Trypanosoma brucei is a single-cell protozoan parasite that causes African sleeping sickness (trypanosomiasis). The parasite cycles between two different hosts, the insect vector, tsetse flies (glossina species), and the mammalian hosts. To adapt to these different environments, the parasite undergoes stages of life-cycle specific differentiation. Bites from infected flies can transmit the parasite into mammalian hosts. In the mammals, the parasite differentiates into a ‘bloodstream form (BF)’ and populates the host’s bloodstream and tissue spaces. My study targets the mammalian infectious form, with aims, firstly, to get knowledges of how cells maintain the genetic information, balancing conservation and variation. This is critical for the battle between pathogen and host, on both sides; the parasite to escape the killing from the host immunity (known as the antigenic variation) and the host to efficiently recognize the pathogens. By better understanding the genome integrity mechanisms that are central to all or unique to T. brucei, next I aim to find therapeutic tools that selectively kill the pathogens. My research focuses on following four areas that are currently at different stages.

Genome-wide functions of replication, chromatin, and transcription, ‘think locally act globally’

Duplication of genome is precisely monitored by and coordinated with a wide range of factors that are involved in DNA replication & repair and in transcription and chromatin biology. Although for the most part, genome duplication must be error free, some regions are programmed to be repaired in an error-prone fashion (e.g., meiotic recombination, AID-mediated somatic hypermutation in antibody diversification); or are prone to replication problems, due to local sequence context (e.g., repetitive sequences: rDNA arrays, telomeres), which then lead to genetic alterations. This dual need for genome maintenance and alteration is especially intensified in trypanosomes, where antigenic surface coat genes, Variant Surface Glycoprotein (VSG) genes are expressed from subtelomere. The parasite mono-allelically expresses a VSG from >2,500 VSG repertoire and occasionally switches to a different one via DNA recombination. The switched parasite is invisible by the immune cells mounted by the old VSG, so can survive from the immune killing. It is further exacerbated by the fact that all transcription units are polycistronic (in T. brucei, over 100 genes on average are transcribed by one promoter), their transcription boundaries marked by specific epigenetic marks. I found evidence that epigenetic marks are important for replication and, here, I am trying to answer a seemingly simple question: why is it that removal of three epigenetic marks that normally coincide at transcription termination sites, causal to symptoms that are characteristic of replication stress? Are these marks required in regulation of DNA replication, and if so, which aspects of regulation are affected in their absence (DNA replication origin recognition, origin choice, replication timing, or replication fork migration)? My hypothesis is that local changes can lead to global restructuring, as abnormalities were detected at loci other than their binding sites, transcription termination sites.

Identification of highly selective small molecule inhibitors with trypanocial activity

One of the most striking phenotypes of MCM-BP deficient mutant and mutants lacking three epigenetics marks is accumulation of sub-G1 and anucleated cells (zoids), which partially and completely lost the DNA, respectively (Kim 2018 NAR & Kim 2013 PLoS ONE). This was observed in population of up to 40-50% of cells at day 1 and day 2 after MCM-BP depletion. As these phenotypes will lead to lethality undoubtedly, not just growth arrest, MCM-BP can be a good target to identify small molecules with trypanocidal activities. Several of other candidates are currently considered for small molecular inhibitor project in collaboration with Dr. Junyong Choi at CUNY Queens College.

Development and application of large-scale screening tools

Of about 9,000 genes in the T. brucei genome database, about 60% are annotated ‘hypothetical’ or ‘hypothetical conserved’. Protein products of these have some or no sequence homology to any known proteins from other organisms. Although genome-wide screens toward protein identification using RNAi library has been developed recently, the converse approach (using over-expression screens to match to specific phenotypes) has been lacking. To fill this gap, I have recently generated an over-expression library for T. brucei with funding from NIH. In collaboration with Drs. Esteban Erben and Nina Papavasiliou at DKFZ (Germany), I am currently trying to validate this tool. In addition to tool development, I have on-going collaborations with Drs. Erben and Papavasiliou with various projects, including studies of control mechanisms for VSG mRNA transcript and VSG protein to understand mono-allelic gene expression as well as immune evasion mechanism.

Biochemical identification of key factors involved in T. brucei survival

 Chromatin factors must interact with proteins involved in replication, transcription and also with other genome or epigenome factors. This interaction is not only important for maintenance of genome integrity (survival of the pathogen) and for diversification of genome (immune evasion). By identifying the network of interaction between key players (protein-protein or protein-RNA interaction) via biochemical approaches, I aim to achieve fundamental understanding of how genome and epigenome contribute to pathogen survival, which then can help designing strategies to develop therapeutics for the disease treatment.