From the environment to the host: re-wiring of the transcriptome of Pseudomonas aeruginosa from 22°C to 37°C

Abstract

Pseudomonas aeruginosa is a highly versatile opportunistic pathogen capable of colonizing multiple ecological niches. This bacterium is responsible for a wide range of both acute and chronic infections in a variety of hosts. The success of this microorganism relies on its ability to adapt to environmental changes and re-program its regulatory and metabolic networks. The study of P. aeruginosa adaptation to temperature is crucial to understanding the pathogenesis upon infection of its mammalian host. We examined the effects of growth temperature on the transcriptome of the P. aeruginosa PAO1. Microarray analysis of PAO1 grown in Lysogeny broth at mid-exponential phase at 22°C and 37°C revealed that temperature changes are responsible for the differential transcriptional regulation of 6.4% of the genome. Major alterations were observed in bacterial metabolism, replication, and nutrient acquisition. Quorum-sensing and exoproteins secreted by type I, II, and III secretion systems, involved in the adaptation of P. aeruginosa to the mammalian host during infection, were up-regulated at 37°C compared to 22°C. Genes encoding arginine degradation enzymes were highly up-regulated at 22°C, together with the genes involved in the synthesis of pyoverdine. However, genes involved in pyochelin biosynthesis were up-regulated at 37°C. We observed that the changes in expression of P. aeruginosa siderophores correlated to an overall increase in Fe²⁺ extracellular concentration at 37°C and a peak in Fe³⁺ extracellular concentration at 22°C. This suggests a distinct change in iron acquisition strategies when the bacterium switches from the external environment to the host. Our work identifies global changes in bacterial metabolism and nutrient acquisition induced by growth at different temperatures. Overall, this study identifies factors that are regulated in genome-wide adaptation processes and discusses how this life-threatening pathogen responds to temperature.

Figure 1. Functional analysis of differential gene expression at 22°C and 37°C.

A. Classifications of the genes showing a significant differential expression in microarray analysis at 22°C or 37°C based on PseudoCAP function class assignments . The percentage of genes down-regulated in PAO1 at 22°C compared to 37°C is indicated in dark grey and the percentage of the genes up-regulated is indicated in light grey. The absolute number of genes differentially regulated under these conditions in each class is indicated on the bars. B. Subcellular localization of the products of the genes differentially regulated at 22°C and 37°C. Data represent the primary localization assignment of the gene products based on PAO1 genome annotation of the Pseudomonas Genome Database (www.pseudomonas.com) . Gene products of unknown location were removed from this analysis.

Figure 2. RT-qPCR analysis of temperature dysregulated genes.

Analysis of the expression of control genes by RT-qPCR to validate microarray analysis. Each gene was analyzed in triplicate and data are represented as fold changes at 22°C compared to 37°C. Data were analyzed using a sample two-tailed t-test. Genes significantly dysregulated are denoted with an asterisk.

Figure 3. Pathways of P. aeruginosa differentially regulated at 22°C and 37°C.

Graphic representation of the global changes in P. aeruginosa PAO1 metabolism detected by microarray analysis. The genes significantly dysregulated at 22°C and 37°C were mapped to metabolic pathways using the Kyoto Encyclopedia of Genes and Genomes database . Genes up-regulated at 22°C (green) and genes up-regulated at 37°C (red) are italicized next to the reaction catalyzed by their products. Green and red areas represent pathways globally up-regulated at 22°C and 37°C, respectively.

Figure 4. Iron availability, pyoverdin production and pH variation during growth at 22°C and 37°C.

Variations of Fe²⁺ (A) and Fe³⁺ (B) levels, pyoverdine production (C) and pH (D) measured during the growth of P. aeruginosa PAO1 in Lysogeny broth at 22°C (green) and 37°C (red). Time frames indicated in grey correspond to the times at which samples were taken for transcriptome analysis. Data collected from six independent cultures were analyzed using a paired two-tailed t-test. Data points that were significantly different are denoted with an asterisk.

Figure 5. Comparison of three different transcriptomes subjected to environmental temperatures.

Venn diagrams comparing datasets proceeding from three different studies: genes observed to be differentially regulated described in this study, genes differentially regulated at 28°C versus 37°C in P. aeruginosa PA14 as determined by RNA-seq and genes differentially regulated in ex vivo samples of burn wound P. aeruginosa infections compared to growth of these isolates at 37°C using Affymetrix microarrays . A. The total number of genes differentially regulated in each dataset is indicated on each arrow. B. The number of genes up-regulated in each condition is indicated in each field. Fold changes in gene expression for the dataset described in this study and the genes present in all datasets are indicated in Table S2 and Table S4, respectively.