Thus AES-1R may be better able to cope with hydrogen peroxide-mediated oxidative stress by induction of the thioredoxin pathway, while chronic AES-1M has adapted by not inducing the host cell-mediated response. Iron uptake is critical in the iron-starved ASMDM and CF sputum [24], thus the synthesis of the siderophores such as www.selleckchem.com/products/Nilotinib.html pyoverdine and pyochelin would be expected to be high in both acute and chronic isolates. Interestingly, in the pyoverdine biosynthesis operon pvcABCD, pvcB was significantly upregulated (3.3fold) while pvcC was significantly downregulated (?7.5fold). A recent study suggests pvcABCD is required not for pyoverdine production but for dihydroxycumarin expression [51]. pvcAB makes an intermediate compound which is oxidized to the catechol form by pvcCD.
It is possible that the final product is not required by AES-1M, leading to either mutation or downregulation of pvcC. Hoboth et al. [23] compared an early mutator with an end-stage non-mutator phenotype from the same patient, seeing a downregulation of virulence-related genes including T3SS, chemotaxis, QS and flagellin genes. While that study did not use isogenic strains, there are some findings in common with our study. Several chemotaxis transducers (wspD, AES_2270 and AES_4899), and T3SS genes (pcrV, pscC and pscI) were downregulated in AES-1M, but most genes in these groups were not differentially expressed. Mutation in any of the chemotaxis genes (wspABCDEF) leads to loss of motility and auto-aggregation in P. aeruginosa [52].
Therefore the downregulation of wspD suggests decreased motility and swarming, possibly leading to less expansion and spread of biofilm microcolonies compared to AES-1R. Our phenotypic data (not shown) demonstrate that AES-1M does swarm less than AES-1R, though not significantly (Pearson’s ��2 test: p=0.062) however it swims and twitches significantly more (p=0.29 and p=0.015, respectively). With respect to expression of metabolic genes, only one TCA cycle metabolism gene (isocitrate dehydrogenase-idh) downregulated in the end-stage mutator [23], was also downregulated in AES-1M, with the remainder not differentially expressed at p<0.05. Possible reasons for the different metabolic profiles include growth media and conditions, and AES-1-strain specific characteristics.
Our study used ASMDM, which resembles the composition of lung sputum, and selected the entire biofilm (anaerobic and aerobic growth) for analysis, compared to microaerobic growth in Luria-Bertani broth used in the Hoboth study. Conclusions The sequencing of the AES-1 isolate AES-1R and the use of a broad-capture array has for the first time enabled detection Cilengitide of the expression of AES-1 genes not found in the reference strain PAO1. Furthermore, the use of ASMDM has provided a profile of differential expression of genes under sputum-like conditions.