Targeting Lysosomal Pathways to Improve Host Immunity Post Coronaviral Infections

Background: Beta-coronavirus lung infections, including those causing common colds such as OC43 and those with pandemic potential such as SARS-CoV-2, are leading causes of morbidity and mortality. Secondary bacterial infections post coronavirus infections represent a major threat to recovery and remain underappreciated despite mounting evidence of their contribution to lethal outcomes. The specific mechanisms that render the host susceptible to bacterial infections post-coronavirus infection remain poorly understood. Given that Respiratory Health is one of the FY24 PRMRP topic areas, this application aims to provide a solution to a key problem affecting respiratory health. A major limitation in understanding the impact of beta-coronaviruses such as SARS-CoV-2 on host immunity is the lack of appropriate mouse models. The most widely used model of SARS-CoV-2 infection utilizes mice that express human ACE2 in an epithelial-specific manner (K-18 mice); however, this model does not allow SARS-CoV-2 to infect immune cells, which is well-documented in humans. Infection of immune cells represents a key pathological mechanism of human coronaviruses such as SARS-CoV-2 and OC43, with potential ramifications for the host's ability to fight off secondary bacterial infections. To overcome this, we used a murine coronavirus (MHV) model, which effectively replicates in mouse immune cells as well as epithelial cells, and mimics various aspects of COVID-19 and host susceptibility to secondary bacterial infection, as shown in our recent publications. Using MHV infection, followed by infection with either a Gram-negative (Pseudomonas) or a Gram-positive (Streptococcus) pathogen, we established the following novel insights: (i) Coronavirus infections impair the host's ability to clear secondary bacterial pathogens and exacerbate tissue injury. (ii) Coronaviruses impair the bacterial killing ability of macrophages by impairing lysosomal acidification, leading to lysosomal swelling, impaired bacterial fusion, rupture, and release of lysosomal components. (iii) Released lysosomal enzymes, especially cathepsin B, contribute to the activation of the inflammasome and pyroptotic cell death. (iv) Blockade of caspase-1 or cathepsin B decreased macrophage cell death and improved in vivo outcomes during post-coronaviral bacterial infections. (v) Coronavirus infection inhibits the key transcription factor TFEB for lysosomal biosynthesis by proteasomal degradation, potentially preventing the recovery of lysosome-mediated host defense. (vi) We have identified that coronavirus degrades TFEB through E3 ligase DCAF7 where kinase PAK2 primes TFEB for the degradation and identified specific inhibitors that block TFEB degradation and restore lysosomal function from a screen of three million compounds. Hypothesis: We hypothesize that coronaviruses impair host immunity by exploiting lysosomal machinery through multiple mechanisms, including blocking de novo lysosomal biosynthesis. Boosting the bactericidal function of existing lysosomes and promoting lysosomal biogenesis will improve outcomes in post-coronaviral bacterial infections. We will test this hypothesis through the following specific aims: Aim 1. To determine the impact of boosting existing lysosomal function/minimizing the deleterious effect of lysosomal rupture on bacterial clearance and lung injury in mouse models of post-coronaviral bacterial infections. Aim 2. To determine the impact of promoting lysosomal biogenesis on antibacterial immunity and tissue injury in mouse models of post-coronaviral bacterial infections. Aim 3. To determine the impact of boosting lysosomal function/biogenesis on antibacterial immunity and pyroptotic cell death in human lung and peripheral blood-derived macrophages. Study Design: To achieve these aims, we will utilize mouse models of MHV and SARS-CoV-2 as well as human lung and peripheral immune cells. We will gain mechanistic insights into lysosomal dysfunction and discover therapeutic approaches that can provide the host with an advantage during secondary bacterial infections. We will use novel mouse models such as humanized mice as well as cell-specific TFEB overexpressing mice. We will test a set of lysosomal modulating agents that are well established for their safety and human use, as well as the novel compounds identified by our team. We will investigate both mechanisms of host defense and those that limit tissue injury during secondary bacterial infections. Impact: Given the extensive threat of coronavirus infections and the contribution of secondary bacterial infection in worsening clinical outcomes, this project addresses a critical unmet need. Relevance to Military Health: Given the rapid spread of infectious diseases in military settings, the success of this proposal will provide novel therapeutic approaches to treat secondary bacterial infections in COVID-19, thereby limiting morbidity and mortality while preserving military preparedness. Less