While AES-1M appeared non-mucoid on HBA and MHA (see Results), there was significant upregulation of most alginate genes in ASMDM, suggesting that this mucus-like medium triggers their enhanced expression. Alginate is considered a chronic infection virulence factor [12] and is involved in biofilm production and resistance to leukocyte killing [37]. The exception was a Ponatinib TNKS1 significant downregulation of algC in AES-1M. algC is transcribed separately from the other alginate genes, which are in a single operon under the control of algD [38] and has another function as a requirement for lipopolysaccharide (LPS) biosynthesis [39]. Thus downregulation of algC would suggest defects in production of A band LPS in these isolates. Dihydroorotase (pyrC-upregulated 12.

1fold) catalyzes the third of six enzymatic steps in the biosynthesis of uracil monophosphate (UMP) from glutamine and aspartate precursors while uridylate kinase pyrH (upregulated 6.4fold) catalyses the conversion of UMP to UDP. Inhibition of the uracil biosynthetic pathway has been demonstrated to repress biofilms and all three QS pathways (Rhl, Las and Pqs) in P. aeruginosa [40] while Staphylococcus aureus pyrC mutants showed a fivefold reduction in virulence in a BALB/c mouse competitive systemic infection model and threefold in a non-competitive systemic infection model [41]. Of the genes with probable virulence roles upregulated in AES-1M, pel genes are involved in P. aeruginosa biofilm formation [42] and enhance a strain’s capacity to persist. Expression of pel genes by P. aeruginosa has also been shown to competitively disrupt S.

aureus biofilms [43]. The phospholipase C precursor plcN is important in extracellular virulence as it degrades phosphatidylcholine, a constituent of lung surfactant [44]. Cardiolipin synthase (cls) plays an important role in membrane fluidity and P. putida cls mutants have shown increased sensitivity to antibiotics [45]. Other genes upregulated in AES-1M included the putative chemotactic transducer pctA (AES_5777 8.2fold) and a P. aeruginosa pathogenicity island-1 (PAPI-1) gene (PA14-59980 5.6fold) also found in UCBPP-PA14. PctA is essential for taxis towards sugars, organic acids and L-amino-acids and in the acquisition of nitrogen [46]. The presence of PAPI-1 pathogenicity island genes in AES1 and their upregulation is of great interest due to the role of this island in UCBPP-PA14 virulence [47] and its mode of spread to CF strains [48].

Amongst genes downregulated in AES-1M, hmgA and thxA stand out. Studies have shown loss of hmgA expression induces pyomelanin production, which in turn leads to increased persistence [49] and we have detected downregulation of HmgA at the protein level in AES-1M. Thioredoxin has been shown to be induced in P. aeruginosa PAO1 biofilms under strict anaerobic conditions [50], yet in our studies Anacetrapib P.