Toxoplasma gondii's Basal Complex: The Other Apicomplexan Business End Is Multifunctional.

The Apicomplexa are famously named for their apical complex, a constellation of organelles at their apical end dedicated to invasion of their host cells. In contrast, at the other end of the cell, the basal complex (BC) has been overshadowed since it is much less prominent and specific functions were not immediately obvious. However, in the past decade a staggering array of functions have been associated with the BC and strides have been made in understanding its structure. Here, these collective insights are supplemented with new data to provide an overview of the understanding of the BC in Toxoplasma gondii. The emerging picture is that the BC is a dynamic and multifunctional complex, with a series of (putative) functions. The BC has multiple roles in cell division: it is the site where building blocks are added to the cytoskeleton scaffold; it exerts a two-step stretch and constriction mechanism as contractile ring; and it is key in organelle division. Furthermore, the BC has numerous putative roles in 'import', such as the recycling of mother cell remnants, the acquisition of host-derived vesicles, possibly the uptake of lipids derived from the extracellular medium, and the endocytosis of micronemal proteins. The latter process ties the BC to motility, whereas an additional role in motility is conferred by Myosin C. Furthermore, the BC acts on the assembly and/or function of the intravacuolar network, which may directly or indirectly contribute to the establishment of chronic tissue cysts. Here we provide experimental support for molecules acting in several of these processes and identify several new BC proteins critical to maintaining the cytoplasmic bridge between divided parasites. However, the dispensable nature of many BC components leaves many questions unanswered regarding its function. In conclusion, the BC in T. gondii is a dynamic and multifunctional structure at the posterior end of the parasite.

The Extracellular Milieu of Toxoplasma's Lytic Cycle Drives Lab Adaptation, Primarily by Transcriptional Reprogramming.

Evolve and resequencing (E&R) was applied to lab adaptation of Toxoplasma gondii for over 1,500 generations with the goal of mapping host-independent in vitro virulence traits. Phenotypic assessments of steps across the lytic cycle revealed that only traits needed in the extracellular milieu evolved. Nonsynonymous single-nucleotide polymorphisms (SNPs) in only one gene, a P4 flippase, fixated across two different evolving populations, whereas dramatic changes in the transcriptional signature of extracellular parasites were identified. Newly developed computational tools correlated phenotypes evolving at different rates with specific transcriptomic changes. A set of 300 phenotype-associated genes was mapped, of which nearly 50% is annotated as hypothetical. Validation of a select number of genes by knockouts confirmed their role in lab adaptation and highlights novel mechanisms underlying in vitro virulence traits. Further analyses of differentially expressed genes revealed the development of a "pro-tachyzoite" profile as well as the upregulation of the fatty acid biosynthesis (FASII) pathway. The latter aligned with the P4 flippase SNP and aligned with a low abundance of medium-chain fatty acids at low passage, indicating this is a limiting factor in extracellular parasites. In addition, partial overlap with the bradyzoite differentiation transcriptome in extracellular parasites indicated that stress pathways are involved in both situations. This was reflected in the partial overlap between the assembled ApiAP2 and Myb transcription factor network underlying the adapting extracellular state with the bradyzoite differentiation program. Overall, E&R is a new genomic tool successfully applied to map the development of polygenic traits underlying in vitro virulence of T. gondii. IMPORTANCE It has been well established that prolonged in vitro cultivation of Toxoplasma gondii augments progression of the lytic cycle. This lab adaptation results in increased capacities to divide, migrate, and survive outside a host cell, all of which are considered host-independent virulence factors. However, the mechanistic basis underlying these enhanced virulence features is unknown. Here, E&R was utilized to empirically characterize the phenotypic, genomic, and transcriptomic changes in the non-lab-adapted strain, GT1, during 2.5 years of lab adaptation. This identified the shutdown of stage differentiation and upregulation of lipid biosynthetic pathways as the key processes being modulated. Furthermore, lab adaptation was primarily driven by transcriptional reprogramming, which rejected the starting hypothesis that genetic mutations would drive lab adaptation. Overall, the work empirically shows that lab adaptation augments T. gondii's in vitro virulence by transcriptional reprogramming and that E&R is a powerful new tool to map multigenic traits.

Isolation and Transfection of Primary Culture Bovine Retinal Pericytes.

This protocol describes an enzymatic approach for isolating homogeneous cultures of pericytes from retinas of bovine source. In summary, retinas are dissected, washed, digested, filtered, cultured in specific media to select for pericytes, and finally expanded for a low passage culture of about 14 million bovine retinal pericytes (BRP) within 4-6 weeks. This protocol also describes a liposomal-based technique for transfection of BRPs.