Nutritional Cues ControlPseudomonas aeruginosaMulticellular Behavior in Cystic Fibrosis Sputum

Abstract

The sputum (mucus) layer of the cystic fibrosis (CF) lung is a complex substrate that provides Pseudomonas aeruginosa with carbon and energy to support high-density growth during chronic colonization. Unfortunately, the CF lung sputum layer has been difficult to mimic in animal models of CF disease, and mechanistic studies of P. aeruginosa physiology during growth in CF sputum are hampered by its complexity. In this study, we performed chromatographic and enzymatic analyses of CF sputum to develop a defined, synthetic CF sputum medium (SCFM) that mimics the nutritional composition of CF sputum. Importantly, P. aeruginosa displays similar phenotypes during growth in CF sputum and in SCFM, including similar growth rates, gene expression profiles, carbon substrate preferences, and cell-cell signaling profiles. Using SCFM, we provide evidence that aromatic amino acids serve as nutritional cues that influence cell-cell signaling and antimicrobial activity of P. aeruginosa during growth in CF sputum.A key concept in bacterial pathogenesis is the ability of invading pathogens to obtain sufficient carbon and energy from the host for in vivo growth. Although Garber originally proposed the host as a growth medium over 40 years ago (12), the nutritional environment of most infection sites is poorly defined and often inadequately modeled by laboratory growth media. This lack of knowledge, combined with the limited utility of many animal models, provides significant challenges for mechanistic studies aimed at examining host nutrients as mediators of colonization and disease. To overcome these challenges, it is critical both to define the nutritional composition of key infection sites and to study bacterial physiology in the context of in vivo-relevant growth substrates.The heritable disease cystic fibrosis (CF) is an archetype for the development of nutritional models with which to study bacterial pathogenesis. A hallmark of CF disease is the accumulation of large volumes of sputum (mucus) within the lungs, which diminishes the host's ability to clear bacterial infections (17,31). The viscous CF lung sputum provides bacteria with a nutritionally rich growth environment composed of host-and bacterial-derived factors (17, 38). The opportunistic pathogen Pseudomonas aeruginosa chronically colonizes the CF lung, where it often grows to high cell densities in CF sputum (Ͼ10 9 cells/ml sputum). Although many other bacterial species persist and grow in the CF lung, chronic P. aeruginosa infection is likely the most clinically relevant, as it is correlated with declining lung function (17). Mechanistically, P. aeruginosa colonization and progression to chronic infection is poorly understood, although potential contributing factors are high-density growth and enhanced fitness of P. aeruginosa in CF sputum. P. aeruginosa fitness has been linked to nutritional components in CF sputum (39), thus necessitating the development of a versatile model that allows examination of CF sputum nutritional cues.The growth environment impacts several...

The Limitations of In Vitro Experimentation in Understanding Biofilms and Chronic Infection

“…Given the issues highlighted previously, how can we better represent in vivo conditions in our in vitro models? It is widely known, for P. aeruginosa at least, that different nutritional cues result in altered biofilm formation, virulence, motility, and QS [46,[113][114][115][116][117]. These differences become increasingly important when factors of clinical relevance, such as virulence and antimicrobial tolerance, are altered [13,118,119].…”

Polymorphonuclear Leukocytes Restrict Growth of Pseudomonas aeruginosa in the Lungs of Cystic Fibrosis Patients

“…Denitrification during chronic lung infection in CF may also be used by other CF pathogens because we recently demonstrated the genetic setup for denitrification as well as anaerobic N 2 O production in Achromobacter xylosoxidans (43), an emerging CF pathogen that induces an inflammatory response resembling the response induced by P. aeruginosa (44). Consumption of glucose by activated PMNs (45,46) may lead to a reduction in available glucose, which may also limit the growth of P. aeruginosa (47,48). However, the high levels of glucose (2 to 4 mM) in CF airway fluids (49) suggest that bacterial growth in infected mucus is not limited by available glucose.…”

Comparative Metabolic Systems Analysis of Pathogenic Burkholderia

“…Growth was evaluated based on the resulting growth curves and maximum change in OD for each carbon source compared to the maximum change in OD of inoculated control wells with no carbon source. Additionally, we evaluated the ability of each organism to catabolize cysteine and tryptophan, which are amino acids present in cystic fibrosis lung sputum but not included in the Biolog plates (35). These amino acids were each added at concentrations of 20 mM to M9 salts to create cysteine and tryptophan minimal media.…”

“…Medium conditions were formulated for lysogeny broth (LB), M9 minimal medium, and synthetic cystic fibrosis medium (SCFM) as in previous publications (34,35). The medium conditions were defined by setting the lower and upper bounds of the exchange reactions to specify metabolites that are available or unavailable.…”