Structural Determination of Functional Units of the Nucleotide Binding Domain (NBD94) of the Reticulocyte Binding Protein Py235 of Plasmodium yoelii

Invasion of the red blood cells (RBC) by the merozoite of malaria parasites involves a large number of receptor ligand interactions. The reticulocyte binding protein homologue family (RH) plays an important role in erythrocyte recognition as well as virulence. Recently, it has been shown that members of RH in addition to receptor binding may also have a role as ATP/ADP sensor. A 94 kDa region named Nucleotide-Binding Domain 94 (NBD94) of Plasmodium yoelii YM, representative of the putative nucleotide binding region of RH, has been demonstrated to bind ATP and ADP selectively. Binding of ATP or ADP induced nucleotide-dependent structural changes in the C-terminal hinge-region of NBD94, and directly impacted on the RBC binding ability of RH.In order to find the smallest structural unit, able to bind nucleotides, and its coupling module, the hinge region, three truncated domains of NBD94 have been generated, termed NBD94(444-547), NBD94(566-663) and NBD94(674-793), respectively. Using fluorescence correlation spectroscopy NBD94(444-547) has been identified to form the smallest nucleotide binding segment, sensitive for ATP and ADP, which became inhibited by 4-Chloro-7-nitrobenzofurazan. The shape of NBD94(444-547) in solution was calculated from small-angle X-ray scattering data, revealing an elongated molecule, comprised of two globular domains, connected by a spiral segment of about 73.1 A in length. The high quality of the constructs, forming the hinge-region, NBD94(566-663) and NBD94(674-793) enabled to determine the first crystallographic and solution structure, respectively. The crystal structure of NBD94(566-663) consists of two helices with 97.8 A and 48.6 A in length, linked by a loop. By comparison, the low resolution structure of NBD94(674-793) in solution represents a chair-like shape with three architectural segments.These structures give the first insight into how nucleotide binding impacts on the overall structure of RH and demonstrates the potential use of this region as a novel drug target

TGF-b2 induction regulates invasiveness of theileria-transformed leukocytes and disease susceptibility

Theileria parasites invade and transform bovine leukocytes causing either East Coast fever (T. parva), or tropical theileriosis (T. annulata). Susceptible animals usually die within weeks of infection, but indigenous infected cattle show markedly reduced pathology, suggesting that host genetic factors may cause disease susceptibility. Attenuated live vaccines are widely used to control tropical theileriosis and attenuation is associated with reduced invasiveness of infected macrophages in vitro. Disease pathogenesis is therefore linked to aggressive invasiveness, rather than uncontrolled proliferation of Theileria-infected leukocytes. We show that the invasive potential of Theileria-transformed leukocytes involves TGF-b signalling. Attenuated live vaccine lines express reduced TGF-b2 and their invasiveness can be rescued with exogenous TGF-b. Importantly, infected macrophages from disease susceptible Holstein-Friesian (HF) cows express more TGF-b2 and traverse Matrigel with great efficiency compared to those from disease-resistant Sahiwal cattle. Thus, TGF-b2 levels correlate with disease susceptibility. Using fluorescence and time-lapse video microscopy we show that Theileria-infected, disease-susceptible HF macrophages exhibit increased actin dynamics in their lamellipodia and podosomal adhesion structures and develop more membrane blebs. TGF-b2-associated invasiveness in HF macrophages has a transcription-independent element that relies on cytoskeleton remodelling via activation of Rho kinase (ROCK). We propose that a TGF-b autocrine loop confers an amoeboid-like motility on Theileria-infected leukocytes, which combines with MMP-dependent motility to drive invasiveness and virulence

Biophysics of Malarial Parasite Exit from Infected Erythrocytes

Upon infection and development within human erythrocytes, P. falciparum induces alterations to the infected RBC morphology and bio-mechanical properties to eventually rupture the host cells through parasitic and host derived proteases of cysteine and serine families. We used previously reported broad-spectrum inhibitors (E64d, EGTA-AM and chymostatin) to inhibit these proteases and impede rupture to analyze mechanical signatures associated with parasite escape. Treatment of late-stage iRBCs with E64d and EGTA-AM prevented rupture, resulted in no major RBC cytoskeletal reconfiguration but altered schizont morphology followed by dramatic re-distribution of three-dimensional refractive index (3D-RI) within the iRBC. These phenotypes demonstrated several-fold increased iRBC membrane flickering. In contrast, chymostatin treatment showed no 3D-RI changes and caused elevated fluctuations solely within the parasitophorous vacuole. We show that E64d and EGTA-AM supported PV breakdown and the resulting elevated fluctuations followed non-Gaussian pattern that resulted from direct merozoite impingement against the iRBC membrane. Optical trapping experiments highlighted reduced deformability of the iRBC membranes upon rupture-arrest, more specifically in the treatments that facilitated PV breakdown. Taken together, our experiments provide novel mechanistic interpretations on the role of parasitophorous vacuole in maintaining the spherical schizont morphology, the impact of PV breakdown on iRBC membrane fluctuations leading to eventual parasite escape and the evolution of membrane stiffness properties of host cells in which merozoites were irreversibly trapped, recourse to protease inhibitors. These findings provide a comprehensive, previously unavailable, body of information on the combined effects of biochemical and biophysical factors on parasite egress from iRBCs.Singapore. Agency for Science, Technology and ResearchSingapore-MIT AllianceGlobal Enterprise for Micro-Mechanics and Molecular MedicineNational University of SingaporeNational Institutes of Health (U.S.) (Grant R01 HL094270-01A1)National Institutes of Health (U.S.) (Grant 1-R01-GM076689-01)National Institutes of Health (U.S.) (P41-RR02594-18-24

Inverted recruitment of autophagy proteins to the <i>Plasmodium berghei</i> parasitophorous vacuole membrane

<div><p>Selective autophagy and related mechanisms can act as variable defense mechanisms against pathogens and can therefore be considered as intracellular immune responses. When in hepatocytes, <i>Plasmodium</i> parasites reside in a parasitophorous vacuole (PV) and the PV membrane (PVM) is the main contact site between host cell and parasite. Early in infection, the PVM is directly labeled with host cell autophagy proteins LC3B and p62 (nucleoporin 62). We investigated the recruitment of different selective autophagy receptors and could show that mainly p62 and NBR1 (neighbour of BRCA1 gene 1) and to a lesser extent NDP52 (nuclear dot protein 52) associate with the PVM. To investigate the recruitment of these receptors to the PVM in <i>Plasmodium</i>-infected cells, we generated LC3B knock out HeLa cells. In these cell lines, autophagosome formation and autophagic flux are not different to those in WT cells. Unexpectedly, p62 and NBR1 recruitment to the PVM was strongly impaired in LC3B-negative host cells, suggesting that LC3B recruits both receptors to the PVM of <i>Plasmodium</i> parasites. We also noticed that LC3B recruited ubiquitin to the PVM. This indicates that, in comparison to classical selective autophagy, in <i>P</i>. <i>berghei</i>-infected cells the order of membrane labeling with autophagy proteins appears to be inverted from canonical ubiquitin-receptor-LC3B recruitment to LC3B-receptor and possibly ubiquitin.</p></div

Generation of LC3B knockout HeLa cell lines.

<p><b>(A)</b> Genomic region of the <i>LC3B</i> gene. Exon 1 is shown in lowercase blue, the 5’-upstream region is printed in black capitals. Binding regions of the two gRNAs are highlighted in green and PAM sequences are shown in red. <b>(B)</b> Western blot of HeLa WT and three LC3B knock out cell lines to confirm the lack of LC3B protein in three clonal cell lines. GAPDH was used as a loading control. <b>(C)</b> IFA analysis of HeLa WT and three LC3B knock out cell lines. Cells were starved for 2 hours in EBSS, fixed, stained with anti-LC3B antibodies (green) and analysed by fluorescence microscopy. DNA was visualised with DAPI (blue). Scale bar 20 μm.</p

Autophagy receptors p62 and NBR1 localise to the parasitophorous vacuole membrane of <i>Plasmodium berghei</i>.

<p><b>(A)</b> HeLa cells were infected with <i>Plasmodium berghei</i>. The parasitophorous vacuole membrane (PVM) of the parasite was stained with antibodies (UIS4, red). To visualise the autophagy receptors, cells were stained with antibodies (p62, green) or transfected 24 hours before infection with plasmids expressing GFP fusion proteins (GFP-NBR1, GFP-NDP52, OPTN-GFP, GFP, all shown in green). Infected cells were fixed 6 hours post-infection, stained with antibodies and analysed by confocal microscopy. DNA was stained with DAPI. Scale bar 10 μm <b>(B)</b> Numbers of receptor-labeled <i>P</i>. <i>berghei</i> parasites were determined by fluorescence microscopy. 60–100 parasites were counted in two separate experiments. Numbers of labeled parasites are expressed as percentages, error bars show standard deviations, p values were calculated using a t-test. <b>(C)</b> Pearson's correlation coefficients were calculated from at least 5 images, except for cells expressing GFP only. Standard deviations are depicted, p values were calculated using a t-test.</p

LC3B knockout cells are able to undergo canonical autophagy.

<p><b>(A)</b> Representative western blot of non-infected HeLa WT, ATG5-knockout cells and one clonal LC3B-knockout cell line left untreated or treated simultaneously with 10 μM chloroquine and 250 ng/ml rapamycin for 4 hours. <b>(B)</b> HeLa WT and HeLa LC3B knockout cells ectopically expressing GFP-Gate16 were left untreated or treated with 10 μM chloroquine and 250 ng/ml rapamycin for 4 hours. Fixed cells were stained with anti-GFP antibodies to visualise Gate16 (green) or anti-LC3B antibodies (red). DNA was stained with DAPI (blue). White arrows in the LC3B panel indicate Gate16-transfected cells. Yellow arrowheads in the enlarged pictures indicate autophagic structures where Gate16 and LC3B colocalise. Scale bar 20 μm. Experiments using other clonal LC3B-knockout cells are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183797#pone.0183797.s001" target="_blank">S1B Fig</a>.</p

LC3B recruits GFP-NBR1 to the PVM.

<p><b>(A)</b> HeLa WT, HeLa LC3B- and ATG5-knockout cells were infected with <i>Pb</i>mCherry (red). 6 hours post-infection, cells were fixed and stained with anti-NBR1 antibodies (green) as a control. Control and knockout cell lines were transfected with RFP-LC3B and GFP-NBR1 expression constructs (two lower panels) and infected 17 hours after transfection with <i>Pb</i>mCherry sporozoites (red). RFP-LC3B (red) and GFP-NBR1 (green) were visualised using mouse monoclonal anti-LC3B and rabbit monoclonal anti-NBR1 antibodies. DNA was labeled with DAPI (blue). Cells were analyzed by confocal microscopy. Scale bar 10 μm. <b>(B)</b> Numbers of GFP-NBR1-labeled <i>P</i>. <i>berghei</i> parasites in non-transfected and in RFP-LC3B-transfected cells were determined by fluorescence microscopy. 84–111 parasites were analysed in the non-transfected HeLa cells and 50 parasites were analysed for the RFP-LC3B-transfected HeLa cells. Two individual experiments were carried out. Labeled parasites are expressed as percentages. Standard deviations are depicted. <b>(C)</b> Pearson’s correlation coefficient of GFP-NBR1 and RFP-LC3B was calculated from at least five individual <i>P</i>. <i>berghei</i> parasites in HeLa WT and LC3B^-/- cells transfected with RFP-LC3B. The mean values of 0.838 (HeLa WT) and 0.910 (LC3B^-/-complemented with RFP-LC3) indicate a strong co-localization of p62 and RFP-LC3B. Depicted are standard deviations.</p

LC3B recruits p62 to the PVM.

<p><b>(A)</b> HeLa WT, HeLa LC3B- and ATG5-knockout cells were infected with <i>Pb</i>mCherry (red). 6 hours post-infection, cells were fixed and stained with α-p62 antibodies (green). All cell lines were transfected with RFP-LC3B (two lower panels) and infected 17 hours after transfection with <i>P</i>. <i>berghei</i> sporozoites expressing mCherry (red). p62 (green) was visualised using a specific monoclonal α-p62 antibody. DNA was labeled with DAPI (blue). Cells were analyzed by confocal microscopy. Scale bar 10 μm. <b>(B)</b> Numbers of p62-labeled <i>P</i>. <i>berghei</i> parasites in non-transfected and in RFP-LC3B-transfected cells were determined by fluorescence microscopy. 100–130 parasites were analyzed in non-transfected HeLa cells and 60–120 parasites were analyzed for RFP-LC3B-transfected HeLa cells. Two individual experiments were carried out. Labeled parasites are expressed as percentages. Standard deviations are depicted. <b>(C)</b> Pearson’s correlation coefficient of p62 and RFP-LC3B were calculated for six individual <i>P</i>. <i>berghei</i> parasites in HeLa WT and LC3B^-/- cells transfected with RFP-LC3B. The mean values of 0.762 (HeLa WT) and 0.715 (LC3B^-/-) indicate a strong co-localization of p62 and RFP-LC3B. Depicted are standard deviations.</p