Exploring Unique Aspects of Apicomplexan Cell Biology Using Molecular Genetic and Small Molecule Approaches

The Phylum Apicomplexa contains a number of devastating pathogens responsible for tremendous human suffering and mortality. Among these are Plasmodium, which is the causative agent of malaria, Cryptosporidium, which causes diarrheal illness in children and immuncompromised people, and Toxoplasma gondii, which causes congenital defects in the developing fetus and severe disease in immunocompromised people. T. gondii also serves as a model organism for other members of this phylum due to the relative ease of parasite culture and manipulation. Although effective treatments exist for some diseases caused by these apicomplexan parasites, drug resistance for others is widespread, perhaps most notably in Plasmodium species. Development of new therapeutic agents is needed to combat this resistance and alleviate disease burden. It is important that the drugs target parasitic cell components not conserved in humans in order to minimize side effects and drug toxicity. However, in order to target unique processes, a better understanding of apicomplexan biology must be gained. One approach toward understanding the unique biological processes of apicomplexan parasites is to study proteins conserved among the Phylum Apicomplexa, but not present in other organisms. One such protein, photosensitized INA-labeled protein 1 (TgPhIL1) was identified previously. The work presented in this dissertation describes targeted disruption of this gene in T. gondii, which results in parasites with an altered shape and a fitness defect in both tissue culture and a mouse model of infection. Another approach to understanding the unique processes of apicomplexan parasites is to perturb them using small molecules. By identifying the targets of the small molecules, a more detailed understanding of the process can be gained. To this end, a small molecule screen was performed in T. gondii in order to identify small molecules that inhibit the apicomplexan-specific and essential process of host-cell invasion. In addition to identifying 24 invasion inhibitors, 6 enhancers were also identified. One of these enhancers, compound 112762, was shown to enhance invasion of other apicomplexan parasites as well. Described herein are attempts to identify the target(s) of this compound. A derivative of this compound was linked to an affinity resin, and TgProfilin was identified as a putative target that may interact covalently with 112762. Additionally, affinity chromatography was used to demonstrate non-covalent binding of a T. gondii FK506-binding protein to 112762. Finally, based on a report in the literature of a compound nearly identical to 112762 that inhibits yeast and mammalian protein arginine methyltransferase 1 (PRMT1), it was hypothesized that 112762 might target TgPRMT1 in T. gondii. Supportive of this hypothesis, 112762 was shown to inhibit TgPRMT1 in vitro, to inhibit parasite protein methylation in vivo, and to bind the 112762 affinity resin. TgPRMT1 knockout parasites are being generated in order to determine whether they show resistance to compound 112762. As a result of this work, three potential targets of 112762 in T. gondii have been identified. This work opens the door for future studies aimed at understanding and controlling invasion by apicomplexan parasites and other processes specific to the Phylum Apicomplexa

Targeted Disruption of TgPhIL1 in Toxoplasma gondii Results in Altered Parasite Morphology and Fitness

The inner membrane complex (IMC), a series of flattened vesicles at the periphery of apicomplexan parasites, is thought to be important for parasite shape, motility and replication, but few of the IMC proteins that function in these processes have been identified. TgPhIL1, a Toxoplasma gondii protein that was previously identified through photosensitized labeling with 5-[125I] iodonapthaline-1-azide, associates with the IMC and/or underlying cytoskeleton and is concentrated at the apical end of the parasite. Orthologs of TgPhIL1 are found in other apicomplexans, but the function of this conserved protein family is unknown. As a first step towards determining the function of TgPhIL1 and its orthologs, we generated a T. gondii parasite line in which the single copy of TgPhIL1 was disrupted by homologous recombination. The TgPhIL1 knockout parasites have a distinctly different morphology than wild-type parasites, and normal shape is restored in the knockout background after complementation with the wild-type allele. The knockout parasites are outcompeted in culture by parasites expressing functional TgPhIL1, and they generate a reduced parasite load in the spleen and liver of infected mice. These findings demonstrate a role for TgPhIL1 in the morphology, growth and fitness of T. gondii tachyzoites

An Energy Feedback System for the MIT/Bates Linear Accelerator

We report the development and implementation of an energy feedback system for the MIT/Bates Linear Accelerator Center. General requirements of the system are described, as are the specific requirements, features, and components of the system unique to its implementation at the Bates Laboratory. We demonstrate that with the system in operation, energy fluctuations correlated with the 60 Hz line voltage and with drifts of thermal origin are reduced by an order of magnitude

The Strange Quark Contribution to the Proton's Magnetic Moment

We report a new determination of the strange quark contribution to the proton's magnetic form factor at a four-momentum transfer Q2 = 0.1 (GeV/c)^2 from parity-violating e-p elastic scattering. The result uses a revised analysis of data from the SAMPLE experiment which was carried out at the MIT-Bates Laboratory. The data are combined with a calculation of the proton's axial form factor GAe to determine the strange form factor GMs(Q2=0.1)=0.37 +- 0.20 +- 0.26 +- 0.07. The extrapolation of GMs to its Q2=0 limit and comparison with calculations is also discussed.Comment: 6 pages, 1 figure, submitted to Phys. Lett.

Parity Violation in Elastic Electron-Proton Scattering and the Proton's Strange Magnetic Form Factor

We report a new measurement of the parity-violating asymmetry in elastic electron scattering from the proton at backward scattering angles. This asymmetry is sensitive to the strange magnetic form factor of the proton as well as electroweak axial radiative corrections. The new measurement of A = -4.92±0.61±0.73 ppm provides a significant constraint on these quantities. The implications for the strange magnetic form factor are discussed in the context of theoretical estimates for the axial corrections

Relativistically invariant analysis of polarization effects in exclusive deuteron electrodisintegration process

A general formalism for the calculation of the differential cross section and polarization observables, for the process of deuteron electrodisintegration, is developed in the framework of relativistic impulse approximation. A detailed analysis of the general structure of the differential cross section and polarization observables for the $e^-+d\to e^-+n+p$ reaction is derived, using the formalism of the structure functions. The obtained expressions have a general nature and they hold in the one--photon--exchange mechanism, assuming P--invariance of the hadron electromagnetic interaction. The model of relativistic impulse approximation is introduced and the final state interaction is taken into account by means of the unitarization of the helicity amplitudes. A detailed description of the unitarization procedure is also presented. Using different parametrizations of the deuteron wave functions, the following polarization observables are calculated in the kinematical region of quasi--elastic deuteron electrodisintegration: the asymmetry for the scattering of longitudinally polarized electrons on a polarized deuteron target, the proton and neutron polarizations (for longitudinally polarized electron beam or vector--polarized deuteron target). The sensitivity to the neutron electric form factor is also thorougly investigated. The predictions of the model are compared with the results of recent polarization measurements and a good agreement with the existing experimental data has been obtained.Comment: 90 pages, 17 figure

A Comparison of Polarization Observables in Electron Scattering from the Proton and Deuteron

Recoil proton polarization observables were measured for both the p($\vec {\rm e}$,e$^\prime\vec{\rm p}\,$) and d($\vec {\rm e}$,e$^\prime\vec{\rm p}\,)$n reactions at two values of Q$^2$ using a newly commissioned proton Focal Plane Polarimeter at the M.I.T.-Bates Linear Accelerator Center. The hydrogen and deuterium spin-dependent observables $D_{\ell\ell}$ and $D_{{\ell}t}$, the induced polarization $P_n$ and the form factor ratio $G^p_E/G^p_M$ were measured under identical kinematics. The deuterium and hydrogen results are in good agreement with each other and with the plane-wave impulse approximation (PWIA).Comment: 9 pages, 1 figure; accepted by Phys. Rev. Let

Measurement of the vector analyzing power in elastic electron-proton scattering as a probe of double photon exchange amplitudes

We report the first measurement of the vector analyzing power in inclusive transversely polarized elastic electron-proton scattering at Q^2 = 0.1 (GeV/c)^2 and large scattering angles. This quantity should vanish in the single virtual photon exchange, plane wave impulse approximation for this reaction, and can therefore provide information on double photon exchange amplitudes for electromagnetic interactions with hadronic systems. We find a non-zero value of A=-15.4+/-5.4 ppm. No calculations of this observable for nuclei other than spin 0 have been carried out in these kinematics, and the calculation using the spin orbit interaction from a charged point nucleus of spin 0 cannot describe these data.Comment: 4 pages, 2 figures, submitted to Phys. Rev. Let

The inner membrane complex through development of Toxoplasma gondii and Plasmodium

Plasmodium spp. and Toxoplasma gondii are important human and veterinary pathogens. These parasites possess an unusual double membrane structure located directly below the plasma membrane named the inner membrane complex (IMC). First identified in early electron micrograph studies, huge advances in genetic manipulation of the Apicomplexa have allowed the visualization of a dynamic, highly structured cellular compartment with important roles in maintaining the structure and motility of these parasites. This review summarizes recent advances in the field and highlights the changes the IMC undergoes during the complex life cycles of the Apicomplexa

Parity Violation in Elastic Electron-Proton Scattering and the Proton's Strange Magnetic Form Factor

We report a new measurement of the parity-violating asymmetry in elastic electron scattering from the proton at backward scattering angles. This asymmetry is sensitive to the strange magnetic form factor of the proton as well as electroweak axial radiative corrections. The new measurement of A=-4.92 +- 0.61 +- 0.73 ppm provides a significant constraint on these quantities. The implications for the strange magnetic form factor are discussed in the context of theoretical estimates for the axial corrections.Comment: 4 pages, 3 figures, submitted to Physical Review Letters, Sept 199