Characterization of a Novel Sensing Mechanism Governing Antigenic Variation in P. Falciparum

Plasmodium falciparum expresses a multi-copy gene family called var in the intraerythroyctic stages of its life cycle in a mutually exclusive manner. var genes encode the chief antigenic and virulence determinant of P. falciparum, PfEMP1, and switching between active genes results in antigenic variation, allowing the parasite to evade the human immune system and cause chronic infections. The molecular mechanisms that control activation and silencing of individual var genes, as well as coordination of the switching process, presently remain incompletely defined. P. falciparum contains only ~60 var gene family members in its genome. Consequently, the question remains as to how this parasite can maintain an antigen-switch rate that allows for the emergence of a new variant when necessary, without rapidly exhausting all 60 members, to sustain chronic infections. The currently held paradigm proposes that antigenic variation follows an intrinsic, programed switching rate, operating independently of any external stimuli. In the following thesis I will present results suggesting the novel possibility that P. falciparum possesses cellular machinery capable of sensing changes in the environment of its host and is able to respond by altering antigen expression. It has been shown that changes in the transcription state of a var gene are controlled epigenetically. The methylation state of histone marks, deposited at active and silent var genes by histone methyltransferases (HMTs), play prominent roles in var gene regulation. Previously, Ukaegbu et al., 2015 showed that manipulating deposition of these marks had a striking impact on var gene expression. Metabolism and epigenetic control of gene expression are linked, as HMT activity is dependent on the intracellular concentrations of methyl donors, which can fluctuate based on nutrient availability. Various studies in other organisms have shown that there is a direct link between the level of intracellular S-adenosylmethionine (SAM), the principle methyl donor in biological methylation modifications, and histone methylation. I explored this connection between metabolism and var gene expression in P. falciparum. Parasites were cultured in growth media containing altered concentrations of nutrients involved in SAM metabolism. Bulk RNA was extracted from cultures, used as a template to synthesize cDNA, and analyzed by qPCR to determine the var gene expression at the population level. Conditions believed to increase SAM pools induced a coordinated switch to one particular var gene, var2csa, over time, phenocopying the results from Ukaegbu et al., 2015. This hypothesis was further tested by modifying expression of key enzymes involved in SAM metabolism. Once again, modifications thought to increase the intracellular level of SAM were found to induce a coordinated switch at the population level to var2csa. Conversely, modifications that lower the level of SAM did not induce expression of var2csa, but instead activated many vars at once across the population. These observations directly challenge the stochastic var switching paradigm by instead suggesting P. falciparum possesses the ability to sense environmental changes. After recognition of a pathogen, activated macrophages modify their microenvironments in various ways. I next tested the effect of two of these immune responses, depletion of amino acids and release of polyamines, on var expression of parasites in vitro. Both perturbations altered var expression, again specifically inducing var2csa. Taking these results together, I propose and discuss two possible models of antigenic variation in P. falciparum. The first centers on intracellular SAM metabolism in describing a promoter competition model governing var switching through var2csa. The second suggests that P. falciparum can sense when the host immune system first begins to recognize it via environmental cues resulting from antibody recognition, and respond by switching var gene expression. This would allow parasites to switch expression of var genes exactly when needed, allowing the most efficient utilization of their limited var gene repertoir