Age-Dependent Glycomic Response to the 2009 Pandemic H1N1 Influenza Virus and Its Association with Disease Severity.

Influenza A viruses cause a spectrum of responses, from mild coldlike symptoms to severe respiratory illness and death. Intrinsic host factors, such as age, can influence disease severity. Glycosylation plays a critical role in influenza pathogenesis; however, the molecular drivers of influenza outcomes remain unknown. In this work, we characterized the host glycomic response to the H1N1 2009 pandemic influenza A virus (H1N1pdm09) as a function of age-dependent severity in a ferret model. Using our dual-color lectin microarray technology, we examined baseline glycosylation and glycomic response to infection in newly weaned and aged animals, models for young children and the elderly, respectively. Compared to adult uninfected ferrets, we observed higher levels of α-2,6-sialosides, the receptor for H1N1pdm09, in newly weaned and aged animals. We also observed age-dependent loss of O-linked α-2,3-sialosides. The loss of these highly charged groups may impact viral clearance by mucins, which corresponds to the lower clearance rates observed in aged animals. Upon infection, we observed dramatic changes in the glycomes of aged animals, a population severely impacted by the virus. In contrast, no significant alterations were observed in the newly weaned animals, which show mild to moderate responses to the H1N1pdm09. High mannose, a glycan recently identified as a marker of severity in adult animals, increased with severity in the aged population. However, the response was delayed, in line with the delayed development of pneumonia observed. Overall, our results may help explain the differential susceptibility to influenza A infection and severity observed as a function of age.

Age-Related Pathology Associated with H1N1 A/California/07/2009 Influenza Virus Infection.

Influenza virus infection causes a spectrum of diseases, ranging from mild upper respiratory tract infection to severe lower respiratory tract infection, that can lead to diffuse alveolar damage, interstitial and airspace inflammation, or acute respiratory failure. Mechanisms instructing disease severity are not completely understood, but host, viral, and bacterial factors influence disease outcome. With age being one host factor associated with a higher risk of severe influenza, we investigated regional pulmonary distribution and severity of pneumonia after 2009 H1N1 influenza virus infection in newly weaned, adult, and aged ferrets to better understand age-dependent susceptibility and pathology. Aged ferrets exhibited greater weight loss and higher rates of mortality than adult ferrets, whereas most newly weaned ferrets did not lose weight but had a lack of weight gain. Newly weaned ferrets exhibited minimal pneumonia, whereas adult and aged ferrets had a spectrum of pneumonia severity. Influenza virus-induced pneumonia peaked earliest in adult ferrets, whereas aged ferrets had delayed presentation. Bronchial severity differed among groups, but bronchial pathology was comparable among all cohorts. Alveolar infection was strikingly different among groups. Newly weaned ferrets had little alveolar cell infection. Adult and aged ferrets had alveolar infection, but aged ferrets were unable to clear infection. These different age-related pneumonia and infection patterns suggest therapeutic strategies to treat influenza should be tailored contingent on age.

Respiratory syncytial virus infection enhances Pseudomonas aeruginosa biofilm growth through dysregulation of nutritional immunity.

Clinical observations link respiratory virus infection and Pseudomonas aeruginosa colonization in chronic lung disease, including cystic fibrosis (CF) and chronic obstructive pulmonary disease. The development of P. aeruginosa into highly antibiotic-resistant biofilm communities promotes airway colonization and accounts for disease progression in patients. Although clinical studies show a strong correlation between CF patients' acquisition of chronic P. aeruginosa infections and respiratory virus infection, little is known about the mechanism by which chronic P. aeruginosa infections are initiated in the host. Using a coculture model to study the formation of bacterial biofilm formation associated with the airway epithelium, we show that respiratory viral infections and the induction of antiviral interferons promote robust secondary P. aeruginosa biofilm formation. We report that the induction of antiviral IFN signaling in response to respiratory syncytial virus (RSV) infection induces bacterial biofilm formation through a mechanism of dysregulated iron homeostasis of the airway epithelium. Moreover, increased apical release of the host iron-binding protein transferrin during RSV infection promotes P. aeruginosa biofilm development in vitro and in vivo. Thus, nutritional immunity pathways that are disrupted during respiratory viral infection create an environment that favors secondary bacterial infection and may provide previously unidentified targets to combat bacterial biofilm formation.

Engineered cationic antimicrobial peptide (eCAP) prevents Pseudomonas aeruginosa biofilm growth on airway epithelial cells.

OBJECTIVES: Chronic infections with the opportunistic pathogen Pseudomonas aeruginosa are responsible for the majority of the morbidity and mortality in patients with cystic fibrosis (CF). While P. aeruginosa infections may initially be treated successfully with standard antibiotics, chronic infections typically arise as bacteria transition to a biofilm mode of growth and acquire remarkable antimicrobial resistance. To address the critical need for novel antimicrobial therapeutics that can effectively suppress chronic bacterial infections in challenging physiological environments, such as the CF lung, we have rationally designed a de novo engineered cationic antimicrobial peptide, the 24-residue WLBU2, with broad-spectrum antibacterial activity for pan-drug-resistant P. aeruginosa in liquid culture. In the current study, we tested the hypothesis that WLBU2 also prevents P. aeruginosa biofilm growth. METHODS: Using abiotic and biotic biofilm assays, co-culturing P. aeruginosa with polarized human airway epithelial cells, we examined the ability of WLBU2 to prevent biofilm biogenesis alone and in combination with currently used antibiotics. RESULTS: We observed a dose-dependent reduction in biofilm growth on an abiotic surface and in association with CF airway epithelial cells. WLBU2 prevented P. aeruginosa biofilm formation when co-cultured with mucus-producing primary human CF airway epithelial cells and using CF clinical isolates of P. aeruginosa, even at low pH and high salt conditions that mimic the CF airway. When used in combination, WLBU2 significantly increases killing by the commonly used antibiotics tobramycin, ciprofloxacin, ceftazidime and meropenem. CONCLUSIONS: While other studies have demonstrated the ability of natural and synthetic antimicrobial peptides to prevent abiotic bacterial biofilm formation, the current studies for the first time demonstrate the effective peptide treatment of a biotic bacterial biofilm in a setting similar to the CF airway, and without negative effects on human airway epithelial cells, thus highlighting the unique potential of this engineered cationic antimicrobial peptide for treatment of human respiratory infections.

Sex disparities in influenza: A multiscale network analysis.

Sex differences in the pathogenesis of infectious diseases because of differential immune responses between females and males have been well-documented for multiple pathogens. However, the molecular mechanism underlying the observed sex differences in influenza virus infection remains poorly understood. In this study, we used a network-based approach to characterize the blood transcriptome collected over the course of infection with influenza A virus from female and male ferrets to dissect sex-biased gene expression. We identified significant differences in the temporal dynamics and regulation of immune responses between females and males. Our results elucidate sex-differentiated pathways involved in the unfolded protein response (UPR), lipid metabolism, and inflammatory responses, including a female-biased IRE1/XBP1 activation and male-biased crosstalk between metabolic reprogramming and IL-1 and AP-1 pathways. Overall, our study provides molecular insights into sex differences in transcriptional regulation of immune responses and contributes to a better understanding of sex biases in influenza pathogenesis.

Simultaneous Antibiofilm and Antiviral Activities of an Engineered Antimicrobial Peptide during Virus-Bacterium Coinfection.

Antimicrobial-resistant infections are an urgent public health threat, and development of novel antimicrobial therapies has been painstakingly slow. Polymicrobial infections are increasingly recognized as a significant source of severe disease and also contribute to reduced susceptibility to antimicrobials. Chronic infections also are characterized by their ability to resist clearance, which is commonly linked to the development of biofilms that are notorious for antimicrobial resistance. The use of engineered cationic antimicrobial peptides (eCAPs) is attractive due to the slow development of resistance to these fast-acting antimicrobials and their ability to kill multidrug-resistant clinical isolates, key elements for the success of novel antimicrobial agents. Here, we tested the ability of an eCAP, WLBU2, to disrupt recalcitrant Pseudomonas aeruginosa biofilms. WLBU2 was capable of significantly reducing biomass and viability of P. aeruginosa biofilms formed on airway epithelium and maintained activity during viral coinfection, a condition that confers extraordinary levels of antibiotic resistance. Biofilm disruption was achieved in short treatment times by permeabilization of bacterial membranes. Additionally, we observed simultaneous reduction of infectivity of the viral pathogen respiratory syncytial virus (RSV). WLBU2 is notable for its ability to maintain activity across a broad range of physiological conditions and showed negligible toxicity toward the airway epithelium, expanding its potential applications as an antimicrobial therapeutic. IMPORTANCE Antimicrobial-resistant infections are an urgent public health threat, making development of novel antimicrobials able to effectively treat these infections extremely important. Chronic and polymicrobial infections further complicate antimicrobial therapy, often through the development of microbial biofilms. Here, we describe the ability of an engineered antimicrobial peptide to disrupt biofilms formed by the ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogen Pseudomonas aeruginosa during coinfection with respiratory syncytial virus. We also observed antiviral activity, indicating the ability of engineered antimicrobial peptides to act as cross-kingdom single-molecule combination therapies.