Protocol to analyze the transmigration efficiency of T. brucei using an in vitro model of the blood-cerebrospinal fluid barrier.

At present, the only approach to investigate the transmigration of Trypanosoma brucei, the causative agent of human African trypanosomiasis, from blood to cerebrospinal fluid is through animal experiments. This protocol details how to analyze the transmigration efficiency using an in vitro model of the blood-cerebrospinal fluid (blood-CSF) barrier. We describe how to grow human choroid plexus epithelial cells on cell culture filter inserts to form the barrier, followed by isolating and quantifying genomic DNA of transmigrated parasites by qPCR. For complete details on the use and execution of this protocol, please refer to Speidel et al. (2022).

Transmigration of Trypanosoma brucei across an in vitro blood-cerebrospinal fluid barrier.

Trypanosoma brucei is the causative agent of human African trypanosomiasis. The parasite transmigrates from blood vessels across the choroid plexus epithelium to enter the central nervous system, a process that leads to the manifestation of second stage sleeping sickness. Using an in vitro model of the blood-cerebrospinal fluid barrier, we investigated the mechanism of the transmigration process. For this, a monolayer of human choroid plexus papilloma cells was cultivated on a permeable membrane that mimics the basal lamina underlying the choroid plexus epithelial cells. Plexus cells polarize and interconnect forming tight junctions. Deploying different T. brucei brucei strains, we observed that geometry and motility are important for tissue invasion. Using fluorescent microscopy, the parasite's moving was visualized between plexus epithelial cells. The presented model provides a simple tool to screen trypanosome libraries for their ability to infect cerebrospinal fluid or to test the impact of chemical substances on transmigration.

A Simple Approach to Perform TEER Measurements Using a Self-Made Volt-Amperemeter with Programmable Output Frequency.

Transepithelial/endothelial electrical resistance (TEER) has been used since the 1980s to determine confluency and permeability of in vitro barrier model systems. In most cases, chopstick electrodes are used to determine the electric impedance between the upper and lower compartment of a cell culture filter insert system containing cellular monolayers. The filter membrane allows the cells to adhere, polarize, and interact by building tight junctions. This technique has been described with a variety of different cell lines (e.g., cells of the blood-brain barrier, blood-cerebrospinal fluid barrier, or gastrointestinal and pulmonary tract). TEER measurement devices can be readily obtained from different laboratory equipment suppliers. However, there are more cost-effective and customizable solutions imaginable if an appropriate voltammeter is self-assembled. The overall aim of this publication is to set up a reliable device with programmable output frequency that can be used with commercially available chopstick electrodes for TEER measurement.

The conserved hypothetical protein Tb427.10.13790 is required for cytokinesis in Trypanosoma brucei.

Trypanosoma brucei, a flagellated protozoan causing the deadly tropical disease Human African Trypanosomiasis (HAT), affects people in sub-Saharan Africa. HAT therapy relies upon drugs which use is limited by toxicity and rigorous treatment regimes, while development of vaccines remains elusive, due to the effectiveness of the parasite´s antigenic variation. Here, we evaluate a hypothetical protein Tb427.10.13790, as a potential drug target. This protein is conserved among all kinetoplastids, but lacks homologs in all other pro- and eukaryotes. Knockdown of Tb427.10.13790 resulted in appearance of monster cells containing multiple nuclei and multiple flagella, a considerable enlargement of the flagellar pocket and eventually a lethal phenotype. Furthermore, analysis of kinetoplast and nucleus division in the knockdown cell line revealed a partial cell cycle arrest and failure to initiate cytokinesis. Likewise, overexpression of the respective protein fused with enhanced green fluorescent protein was also lethal for T. brucei. In these cells, the labelled protein appeared as a single dot near kinetoplast and flagellar pocket. Our results reveal that Tb427.10.13790 is essential for the parasite´s viability and may be a suitable new anti-trypanosomatid drug target candidate. Furthermore, we suggest that it might be worthwhile to investigate also other of the many so far just annotated trypanosome genes as a considerable number of them to lack human homologs but may be of critical importance for the kinetoplastid parasites.

Morphological changes, nitric oxide production, and phagocytosis are triggered in vitro in microglia by bloodstream forms of Trypanosoma brucei.

The flagellated parasite Trypanosoma brucei is the causative agent of Human African Trypanosomiasis (HAT). By a mechanism not well understood yet, trypanosomes enter the central nervous system (CNS), invade the brain parenchyma, and cause a fatal encephalopathy if is not treated. Trypanosomes are fast dividing organisms that, without any immune response, would kill the host in a short time. However, infected individuals survive either 6-12 months or more than 3 years for the acute and chronic forms, respectively. Thus, only when the brain defense collapses a lethal encephalopathy will occur. Here, we evaluated interactions between trypanosomes and microglial cells, which are the primary immune effector cells within the CNS. Using co-cultures of primary microglia and parasites, we found clear evidences of trypanosome phagocytosis by microglial cells. Microglia activation was also evident; analysis of its ultrastructure showed changes that have been reported in activated microglia undergoing oxidative stress caused by infections or degenerative diseases. Accordingly, an increase of the nitric oxide production was detected in supernatants of microglia/parasite co-cultures. Altogether, our results demonstrate that microglial cells respond to the presence of the parasite, leading to parasite's engulfment and elimination.

African trypanosomes and brain infection - the unsolved question.

African trypanosomes induce sleeping sickness. The parasites are transmitted during the blood meal of a tsetse fly and appear primarily in blood and lymph vessels, before they enter the central nervous system. During the latter stage, trypanosomes induce a deregulation of sleep-wake cycles and some additional neurological disorders. Historically, it was assumed that trypanosomes cross the blood-brain barrier and settle somewhere between the brain cells. The brain, however, is a strictly controlled and immune-privileged area that is completely surrounded by a dense barrier that covers the blood vessels: this is the blood-brain barrier. It is known that some immune cells are able to cross this barrier, but this requires a sophisticated mechanism and highly specific cell-cell interactions that have not been observed for trypanosomes within the mammalian host. Interestingly, trypanosomes injected directly into the brain parenchyma did not induce an infection. Likewise, after an intraperitoneal infection of rats, Trypanosoma brucei brucei was not observed within the brain, but appeared readily within the cerebrospinal fluid (CSF) and the meninges. Therefore, the parasite did not cross the blood-brain barrier, but the blood-CSF barrier, which is formed by the choroid plexus, i.e. the part of the ventricles where CSF is produced from blood. While there is no question that trypanosomes are able to invade the brain to induce a deadly encephalopathy, controversy exists about the pathway involved. This review lists experimental results that support crossing of the blood-brain barrier and of the blood-CSF barrier and discuss the implications that either pathway would have on infection progress and on the survival strategy of the parasite. For reasons discussed below, we prefer the latter pathway and suggest the existence of an additional distinct meningeal stage, from which trypanosomes could invade the brain via the Virchow-Robin space thereby bypassing the blood-brain barrier. We also consider healthy carriers, i.e. people living symptomless with the disease for up to several decades, and discuss implications the proposed meningeal stage would have for new anti-trypanosomal drug development. Considering the re-infection of blood, a process called relapse, we discuss the likely involvement of the newly described glymphatic connection between the meningeal space and the lymphatic system, that seems also be important for other infectious diseases.

In vivo protein crystallization in combination with highly brilliant radiation sources offers novel opportunities for the structural analysis of post-translationally modified eukaryotic proteins.

During the last decade, the number of three-dimensional structures solved by X-ray crystallography has increased dramatically. By 2014, it had crossed the landmark of 100 000 biomolecular structures deposited in the Protein Data Bank. This tremendous increase in successfully crystallized proteins is primarily owing to improvements in cloning strategies, the automation of the crystallization process and new innovative approaches to monitor crystallization. However, these improvements are mainly restricted to soluble proteins, while the crystallization and structural analysis of membrane proteins or proteins that undergo major post-translational modifications remains challenging. In addition, the need for relatively large crystals for conventional X-ray crystallography usually prevents the analysis of dynamic processes within cells. Thus, the advent of high-brilliance synchrotron and X-ray free-electron laser (XFEL) sources and the establishment of serial crystallography (SFX) have opened new avenues in structural analysis using crystals that were formerly unusable. The successful structure elucidation of cathepsin B, accomplished by the use of microcrystals obtained by in vivo crystallization in baculovirus-infected Sf9 insect cells, clearly proved that crystals grown intracellularly are very well suited for X-ray analysis. Here, methods by which in vivo crystals can be obtained, isolated and used for structural analysis by novel highly brilliant XFEL and synchrotron-radiation sources are summarized and discussed.

The lane to the brain: how African trypanosomes invade the CNS.

African trypanosomes induce sleeping sickness. Although it is clear that this parasite moves from the blood to the central nervous system (CNS) to induce the second stage of the disease, little is known about the molecular details of this process. Considering new findings of the trypanosome localization, this opinion paper will summarize the current knowledge about CNS infection, propose a different perception of the invasion process, and discuss possible consequences for drug development.

Cyclical appearance of African trypanosomes in the cerebrospinal fluid: new insights in how trypanosomes enter the CNS.

It is textbook knowledge that human infective forms of Trypanosoma brucei, the causative agent of sleeping sickness, enter the brain across the blood-brain barrier after an initial phase of weeks (rhodesiense) or months (gambiense) in blood. Based on our results using an animal model, both statements seem questionable. As we and others have shown, the first infection relevant crossing of the blood brain border occurs via the choroid plexus, i.e. via the blood-CSF barrier. In addition, counting trypanosomes in blood-free CSF obtained by an atlanto-occipital access revealed a cyclical infection in CSF that was directly correlated to the trypanosome density in blood infection. We also obtained conclusive evidence of organ infiltration, since parasites were detected in tissues outside the blood vessels in heart, spleen, liver, eye, testis, epididymis, and especially between the cell layers of the pia mater including the Virchow-Robin space. Interestingly, in all organs except pia mater, heart and testis, trypanosomes showed either a more or less degraded appearance of cell integrity by loss of the surface coat (VSG), loss of the microtubular cytoskeleton and loss of the intracellular content, or where taken up by phagocytes and degraded intracellularly within lysosomes. This is also true for trypanosomes placed intrathecally into the brain parenchyma using a stereotactic device. We propose a different model of brain infection that is in accordance with our observations and with well-established facts about the development of sleeping sickness.

Serial crystallography on in vivo grown microcrystals using synchrotron radiation.

Crystal structure determinations of biological macromolecules are limited by the availability of sufficiently sized crystals and by the fact that crystal quality deteriorates during data collection owing to radiation damage. Exploiting a micrometre-sized X-ray beam, high-precision diffractometry and shutterless data acquisition with a pixel-array detector, a strategy for collecting data from many micrometre-sized crystals presented to an X-ray beam in a vitrified suspension is demonstrated. By combining diffraction data from 80 Trypanosoma brucei procathepsin B crystals with an average volume of 9 µm(3), a complete data set to 3.0 Šresolution has been assembled. The data allowed the refinement of a structural model that is consistent with that previously obtained using free-electron laser radiation, providing mutual validation. Further improvements of the serial synchrotron crystallography technique and its combination with serial femtosecond crystallography are discussed that may allow the determination of high-resolution structures of micrometre-sized crystals.

Natively inhibited Trypanosoma brucei cathepsin B structure determined by using an X-ray laser.

The Trypanosoma brucei cysteine protease cathepsin B (TbCatB), which is involved in host protein degradation, is a promising target to develop new treatments against sleeping sickness, a fatal disease caused by this protozoan parasite. The structure of the mature, active form of TbCatB has so far not provided sufficient information for the design of a safe and specific drug against T. brucei. By combining two recent innovations, in vivo crystallization and serial femtosecond crystallography, we obtained the room-temperature 2.1 angstrom resolution structure of the fully glycosylated precursor complex of TbCatB. The structure reveals the mechanism of native TbCatB inhibition and demonstrates that new biomolecular information can be obtained by the "diffraction-before-destruction" approach of x-ray free-electron lasers from hundreds of thousands of individual microcrystals.

Late stage infection in sleeping sickness.

At the turn of the 19(th) century, trypanosomes were identified as the causative agent of sleeping sickness and their presence within the cerebrospinal fluid of late stage sleeping sickness patients was described. However, no definitive proof of how the parasites reach the brain has been presented so far. Analyzing electron micrographs prepared from rodent brains more than 20 days after infection, we present here conclusive evidence that the parasites first enter the brain via the choroid plexus from where they penetrate the epithelial cell layer to reach the ventricular system. Adversely, no trypanosomes were observed within the parenchyma outside blood vessels. We also show that brain infection depends on the formation of long slender trypanosomes and that the cerebrospinal fluid as well as the stroma of the choroid plexus is a hostile environment for the survival of trypanosomes, which enter the pial space including the Virchow-Robin space via the subarachnoid space to escape degradation. Our data suggest that trypanosomes do not intend to colonize the brain but reside near or within the glia limitans, from where they can re-populate blood vessels and disrupt the sleep wake cycles.