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Transcriptional Responses of Pseudomonas aeruginosa to Inhibition of Lipoprotein Transport by a Small Molecule Inhibitor.

ABSTRACT: Lipoprotein transport from the inner to the outer membrane, carried out by the Lol machinery, is essential for the biogenesis of the Gram-negative cell envelope and, consequently, for bacterial viability. Recently, small molecule inhibitors of the Lol system in Escherichia coli have been identified and shown to inhibit the growth of this organism by interfering with the function of the LolCDE complex. Analysis of the transcriptome of E. coli treated with one such molecule (compound 2) revealed that a number of envelope stress response pathways were induced in response to LolCDE inhibition. However, Pseudomonas aeruginosa is refractory to inhibition by the same small molecule, but we could demonstrate that E. coli lolCDE could be substituted for the P. aeruginosa orthologues, where it functions in the correct transport of Pseudomonas lipoproteins, and the cells are inhibited by the more potent compound 2A. In the present study, we took advantage of the functionality of E. coli LolCDE in P. aeruginosa and determined the P. aeruginosa transcriptional response to LolCDE inhibition by compound 2A. We identified key genes that responded to LolCDE inhibition and also demonstrated that the same genes appeared to be affected by genetic depletion of the native P. aeruginosa LolCDE proteins. Several of the major changes were in an upregulated cluster of genes that encode determinants of alginate biosynthesis and transport, and the levels of alginate were found to be increased either by treatment with the small molecule inhibitor or upon depletion of native LolCDE. Finally, we tested several antibiotics with differing mechanisms of action to identify potential specific reporter genes for the further development of compounds that would inhibit the native P. aeruginosa Lol system.IMPORTANCE A key set of lipoprotein transport components, LolCDE, were inhibited by both a small molecule as well as genetic downregulation of their expression. The data show a unique signature in the Pseudomonas aeruginosa transcriptome in response to perturbation of outer membrane biogenesis. In addition, we demonstrate a transcriptional response in key genes with marked specificity compared to several antibiotic classes with different mechanisms of action. As a result of this work, we identified genes that could be of potential use as biomarkers in a cell-based screen for novel antibiotic inhibitors of lipoprotein transport in P. aeruginosa.

SUBMITTER: Lorenz C

PROVIDER: S-EPMC7685553 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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Transcriptional Responses of Pseudomonas aeruginosa to Inhibition of Lipoprotein Transport by a Small Molecule Inhibitor.

Lorenz Christian C Dougherty Thomas J TJ Lory Stephen S

Journal of bacteriology 20201119 24

Lipoprotein transport from the inner to the outer membrane, carried out by the Lol machinery, is essential for the biogenesis of the Gram-negative cell envelope and, consequently, for bacterial viability. Recently, small molecule inhibitors of the Lol system in <i>Escherichia coli</i> have been identified and shown to inhibit the growth of this organism by interfering with the function of the LolCDE complex. Analysis of the transcriptome of <i>E. coli</i> treated with one such molecule (compound ...[more]

PMID: 32989085

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Project description:Many Pseudomonas aeruginosa infections are derived from residential, recreational, or surface water sources; thus, these environments represent an important preinfection niche. To better understand P. aeruginosa biology in these environments, we quantified transcriptional changes by microarray after exposure to diluted LB, diluted R2B, potable tap water, and freshwater from a eutrophic pond. Quantitative reverse transcription-PCR (qRT-PCR) confirmed the conservation of these responses in other water sources, and competition experiments were used to test the importance of three implicated metabolic pathways. The global transcriptional responses in potable water and freshwater showed strong induction of genes involved in metabolism of the head groups and acyl tails of phospholipids, as well as nucleotide metabolism, with commensurate decreased transcript expression of genes encoding their synthetic pathways. These data suggest that phospholipids and nucleotides are part of the nutritional milieu of these two environments. A unique response in municipal-delivered potable water was to the metals in the piping system, particularly copper. To identify potential nutrient sources used by P. aeruginosa in these environments, we used competition assays between the wild-type and deletion mutant strains in three pathways induced under these conditions. For phospholipid head-group metabolism, ethanolamine utilization (eutB) was important for competition in potable water, while choline oxidation (betBA) was important for competition in freshwater. Nucleotide utilization, particularly pyrimidine metabolism (dht), showed a trend toward importance in freshwater but was not statistically significant. These findings provide new insights into the P. aeruginosa response to potable water and freshwater and led to the identification of potentially important nutrient sources in these environments.IMPORTANCE Much of our knowledge about Pseudomonas aeruginosa comes from the infection niche, and much less is known about its lifestyle in the environment. P. aeruginosa is an adaptable bacterium capable of growing in many environments but is particularly common in potable water systems and freshwater. We used the transcriptional responses of P. aeruginosa to these environments to identify important nutrient sources specific to either of these two environments. Additionally, these environments could provide experimental situations to understand gene function for the large number of transcripts with unknown functions induced under these conditions.

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Transcriptional Responses of Pseudomonas aeruginosa to Inhibition of Lipoprotein Transport by a Small Molecule Inhibitor.

Publications

Transcriptional Responses of Pseudomonas aeruginosa to Inhibition of Lipoprotein Transport by a Small Molecule Inhibitor.

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