Abstract
During cystic fibrosis (CF) chronic lung infections, bacteria of the Burkholderia cepacia complex (Bcc) are exposed for several years to a stressful and changing environment. These environmental challenges results in genetic changes of the initial infecting strain with the consequent diversification of genotypes and phenotypes. The exploitation of functional and comparative genomic approaches has suggested that such diversification is associated with massive metabolic remodeling but these alterations are poorly understood. In the present work, we have explored a high resolution 1H-NMR-based metabolomic approach coupled to multivariate analysis to compare the endometabolome of three B. cenocepacia clonal variants retrieved from a CF patient from the onset of infection (IST439) until death with cepacia syndrome after 3.5 years (IST4113 and IST4134), to complement former proteomic and transcriptomic analyses. A fourth clonal variant (IST4129) retrieved from the same CF patient when the clinical condition worsened during the last months of life, was also examined since it was found to lack the third replicon. The metabolomic profiles obtained, based on the complete 1H-NMR spectra, highlight the separation of the four clonal variants examined, the most distinct profile corresponding to IST4129. Results indicate a variable content of several amino acids in the different isolates examined and suggest that glycolysis and the glyoxylate shunt are favored in late variants. Moreover, the concentration of two metabolites with demonstrated cellular protective functions against stress, glycine-betaine and trehalose, is different in the different isolates examined. However, no clear correlation could be established between their content and stress tolerance. For example, IST4113, previously found to be the most resistant variant to antimicrobials of different classes, exhibits low levels of trehalose and glycine-betaine but the highest resistance to heat and oxidative stress. Also, IST4129, with a high level of glycine-betaine but lacking the third replicon, previously associated with stress resistance and virulence, exhibits the highest susceptibility to all the stresses tested. Taken together, results from this study provide insights into the metabolic diversification of B. cenocepacia clonal variants during long-term infection of the CF airways.
Highlights
Cystic fibrosis (CF) chronic lung infections caused by Burkholderia cepacia complex (Bcc) bacteria are usually associated with a rapid decline in lung function and decreased life expectancy (Mahenthiralingam et al, 2005; Lipuma, 2010)
The 1H-NMR spectra acquired from extracts obtained from B. cenocepacia cells grown 24 h at 37◦C onto the surface of LB agar medium allowed the detection of hundreds of resonances (Figure 1)
Variable Importance in the Projection (VIP) analysis showed that glycine-betaine, trehalose, and glycine are three of the most important identified metabolites contributing to separate IST439 from IST4113 and IST4129, while glycine-betaine, L-valine, and succinic acid are the three identified metabolites that contribute more to separation between IST439 and IST4134
Summary
Cystic fibrosis (CF) chronic lung infections caused by Burkholderia cepacia complex (Bcc) bacteria are usually associated with a rapid decline in lung function and decreased life expectancy (Mahenthiralingam et al, 2005; Lipuma, 2010). The genome sequences of late Burkholderia cenocepacia isolates retrieved from a CF patient after 10 years of chronic infection were compared with the initial infecting strain and mutations in genes involved in metabolism, including genes from amino acid, iron and purine metabolism, were identified (Miller et al, 2015). Another comparative genomic study focusing on B. multivorans isolates retrieved from a CF patient over 20 years of infection has shown that genes involved in gene expression regulation, cell envelope biogenesis, fatty acid and amino acid metabolism exhibit recurrent mutation patterns (Silva et al, 2016). The comparison of genomic expression analyses based on quantitative proteomics and transcriptomics of three B. cenocepacia clonal variants retrieved from the same chronically infected CF patient during 3.5 years is consistent with metabolism diversification
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