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Dual-species proteomic analysis of Pseudomonas aeruginosa and human airway epithelial cells during bacterial adhesion and intracellular infection

ABSTRACT: Pseudomonas aeruginosa can adhere to and be internalized by airway epithelial cells, but the host and bacterial responses during bacterial adhesion and intracellular infection remain incompletely understood. In this study, we characterized the proteomes of P. aeruginosa PAO1 and CuFi-5 human airway epithelial cells carrying the cystic fibrosis-associated CFTR ΔF508 mutation. Experimental groups included uninfected CuFi-5 cells, planktonic PAO1, and CuFi-5 cells with adhered or intracellular PAO1. The experimental design included low- and high-calcium conditions. Proteomic profiles were determined using mass spectrometry-based label-free quantitative proteomics.

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ORGANISM(S): Homo Sapiens (human) Pseudomonas Aeruginosa

TISSUE(S): Lung, Epithelial Cell

SUBMITTER: Yingxin Zhang

LAB HEAD: Erika Lutter

PROVIDER: PXD081656 | Pride | 2026-08-07

REPOSITORIES: Pride

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Project description:Virulence factor production and the development of biofilms in Pseudomonas aeruginosa have been shown to be regulated by two hierarchically organised quorum sensing (QS) systems via small acyl-homoserine lactone (AHL) signal molecules. Recently, a third bacterial signal molecule, the Pseudomonas quinolone signal (PQS), has been identified, which positively regulates a subset of genes dependent on the QS systems. To further dissect the various independent regulation levels for many QS induced genes and to evaluate the impact of PQS on the QS circuitry, we performed a transcriptome analysis of PAO1 cultures supplemented with PQS. The global transcriptional profile in response to PQS revealed a marked up-regulation of genes belonging to the tightly interdependent functional groups of iron acquisition and oxidative stress response. Remarkably, not only most of the differentially regulated genes but also the induction of a lacZ transcriptional fusion of rhlR could be traced back to a iron chelating effect of PQS. Nevertheless, although iron deficiency per se induced rhlR, there seems to be PQS specific effects that are independent of the PQS effect on P. aeruginosa iron homeostasis Experiment Overall Design: For RNA extraction bacteria were harvested at early logarithmic, late logarithmic and stationary phase of growth. Three independent cultures of PQS-treated and untreated PAO1 each were pooled and the RNA was immediately stabilized with RNAprotect Bacteria Reagent (Qiagen, Valencia, CA). RNA was isolated using the RNeasy kit (Qiagen), treated with DNaseI (Roche) for 30 min at 37M-BM-0C and re-purified with the RNeasy spin column. The subsequent steps of cDNA generation and Biotin-ddUTP terminal labelling were performed as described in the manufacturerM-bM-^@M-^Ys instructions for the P. aeruginosa GeneChipM-CM-^R. Fragmented and labelled cDNA was hybridised to a GeneChip at 50 M-BM-0C for 16 h. The washing steps, staining and scanning of the microarrays were performed using the Affymetrix GeneChip system. Data analysis was performed using the Affymetrix Microarray Suite Software 5.0 with Affymetrix default parameters. As expression analysis was performed in duplicate, a total of two GeneChips per culture condition was scanned at 570 nm, 3 M-BM-5m resolution in an Affymetrix GeneChip scanner. The signals were multiplied by a scaling factor to make the average signal for all the arrays equivalent. The data were imported into a Microsoft Access database capable of searching for genes, which were found in all four pairings defined by the Affymetrix Microarray Suite Software as having significant changes in their signal intensities. Data were combined with the latest annotation from the website of the P. aeruginosa PAO1 sequence and the community annotation project provided at www.pseudomonas.com.

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Dual-species proteomic analysis of Pseudomonas aeruginosa and human airway epithelial cells during bacterial adhesion and intracellular infection

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