The Hippo kinases control inflammatory Hippo signaling and restrict bacterial infection in phagocytes.

The Hippo kinases MST1 and MST2 initiate a highly conserved signaling cascade called the Hippo pathway that limits organ size and tumor formation in animals. Intriguingly, pathogens hijack this host pathway during infection, but the role of MST1/2 in innate immune cells against pathogens is unclear. In this report, we generated Mst1/2 knockout macrophages to investigate the regulatory activities of the Hippo kinases in immunity. Transcriptomic analyses identified differentially expressed genes (DEGs) regulated by MST1/2 that are enriched in biological pathways, such as systemic lupus erythematosus, tuberculosis, and apoptosis. Surprisingly, pharmacological inhibition of the downstream components LATS1/2 in the canonical Hippo pathway did not affect the expression of a set of immune DEGs, suggesting that MST1/2 control these genes via alternative inflammatory Hippo signaling. Moreover, MST1/2 may affect immune communication by influencing the release of cytokines, including TNFα, CXCL10, and IL-1ra. Comparative analyses of the single- and double-knockout macrophages revealed that MST1 and MST2 differentially regulate TNFα release and expression of the immune transcription factor MAF, indicating that the two homologous Hippo kinases individually play a unique role in innate immunity. Notably, both MST1 and MST2 can promote apoptotic cell death in macrophages upon stimulation. Lastly, we demonstrate that the Hippo kinases are critical factors in mammalian macrophages and single-cell amoebae to restrict infection by Legionella pneumophila, Escherichia coli, and Pseudomonas aeruginosa. Together, these results uncover non-canonical inflammatory Hippo signaling in macrophages and the evolutionarily conserved role of the Hippo kinases in the anti-microbial defense of eukaryotic hosts. IMPORTANCE: Identifying host factors involved in susceptibility to infection is fundamental for understanding host-pathogen interactions. Clinically, individuals with mutations in the MST1 gene which encodes one of the Hippo kinases experience recurrent infection. However, the impact of the Hippo kinases on innate immunity remains largely undetermined. This study uses mammalian macrophages and free-living amoebae with single- and double-knockout in the Hippo kinase genes and reveals that the Hippo kinases are the evolutionarily conserved determinants of host defense against microbes. In macrophages, the Hippo kinases MST1 and MST2 control immune activities at multiple levels, including gene expression, immune cell communication, and programmed cell death. Importantly, these activities controlled by MST1 and MST2 in macrophages are independent of the canonical Hippo cascade that is known to limit tissue growth and tumor formation. Together, these findings unveil a unique inflammatory Hippo signaling pathway that plays an essential role in innate immunity.

Bacterial effector kinases and strategies to identify their target host substrates.

Post-translational modifications (PTMs) are critical in regulating protein function by altering chemical characteristics of proteins. Phosphorylation is an integral PTM, catalyzed by kinases and reversibly removed by phosphatases, that modulates many cellular processes in response to stimuli in all living organisms. Consequently, bacterial pathogens have evolved to secrete effectors capable of manipulating host phosphorylation pathways as a common infection strategy. Given the importance of protein phosphorylation in infection, recent advances in sequence and structural homology search have significantly expanded the discovery of a multitude of bacterial effectors with kinase activity in pathogenic bacteria. Although challenges exist due to complexity of phosphorylation networks in host cells and transient interactions between kinases and substrates, approaches are continuously being developed and applied to identify bacterial effector kinases and their host substrates. In this review, we illustrate the importance of exploiting phosphorylation in host cells by bacterial pathogens via the action of effector kinases and how these effector kinases contribute to virulence through the manipulation of diverse host signaling pathways. We also highlight recent developments in the identification of bacterial effector kinases and a variety of techniques to characterize kinase-substrate interactions in host cells. Identification of host substrates provides new insights for regulation of host signaling during microbial infection and may serve as foundation for developing interventions to treat infection by blocking the activity of secreted effector kinases.