Unknown,Transcriptomics,Genomics,Proteomics

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Gene expression analysis of wild type Pectobacteriumwasabie SCC3193 wildtype compared to a SCC3193 inactivation mutant of RsmA, inactivation mutant of ExpA and an ExpA RsmA double inactivation mutant.

ABSTRACT: The phytopathogen Pectobacterium wasabiae SCC3193 is a model for research on virulence regulation in soft rot pathogens. Two proteins acting as global regulators of the expression on genes involved in virulence in SCC3193 are ExpA, a DNA binding transcriptional regulator, and RsmA, an RNA binding post transcriptional regulator. Studies of related enterobacterial phytopathogens suggest that homologues of ExpA (GacA) affect gene expression partly by modulating RsmA-like protein activity. However, the exact extent of this regulon overlap has not been mapped. To elucidate the role of RsmA in ExpA controlled gene expression goes through RsmA in P. wasabiae we performed a gene expression microarray analysis of the wild type strain, an expA mutant, an rsmA mutant, and an expA rsmA double mutant. The microarray-based data of various virulence related genes was confirmed through quantitative RT-PCR. Subsequently, assays were made to link observed transcriptomic differences to changes in growth rate, PCWDE production, and virulence in planta between the four strains. We could conclude that the majority of ExpA regulation seems routed via RsmA modulation. However we could identify genes involved in electron transport, oligogalacturonide transport and metabolism, which ExpA seems to influence independently of RsmA, directly or indirectly. Gene expression analysis from P. wasabie SCC3193 wildtype, SCC3193 rsmA mutant, SCC3193 expA mutant and SCC3193 expA rsmA double mutant was performed on RNA samples extraxcted from the strains at late-logarithmic (late-log) and early stationary (stat) growth phase. Strains were grown in liquid hrp inducing minimal medium cultures supplemented with 0.4% polygalacturonic acid at 200 rpm shaking and 28 degrees celsius. Three biological replicates were used from each strain at each growth phase point.

ORGANISM(S): Pectobacterium wasabiae

SUBMITTER: Martin Broberg

PROVIDER: E-GEOD-47545 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Posttranscriptional regulator RsmA is part of a regulatory system that controls production of plant cell wall degrading enzymes (PCWDEs), motility and other virulence factors among the Pectobacterium spp of plant pathogens. We constructed the knockout mutation of rsmA gene in Pectobacterium wasabiae strain SCC3193. The rsmA- strain has three times lower growth rate but increased virulence when compared to wild type. To explain this phenotype we compared wild type and rsmA- mRNA levels using genome wide microarray. We found that the synthesis of all known virulence factors and flagella is upregulated, while cell division and peptidoglycan synthesis is down regulated in rsmA- strain. The TCA cycle is directed fully to energy production, indicating low energetic status of the cell that is a probable reason for the low growth rate of the mutant. Growth experiments showed that the enzymatic activities of extracellular virulence factors polygalacturonase and pectate lyase are very high throughout the growth curve even without induction by plant component. Wild type cells are rod shaped in liquid media, but elongated and hyperflagellated in swarm plate containing potato tuber extract and in potato tuber. The rsmA- strain exhibits the same conditional phenotype, but differentiation into swarmer cells is faster and cell elongation more pronounced. Virulence experiments in potato tuber showed that although rsmA- strain causes bigger tissue maceration, its ability to compete with the natural bacterial community in the host is decreased. We conclude that the lack of RsmA switches the cells to infective phenotype characterized by expression of virulence factors and swarming. The lack of control over these energy costly processes is probably the reason for decreased growth rate and fitness. Gene expression analysis from P. wasabie SCC3193 wildtype and SCC3193 rsmA mutant was performed on RNA samples extraxcted from the rsmA mutant (at 40 and 48 hours) and wildtype (at 24 hours). The strain were grown on 1.5% agar M9 minimal media plates supplemented with 10% potato extract. Three biological replicates were used from each strain at each time point.

Project description:Global expression profiling of airway epithelial cells infected with Pseudomonas aeruginosa and the rsmA mutant. Recent work in our laboratory investigated the impact of RsmA on a number of airway epithelial cell phenotypes including actin depolymerization, cytotoxicity and invasion. These cellular phenotypes were influenced by the positive effect of RsmA on the expression of the TTSS in P. aeruginosa (Mulcahy et al. 2006. Infect. Immun.). Pseudomonas aeruginosa is an important opportunistic pathogen which is capable of causing both acute and chronic infections in immunocompromised patients. Successful adaptation of the bacterium to its host environment relies on the ability of the organism to tightly regulate gene expression. RsmA, a small RNA-binding protein, controls the expression of a large number of virulence-related genes in P. aeruginosa, including those encoding the type III secretion system and associated effector proteins, with important consequences for epithelial cell morphology and cytotoxicity. In order to examine the influence of RsmA-regulated functions in the pathogen on gene expression in the host, we compared global expression profiles of airway epithelial cells in response to infection with P. aeruginosa PAO1 and an rsmA mutant. The RsmA-dependent response of host cells was characterized by significant changes in the global transcriptional pattern, including the increased expression of two Kruppel-like factors, KLF2 and KLF6. This increased expression was mediated by specific type III effector proteins. ExoS was required for the enhanced expression of KLF2, whereas both ExoS and ExoY were required for the enhanced expression of KLF6. Neither ExoT nor ExoU influenced the expression of the transcription factors. Additionally, the increased gene expression of KLF2 and KLF6 was associated with ExoS-mediated cytotoxicity. Therefore, this study identifies for the first time the human transcription factors KLF2 and KLF6 as targets of the P. aeruginosa type III exoenzymes S and Y, with potential importance in host cell death. Keywords: Expression profiling, Pseudomonas aeruginosa, microbial infection, airway epithelial cells, cystic fibrosis, exoenzymes, ExoS, ExoT, ExoU, ExoY, Kruppel-like factors, KLF2, KLF6

Dataset Information

Gene expression analysis of wild type Pectobacteriumwasabie SCC3193 wildtype compared to a SCC3193 inactivation mutant of RsmA, inactivation mutant of ExpA and an ExpA RsmA double inactivation mutant.

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