Regulatory crosstalk between human Caspases & Guanylate Binding Proteins in antimicrobial host-defence

Background: A prompt and proportionate response to an infectious agent is important for clearing infection. The immune system relies on early mechanisms of innate defence which appropriately guide late mechanisms deployed after a few days of infection. Both systems work in tandem and incorrect early responses can result in increased microbial spread in organs, inflammation, and tissue damage. A key role of the early response is to quickly identify the type of infection, for example whether bacterial, viral, or parasitic, and respond in a manner that is effective against the specific type of pathogen. Two families of enzymes play important roles during early immune defence, the Guanylate Binding Proteins (GBPs) and Caspases. GBPs are enzymes that convert the molecule GTP into GDP, and act as molecular on-off switches. Caspases are enzymes that act as molecular scissors and cut a range of important proteins that are involved in immunity. Molecular cutting by caspases is important for the normal functions of many proteins in cells. Together, GBPs and caspases act against diverse infectious agents and protect us.

We previously showed that the family member GBP1 assists in early immune responses against the diarrhoeagenic bacterial pathogen Salmonella as well as the parasitic pathogen Toxoplasma gondii which causes types of brain disease. Others have showed that GBPs and caspases together assist in defending against diseases such as forms of bacterial diarrhoeas, tularemia, Legionnaire's disease, HIV, among others. This reflects the broad protective roles of these proteins and their importance in human infection. Interestingly, these pathogens invade and reside within our own cells, such as immune cells, brain cells, intestinal cells, where they grow and multiply. GBPs and caspases remove infected cells and prevent the growth and spread of pathogens. These enzymes are therefore crucial in human immune responses and their actions need to be better understood. How exactly GBPs and caspases cooperate remains poorly defined.

Aims & approaches: Our overall goal is to understand the molecular and cellular mechanisms of how GBPs and caspases defend us against infection. Their combined actions detect infections by different microbes and set in motion a series of events that result in the loss of the infected host cell, which reduces pathogen multiplication and limits infection. We want to understand how GBPs and caspases cooperate with each other and other molecules and whether collateral damage through loss of host cells can be avoided. We newly discovered that there is a molecular interplay between caspases and GBPs that naturally suppresses inflammation during Salmonella infection. In the proposed work we will broaden this finding to other GBP enzymes and other inflammatory settings. GBPs are anchored by lipids into membranes inside cells to execute their function. Our team includes chemists who have designed new sensitive chemical probes that will help answer important questions such as when/where/how lipids anchors GBPs and control their actions. We will deploy our advanced artificial intelligence-based workflow for microscopic imaging of pathogens for fast analyses of a large amount of data and increased efficiency. We are a team of scientists with many years of experience in immunology, pathogenesis, inflammation, and chemical biology. Our proposal therefore has a high chance of being successful.

Potential benefits: GBPs and caspases protect against major groups of human pathogens. Their beneficial actions could be harnessed to improve our natural defence against infections. Understand what deregulates them will enable us to prevent detrimental inflammation. Our new discoveries on their regulation will have the potential to be exploited in therapies aimed at reducing unwanted inflammation and for a better response against drug-resistant pathogens.

Interferon-inducible Guanylate Binding Proteins (GBPs) are GTPases that mediate cell-intrinsic defence against intracellular bacteria, parasites, and viruses. GBPs promote inflammasome/caspase-mediated cell death and remove replicative niches of intracellular pathogens. Salmonella Typhimurium and Toxoplasma gondii infection of macrophages results in human GBP1-dependent restriction of pathogen replication and programmed cell death through caspase-4 and -8, respectively. Mechanistically, GBP1 required GTPase activity and C-terminal lipidation to traffic to microbial compartments and promote access of cytosolic sensors to Salmonella LPS and Toxoplasma DNA. Moreover, caspase-1 inactivates GBP1 over time by proteolyzing it at a conserved Asp residue and suppresses the GBP1-caspase-4 cell death pathway. We hypothesise that GBP2-5 are also regulated through temporal proteolysis in a feedback loop that balances cell death, cytokine maturation, and antimicrobial defence. Endogenous GBP1/2/5 can be farnesylated or geranylgeranylated as revealed by our sensitive chemical biology probes. We will deploy these new tools to test our hypothesis that differential lipidation controls GBP trafficking to pathogen-specific compartments. With our high-throughput Host Response to Microbe Analysis (HRMAn) workflow, we have discovered new genes that promote GBP-dependent cell-intrinsic immunity independently of cell death, which we will investigate in-depth to dissect new mechanisms of defence orchestrated by these major protein families. Taken together, we will delineate context-specific, family-wide regulatory crosstalk between human GBPs and caspases during infection. Hereditary mutations or deregulation of IFNs/inflammasomes/caspases exacerbates inflammation and are detrimental to host defence. Our elucidation of new regulatory mechanisms will be invaluable in the design of effective therapies directed at boosting innate immunity and preventing unwanted inflammation during infection.