lifespan, while SAO and G6PDd RBCs may have mildly reduced half-lives. Because SAO and G6PDd RBCs come from screened blood donors who are healthy and not anemic, these RBCs have a low-risk for hemolysis and do not need to be removed after the patient recovers from malaria. T-REX could be especially useful if (1) antimalarial medications are not readily available, (2) patients are likely to progress to severe disease, or (3) drug-resistant strains emerge. In conclusion, T-REX is a proposed optimization of manual ET/RBCX that has not yet been utilized but can be considered by physicians to treat malaria patients.
PubMed: 37888606
DOI: 10.3390/tropicalmed8100478 -
Malaria Journal Dec 2023Plasmodium knowlesi is an established experimental model for basic and pre-clinical malaria vaccine research. Historically, rhesus macaques have been the most common...
BACKGROUND
Plasmodium knowlesi is an established experimental model for basic and pre-clinical malaria vaccine research. Historically, rhesus macaques have been the most common host for malaria vaccine studies with P. knowlesi parasites. However, rhesus are not natural hosts for P. knowlesi, and there is interest in identifying alternative hosts for vaccine research. The study team previously reported that pig-tailed macaques (PTM), a natural host for P. knowlesi, could be challenged with cryopreserved P. knowlesi sporozoites (PkSPZ), with time to blood stage infection equivalent to in rhesus. Here, additional exploratory studies were performed to evaluate PTM as potential hosts for malaria vaccine studies. The aim was to further characterize the parasitological and veterinary health outcomes after PkSPZ challenge in this macaque species.
METHODS
Malaria-naïve PTM were intravenously challenged with 2.5 × 10 PkSPZ and monitored for blood stage infection by Plasmodium 18S rRNA RT-PCR and thin blood smears. Disease signs were evaluated by daily observations, complete blood counts, serum chemistry tests, and veterinary examinations. After anti-malarial drug treatment, a subset of animals was re-challenged and monitored as above. Whole blood gene expression analysis was performed on selected animals to assess host response to infection.
RESULTS
In naïve animals, the kinetics of P. knowlesi blood stage replication was reproducible, with parasite burden rising linearly during an initial acute phase of infection from 6 to 11 days post-challenge, before plateauing and transitioning into a chronic low-grade infection. After re-challenge, infections were again reproducible, but with lower blood stage parasite densities. Clinical signs of disease were absent or mild and anti-malarial treatment was not needed until the pre-defined study day. Whole blood gene expression analysis identified immunological changes associated with acute and chronic phases of infection, and further differences between initial challenge versus re-challenge.
CONCLUSIONS
The ability to challenge PTM with PkSPZ and achieve reliable blood stage infections indicate this model has significant potential for malaria vaccine studies. Blood stage P. knowlesi infection in PTM is characterized by low parasite burdens and a benign disease course, in contrast with the virulent P. knowlesi disease course commonly reported in rhesus macaques. These findings identify new opportunities for malaria vaccine research using this natural host-parasite combination.
Topics: Animals; Plasmodium knowlesi; Malaria Vaccines; Macaca nemestrina; Macaca mulatta; Antimalarials; Malaria
PubMed: 38093306
DOI: 10.1186/s12936-023-04788-9 -
Malaria Journal Dec 2023Plasmodium vivax has been more resistant to various control measures than Plasmodium falciparum malaria because of its greater transmissibility and ability to produce...
BACKGROUND
Plasmodium vivax has been more resistant to various control measures than Plasmodium falciparum malaria because of its greater transmissibility and ability to produce latent parasite forms. Therefore, developing P. vivax vaccines and therapeutic monoclonal antibodies (humAbs) remains a high priority. The Duffy antigen receptor for chemokines (DARC) expressed on erythrocytes is central to P. vivax invasion of reticulocytes. P. vivax expresses a Duffy binding protein (PvDBP) on merozoites, a DARC ligand, and the DARC: PvDBP interaction is critical for P. vivax blood stage malaria. Therefore, PvDBP is a leading vaccine candidate for P. vivax and a target for therapeutic human monoclonal antibodies (humAbs).
METHODS
Here, the functional activity of humAbs derived from naturally exposed and vaccinated individuals are compared for the first time using easily cultured Plasmodium knowlesi (P. knowlesi) that had been genetically modified to replace its endogenous PkDBP orthologue with PvDBP to create a transgenic parasite, PkPvDBPOR. This transgenic parasite requires DARC to invade human erythrocytes but is not reticulocyte restricted. This model was used to evaluate the invasion inhibition potential of 12 humAbs (9 naturally acquired; 3 vaccine-induced) targeting PvDBP individually and in combinations using growth inhibition assays (GIAs).
RESULTS
The PvDBP-specific humAbs demonstrated 70-100% inhibition of PkPvDBPOR invasion with the IC values ranging from 51 to 338 µg/mL for the 9 naturally acquired (NA) humAbs and 33 to 99 µg/ml for the 3 vaccine-induced (VI) humAbs. To evaluate antagonistic, additive, or synergistic effects, six pairwise combinations were performed using select humAbs. Of these combinations tested, one NA/NA (099100/094083) combination demonstrated relatively strong additive inhibition between 10 and 100 µg/mL; all combinations of NA and VI humAbs showed additive inhibition at concentrations below 25 µg/mL and antagonism at higher concentrations. None of the humAb combinations showed synergy. Invasion inhibition efficacy by some mAbs shown with PkPvDBPOR was closely replicated using P. vivax clinical isolates.
CONCLUSION
The PkPvDBPOR transgenic model is a robust surrogate of P. vivax to assess invasion and growth inhibition of human monoclonal Abs recognizing PvDBP individually and in combination. There was no synergistic interaction for growth inhibition with the humAbs tested here that target different epitopes or subdomains of PvDBP, suggesting little benefit in clinical trials using combinations of these humAbs.
Topics: Animals; Humans; Plasmodium vivax; Plasmodium knowlesi; Antibodies, Protozoan; Antigens, Protozoan; Protozoan Proteins; Malaria, Vivax; Erythrocytes; Animals, Genetically Modified; Malaria Vaccines; Duffy Blood-Group System
PubMed: 38049801
DOI: 10.1186/s12936-023-04766-1 -
Emerging Infectious Diseases Oct 2023A 55-year-old man sought treatment for an uncomplicated febrile illness after returning to Canada from the Philippines. A suspected diagnosis of Plasmodium knowlesi... (Review)
Review
A 55-year-old man sought treatment for an uncomplicated febrile illness after returning to Canada from the Philippines. A suspected diagnosis of Plasmodium knowlesi infection was confirmed by PCR, and treatment with atovaquone/proguanil brought successful recovery. We review the evolving epidemiology of P. knowlesi malaria in the Philippines, specifically within Palawan Island.
Topics: Male; Humans; Middle Aged; Philippines; Plasmodium knowlesi; Malaria; Canada; Polymerase Chain Reaction
PubMed: 37735805
DOI: 10.3201/eid2910.230809