Identification of Exported Plasmodium falciparum Proteins That Bind to the Erythrocyte Cytoskeleton.

Plasmodium proteins are exported to the erythrocyte cytoplasm to create an environment that supports parasite replication. Although hundreds of proteins are predicted to be exported through Plasmodium export element (PEXEL)-dependent and -independent mechanisms, the functions of exported proteins are largely uncharacterized. In this study, we used a biochemical screening approach to identify putative exported P. falciparum proteins that bound to inside-out vesicles prepared from erythrocytes. Out of 69 P. falciparum PEXEL-motif proteins tested, 18 bound to inside-out vesicles (IOVs) in two or more independent assays. Using co-affinity purifications followed by mass spectrometry, pairwise co-purification experiments, and the split-luciferase assay, we identified 31 putative protein-protein interactions between erythrocyte cytoskeletal proteins and predicted exported P. falciparum proteins. We further showed that PF3D7_1401600 binds to the spectrin-binding domain of erythrocyte ankyrin via its MESA erythrocyte cytoskeleton binding (MEC) motif and to the N-terminal domains of ankyrin and 4.1R through a fragment that required an intact Plasmodium helical interspersed sub-telomeric (PHIST) domain. Introduction of PF3D7_1401600 into erythrocyte ghosts increased retention in the microsphiltration assay, consistent with previous data that reported a reduction of rigidity in red blood cells infected with PF3D7_1401600-deficient parasites.

Multiparametric biophysical profiling of red blood cells in malaria infection.

Biophysical separation promises label-free, less-invasive methods to manipulate the diverse properties of live cells, such as density, magnetic susceptibility, and morphological characteristics. However, some cellular changes are so minute that they are undetectable by current methods. We developed a multiparametric cell-separation approach to profile cells with simultaneously changing density and magnetic susceptibility. We demonstrated this approach with the natural biophysical phenomenon of Plasmodium falciparum infection, which modifies its host erythrocyte by simultaneously decreasing density and increasing magnetic susceptibility. Current approaches have used these properties separately to isolate later-stage infected cells, but not in combination. We present biophysical separation of infected erythrocytes by balancing gravitational and magnetic forces to differentiate infected cell stages, including early stages for the first time, using magnetic levitation. We quantified height distributions of erythrocyte populations-27 ring-stage synchronized samples and 35 uninfected controls-and quantified their unique biophysical signatures. This platform can thus enable multidimensional biophysical measurements on unique cell types.

Erythrocyte CD55 mediates the internalization of Plasmodium falciparum parasites.

Invasion of human erythrocytes by the malaria parasite Plasmodium falciparum is a multi-step process. Previously, a forward genetic screen for P. falciparum host factors identified erythrocyte CD55 as essential for invasion, but its specific role and how it interfaces with the other factors that mediate this complex process are unknown. Using CRISPR-Cas9 editing, antibody-based inhibition, and live cell imaging, here we show that CD55 is specifically required for parasite internalization. Pre-invasion kinetics, erythrocyte deformability, and echinocytosis were not influenced by CD55, but entry was inhibited when CD55 was blocked or absent. Visualization of parasites attached to CD55-null erythrocytes points to a role for CD55 in stability and/or progression of the moving junction. Our findings demonstrate that CD55 acts after discharge of the parasite's rhoptry organelles, and plays a unique role relative to all other invasion receptors. As the requirement for CD55 is strain-transcendent, these results suggest that CD55 or its interacting partners may hold potential as therapeutic targets for malaria.

A common polymorphism in the mechanosensitive ion channel PIEZO1 is associated with protection from severe malaria in humans.

Malaria caused by the apicomplexan parasite Plasmodium falciparum has served as a strong evolutionary force throughout human history, selecting for red blood cell polymorphisms that confer innate protection against severe disease. Recently, gain-of-function mutations in the mechanosensitive ion channel PIEZO1 were shown to ameliorate Plasmodium parasite growth, blood-brain barrier dysfunction, and mortality in a mouse model of malaria. In humans, the gain-of-function allele PIEZO1 E756del is highly prevalent and enriched in Africans, raising the possibility that it is under positive selection due to malaria. Here we used a case-control study design to test for an association between PIEZO1 E756del and malaria severity among children in Gabon. We found that the E756del variant is strongly associated with protection against severe malaria in heterozygotes. In subjects with sickle cell trait, heterozygosity for PIEZO1 E756del did not confer additive protection and homozygosity was associated with an elevated risk of severe disease, suggesting an epistatic relationship between hemoglobin S and PIEZO1 E756del. Using donor blood samples, we show that red cells heterozygous for PIEZO1 E756del are not dehydrated and can support the intracellular growth of P. falciparum similar to wild-type cells. However, surface expression of the P. falciparum virulence protein PfEMP-1 was significantly reduced in infected cells heterozygous for PIEZO1 756del, a phenomenon that has been observed with other protective polymorphisms, such as hemoglobin C. Our findings demonstrate that PIEZO1 is an important innate determinant of malaria susceptibility in humans and suggest that the mechanism of protection may be related to impaired export of P. falciparum virulence proteins.

The Plasmodium falciparum MESA erythrocyte cytoskeleton-binding (MEC) motif binds to erythrocyte ankyrin.

The MESA erythrocyte cytoskeleton binding (MEC) motif is a 13-amino acid sequence found in 14 exported Plasmodium falciparum proteins. First identified in the P. falciparum Mature-parasite-infected Erythrocyte Surface Antigen (MESA), the MEC motif is sufficient to target proteins to the infected red blood cell cytoskeleton. To identify host cell targets, purified MESA MEC motif was incubated with a soluble extract from uninfected erythrocytes, precipitated and subjected to mass spectrometry. The most abundant co-purifying protein was erythrocyte ankyrin (ANK1). A direct interaction between the MEC motif and ANK1 was independently verified using co-purification experiments, the split-luciferase assay, and the yeast two-hybrid assay. A systematic mutational analysis of the core MEC motif demonstrated a critical role for the conserved aspartic acid residue at the C-terminus of the MEC motif for binding to both erythrocyte inside-out vesicles and to ANK1. Using a panel of ANK1 constructs, the MEC motif binding site was localized to the ZU5C domain, which has no known function. The MEC motif had no impact on erythrocyte deformability when introduced into uninfected erythrocyte ghosts, suggesting the MEC motif's primary function is to target exported proteins to the cytoskeleton. Finally, we show that PF3D7_0402100 (PFD0095c) binds to ANK1 and band 4.1, likely through its MEC and PHIST motifs, respectively. In conclusion, we have provided multiple lines of evidence that the MEC motif binds to erythrocyte ANK1.

The Plasmodium falciparum exported protein PF3D7_0402000 binds to erythrocyte ankyrin and band 4.1.

Plasmodium falciparum extensively modifies the infected red blood cell (RBC), resulting in changes in deformability, shape and surface properties. These alterations suggest that the RBC cytoskeleton is a major target for modification during infection. However, the molecular mechanisms leading to these changes are largely unknown. To begin to address this question, we screened for exported P. falciparum proteins that bound to the erythrocyte cytoskeleton proteins ankyrin 1 (ANK1) and band 4.1 (4.1R), which form critical interactions with other cytoskeletal proteins that contribute to the deformability and stability of RBCs. Yeast two-hybrid screens with ANK1 and 4.1R identified eight interactions with P. falciparum exported proteins, including an interaction between 4.1R and PF3D7_0402000 (PFD0090c). This interaction was first identified in a large-scale screen (Vignali et al., Malaria J, 7:211, 2008), which also reported an interaction between PF3D7_0402000 and ANK1. We confirmed the interactions of PF3D7_0402000 with 4.1R and ANK1 in pair-wise yeast two-hybrid and co-precipitation assays. In both cases, an intact PHIST domain in PF3D7_0402000 was required for binding. Complex purification followed by mass spectrometry analysis provided additional support for the interaction of PF3D7_0402000 with ANK1 and 4.1R. RBC ghost cells loaded with maltose-binding protein (MBP)-PF3D7_0402000 passed through a metal microsphere column less efficiently than mock- or MBP-loaded controls, consistent with an effect of PF3D7_0402000 on RBC rigidity or membrane stability. This study confirmed the interaction of PF3D7_0402000 with 4.1R in multiple independent assays, provided the first evidence that PF3D7_0402000 also binds to ANK1, and suggested that PF3D7_0402000 affects deformability or membrane stability of uninfected RBC ghosts.

Soil transmitted helminthiasis in Kathmandu, Nepal.

A total of 200 soil samples taken from different sites and 1,504 stool samples collected from school children (n = 188) and patients (n = 1,316) visiting the health care centres in Kathmandu Valley were included in this study. Soil samples were investigated for the presence of parasitic eggs using sucrose flotation technique. Stool samples were examined by formal-ether concentration and direct smear techniques. The contamination rate of soil with parasitic eggs and larvae was found to be 28.5% (57/ 200). The overall parasitic infection rates in school children and patients were 42.5% and 2.8%, respectively. Four types of parasites were detected from soil samples, of which Ascaris was the most common. Trichuris was most common among school children whereas Ascaris in patients. Females in both study groups had higher prevalence compared with male counterparts with significantly low rate in health care centre visiting patients (p < 0.05).

Microbial study of hospital environment and carrier pattern study among staff in Nepal Medical College Teaching Hospital.

The present study was done to see the microbial flora in the environment (air and surface) of Nepal Medical College Teaching Hospital and the staffs working in the hospital. Altogether 160 environmental (air n = 43, surface n = 117) samples were collected and studied from different wards. Similarly 150 samples (48 nasal swabs, 48 throat swabs and 54 hand samples) from the staffs were collected and studied following the standard microbiological protocols. Gram +ve cocci were the most predominant ones among the bacterial isolates from the environment followed by gram +ve bacilli and gram -ve bacilli. Among fungal isolates, yeast were the most common isolates while Aspergillus spp. were the most frequently occurring mold. Out of 150 samples collected for the study of carrier pattern, 32 out of 54 samples collected were found to have Staphylococcus aureus in their hands, 1 had Escherichia coli. Other isolates were Bacillus spp., Micrococci and coagulase negative staphylococci. Similarly 21 (43.8%) out of 48 nasal samples were found to have S. aureus while none of the staffs were found to have beta-hemolytic streptococci in their throat. In the study, 1.6% environmental isolates and 5.7% carrier isolates of S. aureus were found to be Methicillin resistant.

Intestinal parasitosis among the elderly people in Kathmandu Valley.

Present study was carried out among the elderly people (60+ years of age) from August 2005 to July 2006 in Kathmandu Valley to assess the prevalence of intestinal parasitosis in them. Stool samples were collected from 235 elderly people (122 from government elderly home, 66 from private elderly home and 47 from the households in a rural community). The samples were examined by formal ether sedimentation and Sheather's sucrose floatation followed by Kinyoun's modified Ziehl-Neelsen staining. The overall prevalence of intestinal parasites was found to be 41.7%, out of which 30.6% had multiple parasitism. The government elderly home had significantly higher parasitic prevalence (50.8%) followed by the rural community (46.8%) and the private elderly homes (21.2%) (P<0.05). Males (43.8%) had slightly infection rate than females (40.4%) (P>0.05). There was equal infection rate with protozoa (25.8%) and helminths (27.0%). Trichuris trichiura (39.4%) and Entamoeba histolytica (19.7%) were the commonest helminth and protozoa, respectively.