Ocul Surf. 2024 Jun 11:S1542-0124(24)00065-X. doi: 10.1016/j.jtos.2024.06.002. Online ahead of print.

ABSTRACT

PURPOSE: Ocular surface hydration is critical for eye health and its impairment can lead to dry eye disease. Extracellular calcium-sensing receptor (CaSR) is regulator of ion transport in epithelial cells expressing cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel. CFTR is also a major ion channel in ocular surface epithelia, however the roles of CaSR in ocular surface are not well studied. This study aims to investigate expression and functional roles of CaSR in ocular surface.

METHODS: CaSR immunostaining was performed in mouse and human cornea and conjunctiva. Ocular surface potential difference (OSPD) and tear fluid volume measurements were performed in mice with topically applied cinacalcet (CaSR activator) and NPS-2143 (CaSR inhibitor).

RESULTS: CaSR is expressed in corneal and conjunctival epithelia of mice and humans. Topically administered CaSR activator cinacalcet inhibits cAMP agonist forskolin-induced Cl- secretion and CFTR activity up to 90%. CaSR inhibitor NPS-2143 stimulates CFTR-mediated Cl- secretion in mouse ocular surface, after which cAMP agonist forskolin had minimal additional secretory effects. Single dose NPS-2143 treatment (as an eye drop) increases tear fluid volume in mice by ∼60% compared to vehicle treatment. NPS-2143 effect on tear volume lasts at least eight hours after single dose.

CONCLUSIONS: CaSR is a key regulator of ocular surface ion transport and CaSR inhibition promotes Cl- and tear secretion in the ocular surface. If they are found to be effective in in dry eye models, CaSR inhibitors (currently in clinical development) can potentially be repurposed as novel prosecretory treatments for dry eye disease.

PMID:38871216 | DOI:10.1016/j.jtos.2024.06.002

Sleep Med Rev. 2024 May 29;77:101962. doi: 10.1016/j.smrv.2024.101962. Online ahead of print.

ABSTRACT

Chronic health conditions (CHC; e.g., cystic fibrosis, type 1 diabetes) in children are associated with disease-specific physical symptoms that contribute to a high prevalence of sleep problems. Sleep problems exacerbate other health-related sequelae and can impede therapeutic response to health treatments, increasing the overall complexity of symptom management. Psychosocial sleep interventions (PSI) improve sleep in children with typical development and neurodevelopmental conditions. Yet, the effectiveness of PSI for children with CHC has scarcely been investigated. This systematic review appraises the literature examining the effectiveness and acceptability of PSI for children with CHC. A search identified 20 studies that met inclusion criteria. Data related to participant characteristics, sleep targets, research design and methods, measures, sleep outcomes and collateral effects were extracted. Study rigor was then evaluated. Most studies evaluated youth-directed Cognitive Behavioral Therapy for Insomnia or parent-implemented behavioral sleep interventions. Twelve studies demonstrated positive sleep treatment effects and four demonstrated mixed effects. Collateral improvements were reported in child mental health and parental health and well-being, though physical health benefits for children were not consistently reported. One, five and 14 studies were rated as having strong, adequate, and weak methodological rigor respectively. Recommendations for clinical practice and future research are made.

PMID:38870551 | DOI:10.1016/j.smrv.2024.101962

Science. 2024 Jun 14;384(6701):1175-1176. doi: 10.1126/science.adq0059. Epub 2024 Jun 13.

ABSTRACT

Intravenous targeting of airway basal cells for cystic fibrosis gene therapy overcomes lung barriers.

PMID:38870313 | DOI:10.1126/science.adq0059

Science. 2024 Jun 14;384(6701):1196-1202. doi: 10.1126/science.adk9428. Epub 2024 Jun 13.

ABSTRACT

In vivo genome correction holds promise for generating durable disease cures; yet, effective stem cell editing remains challenging. In this work, we demonstrate that optimized lung-targeting lipid nanoparticles (LNPs) enable high levels of genome editing in stem cells, yielding durable responses. Intravenously administered gene-editing LNPs in activatable tdTomato mice achieved >70% lung stem cell editing, sustaining tdTomato expression in >80% of lung epithelial cells for 660 days. Addressing cystic fibrosis (CF), NG-ABE8e messenger RNA (mRNA)-sgR553X LNPs mediated >95% cystic fibrosis transmembrane conductance regulator (CFTR) DNA correction, restored CFTR function in primary patient-derived bronchial epithelial cells equivalent to Trikafta for F508del, corrected intestinal organoids and corrected R553X nonsense mutations in 50% of lung stem cells in CF mice. These findings introduce LNP-enabled tissue stem cell editing for disease-modifying genome correction.

PMID:38870301 | DOI:10.1126/science.adk9428

JCI Insight. 2024 Jun 13:e172364. doi: 10.1172/jci.insight.172364. Online ahead of print.

ABSTRACT

Duodenal bicarbonate secretion is critical to epithelial protection, nutrient digestion/absorption and is impaired in cystic fibrosis (CF). We examined if linaclotide, typically used to treat constipation, may also stimulate duodenal bicarbonate secretion. Bicarbonate secretion was measured in vivo and in vitro using mouse and human duodenum (biopsies and enteroids). Ion transporter localization was identified with confocal microscopy and de novo analysis of human duodenal single cell RNA sequencing (sc-RNAseq) datasets was performed. Linaclotide increased bicarbonate secretion in mouse and human duodenum in the absence of CFTR expression (Cftr knockout mice) or function (CFTRinh-172). NHE3 inhibition contributed to a portion of this response. Linaclotide-stimulated bicarbonate secretion was eliminated by down-regulated in adenoma (DRA, SLC26A3) inhibition during loss of CFTR activity. Sc-RNAseq identified that 70% of villus cells expressed SLC26A3, but not CFTR, mRNA. Loss of CFTR activity and linaclotide increased apical brush border expression of DRA in non-CF and CF differentiated enteroids. These data provide further insights into the action of linaclotide and how DRA may compensate for loss of CFTR in regulating luminal pH. Linaclotide may be a useful therapy for CF individuals with impaired bicarbonate secretion.

PMID:38869953 | DOI:10.1172/jci.insight.172364

Pediatr Pulmonol. 2024 Jun 13. doi: 10.1002/ppul.27125. Online ahead of print.

ABSTRACT

BACKGROUND: Elexacaftor-tezacaftor-ivacaftor (ETI) is a highly effective cystic fibrosis (CF) transmembrane conductance regulator (CFTR) modulating therapy for people with CF and at least one F508del variant. However, there is limited data about the safety and efficacy of this therapy in pediatric populations and in real-world settings. This study aimed at evaluating the effectiveness, tolerability, and safety of ETI in children with CF.

METHODS: This was a prospective observational study including all children aged 6-11 years who initiated ETI therapy between October 2022 and March 2023 at the Pediatric CF Center of Milan (Italy). Study outcomes included changes in sweat chloride concentration, FEV1, LCI2.5, body mass index (BMI), tolerance, and safety. Mean changes in study outcomes from baseline through 24 weeks were estimated using mixed-effects regression models.

RESULTS: The study included 34 children with CF (median age: 8.3 years). At Week 12, we observed an average decrease in LCI2.5 of 2.3 units (95% confidence interval [CI]: -3.1; -1.5). At Week 24, sweat chloride concentration decreased by 63 mEq/L (95% CI: -69; -58), FEV1 increased by 8.8 percentage point (95% CI: 3.7; 13.9) and BMI increased by 0.15 standard deviation scores (95% CI: 0.04; 0.25). Skin rashes appeared in 6 patients which spontaneously resolved within a few days. One month after treatment initiation, one patient experienced an elevation in liver function test results, which subsequently decreased during follow-up visits without necessitating discontinuation of therapy.

CONCLUSIONS: Our data indicate that ETI therapy is well tolerated by children with CF and is effective in improving signs of lung function abnormalities from early childhood.

PMID:38869349 | DOI:10.1002/ppul.27125

Pediatr Pulmonol. 2024 Jun 13. doi: 10.1002/ppul.27130. Online ahead of print.

NO ABSTRACT

PMID:38869247 | DOI:10.1002/ppul.27130

iScience. 2024 May 17;27(6):110009. doi: 10.1016/j.isci.2024.110009. eCollection 2024 Jun 21.

ABSTRACT

Continuous assessment of the impact of SARS-CoV-2 on the host at the cell-type level is crucial for understanding key mechanisms involved in host defense responses to viral infection. We investigated host response to ancestral-strain and Alpha-variant SARS-CoV-2 infections within air-liquid-interface human nasal epithelial cells from younger adults (26-32 Y) and older children (12-14 Y) using single-cell RNA-sequencing. Ciliated and secretory-ciliated cells formed the majority of highly infected cell-types, with the latter derived from ciliated lineages. Strong innate immune responses were observed across lowly infected and uninfected bystander cells and heightened in Alpha-infection. Alpha highly infected cells showed increased expression of protein-refolding genes compared with ancestral-strain-infected cells in children. Furthermore, oxidative phosphorylation-related genes were down-regulated in bystander cells versus infected and mock-control cells, underscoring the importance of these biological functions for viral replication. Overall, this study highlights the complexity of cell-type-, age- and viral strain-dependent host epithelial responses to SARS-CoV-2.

PMID:38868206 | PMC:PMC11166695 | DOI:10.1016/j.isci.2024.110009

Pediatr Infect Dis J. 2024 Jun 11. doi: 10.1097/INF.0000000000004427. Online ahead of print.

ABSTRACT

OBJECTIVES: Mucor within the airways of immunocompromised patients often signifies an invasive life-threatening infection. However, its significance in immunocompetent patients with chronic lung diseases is less clear. We aimed to assess the clinical implication of mucor in airway-secretion cultures of these patients.

METHODS: A single-center retrospective cohort study was performed. Patients with cystic fibrosis (CF), primary ciliary dyskinesia (PCD) or non-CF/non-PCD bronchiectasis followed in our Pediatric Pulmonary Institute, with sputum or bronchoalveolar lavage cultures growing Mucorales molds in the years 2010-2022, were included. Demographic and clinical parameters such as body mass index and spirometry values (forced expiratory volume at 1 second) were collected and compared with values up to 12 months prior to and following the index (positive culture) visit.

RESULTS: A total of 27 patients of whom 22 (82%) patients were with CF, 3 with PCD (11%) and 2 (7%) with non-CF/non-PCD bronchiectasis were included. Median age was 21.8 (14.9-32.1) years, with forced expiratory volume at 1 second of 62.8% ± 21.9% at the index visit. None of the patients developed disseminated disease, none had clinical or radiological evidence of fungal disease and none required antifungal therapy. Throughout the 12 months prior to and following the positive cultures, no significant changes were noted in body mass index, forced expiratory volume at 1 second, frequency of pulmonary exacerbations, days of hospitalization or days of antibiotic treatment.

CONCLUSIONS: Evidence of mucor in airway cultures of immunocompetent patients with chronic lung disease does not necessarily signify clinical deterioration nor suggests invasive fungal disease. Larger, long-term prospective studies are required to obviate the need for a thorough evaluation in these patients.

PMID:38865559 | DOI:10.1097/INF.0000000000004427

Cureus. 2024 May 12;16(5):e60140. doi: 10.7759/cureus.60140. eCollection 2024 May.

ABSTRACT

A 15-month-old African American male patient presented to the pediatric clinic to establish care. The patient had been seen and treated by a previous pediatrician who had diagnosed him with failure to thrive, anemia, and hepatosplenomegaly, according to the patient's parents. Upon physical examination, the patient was determined to be less than the first percentile for height and in the eighth percentile for weight. Frontal bossing was also observed. The patient's hemoglobin level was measured in the office to help confirm the previous anemia diagnosis and was determined to be 6.3 g/dL (normal: 10.5-13.0 g/dL). At this point, the patient was sent to a pediatric emergency department for continued treatment and workup. At the emergency department, the patient received an extensive laboratory workup for the evaluation of anemia, revealing iron deficiency anemia (hemoglobin: 5.6 g/dL (normal: 10.5-13 g/dL), mean corpuscular volume: 51.4 fl (normal: 70-84 fl), iron: 18 mcg/dL (normal: 30-70 mcg/dL), total iron binding capacity: 598 mcg/dL (normal: 100-400 mcg/dL), and hematocrit: 23.7% (normal: 33-38%)) and decreased levels of vitamin D (<6 ng/mL, normal: >30 ng/mL), ionized calcium (1.17 mg/dL, normal: 4.4-5.2 mg/dL), and phosphorus (2.4 mg/dL, normal: 2.9-5.9 mg/dL). These studies, paired with X-ray images of the patient's shoulders and wrists, further confirmed the diagnosis of rickets. Rickets is a disease in pediatric patients defined as a condition in which the mineralization of epiphyseal plates is defective. A nutritional deficiency in vitamin D, calcium, or phosphate causes acquired rickets. This condition is most commonly found in developing countries; some predisposing factors include poor sun exposure, high altitude, and breastfeeding. The patient was seen in the outpatient pediatric setting after the hospitalization, in which he received a blood transfusion, where he was managed on supplementation of calcium carbonate suspension, polysaccharide iron complex/novaferrum drops, and cholecalciferol drops with referral to endocrinology, hematology, and dietetics. This case serves as an example of how the diagnosis of nutritional deficiencies, such as rickets, can also be found in developed countries like the United States. Other conditions considered in the differential diagnosis were cystic fibrosis, necrotizing enterocolitis, metabolic disorders, inadequate absorption, and mechanical feeding difficulties, each of which must be ruled out to ensure that even an unlikely finding was not missed.

PMID:38864063 | PMC:PMC11165436 | DOI:10.7759/cureus.60140

Front Pharmacol. 2024 May 28;15:1428213. doi: 10.3389/fphar.2024.1428213. eCollection 2024.

ABSTRACT

[This corrects the article DOI: 10.3389/fphar.2024.1362325.].

PMID:38863980 | PMC:PMC11165198 | DOI:10.3389/fphar.2024.1428213

J Breath Res. 2024 Jun 11. doi: 10.1088/1752-7163/ad56bc. Online ahead of print.

ABSTRACT

Diagnosing lung infections is often challenging because of the lack of a high-quality specimen from the diseased lung. Since persons with cystic fibrosis are subject to chronic lung infection, there is frequently a need for a lung specimen. In this small, proof of principle study, we determined that PneumoniaCheckTM, a non-invasive device that captures coughed droplets from the lung on a filter, might help meet this need. We obtained 10 PneumoniaCheckTM coughed specimens and 2 sputum specimens from adult CF patients hospitalized with an exacerbation of their illness. We detected amylase (upper respiratory tract) with an enzymatic assay, surfactant A (lower respiratory tract) with an immunoassay, pathogenic bacteria by PCR, and markers of inflammation by a Luminex multiplex immunoassay. The amylase and surfactant A levels suggested that 9/10 coughed specimens were from lower respiratory tract with minimal upper respiratory contamination. The PCR assays detected pathogenic bacteria in 7 of 9 specimens and multiplex Luminex assay detected a variety of cytokines or chemokines. These data indicate that the PneumoniaCheckTM coughed specimens can capture good quality lower respiratory tract specimens that have the potential to help in diagnosis, management and understanding of CF exacerbations and other lung disease.

PMID:38861972 | DOI:10.1088/1752-7163/ad56bc

Pediatr Pulmonol. 2024 Jun 11. doi: 10.1002/ppul.27100. Online ahead of print.

ABSTRACT

INTRODUCTION: Cystic fibrosis transmembrane conductance regulator (CFTR) modulator elexacaftor/tezacaftor/ivacaftor (ETI) significantly improves lung function but its effect on mental health and sleep remains poorly understood. We report on mental health, sleep, and respiratory health outcomes of adolescents with CF commenced on ETI therapy, and monitored the prevalence of neuropsychiatric issues through the coronavirus disease 2019 pandemic.

METHODS: We conducted a prospective longitudinal study of 31 adolescents (aged 10-18 years) from July 2021 to October 2022. Data collected include demographics, Patient Health Questionnaire-9 (PHQ-9), General Anxiety Disorder-7 (GAD-7), Pediatric Daytime Sleepiness Scale (PDSS), Sleep Disturbance Scale for Children (SDSC) scores, and FEV1 percent predicted. Twenty of 31 adolescents had data before and after ETI initiation. Mean differences (MD) in mental health, sleep, and respiratory health pre- and post-ETI therapy commencement were estimated using paired t-tests. The prevalence and trajectories of anxiety, depression, and sleep disturbance were described between the ETI epochs, and over the pandemic period.

RESULTS: FEV1 improved following ETI therapy commencement (MD, 95% confidence interval [CI]: 7.1% (4.7%-9.6%) whereas PHQ-9, GAD-7, PDSS, and SDSC scores did not change significantly. Ten percent of participants developed new-onset anxiety/depression concerns and 10% developed new sleep concerns following ETI initiation.

CONCLUSION: This is the first prospective longitudinal study of mental health and sleep changes after ETI commencement in adolescents with CF. Although respiratory outcomes improved, ETI did not improve anxiety, depression or sleep.

PMID:38860602 | DOI:10.1002/ppul.27100

Pediatr Pulmonol. 2024 Jun 11. doi: 10.1002/ppul.27128. Online ahead of print.

NO ABSTRACT

PMID:38860588 | DOI:10.1002/ppul.27128

Am J Physiol Lung Cell Mol Physiol. 2024 Jun 11. doi: 10.1152/ajplung.00392.2023. Online ahead of print.

ABSTRACT

The mucociliary transport apparatus is critical for maintaining lung health via the coordinated movement of cilia to clear mucus and particulates. A metachronal wave propagates across the epithelium when cilia on adjacent multiciliated cells beat slightly out of phase along the proximal-distal axis of the airways in alignment with anatomically directed mucociliary clearance. We hypothesized that metachrony optimizes mucociliary transport (MCT) and that disruptions of calcium signaling would abolish metachrony and decrease MCT. We imaged bronchi from human explants and ferret tracheae using micro-Optical Coherence Tomography (µOCT) to evaluate airway surface liquid depth (ASL), periciliary liquid depth (PCL), cilia beat frequency (CBF), MCT, and metachrony in situ. We developed statistical models that included covariates of MCT. Ferret tracheae were treated with BAPTA-AM (chelator of intracellular Ca2+), lanthanum chloride (nonpermeable Ca2+channel competitive antagonist), and repaglinide (inhibitor of calaxin) to test calcium-dependence of metachrony. We demonstrated metachrony contributes to mucociliary transport of human and ferret airways. MCT was augmented in regions of metachrony compared to non-metachronous regions by 48.1%, P=0.0009 or 47.5%, P<0.0020 in humans and ferrets, respectively. PCL and metachrony were independent contributors to MCT rate in humans; ASL, CBF, and metachrony contribute to ferret MCT rates. Metachrony can be disrupted by interference with calcium signaling including intracellular, mechanosensitive channels, and calaxin. Our results support that the presence of metachrony augments MCT in a calcium-dependent mechanism.

PMID:38860289 | DOI:10.1152/ajplung.00392.2023

J Vasc Access. 2024 Jun 10:11297298241258257. doi: 10.1177/11297298241258257. Online ahead of print.

ABSTRACT

INTRODUCTION: The use of midline catheters for patients requiring a peripheral IV infusion is sometimes limited by their cost. Although decision trees allow them to be positioned in relation to short peripheral cannulas (SPC), Midlines, and PICCs, their economic impact has not yet been evaluated. A study was conducted to estimate and compare the actual costs of using the three types of catheters for durations of 7, 14, and 21 days.

METHODS: A budget impact analysis compared midlines or mini-midlines/long peripheral cannulas (LPCs) with SPCs and PICCs for typical medical indications excluding indications requiring central line (infusion of irritant or vesicant drugs): treatment of peritonitis over 7 days, cystic fibrosis infection over 14 days, and meningitis over 21 days. A micro-costing study identified resources used during catheter care procedures (consumables, medical/nursing care, examinations, mechanical complications). The cost of remote systemic complications was estimated from the French national cost study. Literature review compared data based on published complication frequencies.

RESULTS: Midline is more economic than the SPC (saving of 39€ over 7 days and 174€ over 14 days), and than the PICC (saving of 102€ over 14 days and 95€ over 21 days).

DISCUSSION: Despite a much higher acquisition cost of the Midline than a SPC, the cost of using a Midline is lower. Although this approach cannot be the only argument for choosing a medical device, it can contribute to it in a tense economic context. The micro-costing has been performed in a center placing PICCline using fluoroscopy for catheter tip positioning. The implantation of a PICC with ECG technique does not require an interventional radiology facility and involves significantly lower logistical and personnel costs. This factor is a limitation in this study. However, even with the use of EGC, the cost difference is in favor of Midline.

PMID:38855974 | DOI:10.1177/11297298241258257

Ear Nose Throat J. 2024 Jun 10:1455613241261563. doi: 10.1177/01455613241261563. Online ahead of print.

ABSTRACT

Patients with chronic rhinosinusitis (CRS) that is refractory to maximal medical and surgical therapy should be evaluated for other primary conditions. Cystic fibrosis (CF), primary immunodeficiency (PID), and primary ciliary dyskinesia (PCD) are potential risk factors for refractory CRS. These conditions present with variable disease severity and diagnosis may be delayed into adulthood. We report a case of a mother-daughter pair with CRS refractory to maximal medical management. The patients were further evaluated and found to have features consistent with CF, PID, and PCD. All 3 are rare disorders and thought to cause CRS in isolation. Patients with refractory CRS should be further evaluated to identify alternative diagnoses and ensure proper management. Refractory CRS may be multifactorial, with different risk factors simultaneously contributing to its persistence.

PMID:38855824 | DOI:10.1177/01455613241261563

Front Cell Infect Microbiol. 2024 May 24;14:1411333. doi: 10.3389/fcimb.2024.1411333. eCollection 2024.

ABSTRACT

Mycobacterium abscessus (Mab) is an opportunistic pathogen afflicting individuals with underlying lung disease such as Cystic Fibrosis (CF) or immunodeficiencies. Current treatment strategies for Mab infections are limited by its inherent antibiotic resistance and limited drug access to Mab in its in vivo niches resulting in poor cure rates of 30-50%. Mab's ability to survive within macrophages, granulomas and the mucus laden airways of the CF lung requires adaptation via transcriptional remodeling to counteract stresses like hypoxia, increased levels of nitrate, nitrite, and reactive nitrogen intermediates. Mycobacterium tuberculosis (Mtb) is known to coordinate hypoxic adaptation via induction of respiratory nitrate assimilation through the nitrate reductase narGHJI. Mab, on the other hand, does not encode a respiratory nitrate reductase. In addition, our recent study of the transcriptional responses of Mab to hypoxia revealed marked down-regulation of a locus containing putative nitrate assimilation genes, including the orphan response regulator nnaR (nitrate/nitrite assimilation regulator). These putative nitrate assimilation genes, narK3 (nitrate/nitrite transporter), nirBD (nitrite reductase), nnaR, and sirB (ferrochelatase) are arranged contiguously while nasN (assimilatory nitrate reductase identified in this work) is encoded in a different locus. Absence of a respiratory nitrate reductase in Mab and down-regulation of nitrogen metabolism genes in hypoxia suggest interplay between hypoxia adaptation and nitrate assimilation are distinct from what was previously documented in Mtb. The mechanisms used by Mab to fine-tune the transcriptional regulation of nitrogen metabolism in the context of stresses e.g. hypoxia, particularly the role of NnaR, remain poorly understood. To evaluate the role of NnaR in nitrate metabolism we constructed a Mab nnaR knockout strain (MabΔnnaR ) and complement (MabΔnnaR+C ) to investigate transcriptional regulation and phenotypes. qRT-PCR revealed NnaR is necessary for regulating nitrate and nitrite reductases along with a putative nitrate transporter. Loss of NnaR compromised the ability of Mab to assimilate nitrate or nitrite as sole nitrogen sources highlighting its necessity. This work provides the first insights into the role of Mab NnaR setting a foundation for future work investigating NnaR's contribution to pathogenesis.

PMID:38854658 | PMC:PMC11162112 | DOI:10.3389/fcimb.2024.1411333

bioRxiv [Preprint]. 2024 May 28:2024.05.28.596254. doi: 10.1101/2024.05.28.596254.

ABSTRACT

In Pseudomonas aeruginosa, alginate biosynthesis gene expression is inhibited by the transmembrane anti-sigma factor MucA, which sequesters the AlgU sigma factor. Cell envelope stress initiates cleavage of the MucA periplasmic domain by site-1 protease AlgW, followed by further MucA degradation to release AlgU. However, after colonizing the lungs of people with cystic fibrosis, P. aeruginosa converts to a mucoid form that produces alginate constitutively. Mucoid isolates often have mucA mutations, with the most common being mucA22 , which truncates the periplasmic domain. MucA22 is degraded constitutively, and genetic studies suggested that the Prc protease is responsible. Some studies also suggested that Prc contributes to induction in strains with wild type MucA, whereas others suggested the opposite. However, missing from all previous studies is a demonstration that Prc cleaves any protein directly, which leaves open the possibility that the effect of a prc null mutation is indirect. To address the ambiguities and shortfalls, we reevaluated the roles of AlgW and Prc as MucA and MucA22 site-1 proteases. In vivo analyses using three different assays, and two different inducing conditions, all suggested that AlgW is the only site-1 protease for wild type MucA in any condition. In contrast, genetics suggested that AlgW or Prc act as MucA22 site-1 proteases in inducing conditions, whereas Prc is the only MucA22 site-1 protease in non-inducing conditions. For the first time, we also show that Prc is unable to degrade the periplasmic domain of wild type MucA, but does degrade the mutated periplasmic domain of MucA22 directly.

IMPORTANCE: After colonizing the lungs of individuals with cystic fibrosis, P. aeruginosa undergoes mutagenic conversion to a mucoid form, worsening the prognosis. Most mucoid isolates have a truncated negative regulatory protein MucA, which leads to constitutive production of the extracellular polysaccharide alginate. The protease Prc has been implicated, but not shown, to degrade the most common MucA variant, MucA22, to trigger alginate production. This work provides the first demonstration that the molecular mechanism of Prc involvement is direct degradation of the MucA22 periplasmic domain, and perhaps other truncated MucA variants as well. MucA truncation and degradation by Prc might be the predominant mechanism of mucoid conversion in cystic fibrosis infections, suggesting that Prc activity could be a useful therapeutic target.

PMID:38854061 | PMC:PMC11160602 | DOI:10.1101/2024.05.28.596254

Res Sq [Preprint]. 2024 May 31:rs.3.rs-4421613. doi: 10.21203/rs.3.rs-4421613/v1.

ABSTRACT

Mucus stasis is a pathologic hallmark of muco-obstructive diseases, including cystic fibrosis (CF). Mucins, the principal component of mucus, are extensively modified with hydroxyl (O)-linked glycans, which are largely terminated by sialic acid. Sialic acid is a negatively charged monosaccharide and contributes to the biochemical/biophysical properties of mucins. Reports suggest that mucin sialylation may be altered in CF; however, the consequences of reduced sialylation on mucus clearance have not been fully determined. Here, we investigated the consequences of reduced sialylation on the charge state and conformation of the most prominent airway mucin, MUC5B, and defined the functional consequences of reduced sialylation on mucociliary transport (MCT). Reduced sialylation contributed to a lower charged MUC5B form and decreased polymer expansion. The inhibition of total mucin sialylation de novo impaired MCT in primary human bronchial epithelial cells and rat airways, and specific α-2,3 sialylation blockade was sufficient to recapitulate these findings. Finally, we show that ST3 beta-galactoside alpha-2,3-sialyltransferase (ST3Gal1) expression is downregulated in CF and partially restored by correcting CFTR via Elexacaftor/Tezacaftor/Ivacaftor treatment. Overall, this study demonstrates the importance of mucin sialylation in mucus clearance and identifies decreased sialylation by ST3Gal1 as a possible therapeutic target in CF and potentially other muco-obstructive diseases.

PMID:38853971 | PMC:PMC11160914 | DOI:10.21203/rs.3.rs-4421613/v1