PROJECT SUMMARY Influenza A virus (IAV) is commonly complicated by secondary infections with Streptococcus pneumoniae (Spn) and this increases the morbidity and mortality of IAV, particularly during IAV pandemics. Highly pathogenic avian influenza (HPAI) currently poses a risk as the etiological agent for an influenza pandemic. However, little is known about how this pathogen may increase susceptibility to secondary bacterial infections. The airway epithelium serves as the first line of defense against bacterial pathogens in the lungs and it is severely damaged during IAV infection. Despite this, the role of the airway epithelium in increased susceptibility to secondary bacterial infections after IAV is not well defined. IAV induces a robust endoplasmic reticulum (ER) stress response in the airway, contributing to a disruption in host protein production during IAV infection. Interestingly, induced ER stress in the airway epithelium leads to a reduction in cystic fibrosis transmembrane conductance regulator (CFTR) function. Prior work from our lab demonstrates that IAV causes CFTR dysfunction, disrupting the airway surface liquid and subsequently increasing the burden of Spn in primary differentiated human bronchial epithelial cells (HBECs). Our preliminary data indicates that correction of ER stress during IAV infection reduces the burden of Spn in the airway epithelium without interfering with IAV replication. The broad objective of this proposal is to determine how ER stress during IAV infection is involved in both a loss of CFTR and an increase in susceptibility to secondary Spn infections. Aim 1 will test the hypothesis that ER stress during IAV disrupts host protein production, damaging the airway epithelium by causing a loss of CFTR and a reduction in secreted host proteins. To specifically study IAV infection in the airway epithelium, we will use an HBEC culture system. To identify the role of ER stress in a loss of CFTR after IAV, we will infect cells with IAV and treat them with pharmacological agents to manipulate the ER stress response and then quantify CFTR abundance and function by Western blot, Ussing chamber analysis and micro-optical coherence tomography. Additionally, we will evaluate the effect of ER stress during IAV on the secretion of host proteins by proteomic analysis of the airway surface liquid. Aim 2 will test the hypothesis that ER stress during IAV increases susceptibility to Spn and disrupts the host response to bacterial infection. To evaluate the role of ER stress in increased susceptibility to Spn, we will coinfect HBECs with IAV and Spn and treat them with pharmacological agents to manipulate the ER stress response. We will quantify Spn in both the airway surface liquid and the airway epithelium. To determine the effect of IAV infection on the ER stress response to Spn, we will coinfect HBECs with IAV and Spn and measure ER stress markers by Western blot. We will evaluate various strains of Spn to identify strain-...