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Dictyostelium transcriptional responses to Pseudomonas aeruginosa: common and specific effects from PAO1 and PA14 strains.

ABSTRACT: Pseudomonas aeruginosa is one of the most relevant human opportunistic bacterial pathogens. Two strains (PAO1 and PA14) have been mainly used as models for studying virulence of P. aeruginosa. The strain PA14 is more virulent than PAO1 in a wide range of hosts including insects, nematodes and plants. Whereas some of the differences might be attributable to concerted action of determinants encoded in pathogenicity islands present in the genome of PA14, a global analysis of the differential host responses to these P. aeruginosa strains has not been addressed. Little is known about the host response to infection with P. aeruginosa and whether or not the global host transcription is being affected as a defense mechanism or altered in the benefit of the pathogen. Since the social amoeba Dictyostelium discoideum is a suitable host to study virulence of P. aeruginosa and other pathogens, we used available genomic tools in this model system to study the transcriptional host response to P. aeruginosa infection.We have compared the virulence of the P. aeruginosa PAO1 and PA14 using D. discoideum and studied the transcriptional response of the amoeba upon infection. Our results showed that PA14 is more virulent in Dictyostelium than PA01using different plating assays. For studying the differential response of the host to infection by these model strains, D. discoideum cells were exposed to either P. aeruginosa PAO1 or P. aeruginosa PA14 (mixed with an excess of the non-pathogenic bacterium Klebsiella aerogenes as food supply) and after 4 hours, cellular RNA extracted. A three-way comparison was made using whole-genome D. discoideum microarrays between RNA samples from cells treated with the two different strains and control cells exposed only to K. aerogenes. The transcriptomic analyses have shown the existence of common and specific responses to infection. The expression of 364 genes changed in a similar way upon infection with one or another strain, whereas 169 genes were differentially regulated depending on whether the infecting strain was either P. aeruginosa PAO1 or PA14. Effects on metabolism, signalling, stress response and cell cycle can be inferred from the genes affected.Our results show that pathogenic Pseudomonas strains invoke both a common transcriptional response from Dictyostelium and a strain specific one, indicating that the infective process of bacterial pathogens can be strain-specific and is more complex than previously thought.

SUBMITTER: Carilla-Latorre S

PROVIDER: S-EPMC2474670 | biostudies-literature | 2008 Jun

REPOSITORIES: biostudies-literature

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Dictyostelium transcriptional responses to Pseudomonas aeruginosa: common and specific effects from PAO1 and PA14 strains.

Carilla-Latorre Sergio S Calvo-Garrido Javier J Bloomfield Gareth G Skelton Jason J Kay Robert R RR Ivens Alasdair A Martinez José L JL Escalante Ricardo R

BMC microbiology 20080630

<h4>Background</h4>Pseudomonas aeruginosa is one of the most relevant human opportunistic bacterial pathogens. Two strains (PAO1 and PA14) have been mainly used as models for studying virulence of P. aeruginosa. The strain PA14 is more virulent than PAO1 in a wide range of hosts including insects, nematodes and plants. Whereas some of the differences might be attributable to concerted action of determinants encoded in pathogenicity islands present in the genome of PA14, a global analysis of the ...[more]

PMID: 18590548

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Project description:Pseudomonas aeruginosa is an opportunistic pathogen that causes difficult-to-treat infections. Two well-studied divergent P. aeruginosa strain types, PAO1 and PA14, have significant genomic heterogeneity, including diverse accessory genes present in only some strains. Genome content comparisons find core genes that are conserved across both PAO1 and PA14 strains and accessory genes that are present in only a subset of PAO1 and PA14 strains. Here, we use recently assembled transcriptome compendia of publicly available P. aeruginosa RNA sequencing (RNA-seq) samples to create two smaller compendia consisting of only strain PAO1 or strain PA14 samples with each aligned to their cognate reference genome. We confirmed strain annotations and identified other samples for inclusion by assessing each sample's median expression of PAO1-only or PA14-only accessory genes. We then compared the patterns of core gene expression in each strain. To do so, we developed a method by which we analyzed genes in terms of which genes showed similar expression patterns across strain types. We found that some core genes had consistent correlated expression patterns across both compendia, while others were less stable in an interstrain comparison. For each accessory gene, we also determined core genes with correlated expression patterns. We found that stable core genes had fewer coexpressed neighbors that were accessory genes. Overall, this approach for analyzing expression patterns across strain types can be extended to other groups of genes, like phage genes, or applied for analyzing patterns beyond groups of strains, such as samples with different traits, to reveal a deeper understanding of regulation. IMPORTANCE Pseudomonas aeruginosa is a ubiquitous pathogen. There is much diversity among P. aeruginosa strains, including two divergent but well-studied strains, PAO1 and PA14. Understanding how these different strain-level traits manifest is important for identifying targets that regulate different traits of interest. With the availability of thousands of PAO1 and PA14 samples, we created two strain-specific RNA-seq compendia where each one contains hundreds of samples from PAO1 or PA14 strains and used them to compare the expression patterns of core genes that are conserved in both strain types and to determine which core genes have expression patterns that are similar to those of accessory genes that are unique to one strain or the other using an approach that we developed. We found a subset of core genes with different transcriptional patterns across PAO1 and PA14 strains and identified those core genes with expression patterns similar to those of strain-specific accessory genes.

Project description:Two clinical isolates of the opportunist pathogen Pseudomonas aeruginosa named PAO1 and PA14 are commonly studied in research laboratories. Despite the isolates being closely related, PA14 exhibits increased virulence compared to that of PAO1 in various models. To determine which players are responsible for the hypervirulence phenotype of the PA14 strain, we elected a transcriptomic approach through RNA sequencing. We found 2,029 genes that are differentially expressed between the two strains, including several genes that are involved with or regulated by quorum sensing (QS), known to control most of the virulence factors in P. aeruginosa Among them, we chose to focus our study on QslA, an antiactivator of QS whose expression was barely detectable in the PA14 strain according our data. We hypothesized that lack of expression of qslA in PA14 could be responsible for higher QS expression in the PA14 strain, possibly explaining its hypervirulence phenotype. After confirming that QslA protein was highly produced in PAO1 but not in the PA14 strain, we obtained evidence showing that a PAO1 deletion strain of qslA has faster QS gene expression kinetics than PA14. Moreover, known virulence factors activated by QS, such as (i) pyocyanin production, (ii) H2-T6SS (type VI secretion system) gene expression, and (iii) Xcp-T2SS (type II secretion system) machinery production and secretion, were all lower in PAO1 than in PA14, due to higher qslA expression. However, biofilm formation and cytotoxicity toward macrophages, although increased in PA14 compared to PAO1, were independent of QslA control. Together, our findings implicated differential qslA expression as a major determinant of virulence factor expression in P. aeruginosa strains PAO1 and PA14.IMPORTANCEPseudomonas aeruginosa is an opportunistic pathogen responsible for acute nosocomial infections and chronic pulmonary infections. P. aeruginosa strain PA14 is known to be hypervirulent in different hosts. Despite several studies in the field, the underlining molecular mechanisms sustaining this phenotype remain enigmatic. Here we provide evidence that the PA14 strain has faster quorum sensing (QS) kinetics than the PAO1 strain, due to the lack of QslA expression, an antiactivator of QS. QS is a major regulator of virulence factors in P. aeruginosa; therefore, we propose that the hypervirulent phenotype of the PA14 strain is, at least partially, due to the lack of QslA expression. This mechanism could be of great importance, as it could be conserved among other P. aeruginosa isolates.

Project description:Plasticity of Pseudomonas aeruginosa chromosomes is mainly driven by an extended accessory genome that is shaped by insertion and deletion events. Further modification of the genome composition can be induced by chromosomal inversion events which lead to relocation of genes in the affected genomic DNA segments, modify the otherwise highly conserved core genome synteny and could even alter the location of the replication terminus. Although the genome of the first sequenced strain, PAO1, displayed such a large genomic inversion, knowledge on such recombination events in the P. aeruginosa population is limited. Several large inversions had been discovered in the late 1990s in cystic fibrosis isolates of the major clonal lineage C by physical genome mapping, and subsequent work on these examples led to the characterization of the DNA at the recombination breakpoints and a presumed recombination mechanism. Since then, the topic was barely addressed in spite of the compilation of thousands of P. aeruginosa genome sequences that are deposited in databases. Due to the use of second-generation sequencing, genome contig assembly had usually followed synteny blueprints provided by the existing reference genome sequences. Inversion detection was not feasible by these approaches, as the respective read lengths did not allow reliable resolution of sequence repeats that are typically found at the borders of inverted segments. In this study, we applied PacBio and MinION long-read sequencing to isolates of the mentioned clone C collection. Confirmation of inversions predicted from the physical mapping data demonstrated that unbiased sequence assembly of such read datasets allows the detection of genomic inversions and the resolution of the recombination breakpoint regions. Additional long-read sequencing of representatives of the other major clonal lineage, PA14, revealed large inversions in several isolates, from cystic fibrosis origin as well as from other sources. These findings indicated that inversion events are not restricted to strains from chronic infection background, but could be widespread in the P. aeruginosa population and contribute to genome plasticity. Moreover, the monitored examples emphasized the role of small mobile DNA units, such as IS elements or transposons, and accessory DNA elements in the inversion-related recombination processes.

Dataset Information

Dictyostelium transcriptional responses to Pseudomonas aeruginosa: common and specific effects from PAO1 and PA14 strains.

Publications

Dictyostelium transcriptional responses to Pseudomonas aeruginosa: common and specific effects from PAO1 and PA14 strains.

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