ABSTRACT: It has been proposed that the gastro-intestinal tract environment containing high levels of neuroendocrine hormones is important for gut-derived P. aeruginosa infections. In this study, we report that the hormone norepinephrine increases P. aeruginosa PA14 growth, virulence factor production, invasion of HCT-8 epithelial cells, and swimming motility in a concentration-dependent manner. Transcriptome analysis of P. aeruginosa exposed to 500 µM, but not 50 µM, norepinephrine for 7 h showed that genes involved in the regulation of the virulence determinants pyocyanin, elastase, and Pseudomonas quinolone signal (PQS, 2-heptyl-3-hydroxy-4-quinolone) were up-regulated. The production of rhamnolipids, which are also important in P. aeruginosa infections, was significantly decreased on semi-solid surfaces, but not in planktonic cultures, upon exposure to norepinephrine. Swarming motility, a phenotype that is directly influenced by rhamnolipids, was also decreased upon 500 µM norepinephrine exposure. The increase in the transcriptional activation of lasR, but not that of rhlR, suggest that the effects of norepinephrine are mediated primarily through the las quorum sensing pathway. Together, our data strongly suggests that norepinephrine can play an important role in gut-derived infections by increasing the pathogenicity of P. aeruginosa PA14. For growth rate measurements, a single colony of PA14 was grown overnight in Luria Bertini (LB) broth, and re-inoculated in serum-RPMI medium in the presence of 50 µM and 500 µM NE to an initial turbidity of ~ 0.05 at 600 nm. The turbidity of the cultures at 600 nm was monitored every hour and growth rate of the exponentially growing cultures was calculated. Growth curves were obtained in triplicate using three independent cultures, and the statistical significance of specific growth rate was determined using the unpaired Student’s t-test. Total RNA isolation and microarray analysis PA14 was grown overnight in LB to turbidity at 600 nm of ~5.0 and diluted in 100 mL of serum-RPMI medium to an initial turbidity at 600 nm of 0.05. Different concentrations of NE (50 µM or 500 µM) were added and the cultures grown for 7 hours till late exponential phase (turbidity at 600 nm of ~0.5, 1.1 and 1.5 with 0 µM, 50 µM and 500 µM NE, respectively). Cell pellets were prepared and RNA was isolated as described previously (Ren et al. 2004a). The P. aeruginosa Genome Array (Affymetrix, P/N 510596) containing 5,500 of the 5,570 open reading frames of P. aeruginosa PA01, was used to analyze changes in the PA14 transcriptome. cDNA synthesis, fragmentation and hybridizations were as described previously (Gonzalez Barrios et al. 2006). Hybridization was performed for 16 h at 50°C, and the total cell intensity was scaled to an average value of 500. The probe array images were inspected for any image artifact. Background values, noise values and scaling factors of all the arrays were examined and were comparable. The intensities of polyadenosine RNA control were used to monitor the labelling process. For each binary microarray comparison of differential genes expression, if the gene with the larger transcription rate did not have a consistent transcription rate based on the 13 probe pairs (P-value less than 0.05), these genes were discarded. A gene was considered differentially expressed when the P-value for comparing two chips was lower than 0.05 (to assure that the change in gene expression was statistically significant and that false positives arise less than 5%), and the expression ratio (between cells treated with NE and control) was greater than 4.0 for the 50 µM NE array and 2.0 for 500 µM NE array (based on the standard deviation of fold-change values) (Ren et al. 2004b). Gene functions and locus tag numbers were obtained from the Pseudomonas Genome Database (http://www.pseudomonas.com/download.jsp).
