Legionella pneumophila and related species are emerging opportunistic human pathogens causing the potentially fatal pneumonia Legionnaires’ disease. The virulence of Legionella species and ability to exploit eukaryotic host cell processes for intracellular replication depends on the Dot/Icm type IV secretion system (T4SS), translocating >300 effector proteins into host cells. Although several effectors have been functionally characterised, the molecular activities and host targets of many remain unclear.  Through comparative genomics we identified a family of peptidase C58-like T4SS effectors encoded arcross a variety of Legionella species. Functional characterisation of the most conserved member of the family revealed that it is a protease with autoproteolytic activity and, upon ectopic expression, has profound effects on the physiology of eukaryotic cells, inducing changes to ER structure and nuclear morphology.  Interactome profiling using the inactive protease identified association with proteins involved in nucleolar rRNA transcription, and ribosome biogenesis, among them Nucleolin (NCL). Subsequent experiments using the wildtype protease showed cleavage of NCL and complete loss of the nucleoli and a termination of translation.    Infection experiments using L. pneumophila 130b wildtype and a strain lacking the protease effector revealed the disruption of the nucleoli and nucleolar protein redistribution to the nucleoplasm as early as 6 h post infection; however, this phenotype was not dependent on the protease suggesting that redundant effectors targeting the nucleoli exist. Nevertheless, attenuated replication of the L. pneumophila protease effector mutant strain in host cells showed that it plays a significant role for the manipulation of host cells by the bacteria.