Toxicogenomic response of Pseudomonas aeruginosa to ortho-phenylphenol

Abstract

Background: Pseudomonas aeruginosa (P. aeruginosa) is the most common opportunistic pathogen implicated in nosocomial infections and in chronic lung infections in cystic fibrosis patients. Ortho-phenylphenol (OPP) is an antimicrobial agent used as an active ingredient in several EPA registered disinfectants. Despite its widespread use, there is a paucity of information on its target molecular pathways and the cellular responses that it elucidates in bacteria in general and in P. aeruginosa in particular. An understanding of the OPP-driven gene regulation and cellular response it elicits will facilitate more effective utilization of this antimicrobial and possibly lead to the development of more effective disinfectant treatments.

Results: Herein, we performed a genome-wide transcriptome analysis of the cellular responses of P. aeruginosa exposed to 0.82 mM OPP for 20 and 60 minutes. Our data indicated that OPP upregulated the transcription of genes encoding ribosomal, virulence and membrane transport proteins after both treatment times. After 20 minutes of exposure to 0.82 mM OPP, genes involved in the exhibition of swarming motility and anaerobic respiration were upregulated. After 60 minutes of OPP treatment, the transcription of genes involved in amino acid and lipopolysaccharide biosynthesis were upregulated. Further, the transcription of the ribosome modulation factor (rmf) and an alternative sigma factor (rpoS) of RNA polymerase were downregulated after both treatment times.

Conclusion: Results from this study indicate that after 20 minutes of exposure to OPP, genes that have been linked to the exhibition of anaerobic respiration and swarming motility were upregulated. This study also suggests that the downregulation of the rmf and rpoS genes may be indicative of the mechanism by which OPP causes decreases in cell viability in P. aeruginosa. Consequently, a protective response involving the upregulation of translation leading to the increased synthesis of membrane related proteins and virulence proteins is possibly induced after both treatment times. In addition, cell wall modification may occur due to the increased synthesis of lipopolysaccharide after 60 minutes exposure to OPP. This gene expression profile can now be utilized for a better understanding of the target cellular pathways of OPP in P. aeruginosa and how this organism develops resistance to OPP.

Figure 1

Growth inhibition of P. aeruginosa treated with orthophenylphenol (OPP). Cell density was monitored as the OD₆₀₀ in ten minute intervals. The OPP concentrations were as follows: 0 mM control with DMSO (filled square), 0.58 mM (filled triangle), 0.82 mM (inverted filled triangle), 0.94 mM (filled diamond), 0.99 mM (filled circle), 1.05 mM (empty square), 1.18 mM (empty triangle). Each data point was derived as the average of three separate experiments and the error bars represent the standard deviation obtained.

Figure 2

Functional classification of statistically significant upregulated (filled bars) and downregulated (empty bars) genes after 20 minutes and 60 minutes exposure to 0.82 mM OPP. The numbers in parentheses indicate the total number of genes for each functional class in both groups (a total of 509 genes).

Figure 3

Classification of differentially regulated 372 genes into six groups based on their transcription directions after 20 and 60 minutes exposure to OPP. Note that genes belonging to the functional class "hypothetical, unclassified, unknown" (137 genes) are not represented in this figure. Filled bars indicate upregulation either after one or both treatment times. Empty bars indicate downregulation either after one or both treatment times. Group I is made up of genes upregulated after both exposure times. Group II contains genes upregulated at 20 minutes, with no significant changes after 60 minute exposure. Group III consists of genes downregulated after 20 minutes, with no significant changes upon 60 minutes of treatment. Group IV is made up of genes that were upregulated in response to 60 minutes of treatment. Group V is made up of genes that were downregulated upon 60 minutes of treatment. Group VI is made up of genes that were downregulated upon both exposure times.