Legionella pneumophila, the etiological agent of Legionnaires’ disease, employs an arsenal of hundreds of Dot/Icm-translocated effector proteins to facilitate replication within eukaryotic phagocytes. Several effectors, called metaeffectors, function to regulate the activity of other Dot/Icm-translocated effectors during infection. The metaeffector Lpg2505 is essential for L. pneumophila intracellular replication only when its cognate effector, SidI, is present. SidI is a cytotoxic effector that interacts with the host translation factor eEF1A and potently inhibits eukaryotic protein translation by an unknown mechanism. Here, we evaluated the impact of Lpg2505 on SidI-mediated phenotypes and investigated the mechanism of SidI function. We determined that Lpg2505 binds with nanomolar affinity to SidI and suppresses SidI-mediated inhibition of protein translation. SidI binding to eEF1A and Lpg2505 is not mutually exclusive, and the proteins bind distinct regions of SidI. We also discovered that SidI possesses GDP-dependent glycosyl hydrolase activity and that this activity is regulated by Lpg2505. We have therefore renamed Lpg2505 MesI (metaeffector of SidI). This work reveals novel enzymatic activity for SidI and provides insight into how intracellular replication of L. pneumophila is regulated by a metaeffector.

19Legionella pneumophila, the etiological agent of Legionnaires Disease, employs an arsenal of 20 hundreds of Dot/Icm-translocated effector proteins to facilitate replication within eukaryotic 21 phagocytes. Several effectors, called metaeffectors, function regulate the activity of other 22 Dot/Icm-translocated effectors during infection. The metaeffector Lpg2505 is essential for L. 23 pneumophila intracellular replication only when its cognate effector, SidI, is present. SidI is a 24 cytotoxic effector that interacts with the host translation factor eEF1A and potently inhibits 25 eukaryotic protein translation by an unknown mechanism. Here, we evaluated the impact of 26Lpg2505 on SidI-mediated phenotypes and investigated the mechanism of SidI function. We 27 determined that Lpg2505 binds with nanomolar affinity to SidI and suppresses SidI-mediated 28 inhibition of protein translation. SidI binding to eEF1A and SusF is not mutually exclusive and 29 these proteins bind distinct regions of SidI. We also discovered that SidI possesses GDP-30 dependent glycosyltransferase activity and that this activity is regulated by Lpg2505. We have 31 therefore renamed Lpg2505, SusF (suppressor of SidI function). This work reveals novel 32 enzymatic activity for SidI and provides insight into how intracellular replication of L. 33 pneumophila is regulated by a metaeffector. 34 35 Introduction 36Legionella pneumophila is the etiological agent of Legionnaires' Disease, a severe 37 inflammatory pneumonia that results from uncontrolled bacterial replication within alveolar 38 macrophages. Upon phagocytosis, L. pneumophila avoids lysosomal degradation through 39 establishment of an endoplasmic reticulum-derived compartment called the Legionella-containing 40 vacuole (LCV) (1). For biogenesis of the LCV and acquisition of nutrients from the host cell, L. 41 pneumophila is dependent on a massive arsenal of over 300 individual effector proteins that are 42 translocated directly into the host cell through a Dot/Icm Type IVB secretion system (T4BSS) (2). 43The cellular functions of the majority of effectors have yet to be elucidated, due in part to their 44 functional redundancy within macrophages (3). 45Metaeffectors have emerged as a common theme in L. pneumophila pathogenesis and are 46 used by L. pneumophila to regulate effector function (4). The first metaeffector described was LubX, 47 which temporally regulates function of its cognate effector SidH by hijacking host ubiquitination 48 machinery to facilitate proteasomal degradation of SidH (5). At least 20 of the over 300 identified 49 L. pneumophila effectors are metaeffectors (4) and two of these -SidJ and Lpg2505 -are members 50 of a small group of effectors that are individually important for L. pneumophila intracellular 51 replication within macrophages (6-8). SidJ is a glutamylase that covalently modifies and 52 abrogates the function of the SidE family of effector ubiquitin ligases (9-11). Lpg2505 is a 53 metaeffector of unknown function that suppresses toxicity of its cognate...