Susan E. Birket

Muco-obstructive airway diseases result in an increase in mucus accumulation and a decrease in mucus clearance. MUC5B is the most abundant secreted mucin in the human airways, and MUC5B mucin strands dimerize to create the mucus mesh network in the healthy respiratory tract. In muco-obstructive airway diseases like cystic fibrosis (CF), immune cells and bacteria release enzymes that degrade MUC5B into smaller fragments that become entangled and compacted, contributing to pathogenesis. We utilized synthetic cystic fibrosis sputum media (SCFM) to examine how mucin polymers can impact Staphylococcus aureus , a common CF pathogen that persists despite highly effective modulator therapies to correct CF disease. We found that low-molecular-weight (LMW) mucin negatively impacts S. aureus survival and biofilm biomass compared to high-molecular-weight (HMW) mucin. Adding extracellular DNA to SCFM with LMW mucin was not sufficient to restore growth. LMW mucin had a broad negative impact on S. aureus laboratory strains and CF clinical isolates. We next tested other CF pathogens, including Pseudomonas aeruginosa and nontypeable Haemophilus influenzae , and saw no significant differences in growth in HMW or LMW mucin. LMW mucin did not significantly impact Staphylococcus epidermidis growth, indicating that there may be specific interactions with S. aureus . Overall, this work highlights how interactions with pathogenic mucins may limit S. aureus growth in the diseased airways while supporting low-level persistence, and its ability to thrive in the presence of longer mucin strands may help explain why S. aureus is well adapted to survive in the healthy respiratory tract.

The mucosa of the airways is under a near-constant barrage of contaminants, allergens, and pathogens that can accumulate and cause irritation or infection if not promptly removed. Mucus, composed of a mucin glycoprotein mesh, protects the airways from these contaminants by entrapment and removal. Several diseases stall the protection provided by the mucus by altering its components and biophysical properties. This review provides an overview of how the defensive mucus of the airways functions in health, how the mucus can fail to clear with muco-obstructive lung diseases, how mucins respond to pathogens that infect the mucus, how the molecular signals of mucin secretion function, and how therapeutics may improve morbidity and mortality for people with muco-obstructive lung disease. Recent studies of the spatial organization of mucin-producing cells have led to new understanding of the difference between constitutive MUC5B production by superficial epithelial cells and between trigger-induced production and secretion by the goblet cells and submucosal glands. The coordination of mucin production and secretion with ion and water homeostasis is discussed to evaluate how changes in sodium, calcium, bicarbonate, and chloride are responsible for failures of mucin unfolding and the subsequent biophysical properties of the mucus. Rheological and inflammatory characteristics of the muco-obstructive lung diseases are compared to determine how the defect in mucus clearance leads to distinct morbidity and mortality with attention paid to the microbial pathogens prevalent between groups. The distinctions between the molecular mechanisms of mucin production and secretion within muco-obstructive lung diseases are discussed with a focus on several cytokines such as IL-1β, secretagogues like ATP, and protein chaperones such as ERN2. Pathogenic induction of mucin production and secretion are discussed with primary focus on bacterial mediators. Finally, established and novel therapies for muco-obstructive lung diseases are discussed for potential at improving mucociliary clearance defects and for reducing exacerbation.

Muco-obstructive airway diseases result in an increase in mucus accumulation and a decrease in mucus clearance. MUC5B is the most abundant secreted mucin in the human airways, and MUC5B mucin strands dimerize to create the mucus mesh network in the healthy respiratory tract. In muco-obstructive airway diseases like cystic fibrosis (CF), immune cells and bacteria release enzymes that degrade MUC5B into smaller fragments that become entangled and compacted, contributing to pathogenesis. We utilized synthetic cystic fibrosis sputum media (SCFM) to examine how mucin polymers can impact Staphylococcus aureus , a common CF pathogen that persists despite highly effective modulator therapies to correct CF disease. We found low molecular weight (LMW) mucin negatively impacts S. aureus survival and biofilm biomass compared to high molecular weight (HMW) mucin. Adding extracellular DNA to SCFM with LMW mucin was not sufficient to restore growth. LMW mucin had a broad negative impact on S. aureus laboratory strains and CF clinical isolates. We next tested other CF pathogens, including Pseudomonas aeruginosa and nontypeable Haemophilus influenzae , and saw no significant differences in growth in HMW or LMW mucin. LMW mucin did not significantly impact Staphylococcus epidermidis growth, indicating there may be specific interactions with S. aureus . Overall, this work highlights how interactions with pathogenic mucins may limit S. aureus growth in the diseased airways while supporting low-level persistence, and its ability to thrive in the presence of longer mucin strands may help to explain why S. aureus is well adapted to survive in the healthy respiratory tract. IMPORTANCE: Staphylococcus aureus is a common colonizer of the healthy human airways that is also known to establish persistent infections in the lungs of people with muco-obstructive airway diseases, including cystic fibrosis. Yet, few studies have investigated how S. aureus interactions with airway mucins in chronic airway disease may regulate bacterial persistence or pathogenesis. In this study, we report that low molecular weight mucins representative of short, degraded mucin polymers found at high abundance in muco-obstructive airway disease suppress S. aureus growth and limit bacterial biofilm formation and aggregation. Longer mucin polymers did not negatively affect S. aureus survival. Other common CF pathogens were not significantly affected by low molecular weight mucins, suggesting there are species-specific interactions between airway mucins and S. aureus in the chronically diseased lung. This work highlights how pathogenic mucins found in muco-obstructive disease can affect S. aureus growth and promote phenotypes associated with airway persistence.

Staphylococcus aureus infections remain an ongoing challenge for people with cystic fibrosis (PwCF), with the increased global prevalence of multidrug-resistant strains requiring new therapeutic approaches. Our previous studies demonstrated anti-inflammatory effects of several MEK1/2 inhibitor compounds, including PD0325901, CI-1040, and trametinib, in human phagocytes from PwCF and a murine S. aureus pulmonary infection model (M. De, G. Serpa, E. Zuiker, K. B. Hisert, et al., Front Cell Infect Microbiol 14:1275940, 2024, https://doi.org/10.3389/fcimb.2024.1275940 ). A recently developed MEK1/2 inhibitor compound, ATR-002, has been recognized for its ability to exert direct antibacterial effects on gram-positive bacterial species, including S. aureus (C. Bruchhagen, M. Jarick, C. Mewis, T. Hertlein, et al., Sci Rep 8:9114, 2018, https://doi.org/10.1038/s41598-018-27445-7 ). However, whether ATR-002 elicits antibacterial effects on clinically relevant strains of S. aureus or anti-inflammatory effects is unknown. In this study, the effects of ATR-002 on human CF macrophage TLR2-induced pro-inflammatory cytokine secretion were evaluated, demonstrating that ATR-002 reduced TNF-α and IL-8 secretion induced by the TLR2 agonists FSL-1 or Pam3CSK4. The antibacterial effects of ATR-002 were evaluated by minimum inhibitory concentration testing using S. aureus clinical isolates obtained from PwCF. Utilization of a murine methicillin-resistant S. aureus (MRSA) pulmonary infection model further confirmed the in vivo anti-inflammatory and antibacterial effects of ATR-002. Finally, infection of wild-type and Mek2 ^KO mice revealed that loss of MEK2 was host-protective during MRSA pulmonary infection by reducing neutrophil-mediated inflammation without altering bacterial clearance. In summary, this study highlights the therapeutic potential of targeting the MEK1/2 pathway to combat MRSA pulmonary infections. IMPORTANCE: Staphylococcus aureus infections pose a significant burden on global healthcare systems. Community-associated transmission of methicillin-resistant S. aureus (MRSA) and the increasing prevalence of other drug-resistant S. aureus isolates limit therapeutic options to combat this opportunistic pathogen. Infection-induced inflammation is a significant driver of tissue damage, especially in cystic fibrosis pulmonary infections. However, therapeutic strategies that can reduce inflammation without compromising host defense and bacterial clearance mechanisms are lacking. This study investigates the dual anti-inflammatory and antibacterial effects of a MEK1/2 inhibitor as a therapeutic strategy to target both host and pathogen with a single compound. This work also identifies host MEK2 as a specific target that can be modulated to reduce inflammation without impairing host defense against MRSA pulmonary infection. Results from this study can inform future human clinical trials to evaluate the ability of the MEK1/2 inhibitor compound ATR-002 to both combat S. aureus infections and reduce inflammation that accompanies these infections.

Cystic fibrosis (CF) is a genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator ( CFTR ) gene, resulting in CFTR protein dysfunction. CFTR dysfunction has multi-organ consequences, leading to dehydrated mucus that is adherent to epithelia. In the lungs, this leads to recalcitrant infections with bacteria such as Pseudomonas aeruginosa . In the gut, mucus-laden feces can adhere to the intestines, resulting in distal intestinal obstruction syndrome (DIOS). There is limited information on how lung colonization and DIOS are correlated in people with CF (pwCF). In this novel work, we describe the development of spontaneous lung colonization of CF pathogens in young (P. aeruginosa in the CF rats reflects data found in pwCF, as once CF rats are infected, they retain a higher relative abundance of P. aeruginosa than their healthy agemates. Finally, we found that ivacaftor treatment favors a healthier gut microbiome in CF rats, decreasing the relative abundance of Escherichia coli . These results indicate that the CF rat model is recapitulative of human CF disease with the spontaneous lung colonization of traditional CF pathogens and maintenance of P. aeruginosa after induced infection. Furthermore, these results indicate a possible role for the gut-lung axis in lung colonization and DIOS in CF. IMPORTANCE: These data describe for the first time the development of spontaneous lung colonization in the cystic fibrosis (CF) rat model, a hallmark aspect of human CF disease. We also find that CF rats infected with Pseudomonas aeruginosa maintain higher relative abundance following chronic infection as compared to healthy rats, similar to those is seen in people with CF. Additionally, we describe the possible contribution of the gut-lung axis linking lung health with distal intestinal obstruction syndrome, a relationship largely unexplored in the context of CF.

Newly approved highly effective modulation therapies (HEMT) have been life-changing for people with CF. Although these drugs have resulted in significant improvements in lung function and exacerbation rate, bacterial populations in the lung have not been eradicated. The mechanisms behind the continued colonization are not completely clear. We used a humanized rat to assess the effects of ivacaftor therapy on infection outcomes. Rats harbor an insert expressing humanized CFTR cDNA, including the G551D mutation. hG551D rats were treated with ivacaftor either during or before infection with P. aeruginosa. The response to infection was assessed by bacterial burden in the lung and mucus burden in the lung. We found that hG551D rats treated with ivacaftor had reduced bacteria present in the lung in the acute phase of the infection but were not different than vehicle control in the chronic phase of the infection. Similarly, the percentage of neutrophils in the airways were reduced at the acute, but not chronic, timepoints. Overall weight data indicated that the hG551D rats had significantly better weight recovery during the course of infection when treated with ivacaftor. Potentiation of the G551D mutation with ivacaftor resultant in short-circuit current measurements equal to WT, even during the chronic phase of the infection. Despite the persistent infection, hG551D rats treated with ivacaftor had fewer airways with mucus plugs during the chronic infection. The data indicate that the hG551D rats have better outcomes during infection when treated with ivacaftor compared to the vehicle group. Rats have increased weight gain, increased CFTR protein function, and decreased mucus accumulation, despite the persistence of infection and inflammation. These data suggest that ivacaftor improves tolerance of infection, rather than eradication, in this rat model.

Although highly effective modulator therapies for cystic fibrosis (CF) have improved mucus-related outcomes of disease for people with CF, eradication of Pseudomonas aeruginosa (PA) infection has not been achieved in this population. In addition, current therapies for CF do not target mucin hypersecretion directly. Here, we show that a novel approach of normalizing airway Muc5b hypersecretion ameliorates infection-induced mucus plugging and neutrophilic inflammation during persistent PA infection in CFTR^–/– rats.

Background: Ivacaftor exhibits anti-staphylococcal properties but does not clear Staphylococcus aureus from the lungs of people with cystic fibrosis (pwCF). We assessed whether exposure to therapeutic concentrations of ivacaftor could allow S. aureus to form small colony variants (SCVs), a phenotype commonly associated with bacterial persistence. Methods: Humanized G551D-CFTR (hG551D) rats were treated with ivacaftor for 7 days. Concentrations in the plasma, epithelial lining fluid and lung tissue lysate were measured using LC-MS/MS. Survival of S. aureus during ivacaftor treatment was assessed in an hG551D rat model of lung infection. S. aureus adaptation to therapeutic concentrations of ivacaftor was investigated in vitro by serial passage in the presence of 10 µM ivacaftor. Bacterial survival in the presence of antimicrobials was evaluated using growth curves and density assays. Results: Ivacaftor plasma concentrations of treated hG551D rats reached 3.488 ± 1.118 µM, with more variable concentrations in the epithelial lining fluid and lung tissue lysate. During S. aureus infection, ivacaftor-treated hG551D rats returned similar numbers of bacteria from the lung, compared with vehicle-treated controls. Exposure of S. aureus to ivacaftor in vitro led to the formation of ivacaftor-tolerant SCVs with an unstable phenotype and increased antibiotic tolerance. Conclusions: Treatment with ivacaftor did not alter S. aureus burden in the cystic fibrosis rat and led to the formation of tolerant SCVs in vitro, suggesting that development of an SCV phenotype may allow S. aureus to persist in the cystic fibrosis lung during ivacaftor therapy.