Phenylalanine induces Burkholderia cenocepacia phenylacetic acid catabolism through degradation to phenylacetyl-CoA in synthetic cystic fibrosis sputum medium

a b s t r a c t Synthetic cystic fibrosis sputum medium (SCFM) is rich in amino acids and supports robust growth of Burkholderia cenocepacia, a member of the Burkholderia cepacia complex (Bcc). Previous work demonstrated that B. cenocepacia phenylacetic acid (PA) catabolic genes are up-regulated during growth in SCFM and are required for full virulence in a Caenorhabditis elegans host model. In this work, we investigated the role of phenylalanine, one of the aromatic amino acids present in SCFM, as an inducer of the PA catabolic pathway. Phenylalanine degradation intermediates were used as sole carbon sources for growth and gene reporter experiments. In addition to phenylalanine and PA, phenylethylamine, phenylpyruvate, and 2-phenylacetamide were usable as sole carbon sources by wild type B. cenocepacia K56-2, but not by a PA catabolism-defective mutant. EMSA analysis showed that the binding of PaaR, the negative regulator protein of B. cenocepacia PA catabolism, to PA regulatory DNA could only be relieved by phenylacetyl-Coenzyme A (PA-CoA), but not by any of the putative phenylalanine degradation intermediates. Taken together, our results show that in B. cenocepacia, phenylalanine is catabolized to PA and induces PA catabolism through PA activation to PA-CoA. Thus, PaaR shares the same inducer with PaaX, the regulator of PA catabolism in Escherichia coli, despite belonging to a different protein family

Understanding the Pathogenicity of Burkholderia contaminans, an Emerging Pathogen in Cystic Fibrosis.

Several bacterial species from the Burkholderia cepacia complex (Bcc) are feared opportunistic pathogens that lead to debilitating lung infections with a high risk of developing fatal septicemia in cystic fibrosis (CF) patients. However, the pathogenic potential of other Bcc species is yet unknown. To elucidate clinical relevance of Burkholderia contaminans, a species frequently isolated from CF respiratory samples in Ibero-American countries, we aimed to identify its key virulence factors possibly linked with an unfavorable clinical outcome. We performed a genome-wide comparative analysis of two isolates of B. contaminans ST872 from sputum and blood culture of a female CF patient in Argentina. RNA-seq data showed significant changes in expression for quorum sensing-regulated virulence factors and motility and chemotaxis. Furthermore, we detected expression changes in a recently described low-oxygen-activated (lxa) locus which encodes stress-related proteins, and for two clusters responsible for the biosynthesis of antifungal and hemolytic compounds pyrrolnitrin and occidiofungin. Based on phenotypic assays that confirmed changes in motility and in proteolytic, hemolytic and antifungal activities, we were able to distinguish two phenotypes of B. contaminans that coexisted in the host and entered her bloodstream. Whole genome sequencing revealed that the sputum and bloodstream isolates (each representing a distinct phenotype) differed by over 1,400 mutations as a result of a mismatch repair-deficient hypermutable state of the sputum isolate. The inferred lack of purifying selection against nonsynonymous mutations and the high rate of pseudogenization in the derived isolate indicated limited evolutionary pressure during evolution in the nutrient-rich, stable CF sputum environment. The present study is the first to examine the genomic and transcriptomic differences between longitudinal isolates of B. contaminans. Detected activity of a number of putative virulence factors implies a genuine pathogenic nature of this novel Bcc species

Summary of the genetic differences between <i>B</i>. <i>contaminans</i> isolates 467_S and MF16_B.

<p>Summary of the genetic differences between <i>B</i>. <i>contaminans</i> isolates 467_S and MF16_B.</p

<i>B</i>. <i>contaminans</i> isolates used in the study.

<p><i>B</i>. <i>contaminans</i> isolates used in the study.</p

Genetic relationship of sequenced <i>B</i>. <i>contaminans</i>.

<p>The overall tree (<i>top</i>) depicts whole-genome phylogeny inferred from SNPs. Multilocus sequence types of the isolates are denoted in parentheses. <i>Bottom</i>–the subtree containing only ST872 isolates.</p

Agar plate phenotypic assays.

<p>A) swimming motility, B) proteolysis, C) hemolysis and D) antifungal activity against <i>Geotrichum candidum</i>. <i>Left</i>–isolate 467_S, <i>right–</i>isolate MF16_B.</p

The <i>lxa</i> locus, putative regulators and co-regulated genes as derived from <i>B</i>. <i>contaminans</i> genomic and transcriptomic data.

<p>The <i>lxa</i> locus, putative regulators and co-regulated genes as derived from <i>B</i>. <i>contaminans</i> genomic and transcriptomic data.</p