Dissecting the role of glutathione biosynthesis in Plasmodium falciparum

Glutathione (γ-glutamylcysteinyl-glycine, GSH) has vital functions as thiol redox buffer and cofactor of antioxidant and detoxification enzymes. Plasmodium falciparum possesses a functional GSH biosynthesis pathway and contains mM concentrations of the tripeptide. It was impossible to delete in P. falciparum the genes encoding γ-glutamylcysteine synthetase (γGCS) or glutathione synthetase (GS), the two enzymes synthesizing GSH, although both gene loci were not refractory to recombination. Our data show that the parasites cannot compensate for the loss of GSH biosynthesis via GSH uptake. This suggests an important if not essential function of GSH biosynthesis pathway for the parasites. Treatment with the irreversible inhibitor of γGCS L-buthionine sulfoximine (BSO) reduced intracellular GSH levels in P. falciparum and was lethal for their intra-erythrocytic development, corroborating the suggestion that GSH biosynthesis is important for parasite survival. Episomal expression of γgcs in P. falciparum increased tolerance to BSO attributable to increased levels of γGCS. Concomitantly expression of glutathione reductase was reduced leading to an increased GSH efflux. Together these data indicate that GSH levels are tightly regulated by a functional GSH biosynthesis and the reduction of GSSG

Conditional expression of apical membrane antigen 1 in Plasmodium falciparum shows it is required for erythrocyte invasion by merozoites

Malaria is caused by obligate intracellular parasites, of which Plasmodium falciparum is the most lethal species. In humans, P. falciparum merozoites (invasive forms of the parasite) employ a host of parasite proteins to rapidly invade erythrocytes. One of these is the P. falciparum apical membrane antigen 1 (PfAMA1) which forms a complex with rhoptry neck proteins at the tight junction. Here, we have placed the Pfama1 gene under conditional control using dimerizable Cre recombinase (DiCre) in P. falciparum. DiCre‐mediated excision of the loxP‐flanked Pfama1 gene results in approximately 80% decreased expression of the protein within one intraerythrocytic growth cycle. This reduces growth by 40%, due to decreased invasion efficiency characterized by a post‐invasion defect in sealing of the parasitophorous vacuole. These results show that PfAMA1 is an essential protein for merozoite invasion in P. falciparum and either directly or indirectly plays a role in resealing of the red blood cell at the posterior end of the invasion event.Alan Yap, Mauro F. Azevedo, Paul R. Gilson, Greta E. Weiss, Matthew T. O’Neill, Danny W. Wilson, Brendan S. Crabb and Alan F. Cowma

Defining species-specific and conserved interactions of apical membrane protein 1 during erythrocyte invasion in malaria to inform multi-species vaccines

Published online: 27 February 2023Plasmodium falciparum and P. vivax are the major causes of human malaria, and P. knowlesi is an important additional cause in SE Asia. Binding of apical membrane antigen 1 (AMA1) to rhoptry neck protein 2 (RON2) was thought to be essential for merozoite invasion of erythrocytes by Plasmodium spp. Our findings reveal that P. falciparum and P. vivax have diverged and show species-specific binding of AMA1 to RON2, determined by a β-hairpin loop in RON2 and specific residues in AMA1 Loop1E. In contrast, cross-species binding of AMA1 to RON2 is retained between P. vivax and P. knowlesi. Mutation of specific amino acids in AMA1 Loop1E in P. falciparum or P. vivax ablated RON2 binding without impacting erythrocyte invasion. This indicates that the AMA1–RON2-loop interaction is not essential for invasion and additional AMA1 interactions are involved. Mutations in AMA1 that disrupt RON2 binding also enable escape of invasion inhibitory antibodies. Therefore, vaccines and therapeutics will need to be broader than targeting only the AMA1–RON2 interaction. Antibodies targeting AMA1 domain 3 had greater invasion-inhibitory activity when RON2-loop binding was ablated, suggesting this domain is a promising additional target for vaccine development. Targeting multiple AMA1 interactions involved in invasion may enable vaccines that generate more potent inhibitory antibodies and address the capacity for immune evasion. Findings on specific residues for invasion function and species divergence and conservation can inform novel vaccines and therapeutics against malaria caused by three species, including the potential for cross-species vaccines.Damien R. Drew, Danny W. Wilson, Gretchen E. Weiss, Lee M. Yeoh, Isabelle G. Henshall, Brendan S. Crabb, Sheetij Dutta, Paul R. Gilson, James G. Beeso

Electron Scattering From High-Momentum Neutrons in Deuterium

We report results from an experiment measuring the semi-inclusive reaction $d(e,e'p_s)$ where the proton $p_s$ is moving at a large angle relative to the momentum transfer. If we assume that the proton was a spectator to the reaction taking place on the neutron in deuterium, the initial state of that neutron can be inferred. This method, known as spectator tagging, can be used to study electron scattering from high-momentum (off-shell) neutrons in deuterium. The data were taken with a 5.765 GeV electron beam on a deuterium target in Jefferson Laboratory's Hall B, using the CLAS detector. A reduced cross section was extracted for different values of final-state missing mass $W^{*}$, backward proton momentum $\vec{p}_{s}$ and momentum transfer $Q^{2}$. The data are compared to a simple PWIA spectator model. A strong enhancement in the data observed at transverse kinematics is not reproduced by the PWIA model. This enhancement can likely be associated with the contribution of final state interactions (FSI) that were not incorporated into the model. A ``bound neutron structure function'' $F_{2n}^{eff}$ was extracted as a function of $W^{*}$ and the scaling variable $x^{*}$ at extreme backward kinematics, where effects of FSI appear to be smaller. For $p_{s}>400$ MeV/c, where the neutron is far off-shell, the model overestimates the value of $F_{2n}^{eff}$ in the region of $x^{*}$ between 0.25 and 0.6. A modification of the bound neutron structure function is one of possible effects that can cause the observed deviation.Comment: 33 pages RevTeX, 9 figures, to be submitted to Phys. Rev. C. Fixed 1 Referenc

A Bayesian analysis of pentaquark signals from CLAS data

We examine the results of two measurements by the CLAS collaboration, one of which claimed evidence for a $\Theta^{+}$ pentaquark, whilst the other found no such evidence. The unique feature of these two experiments was that they were performed with the same experimental setup. Using a Bayesian analysis we find that the results of the two experiments are in fact compatible with each other, but that the first measurement did not contain sufficient information to determine unambiguously the existence of a $\Theta^{+}$. Further, we suggest a means by which the existence of a new candidate particle can be tested in a rigorous manner.Comment: 5 pages, 3 figure

A Kinematically Complete Measurement of the Proton Structure Function F2 in the Resonance Region and Evaluation of Its Moments

We measured the inclusive electron-proton cross section in the nucleon resonance region (W < 2.5 GeV) at momentum transfers Q**2 below 4.5 (GeV/c)**2 with the CLAS detector. The large acceptance of CLAS allowed for the first time the measurement of the cross section in a large, contiguous two-dimensional range of Q**2 and x, making it possible to perform an integration of the data at fixed Q**2 over the whole significant x-interval. From these data we extracted the structure function F2 and, by including other world data, we studied the Q**2 evolution of its moments, Mn(Q**2), in order to estimate higher twist contributions. The small statistical and systematic uncertainties of the CLAS data allow a precise extraction of the higher twists and demand significant improvements in theoretical predictions for a meaningful comparison with new experimental results.Comment: revtex4 18 pp., 12 figure

eta-prime photoproduction on the proton for photon energies from 1.527 to 2.227 GeV

Differential cross sections for the reaction gamma p -> eta-prime p have been measured with the CLAS spectrometer and a tagged photon beam with energies from 1.527 to 2.227 GeV. The results reported here possess much greater accuracy than previous measurements. Analyses of these data indicate for the first time the coupling of the etaprime N channel to both the S_11(1535) and P_11(1710) resonances, known to couple strongly to the eta N channel in photoproduction on the proton, and the importance of j=3/2 resonances in the process.Comment: 6 pages, 3 figure

Measurement of the Deuteron Structure Function F2 in the Resonance Region and Evaluation of Its Moments

Inclusive electron scattering off the deuteron has been measured to extract the deuteron structure function F2 with the CEBAF Large Acceptance Spectrometer (CLAS) at the Thomas Jefferson National Accelerator Facility. The measurement covers the entire resonance region from the quasi-elastic peak up to the invariant mass of the final-state hadronic system W~2.7 GeV with four-momentum transfers Q2 from 0.4 to 6 (GeV/c)^2. These data are complementary to previous measurements of the proton structure function F2 and cover a similar two-dimensional region of Q2 and Bjorken variable x. Determination of the deuteron F2 over a large x interval including the quasi-elastic peak as a function of Q2, together with the other world data, permit a direct evaluation of the structure function moments for the first time. By fitting the Q2 evolution of these moments with an OPE-based twist expansion we have obtained a separation of the leading twist and higher twist terms. The observed Q2 behaviour of the higher twist contribution suggests a partial cancellation of different higher twists entering into the expansion with opposite signs. This cancellation, found also in the proton moments, is a manifestation of the "duality" phenomenon in the F2 structure function

First measurement of direct f0(980)f_0(980) photoproduction on the proton

We report on the results of the first measurement of exclusive $f_0(980)$ meson photoproduction on protons for $E_\gamma=3.0 - 3.8$ GeV and $-t = 0.4-1.0$ GeV$^2$. Data were collected with the CLAS detector at the Thomas Jefferson National Accelerator Facility. The resonance was detected via its decay in the $\pi^+ \pi^-$ channel by performing a partial wave analysis of the reaction $\gamma p \to p \pi^+ \pi^-$. Clear evidence of the $f_0(980)$ meson was found in the interference between $P$ and $S$ waves at $M_{\pi^+ \pi^-}\sim 1$ GeV. The $S$-wave differential cross section integrated in the mass range of the $f_0(980)$ was found to be a factor of 50 smaller than the cross section for the $\rho$ meson. This is the first time the $f_0(980)$ meson has been measured in a photoproduction experiment

Deeply Virtual Compton Scattering Beam-Spin Asymmetries

The beam spin asymmetries in the hard exclusive electroproduction of photons on the proton (ep -> epg) were measured over a wide kinematic range and with high statistical accuracy. These asymmetries result from the interference of the Bethe-Heitler process and of deeply virtual Compton scattering. Over the whole kinematic range (x_B from 0.11 to 0.58, Q^2 from 1 to 4.8 GeV^2, -t from 0.09 to 1.8 GeV^2), the azimuthal dependence of the asymmetries is compatible with expectations from leading-twist dominance, A = a*sin(phi)/[1+c*cos(phi)]. This extensive set of data can thus be used to constrain significantly the generalized parton distributions of the nucleon in the valence quark sector.Comment: 1 tex file (6 pages), 4 (eps) figure