RESUMO
In a healthy colon, the stratified mucus layer serves as a crucial innate immune barrier to protect the epithelium from microbes. Mucins are complex glycoproteins that serve as a nutrient source for resident microflora and can be exploited by pathogens. We aimed to understand how the intestinal pathogen, Clostridioides difficile, independently uses or manipulates mucus to its benefit, without contributions from members of the microbiota. Using a 2-D primary human intestinal epithelial cell model to generate physiologic mucus, we assessed C. difficile-mucus interactions through growth assays, RNA-Seq, biophysical characterization of mucus, and contextualized metabolic modeling. We found that host-derived mucus promotes C. difficile growth both in vitro and in an infection model. RNA-Seq revealed significant upregulation of genes related to central metabolism in response to mucus, including genes involved in sugar uptake, the Wood-Ljungdahl pathway, and the glycine cleavage system. In addition, we identified differential expression of genes related to sensing and transcriptional control. Analysis of mutants with deletions in highly upregulated genes reflected the complexity of C. difficile-mucus interactions, with potential interplay between sensing and growth. Mucus also stimulated biofilm formation in vitro, which may in turn alter the viscoelastic properties of mucus. Context-specific metabolic modeling confirmed differential metabolism and the predicted importance of enzymes related to serine and glycine catabolism with mucus. Subsequent growth experiments supported these findings, indicating mucus is an important source of serine. Our results better define responses of C. difficile to human gastrointestinal mucus and highlight flexibility in metabolism that may influence pathogenesis. IMPORTANCE: Clostridioides difficile results in upward of 250,000 infections and 12,000 deaths annually in the United States. Community-acquired infections continue to rise, and recurrent disease is common, emphasizing a vital need to understand C. difficile pathogenesis. C. difficile undoubtedly interacts with colonic mucus, but the extent to which the pathogen can independently respond to and take advantage of this niche has not been explored extensively. Moreover, the metabolic complexity of C. difficile remains poorly understood but likely impacts its capacity to grow and persist in the host. Here, we demonstrate that C. difficile uses native colonic mucus for growth, indicating C. difficile possesses mechanisms to exploit the mucosal niche. Furthermore, mucus induces metabolic shifts and biofilm formation in C. difficile, which has potential ramifications for intestinal colonization. Overall, our work is crucial to better understand the dynamics of C. difficile-mucus interactions in the context of the human gut.
Assuntos
Biofilmes , Clostridioides difficile , Regulação Bacteriana da Expressão Gênica , Muco , Clostridioides difficile/genética , Clostridioides difficile/fisiologia , Clostridioides difficile/metabolismo , Biofilmes/crescimento & desenvolvimento , Humanos , Muco/microbiologia , Muco/metabolismo , Células Epiteliais/microbiologia , Mucosa Intestinal/microbiologia , Mucosa Intestinal/metabolismo , Infecções por Clostridium/microbiologiaRESUMO
The airway milieu of individuals with muco-obstructive airway diseases (MADs) is defined by the accumulation of dehydrated mucus due to hyperabsorption of airway surface liquid and defective mucociliary clearance. Pathological mucus becomes progressively more viscous with age and disease severity due to the concentration and overproduction of mucin and accumulation of host-derived extracellular DNA (eDNA). Respiratory mucus of MADs provides a niche for recurrent and persistent colonization by respiratory pathogens, including Pseudomonas aeruginosa, which is responsible for the majority of morbidity and mortality in MADs. Despite high concentration inhaled antibiotic therapies and the absence of antibiotic resistance, antipseudomonal treatment failure in MADs remains a significant clinical challenge. Understanding the drivers of antibiotic tolerance is essential for developing more effective treatments that eradicate persistent infections. The complex and dynamic environment of diseased airways makes it difficult to model antibiotic efficacy in vitro. We aimed to understand how mucin and eDNA concentrations, the two dominant polymers in respiratory mucus, alter the antibiotic tolerance of P. aeruginosa. Our results demonstrate that polymer concentration and molecular weight affect P. aeruginosa survival post antibiotic challenge. Polymer-driven antibiotic tolerance was not explicitly associated with reduced antibiotic diffusion. Lastly, we established a robust and standardized in vitro model for recapitulating the ex vivo antibiotic tolerance of P. aeruginosa observed in expectorated sputum across age, underlying MAD etiology, and disease severity, which revealed the inherent variability in intrinsic antibiotic tolerance of host-evolved P. aeruginosa populations. IMPORTANCE: Antibiotic treatment failure in Pseudomonas aeruginosa chronic lung infections is associated with increased morbidity and mortality, illustrating the clinical challenge of bacterial infection control. Understanding the underlying infection environment, as well as the host and bacterial factors driving antibiotic tolerance and the ability to accurately recapitulate these factors in vitro, is crucial for improving antibiotic treatment outcomes. Here, we demonstrate that increasing concentration and molecular weight of mucin and host eDNA drive increased antibiotic tolerance to tobramycin. Through systematic testing and modeling, we identified a biologically relevant in vitro condition that recapitulates antibiotic tolerance observed in ex vivo treated sputum. Ultimately, this study revealed a dominant effect of in vivo evolved bacterial populations in defining inter-subject ex vivo antibiotic tolerance and establishes a robust and translatable in vitro model for therapeutic development.
Assuntos
Antibacterianos , Muco , Infecções por Pseudomonas , Pseudomonas aeruginosa , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/genética , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Infecções por Pseudomonas/tratamento farmacológico , Infecções por Pseudomonas/microbiologia , Muco/microbiologia , Muco/metabolismo , Humanos , Mucinas/metabolismo , Farmacorresistência Bacteriana , Polímeros/metabolismo , Infecção Persistente/microbiologia , Pulmão/microbiologia , Infecções Respiratórias/microbiologia , Infecções Respiratórias/tratamento farmacológico , Adaptação FisiológicaRESUMO
In a healthy colon, the stratified mucus layer serves as a crucial innate immune barrier to protect the epithelium from microbes. Mucins are complex glycoproteins that serve as a nutrient source for resident microflora and can be exploited by pathogens. We aimed to understand how the intestinal pathogen, Clostridioides diffiicile, independently uses or manipulates mucus to its benefit, without contributions from members of the microbiota. Using a 2-D primary human intestinal epithelial cell model to generate physiologic mucus, we assessed C. difficile-mucus interactions through growth assays, RNA-Seq, biophysical characterization of mucus, and contextualized metabolic modeling. We found that host-derived mucus promotes C. difficile growth both in vitro and in an infection model. RNA-Seq revealed significant upregulation of genes related to central metabolism in response to mucus, including genes involved in sugar uptake, the Wood-Ljungdahl pathway, and the glycine cleavage system. In addition, we identified differential expression of genes related to sensing and transcriptional control. Analysis of mutants with deletions in highly upregulated genes reflected the complexity of C. difficile-mucus interactions, with potential interplay between sensing and growth. Mucus also stimulated biofilm formation in vitro, which may in turn alter viscoelastic properties of mucus. Context-specific metabolic modeling confirmed differential metabolism and predicted importance of enzymes related to serine and glycine catabolism with mucus. Subsequent growth experiments supported these findings, indicating mucus is an important source of serine. Our results better define responses of C. difficile to human gastrointestinal mucus and highlight a flexibility in metabolism that may influence pathogenesis.
RESUMO
People with muco-obstructive pulmonary diseases such as cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) often have acute or chronic respiratory infections that are difficult to treat due in part to the accumulation of hyperconcentrated mucus within the airway. Mucus accumulation and obstruction promote chronic inflammation and infection and reduce therapeutic efficacy. Bacterial aggregates in the form of biofilms exhibit increased resistance to mechanical stressors from the immune response (e.g., phagocytosis) and chemical treatments including antibiotics. Herein, combination treatments designed to disrupt the mechanical properties of biofilms and potentiate antibiotic efficacy are investigated against mucus-grown Pseudomonas aeruginosa biofilms and optimized to 1) alter biofilm viscoelastic properties, 2) increase mucociliary transport rates, and 3) reduce bacterial viability. A disulfide bond reducing agent (tris(2-carboxyethyl)phosphine, TCEP), a surfactant (NP40), a biopolymer (hyaluronic acid, HA), a DNA degradation enzyme (DNase), and an antibiotic (tobramycin) are tested in various combinations to maximize biofilm disruption. The viscoelastic properties of biofilms are quantified with particle tracking microrheology and transport rates are quantified in a mucociliary transport device comprised of fully differentiated primary human bronchial epithelial cells. The combination of the NP40 with hyaluronic acid and tobramycin was the most effective at increasing mucociliary transport rates, decreasing the viscoelastic properties of mucus, and reducing bacterial viability. Multimechanistic targeting of biofilm infections may ultimately result in improved clinical outcomes, and the results of this study may be translated into future in vivo infection models.
Assuntos
Depuração Mucociliar , Infecções por Pseudomonas , Humanos , Pseudomonas aeruginosa , Ácido Hialurônico/farmacologia , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Tobramicina/farmacologia , Tobramicina/uso terapêutico , Infecções por Pseudomonas/tratamento farmacológico , Infecções por Pseudomonas/microbiologia , BiofilmesRESUMO
People with muco-obstructive pulmonary diseases such as cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) often have acute or chronic respiratory infections that are difficult to treat due in part to the accumulation of hyperconcentrated mucus within the airway. Mucus accumulation and obstruction promote chronic inflammation and infection and reduce therapeutic efficacy. Bacterial aggregates in the form of biofilms exhibit increased resistance to mechanical stressors from the immune response (e.g., phagocytosis) and chemical treatments including antibiotics. Herein, combination treatments designed to disrupt the mechanical properties of biofilms and potentiate antibiotic efficacy are investigated against mucus-grown Pseudomonas aeruginosa biofilms and optimized to 1) alter biofilm viscoelastic properties, 2) increase mucociliary transport rates, and 3) reduce bacterial viability. A disulfide bond reducing agent (tris(2-carboxyethyl)phosphine, TCEP), a surfactant (NP40), a biopolymer (hyaluronic acid, HA), a DNA degradation enzyme (DNase), and an antibiotic (tobramycin) are tested in various combinations to maximize biofilm disruption. The viscoelastic properties of biofilms are quantified with particle tracking microrheology and transport rates are quantified in a mucociliary transport device comprised of fully differentiated primary human bronchial epithelial cells. The combination of the NP40 with hyaluronic acid and tobramycin was the most effective at increasing mucociliary transport rates, decreasing the viscoelastic properties of mucus, and reducing bacterial viability. Multimechanistic targeting of biofilm infections may ultimately result in improved clinical outcomes, and the results of this study may be translated into future in vivo infection models.
RESUMO
Across the globe, millions of people are affected by muco-obstructive pulmonary diseases like cystic fibrosis, asthma, and chronic obstructive pulmonary disease. In MOPDs, the airway mucus becomes hyperconcentrated, increasing viscoelasticity and impairing mucus clearance. Research focused on treatment of MOPDs requires relevant sources of airway mucus both as a control sample type and as a basis for manipulation to study the effects of additional hyperconcentration, inflammatory milieu, and biofilm growth on the biochemical and biophysical properties of mucus. Endotracheal tube mucus has been identified as a prospective source of native airway mucus given its several advantages over sputum and airway cell culture mucus such as ease of access and in vivo production that includes surface airway and submucosal gland secretions. Still, many ETT samples suffer from altered tonicity and composition from either dehydration, salivary dilution, or other contamination. Herein, the biochemical compositions of ETT mucus from healthy human subjects were determined. Samples were characterized in terms of tonicity, pooled, and restored to normal tonicity. Salt-normalized ETT mucus exhibited similar concentration-dependent rheologic properties as originally isotonic mucus. This rheology agreed across spatial scales and with previous reports of the biophysics of ETT mucus. This work affirms previous reports of the importance of salt concentration on mucus rheology and presents methodology to increase yield native airway mucus samples for laboratory use and manipulation.
RESUMO
The viscoelastic properties of biofilms are correlated with their susceptibility to mechanical and chemical stress, and the airway environment in muco-obstructive pulmonary diseases (MOPD) facilitates robust biofilm formation. Hyperconcentrated, viscoelastic mucus promotes chronic inflammation and infection, resulting in increased mucin and DNA concentrations. The viscoelastic properties of biofilms are regulated by biopolymers, including polysaccharides and DNA, and influence responses to antibiotics and phagocytosis. We hypothesize that targeted modulation of biofilm rheology will compromise structural integrity and increase antibiotic susceptibility and mucociliary transport. We evaluate biofilm rheology on the macro, micro, and nano scale as a function of treatment with a reducing agent, a biopolymer, and/or tobramycin to define the relationship between the viscoelastic properties of biofilms and susceptibility. Disruption of the biofilm architecture is associated with altered macroscopic and microscopic moduli, rapid vector permeability, increased antibiotic susceptibility, and improved mucociliary transport, suggesting that biofilm modulating therapeutics will improve the treatment of chronic respiratory infections in MOPD.
RESUMO
The airway milieu of individuals with muco-obstructive airway diseases (MADs) is defined by the accumulation of dehydrated mucus due to hyperabsorption of airway surface liquid and defective mucociliary clearance. Pathological mucus becomes progressively more viscous with age and disease severity due to the concentration and overproduction of mucin and accumulation of host-derived extracellular DNA (eDNA). Respiratory mucus of MADs provides a niche for recurrent and persistent colonization by respiratory pathogens, including Pseudomonas aeruginosa , which is responsible for the majority of morbidity and mortality in MADs. Despite high concentration inhaled antibiotic therapies and the absence of antibiotic resistance, antipseudomonal treatment failure in MADs remains a significant clinical challenge. Understanding the drivers of antibiotic recalcitrance is essential for developing more effective treatments that eradicate persistent infections. The complex and dynamic environment of diseased airways makes it difficult to model antibiotic efficacy in vitro . We aimed to understand how mucin and eDNA concentrations, the two dominant polymers in respiratory mucus, alter the antibiotic tolerance of P. aeruginosa . Our results demonstrate that polymer concentration and molecular weight affect P. aeruginosa survival post antibiotic challenge. Polymer-driven antibiotic tolerance was not explicitly associated with reduced antibiotic diffusion. Lastly, we established a robust and standardized in vitro model for recapitulating the ex vivo antibiotic tolerance of P. aeruginosa observed in expectorated sputum across age, underlying MAD etiology, and disease severity, which revealed the inherent variability in intrinsic antibiotic tolerance of host-evolved P. aeruginosa populations. Importance: Antibiotic treatment failure in Pseudomonas aeruginosa chronic lung infections is associated with increased morbidity and mortality, illustrating the clinical challenge of bacterial infection control. Understanding the underlying infection environment, as well as the host and bacterial factors driving antibiotic tolerance and the ability to accurately recapitulate these factors in vitro , is crucial for improving antibiotic treatment outcomes. Here, we demonstrate that increasing concentration and molecular weight of mucin and host eDNA drive increased antibiotic tolerance to tobramycin. Through systematic testing and modeling, we identified a biologically relevant in vitro condition that recapitulates antibiotic tolerance observed in ex vivo treated sputum. Ultimately, this study revealed a dominant effect of in vivo evolved bacterial populations in defining inter-subject ex vivo antibiotic tolerance and establishes a robust and translatable in vitro model for therapeutic development.