Rauh, F. | bioRxiv Science

bioRxiv2025

Systematic Discovery of Pathogen Effector Functions across Human Pathogens and Pathways

Pathogens deploy effector proteins to exploit host cell biology, and most pathogen open reading frames (ORFs) are rapidly evolving and lack functional annotation. We developed the eORFeome, a scalable functional genomics platform encompassing 3,835 effector ORFs from diverse viruses, bacteria, and parasites. High-throughput barcoded screens across NF{kappa}B, apoptosis, p53, cGAS-STING and MHC-I pathways revealed functions for hundreds of uncharacterized eORFs, unexpected new activities for known effectors, and distinct pathway-specific functions encoded by single ORFs. Illustrating the power of the approach, we identify HHV6A U14 as a p53 antagonist, HHV7 U21 as a dual-function STING antagonist and MHC-I antigen display inhibitor, and adenoviral 13.6K/i-leader protein as a de novo evolved TAP inhibitor that suppresses MHC-I display. These results establish a general framework for systematic effector annotation, uncover new mechanisms of host-pathogen interaction across kingdoms, and highlight pathogen effectors as a versatile toolkit for rewiring and probing human cellular pathways.

systems biology↗

bioRxiv2019

Mechanism of in vivo activation of the MutLγ-Exo1 complex for meiotic crossover formation

Crossovers generated during the repair of programmed double-strand breaks (DSBs) during homologous recombination are essential for fertility to allow accurate homolog segregation during the first meiotic division. Most crossovers arise through the cleavage of recombination intermediates by the Mlh1-Mlh3 (MutL{gamma}) endonuclease and an elusive non-catalytic function of Exo1, and require the Polo kinase Cdc5. Here we show in budding yeast that MutL{gamma} forms a constitutive complex with Exo1, and in meiotic cells transiently contacts the Msh4-Msh5 (MutS{gamma}) heterodimer, also required for crossover formation. We further show that MutL{gamma}-Exo1 associates with recombination intermediates once they are committed to the crossover repair pathway, and then Exo1 recruits Cdc5 through a direct interaction that is required for activating MutL{gamma} and crossover formation. Exo1 therefore serves as a non-catalytic matchmaker between Cdc5 and MutL{gamma}. We finally show that in vivo, MutL{gamma} associates with the vast majority of DSB hotspots, but at a lower frequency near centromeres, consistent with a strategy to reduce at-risk crossover events in these regions. Our data highlight the tight temporal and spatial control of the activity of a constitutive, potentially harmful, nuclease.

molecular biology↗