Gwilt, L. L. | bioRxiv Science

bioRxiv2025

Pseudomonas aeruginosa lasR Mutants Resist Phagocytosis and Alter Inflammatory Cytokine Production by Cystic Fibrosis Macrophages

Cystic Fibrosis is characterized by chronic muco-obstructive lung disease and infection. People with CF (pwCF) are often colonized with Pseudomonas aeruginosa for years to decades, allowing for evolutionary adaptation. In chronic P. aeruginosa lung isolates from pwCF, the quorum sensing regulator LasR frequently is nonfunctional, however the factors enabling lasR loss-of-function (LOF) mutant selection are incompletely understood. We hypothesized that LOF mutations in lasR could allow P. aeruginosa to resist the selective pressure of phagocytosis. We found that in multiple strain backgrounds, LasR LOF decreased phagocytosis by both model THP-1 and primary monocyte-derived macrophages. While exogenous administration of the quorum-sensing autoinducer 3-oxo-C12-homoserine-lactone (3OC12HSL) that is made by an enzyme regulated by LasR activity inhibited phagocytosis and mitochondrial respiration, the phagocytosis resistance seen with lasR mutants appears to be bacterial cell intrinsic rather than due to secreted factors. Finally, we found that lasR LOF mutations altered the inflammatory profile upon infection of CF macrophages, with a shift from IL-1 family cytokine expression towards canonical inflammatory markers including IL-6 and TNF. Collectively these data provide a potential explanation for both the prevalence of lasR mutants in the CF lung as well as their association with worse outcomes. ImportanceCystic Fibrosis (CF) is a genetically inherited disease that leads to chronic lung infections. Pseudomonas aeruginosa is often implicated with worsening of lung disease, and it evolves in the lung over time to resist eradication. One of the most commonly disrupted gene targets identified in Pseudomonas aeruginosa isolates from chronically infected CF lungs is lasR, which encodes a transcription factor which regulates multiple virulence factors. What contributes to the apparent fitness of lasR mutants in the CF lung is not well known. Our study shows that lasR loss-of-function (LOF) mutants resist phagocytosis by macrophages, one of the fundamental mechanisms of clearance by the immune system. We identify mechanisms promoting resistance to phagocytosis, and explore the downstream consequences on inflammatory responses. Understanding why lasR mutations arise could inform strategies to eradicate them from the CF lung and improve outcomes.

microbiology↗

bioRxiv2023

Characterization of SARS-CoV-2 Convalescent Patients' Serological Repertoire Reveals High Prevalence of Iso-RBD Antibodies

While our understanding of SARS-CoV-2 pathogenesis and antibody responses following infection and vaccination has improved tremendously since the outbreak in 2019, the sequence identities and relative abundances of the individual constituent antibody molecules in circulation remain understudied. Using Ig-Seq, we proteomically profiled the serological repertoire specific to the whole ectodomain of SARS-CoV-2 prefusion-stabilized spike (S) as well as to the receptor binding domain (RBD) over a 6-month period in four subjects following SARS-CoV-2 infection before SARS-CoV-2 vaccines were available. In each individual, we identified between 59 and 167 unique IgG clonotypes in serum. To our surprise, we discovered that [~]50% of serum IgG specific for RBD did not recognize prefusion-stabilized S (referred to as iso-RBD antibodies), suggesting that a significant fraction of serum IgG targets epitopes on RBD inaccessible on the prefusion-stabilized conformation of S. On the other hand, the abundance of iso-RBD antibodies in nine individuals who received mRNA-based COVID-19 vaccines encoding prefusion-stabilized S was significantly lower ([~]8%). We expressed a panel of 12 monoclonal antibodies (mAbs) that were abundantly present in serum from two SARS-CoV-2 infected individuals, and their binding specificities to prefusion-stabilized S and RBD were all in agreement with the binding specificities assigned based on the proteomics data, including 1 iso-RBD mAb which bound to RBD but not to prefusion-stabilized S. 2 of 12 mAbs demonstrated neutralizing activity, while other mAbs were non-neutralizing. 11 of 12 mAbs also bound to S (B.1.351), but only 1 maintained binding to S (B.1.1.529). This particular mAb binding to S (B.1.1.529) 1) represented an antibody lineage that comprised 43% of the individuals total S-reactive serum IgG binding titer 6 months post-infection, 2) bound to the S from a related human coronavirus, HKU1, and 3) had a high somatic hypermutation level (10.9%), suggesting that this antibody lineage likely had been elicited previously by pre-pandemic coronavirus and was re-activated following the SARS-CoV-2 infection. All 12 mAbs demonstrated their ability to engage in Fc-mediated effector function activities. Collectively, our study provides a quantitative overview of the serological repertoire following SARS-CoV-2 infection and the significant contribution of iso-RBD antibodies, demonstrating how vaccination strategies involving prefusion-stabilized S may have reduced the elicitation of iso-RBD serum antibodies which are unlikely to contribute to protection.

immunology↗