Generation of Mature Toxoplasma gondii Bradyzoites in Human Immortalized Myogenic KD3 Cells.

Toxoplasma gondii is a zoonotic protozoan parasite and one of the most successful foodborne pathogens. Upon infection and dissemination, the parasites convert into the persisting, chronic form called bradyzoites, which reside within cysts in muscle and brain tissue. Despite their importance, bradyzoites remain difficult to investigate directly, owing to limited in vitro models. In addition, the need for new drugs targeting the chronic stage, which is underlined by the lack of eradicating treatment options, remains difficult to address since in vitro access to drug-tolerant bradyzoites remains limited. We recently published the use of a human myotube-based bradyzoite cell culture system and demonstrated its applicability to investigate the biology of T. gondii bradyzoites. Encysted parasites can be functionally matured during long-term cultivation in these immortalized cells and possess many in vivo-like features, including pepsin resistance, oral infectivity, and antifolate resistance. In addition, the system is scalable, enabling experimental approaches that rely on large numbers, such as metabolomics. In short, we detail the cultivation of terminally differentiated human myotubes and their subsequent infection with tachyzoites, which then mature to encysted bradyzoites within four weeks at ambient CO2 levels. We also discuss critical aspects of the procedure and suggest improvements. Key features • This protocol describes a scalable human myotube-based in vitro system capable of generating encysted bradyzoites featuring in vivo hallmarks. • Bradyzoite differentiation is facilitated through CO2 depletion but without additional artificial stress factors like alkaline pH. • Functional maturation occurs over four weeks.

Smaller size packs a stronger punch - Recent advances in small antibody fragments targeting tumour-associated carbohydrate antigens.

Attached to proteins, lipids, or forming long, complex chains, glycans represent the most versatile post-translational modification in nature and surround all human cells. Unique glycan structures are monitored by the immune system and differentiate self from non-self and healthy from malignant cells. Aberrant glycosylations, termed tumour-associated carbohydrate antigens (TACAs), are a hallmark of cancer and are correlated with all aspects of cancer biology. Therefore, TACAs represent attractive targets for monoclonal antibodies for cancer diagnosis and therapy. However, due to the thick and dense glycocalyx as well as the tumour micro-environment, conventional antibodies often suffer from restricted access and limited effectiveness in vivo. To overcome this issue, many small antibody fragments have come forth, showing similar affinity with better efficiency than their full-length counterparts. Here we review small antibody fragments against specific glycans on tumour cells and highlight their advantages over conventional antibodies.

Advances towards the complete in vitro life cycle of Toxoplasma gondii.

The full life cycle of Toxoplasma gondii cannot be recapitulated in vitro, and access to certain stages, such as mature tissue cysts (bradyzoites) and oocysts (sporozoites), traditionally requires animal experiments. This has greatly hindered the study of the biology of these morphologically and metabolically distinct stages, which are essential for the infection of humans and animals. However, several breakthrough advances have been made in recent years towards obtaining these life stages in vitro, such as the discovery of several molecular factors that induce differentiation and commitment to the sexual cycle, and different culture methods that use, for example, myotubes and intestinal organoids to obtain mature bradyzoites and different sexual stages of the parasite. We review these novel tools and approaches, highlight their limitations and challenges, and discuss what research questions can already be answered with these models. We finally identify future routes for recapitulating the entire sexual cycle in vitro.

Late Embryogenesis Abundant Proteins Contribute to the Resistance of Toxoplasma gondii Oocysts against Environmental Stresses.

Toxoplasma gondii oocysts, which are shed in large quantities in the feces from infected felines, are very stable in the environment, resistant to most inactivation procedures, and highly infectious. The oocyst wall provides an important physical barrier for sporozoites contained inside oocysts, protecting them from many chemical and physical stressors, including most inactivation procedures. Furthermore, sporozoites can withstand large temperature changes, even freeze-thawing, as well as desiccation, high salinity, and other environmental insults; however, the genetic basis for this environmental resistance is unknown. Here, we show that a cluster of four genes encoding Late Embryogenesis Abundant (LEA)-related proteins are required to provide Toxoplasma sporozoites resistance to environmental stresses. Toxoplasma LEA-like genes (TgLEAs) exhibit the characteristic features of intrinsically disordered proteins, explaining some of their properties. Our in vitro biochemical experiments using recombinant TgLEA proteins show that they have cryoprotective effects on the oocyst-resident lactate dehydrogenase enzyme and that induced expression in E. coli of two of them leads to better survival after cold stress. Oocysts from a strain in which the four LEA genes were knocked out en bloc were significantly more susceptible to high salinity, freezing, and desiccation compared to wild-type oocysts. We discuss the evolutionary acquisition of LEA-like genes in Toxoplasma and other oocyst-producing apicomplexan parasites of the Sarcocystidae family and discuss how this has likely contributed to the ability of sporozoites within oocysts to survive outside the host for extended periods. Collectively, our data provide a first molecular detailed view on a mechanism that contributes to the remarkable resilience of oocysts against environmental stresses. IMPORTANCE Toxoplasma gondii oocysts are highly infectious and may survive in the environment for years. Their resistance against disinfectants and irradiation has been attributed to the oocyst and sporocyst walls by acting as physical and permeability barriers. However, the genetic basis for their resistance against stressors like changes in temperature, salinity, or humidity, is unknown. We show that a cluster of four genes encoding Toxoplasma Late Embryogenesis Abundant (TgLEA)-related proteins are important for this resistance to environmental stresses. TgLEAs have features of intrinsically disordered proteins, explaining some of their properties. Recombinant TgLEA proteins show cryoprotective effects on the parasite's lactate dehydrogenase, an abundant enzyme in oocysts, and expression in E. coli of two TgLEAs has a beneficial effect on growth after cold stress. Moreover, oocysts from a strain lacking all four TgLEA genes were more susceptible to high salinity, freezing, and desiccation compared to wild-type oocysts, highlighting the importance of the four TgLEAs for oocyst resilience.

Generation of glycan-specific nanobodies.

The development of antibodies that target specific glycan structures on cancer cells or human pathogens poses a significant challenge due to the immense complexity of naturally occurring glycans. Automated glycan assembly enables the production of structurally homogeneous glycans in amounts that are difficult to derive from natural sources. Nanobodies (Nbs) are the smallest antigen-binding domains of heavy-chain-only antibodies (hcAbs) found in camelids. To date, the development of glycan-specific Nbs using synthetic glycans has not been reported. Here, we use defined synthetic glycans for alpaca immunization to elicit glycan-specific hcAbs, and describe the identification, isolation, and production of a Nb specific for the tumor-associated carbohydrate antigen Globo-H. The Nb binds the terminal fucose of Globo-H and recognizes synthetic Globo-H in solution and native Globo-H on breast cancer cells with high specificity. These results demonstrate the potential of our approach for generating glycan-targeting Nbs to be used in biomedical and biotechnological applications.

From 3D to 2D: Harmonization of Protocols for Two-dimensional Cultures on Cell Culture Inserts of Intestinal Organoids from Various Species.

In the expanding field of intestinal organoid research, various protocols for three- and two-dimensional organoid-derived cell cultures exist. Two-dimensional organoid-derived monolayers are used to overcome some limitations of three-dimensional organoid cultures. They are increasingly used also in infection research, to study physiological processes and tissue barrier functions, where easy experimental access of pathogens to the luminal and/or basolateral cell surface is required. This has resulted in an increasing number of publications reporting different protocols and media compositions for organoid manipulation, precluding direct comparisons of research outcomes in some cases. With this in mind, here we describe a protocol aimed at the harmonization of seeding conditions for three-dimensional intestinal organoids of four commonly used research species onto cell culture inserts, to create organoid-derived monolayers that form electrophysiologically tight epithelial barriers. We give an in-depth description of media compositions and culture conditions for creating these monolayers, enabling also the less experienced researchers to obtain reproducible results within a short period of time, and which should simplify the comparison of future studies between labs, but also encourage others to consider these systems as alternative cell culture models in their research. Graphic abstract: Schematic workflow of organoid-derived monolayer generation from intestinal spheroid cultures. ECM, extracellular matrix; ODM, organoid-derived monolayer.