Acquisition of human plasminogen facilitates complement evasion by the malaria parasite Plasmodium falciparum.

We show that the intraerythrocytic stages of the malaria parasite Plasmodium falciparum bind plasminogen and mediate its conversion into plasmin to inactivate parasite-bound C3b. This complement evasion mechanism counteracts terminal complex formation and hence promotes parasite survival in human blood.

Cutting Edge: FHR-1 Binding Impairs Factor H-Mediated Complement Evasion by the Malaria Parasite Plasmodium falciparum.

Human complement is the first line of defense against invading pathogens, including the malaria parasite Plasmodium falciparum We previously demonstrated that human complement represents a particular threat for the clinically relevant blood stages of the parasite. To evade complement-mediated destruction, the parasites acquire factor H (FH) via specific receptors. We now report that the FH-related protein FHR-1 competes with FH for binding to the parasites. FHR-1, which is composed of five complement control protein domains with variable homology to FH but lacks C3b regulatory activity, accumulates on the surfaces of intraerythrocytic schizonts and free merozoites. Although binding of FH to schizont-infected RBCs and merozoites is increased in FHR-1-deficient human serum, the addition of recombinant FHR-1 decreases FH binding. The presence of FHR-1 consequently impairs C3b inactivation and parasite viability. We conclude that FHR-1 acts as a protective factor in human immunity by counteracting FH-mediated microbial complement evasion.

Plasmodium falciparum Gametes and Sporozoites Hijack Plasmin and Factor H To Evade Host Complement Killing.

Plasmodium parasites are the etiological agents of malaria, a disease responsible for over half a million deaths annually. Successful completion of the parasite's life cycle in the vertebrate host and transmission to a mosquito vector is contingent upon the ability of the parasite to evade the host's defenses. The extracellular stages of the parasite, including gametes and sporozoites, must evade complement attack in both the mammalian host and in the blood ingested by the mosquito vector. Here, we show that Plasmodium falciparum gametes and sporozoites acquire mammalian plasminogen and activate it into the serine protease plasmin to evade complement attack by degrading C3b. Complement-mediated permeabilization of gametes and sporozoites was higher in plasminogen-depleted plasma, suggesting that plasminogen is important for complement evasion. Plasmin also facilitates gamete exflagellation through complement evasion. Furthermore, supplementing serum with plasmin significantly increased parasite infectivity to mosquitoes and lowered the transmission-blocking activity of antibodies to Pfs230, a potent vaccine candidate currently in clinical trials. Finally, we show that human factor H, previously shown to facilitate complement evasion by gametes, also facilitates complement evasion by sporozoites. Plasmin and factor H simultaneously cooperate to enhance complement evasion by gametes and sporozoites. Taken together, our data show that Plasmodium falciparum gametes and sporozoites hijack the mammalian serine protease plasmin to evade complement attack by degrading C3b. Understanding of the mechanisms of complement evasion by the parasite is key to the development of novel effective therapeutics. IMPORTANCE Current approaches to control malaria are complicated by the development of antimalarial-resistant parasites and insecticide-resistant vectors. Vaccines that block transmission to mosquitoes and humans are a plausible alternative to overcome these setbacks. To inform the development of efficacious vaccines, it is imperative to understand how the parasite interacts with the host immune response. In this report, we show that the parasite can co-opt host plasmin, a mammalian fibrinolytic protein to evade host complement attack. Our results highlight a potential mechanism that may reduce efficacy of potent vaccine candidates. Taken together, our results will inform future studies in developing novel antimalarial therapeutics.

The impact of human complement on the clinical outcome of malaria infection.

The tropical disease malaria remains a major cause of global morbidity. Once transmitted to the human by a blood-feeding mosquito, the unicellular malaria parasite comes into contact with the complement system and continues to interact with human complement during its intraerythrocytic replication cycles. In the course of infection, both the classical and the alternative pathway of complement are activated, leading to parasite opsonization and lysis as well as the induction of complement-binding antibodies. While complement activity can be linked to the severity of malaria, it remains to date unclear, whether human complement is beneficial for protective immunity or if extensive complement reactions may rather enhance pathogenesis. In addition, the parasite has evolved molecular strategies to circumvent attack by human complement and has even developed means to utilize complement factors as mediators of host cell infection. In this review, we highlight current knowledge on the role of human complement for the progression of malaria infection. We discuss the various types of interactions between malaria parasites and complement factors with regard to immunity and infection outcome and set a special emphasis on the dual role of complement in the context of parasite fitness.

Exploring the limits of polarization transfer efficiency in homonuclear three spin systems.

The limits of polarization transfer efficiency are explored for systems consisting of three isotropically coupled spins 1/2 in the absence of relaxation. An idealized free evolution and control Hamiltonian is studied, which provides an upper limit of transfer efficiency (in terms of transfer amplitude and transfer time) for realistic homonuclear spin systems with arbitrary Heisenberg-type coupling constants J12, J13, and J23. It is shown that optimal control based pulse sequences have significantly improved transfer efficiencies compared to conventional transfer schemes. An experimental demonstration of optimal polarization transfer is given for the case of the carbon spin system of fully 13C labelled alanine at 62.5 MHz Larmor frequency.

Tailoring the optimal control cost function to a desired output: application to minimizing phase errors in short broadband excitation pulses.

The de facto standard cost function has been used heretofore to characterize the performance of pulses designed using optimal control theory. The freedom to choose new, creative quality factors designed for specific purposes is demonstrated. While the methodology has more general applicability, its utility is illustrated by comparison to a consistently chosen example--broadband excitation. The resulting pulses are limited to the same maximum RF amplitude used previously and tolerate the same variation in RF homogeneity deemed relevant for standard high-resolution NMR probes. Design criteria are unchanged: transformation of I(z)--> I(x) over resonance offsets of +/-20 kHz and RF variability of +/-5%, with a peak RF amplitude equal to 17.5 kHz. However, the new cost effectively trades a small increase in residual z magnetization for improved phase in the transverse plane. Compared to previous broadband excitation by optimized pulses (BEBOP), significantly shorter pulses are achievable, with only marginally reduced performance. Simulations transform I(z) to greater than 0.98 I(x), with phase deviations of the final magnetization less than 2 degrees, over the targeted ranges of resonance offset and RF variability. Experimental performance is in excellent agreement with the simulations.

Optimal control of coupled spin dynamics: design of NMR pulse sequences by gradient ascent algorithms.

In this paper, we introduce optimal control algorithm for the design of pulse sequences in NMR spectroscopy. This methodology is used for designing pulse sequences that maximize the coherence transfer between coupled spins in a given specified time, minimize the relaxation effects in a given coherence transfer step or minimize the time required to produce a given unitary propagator, as desired. The application of these pulse engineering methods to design pulse sequences that are robust to experimentally important parameter variations, such as chemical shift dispersion or radiofrequency (rf) variations due to imperfections such as rf inhomogeneity is also explained.

Broadband geodesic pulses for three spin systems: time-optimal realization of effective trilinear coupling terms and indirect SWAP gates.

Broadband implementations of time-optimal geodesic pulse elements are introduced for the efficient creation of effective trilinear coupling terms for spin systems consisting of three weakly coupled spins 1/2. Based on these pulse elements, the time-optimal implementation of indirect SWAP operations is demonstrated experimentally. The duration of indirect SWAP gates based on broadband geodesic sequence is reduced by 42.3% compared to conventional approaches.

Time-optimal coherence-order-selective transfer of in-phase coherence in heteronuclear IS spin systems.

Based on principles of geometric optimal control theory, coherence transfer building blocks can be derived which achieve optimal sensitivity. Here, experimental pulse sequences are presented that achieve the best possible coherence-order-selective in-phase transfer (S(-)-->I(-)) for a heteronuclear 2-spin system for any given mixing time in the absence of relaxation. For short mixing times, the optimal experiment improves the sensitivity of isotropic mixing by up to 12.5%.

Optimal control of spin dynamics in the presence of relaxation.

Experiments in coherent spectroscopy correspond to control of quantum mechanical ensembles guiding them from initial to final target states. The control inputs (pulse sequences) that accomplish these transformations should be designed to minimize the effects of relaxation and to optimize the sensitivity of the experiments. For example in nuclear magnetic resonance (NMR) spectroscopy, a question of fundamental importance is what is the maximum efficiency of coherence or polarization transfer between two spins in the presence of relaxation. Furthermore, what is the optimal pulse sequence which achieves this efficiency? In this paper, we give analytical answers to the above questions. Unexpected gains in sensitivity are reported for one of the most commonly used experimental building blocks in NMR spectroscopy. Surprisingly, in the case when longitudinal relaxation is small, the relaxation optimized pulse elements (ROPE) that transfer maximum polarization between coupled spins are longer than conventional sequences.

Reducing the duration of broadband excitation pulses using optimal control with limited RF amplitude.

Combining optimal control theory with a new RF limiting step produces pulses with significantly reduced duration and improved performance for a given maximum RF amplitude compared to previous broadband excitation by optimized pulses (BEBOP). The resulting pulses tolerate variations in RF homogeneity relevant for standard high-resolution NMR probes. Design criteria were transformation of Iz-->Ix over resonance offsets of +/-20kHz and RF variability of +/-5%, with a pulse length of 500 micros and peak RF amplitude equal to 17.5 kHz. Simulations transform Iz to greater than 0.995 Ix, with phase deviations of the final magnetization less than 2 degrees, over ranges of resonance offset and RF variability that exceed the design targets. Experimental performance of the pulse is in excellent agreement with the simulations. Performance tradeoffs for yet shorter pulses or pulses with decreased digitization are also investigated.

Application of optimal control theory to the design of broadband excitation pulses for high-resolution NMR.

Optimal control theory is considered as a methodology for pulse sequence design in NMR. It provides the flexibility for systematically imposing desirable constraints on spin system evolution and therefore has a wealth of applications. We have chosen an elementary example to illustrate the capabilities of the optimal control formalism: broadband, constant phase excitation which tolerates miscalibration of RF power and variations in RF homogeneity relevant for standard high-resolution probes. The chosen design criteria were transformation of I(z)-->I(x) over resonance offsets of +/- 20 kHz and RF variability of +/-5%, with a pulse length of 2 ms. Simulations of the resulting pulse transform I(z)-->0.995I(x) over the target ranges in resonance offset and RF variability. Acceptably uniform excitation is obtained over a much larger range of RF variability (approximately 45%) than the strict design limits. The pulse performs well in simulations that include homonuclear and heteronuclear J-couplings. Experimental spectra obtained from 100% 13C-labeled lysine show only minimal coupling effects, in excellent agreement with the simulations. By increasing pulse power and reducing pulse length, we demonstrate experimental excitation of 1H over +/-32 kHz, with phase variations in the spectra <8 degrees and peak amplitudes >93% of maximum. Further improvements in broadband excitation by optimized pulses (BEBOP) may be possible by applying more sophisticated implementations of the optimal control formalism.

Improving solid-state NMR dipolar recoupling by optimal control.

We present the first solid-state NMR experiments developed using optimal control theory. Taking heteronuclear dipolar recoupling in magic-angle-spinning NMR as an example, it proves possible to significantly improve the efficiency of the experiments while introducing robustness toward instrumental imperfections such as radio frequency inhomogeneity. The improvements are demonstrated by numerical simulations as well as practical experiments on a 13Calpha,15N-labeled powder of glycine. The experiments demonstrate a gain of 53% in the efficiency for 15N to 13Calpha coherence transfer relative to the typically double-cross-polarization experiments.