UC Berkeley

Initial detection of invading microorganisms is one of the primary tasks of the innate immune system. However, the molecular mechanisms by which pathogens are recognized remain incompletely understood. I used the intracellular gram-negative Legionella pneumophila to study mechanisms by which the innate immune system distinguishes virulent bacteria from avirulent bacteria. I have made the surprising observation that a cytosolic RNA immunosurveillance pathway (called the RIG-I/MDA5 pathway), thought primarily to detect viruses, is also involved in the innate immune response to the intracellular vacuolar bacterial pathogen, Legionella pneumophila. In the response to viruses, the RIG-I/MDA5 immunosurveillance pathway has been shown to respond to viral RNA or DNA. We found that the RIG-I pathway was required for the response to L. pneumophila RNA, but was not required for the response to L. pneumophila DNA. Thus one explanation of my results is that L. pneumophila RNA accesses the host cell cytosol via its type IV secretion system, where it triggers the RIG-I/MDA5 pathway. This is unexpected since bacteria have not previously been thought to translocate RNA into host cells. I was able to isolate IFN-stimulatory activity by immunoprecipitating RIG-I from cells infected with T4SS-competent Legionella. In the future, I will utilize deep sequencing technology to pinpoint the origin and identity of RIG-I bound ligands during T4SS+ L. pneumophila infection.

I also found that L. pneumophila suppresses the RIG-I/MDA5 pathway by a translocated effector protein, SdhA. Several viral repressors of the RIG-I/MDA5 pathway have been described, but bacterial repressors of RIG-I/MDA5 are not known. Thus, this study provides novel insights into the molecular mechanisms by which the immune system detects bacterial infection, and conversely, by which bacteria suppress innate immune responses.

While all bacteria are capable of inducing type I interferon, many species do so independently of the cytosolic RNA sensing pathway that responds to Legionella. Therefore, I, along with many Vance lab members, investigated the mechanism by which cyclic dinucleotides (c-di-GMP and c-di-AMP), bacterial second-messenger molecules, activate a robust and specific host response in macrophages. c-di-GMP has been shown to activate TBK-1, IRF3, NF-κB, and MAP kinases to induce type I interferon, in manner independently of known TLR and cytosolic nucleic acid sensing pathways. In parallel studies in the lab, ENU mutagenesis of mice identified a mouse mutant that completely abrogates the host response to cyclic dinucleotides. Sequencing identified a missense mutation in the open reading frame of Sting, which converts an isoleucine to aspargine in the C-terminal globular domain, rendering STING protein undetectable in mutant macrophages. Previous reports of Sting demonstrated a role for this multiple transmembrane domain containing protein that localizes to the endoplasmic reticulum and/or mitochondrial associated-membrane (MAM) in cytosolic DNA and RNA sensing pathways. I found that overexpression of the ENU-induced mutant Sting allele failed to induce type I interferon, despite robust expression of the protein. Surprisingly, studies in the Vance lab have shown that wild type STING is capable of binding c-di-GMP, in contrast to the ENU mutant allele, which does not bind. I found that a soluble C-terminal truncation of STING (amino acids 138-379), which removes most predicted transmembrane domains, is sufficient to bind c-di-GMP. Taken together, genetic studies have demonstrated an essential role for Sting in the innate immune response to cyclic dinucleotides and biochemical data shows that the C-terminal region of the protein functions as the direct sensor of cyclic dinucleotides.

The experimental line of investigation presented in my thesis dissects the pathways by which the innate immune system recognizes infection of virulent bacteria. Both stories discussed herein demonstrate the importance of innate immune detection of nucleic acids; molecules microbes cannot live without. Interestingly, it is the compartment in which nucleic acids are present that ultimately triggers innate immune receptors. The demonstration that cytosolic RNA sensors detect secretion system competent Legionella illustrates the consequence of breaching the phagosomal barrier. While bacterial replication may be restricted to membrane bound compartments, accessing the host cell cytosol via the T4SS alerts host sensors to the pathogen's presence. Previously thought only to recognize viral infection, I have demonstrated the breadth of cytosolic RNA sensors in recognizing not only viruses, but also bacteria. STING's ability to detect cyclic dinucleotides delivered to the host cell cytosol exemplifies how the innate immune system has honed in on a uniquely bacterial nucleic acid molecule. Cyclic dinucleotides are not only structurally distinct, but their role in regulating virulence factor expression makes them an excellent target for innate immune detection of pathogens.