Citation: Nikolka, F.; Karagöz, M.S.; Nassef, M.Z.; Hiller, K.; Steinert, M.; Cordes, T. The Virulence Factor Macrophage Infectivity Potentiator (Mip) Influences Branched-Chain Amino Acid Metabolism and Pathogenicity of Legionella pneumophila. Metabolites 2023, 13, 834. https://doi.org/10.3390/ metabo13070834 Academic Editors: Nicole Strittmatter and Regina Verena Taudte Received: 28 April 2023 Revised: 15 June 2023 Accepted: 3 July 2023 Published: 11 July 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). metabolites H OH OH Article The Virulence Factor Macrophage Infectivity Potentiator (Mip) Influences Branched-Chain Amino Acid Metabolism and Pathogenicity of Legionella pneumophila Fabian Nikolka 1 , Mustafa Safa Karagöz 2 , Mohamed Zakaria Nassef 1 , Karsten Hiller 1 , Michael Steinert 2 and Thekla Cordes 1,3, * 1 Department of Bioinformatics and Biochemistry, Braunschweig Integrated Centre of Systems Biology (BRICS), Technische Universität Braunschweig, 38106 Braunschweig, Germany 2 Institut für Mikrobiologie, Braunschweig Integrated Centre of Systems Biology (BRICS), Technische Universität Braunschweig, 38106 Braunschweig, Germany 3 Research Group Cellular Metabolism in Infection, Helmholtz Centre for Infection Research, 38124 Braunschweig, Germany * Correspondence: thekla.cordes@tu-braunschweig.de Abstract: Legionella pneumophila (Lp) is a common etiological agent of bacterial pneumonia that causes Legionnaires’ disease (LD). The bacterial membrane-associated virulence factor macrophage infectiv- ity potentiator (Mip) exhibits peptidyl-prolyl-cis/trans-isomerase (PPIase) activity and contributes to the intra- and extracellular pathogenicity of Lp. Though Mip influences disease outcome, little is known about the metabolic consequences of altered Mip activity during infections. Here, we established a metabolic workflow and applied mass spectrometry approaches to decipher how Mip activity influences metabolism and pathogenicity. Impaired Mip activity in genetically engineered Lp strains decreases intracellular replication in cellular infection assays, confirming the contribution of Mip for Lp pathogenicity. We observed that genetic and chemical alteration of Mip using the PPIase inhibitors rapamycin and FK506 induces metabolic reprogramming in Lp, specifically branched-chain amino acid (BCAA) metabolism. Rapamycin also inhibits PPIase activity of mammalian FK506 binding proteins, and we observed that rapamycin induces a distinct metabolic signature in human macrophages compared to bacteria, suggesting potential involvement of Mip in normal bacteria and in infection. Our metabolic studies link Mip to alterations in BCAA metabolism and may help to decipher novel disease mechanisms associated with LD. Keywords: Legionella pneumophila; infection model; metabolism; tracing; mass spectrometry; branched- chain amino acid; macrophage; macrophage infectivity potentiator (Mip); virulence factor; mass spectrometry 1. Introduction Legionnaires’ disease (LD) is a severe form of pneumonia caused by the bacterium Legionella pneumophila (Lp). The bacterium is characterized by various virulence factors that enable it to survive and multiply within host cells. One such virulence factor is the bacterial membrane-associated virulence factor macrophage infectivity potentiator (Mip) which exhibits peptidyl-prolyl-cis/trans-isomerase (PPIase) activity. Mip contributes to the intracellular pathogenicity in macrophages and extracellular tissue dissemination of Lp [1]. However, how the surface protein Mip helps the bacterium to evade the host immune system is not well understood. To protect the body against pathogens, immune cells must detect and respond to signals from their surrounding environment. Macrophages undergo significant changes in their metabolism that are crucial for the host’s defense against bacterial infections. Intracellular metabolites, such as mitochondrial TCA cycle-related small molecules, emerge Metabolites 2023, 13, 834. https://doi.org/10.3390/metabo13070834 https://www.mdpi.com/journal/metabolites