Abstract 1246 Comparative Transcriptomic Approach to Find Cystogenic Regulators of Toxoplasma gondii

Intracellular bacterial pathogens have evolved to avoid detection and destruction by the host cells they infect.Mycobacterium tuberculosis (Mtb), the bacterium that causes tuberculosis and kills 1.5 million people every year, has co-evolved with humans for millennia, and as a result, has an impressive collection of host manipulation strategies.Central to Mtb's subversion of host defenses is its arsenal of effector proteins that it secretes into host cells during infection.One class of Mtb effectors is the massive family of PE/PPE proteins, which includes 170 proteins encoded by an impressive 10% of the Mtb genome.PE/PPE proteins have conserved N-termini containing Pro-Glu (PE) or Pro-Pro-Glu (PPE) motifs and diverse C-terminal domains predicted to mediate host-pathogen interactions.However, PE/PPEs' exceedingly repetitive sequences have made them challenging to study, leaving their precise contributions to Mtb virulence a major unanswered question.To understand how Mtb uses PE/PPEs to manipulate host cell biology, we are systematically expressing each PE/PPE in macrophages and using immunoprecipitations and mass spectrometry to catalog their protein interactions and infer their functions.For one protein, PE5, we identified a specific interaction with the ubiquitin ligase complex CRL2 and one of CRL2's substrate receptors, KLHDC2.KLHDC2 binds to substrates with C-terminal Gly-Gly motifs, which PE5 in fact contains.Intriguingly, PE5 does not appear to be ubiquitinated, suggesting it may be an inhibitor of CRL2-KLHDC2 rather than a substrate.We also discovered that expression of PE5 in macrophages led to mislocalization of plasma membrane proteins, decreased phagocytosis, and increased survival of intracellular bacteria, suggesting PE5 blocks endosomal trafficking pathways.This appears to require PE5's interaction with CRL2-KLHDC2 since expressing a PE5 mutant lacking its C-terminal Gly-Gly does not affect trafficking.Together, our data indicate that PE5 is a bona fide Mtb virulence factor that modulates CRL2-KLHDC2 to alter endosomal trafficking and promote replication of intracellular bacterial pathogens.Our ongoing work seeks to understand precisely how PE5 binding to CRL2-KLHDC2 leads to changes in trafficking.We expect these findings to deepen our understanding of Mtb virulence and point to new therapies for tuberculosis.