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

Carilla-Latorre S, Calvo-Garrido J, Bloomfield G, Skelton J, Kay RR, Ivens A, Martinez JL, Escalante R - BMC Microbiol. (2008)

Bottom Line: Our results showed that PA14 is more virulent in Dictyostelium than PA01using different plating assays.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.

Affiliation: Instituto de Investigaciones Biomédicas Alberto Sols, Universidad Autónoma de Madrid-Consejo Superior de Investigaciones Científicas, Madrid, Spain. scarilla@iib.uam.es

ABSTRACT

Background: 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.

Results: 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.

Conclusion: 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.

PA14 is more virulent than PAO1 in PDF-agar plating assay. D. discoideum cells were cultivated in non-nutrient agar on a lawn of Klebsiella and Pseudomonas (PAO1 and PA14) at the indicated proportion. Under these conditions PA14 maintain a high virulence as seen by the strong inhibition of D. discoideum growth. Heat inactivated PA14 is used as a control.
© Copyright Policy - open-access

Figure 2: PA14 is more virulent than PAO1 in PDF-agar plating assay. D. discoideum cells were cultivated in non-nutrient agar on a lawn of Klebsiella and Pseudomonas (PAO1 and PA14) at the indicated proportion. Under these conditions PA14 maintain a high virulence as seen by the strong inhibition of D. discoideum growth. Heat inactivated PA14 is used as a control.

Mentions: PAO1 and PA14 are two clinical isolates of P. aeruginosa frequently used as model strains to analyze the virulence of this bacterial pathogen. Since they behave differently in some aspects dealing with the expression of virulence determinants, we wanted to compare the differential response of the host to these strains. For this purpose, we made use of D. discoideum as a model for virulence. As a first step a plating assay of virulence was set up. Figure 1 shows a representative experiment of three independent assays in which D. discoideum cells were grown in association with bacteria on nutrient SM plates. Klebsiella aerogenes, a non -pathogenic bacteria, was used as an appropriate food supply and P. aeruginosa mixed at the indicated proportions. An effect in the size of the clearing plaques could already be seen when only 3.5% of P. aeruginosa cells were mixed with 96.5% of K. aerogenes cells and this effect was even clearer using 17% of P. aeruginosa cells. When the behavior of the strains was analyzed in more detail, it was found that PAO1 is reproducibly more permissive than PA14 as observed by the higher growth of D. discoideum on PAO1. The differences in the area of the cleared bacterial lawn between PAO1 and PA14 were measured for the condition corresponding to the 3.5 % mixture. The average area and the standard deviation were 1.65 ± 1.2 mm2 for PAO1 and 0.11 ± 0.07 mm2 for PA14 (the number of clear plaques measured in each condition was 50). The significance of differences between groups as determined by Student's t-test was p < 10-8. To further confirm these results a different plating assay was performed on non-nutrient agar. PAO1, PA14 and K. aerogenes were previously grown in LB overnight, washed out of the media by centrifugation and deposited with D. discoideum cells in agar plates at the indicated proportions. Under these conditions the difference in the virulence between PAO1 and PA14 was even more evident as shown in a representative experiment in Figure 2. Interestingly PAO1 is permissive to D. discoideum growth under these non-nutrient conditions. However, PA14 still shows a strong virulence against D. discoideum. All together these results suggest that PA14 is more virulent than PAO1 in the D. discoideum model of virulence.

View Similar Images In: Results  - Collection
View Article: PubMed Central - HTML -  PubMed
Show All Figures - Show MeSH
getmorefigures.php?pmc=2474670&rFormat=json&query=null&req=5
Dictyostelium transcriptional responses to Pseudomonas aeruginosa: common and specific effects from PAO1 and PA14 strains

Carilla-Latorre S, Calvo-Garrido J, Bloomfield G, Skelton J, Kay RR, Ivens A, Martinez JL, Escalante R - BMC Microbiol. (2008)

PA14 is more virulent than PAO1 in PDF-agar plating assay. D. discoideum cells were cultivated in non-nutrient agar on a lawn of Klebsiella and Pseudomonas (PAO1 and PA14) at the indicated proportion. Under these conditions PA14 maintain a high virulence as seen by the strong inhibition of D. discoideum growth. Heat inactivated PA14 is used as a control.
© Copyright Policy - open-access
Figure 2: PA14 is more virulent than PAO1 in PDF-agar plating assay. D. discoideum cells were cultivated in non-nutrient agar on a lawn of Klebsiella and Pseudomonas (PAO1 and PA14) at the indicated proportion. Under these conditions PA14 maintain a high virulence as seen by the strong inhibition of D. discoideum growth. Heat inactivated PA14 is used as a control.
Mentions: PAO1 and PA14 are two clinical isolates of P. aeruginosa frequently used as model strains to analyze the virulence of this bacterial pathogen. Since they behave differently in some aspects dealing with the expression of virulence determinants, we wanted to compare the differential response of the host to these strains. For this purpose, we made use of D. discoideum as a model for virulence. As a first step a plating assay of virulence was set up. Figure 1 shows a representative experiment of three independent assays in which D. discoideum cells were grown in association with bacteria on nutrient SM plates. Klebsiella aerogenes, a non -pathogenic bacteria, was used as an appropriate food supply and P. aeruginosa mixed at the indicated proportions. An effect in the size of the clearing plaques could already be seen when only 3.5% of P. aeruginosa cells were mixed with 96.5% of K. aerogenes cells and this effect was even clearer using 17% of P. aeruginosa cells. When the behavior of the strains was analyzed in more detail, it was found that PAO1 is reproducibly more permissive than PA14 as observed by the higher growth of D. discoideum on PAO1. The differences in the area of the cleared bacterial lawn between PAO1 and PA14 were measured for the condition corresponding to the 3.5 % mixture. The average area and the standard deviation were 1.65 ± 1.2 mm2 for PAO1 and 0.11 ± 0.07 mm2 for PA14 (the number of clear plaques measured in each condition was 50). The significance of differences between groups as determined by Student's t-test was p < 10-8. To further confirm these results a different plating assay was performed on non-nutrient agar. PAO1, PA14 and K. aerogenes were previously grown in LB overnight, washed out of the media by centrifugation and deposited with D. discoideum cells in agar plates at the indicated proportions. Under these conditions the difference in the virulence between PAO1 and PA14 was even more evident as shown in a representative experiment in Figure 2. Interestingly PAO1 is permissive to D. discoideum growth under these non-nutrient conditions. However, PA14 still shows a strong virulence against D. discoideum. All together these results suggest that PA14 is more virulent than PAO1 in the D. discoideum model of virulence.

Bottom Line: Our results showed that PA14 is more virulent in Dictyostelium than PA01using different plating assays.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.

Affiliation: Instituto de Investigaciones Biomédicas Alberto Sols, Universidad Autónoma de Madrid-Consejo Superior de Investigaciones Científicas, Madrid, Spain. scarilla@iib.uam.es

ABSTRACT

Background: 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.

Results: 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.

Conclusion: 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.

View Similar Images In: Results  - Collection
View Article: PubMed Central - HTML -  PubMed
Show All Figures - Show MeSH
getmorefigures.php?pmc=2474670&rFormat=json&query=null&req=5