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Extracellular ATP inhibits twitching motility-mediated biofilm expansion by Pseudomonas aeruginosa.

Nolan LM, Cavaliere R, Turnbull L, Whitchurch CB - BMC Microbiol. (2015)

Bottom Line: We have determined that eATP does not inhibit expression of the tfp major subunit, PilA, but reduces the levels of surface assembled tfp.Endogenous eATP produced by P. aeruginosa serves as a signalling molecule to co-ordinate complex multicellular behaviours of this pathogen.Our findings have significant implications in the development and pathogenesis of P. aeruginosa biofilm infections.

View Article: PubMed Central - PubMed

Affiliation: The ithree institute, University of Technology Sydney, PO Box 123, Broadway, NSW, 2007, Australia. laura.nolan25@gmail.com.

ABSTRACT

Background: Pseudomonas aeruginosa is an opportunistic pathogen that exploits damaged epithelia to cause infection. Type IV pili (tfp) are polarly located filamentous structures which are the major adhesins for attachment of P. aeruginosa to epithelial cells. The extension and retraction of tfp powers a mode of surface translocation termed twitching motility that is involved in biofilm development and also mediates the active expansion of biofilms across surfaces. Extracellular adenosine triphosphate (eATP) is a key "danger" signalling molecule that is released by damaged epithelial cells to alert the immune system to the potential presence of pathogens. As P. aeruginosa has a propensity for infecting damaged epithelial tissues we have explored the influence of eATP on tfp biogenesis and twitching motility-mediated biofilm expansion by P. aeruginosa.

Results: In this study we have found that eATP inhibits P. aeruginosa twitching motility-mediated expansion of interstitial biofilms at levels that are not inhibitory to growth. We have determined that eATP does not inhibit expression of the tfp major subunit, PilA, but reduces the levels of surface assembled tfp. We have also determined that the active twitching zone of expanding P. aeruginosa interstitial biofilms contain large quantities of eATP which may serve as a signalling molecule to co-ordinate cell movements in the expanding biofilm. The inhibition of twitching motility-mediated interstitial biofilm expansion requires eATP hydrolysis and does not appear to be mediated by the Chp chemosensory system.

Conclusions: Endogenous eATP produced by P. aeruginosa serves as a signalling molecule to co-ordinate complex multicellular behaviours of this pathogen. Given the propensity for P. aeruginosa to infect damaged epithelial tissues, our observations suggest that eATP released by damaged cells may provide a cue to reduce twitching motility of P. aeruginosa in order to establish infection at the site of damage. Furthermore, eATP produced by P. aeruginosa biofilms and by damaged epithelial cells may play a role in P. aeruginosa pathogenesis by inducing inflammatory damage and fibrosis. Our findings have significant implications in the development and pathogenesis of P. aeruginosa biofilm infections.

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Examination of twitching motility-mediated biofilm expansion in response to eATP by wildtypeP. aeruginosastrains and mutants ofcyaA,cyaB,cpdAor the Chp chemosensory system. The twitching motility response of P. aeruginosa strains to eATP at the interstitial space between a solidified nutrient media-coated microscope slide and a coverslip after incubation for 15 h at 37°C. (A) Phase-contrast microscopy of interstitial biofilms of wildtype P. aeruginosa strains PAK, PAO1, PA14 and PA103 in the presence of a H2O-saturated disc (upper images) or ATP-saturated disc (lower images). Scalebar is 50 μm and the arrows indicate the direction of expansion towards the disc. Images are representative of three independent experiments. (B) Surface areas of interstitial biofilms formed in the absence of eATP (black bars) or a constant concentration of 7.5 mM eATP (grey bars); (C) the distances expanded towards a H2O-saturated disc (black bars) or ATP-saturated disc (grey bars). The data are represented as the mean ± SD for three independent experiments.
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Fig4: Examination of twitching motility-mediated biofilm expansion in response to eATP by wildtypeP. aeruginosastrains and mutants ofcyaA,cyaB,cpdAor the Chp chemosensory system. The twitching motility response of P. aeruginosa strains to eATP at the interstitial space between a solidified nutrient media-coated microscope slide and a coverslip after incubation for 15 h at 37°C. (A) Phase-contrast microscopy of interstitial biofilms of wildtype P. aeruginosa strains PAK, PAO1, PA14 and PA103 in the presence of a H2O-saturated disc (upper images) or ATP-saturated disc (lower images). Scalebar is 50 μm and the arrows indicate the direction of expansion towards the disc. Images are representative of three independent experiments. (B) Surface areas of interstitial biofilms formed in the absence of eATP (black bars) or a constant concentration of 7.5 mM eATP (grey bars); (C) the distances expanded towards a H2O-saturated disc (black bars) or ATP-saturated disc (grey bars). The data are represented as the mean ± SD for three independent experiments.

Mentions: To determine if the observed inhibition by eATP of twitching motility-mediated biofilm expansion by P. aeruginosa strain PAK is likely to be conserved in other P. aeruginosa strains, we examined the influence of eATP gradients on interstitial biofilm expansion by the wild-type P. aeruginosa strains PAO1, PA14 and PA103. These assays showed all strains were similarly inhibited by gradients of eATP (Figure 4A).Figure 4


Extracellular ATP inhibits twitching motility-mediated biofilm expansion by Pseudomonas aeruginosa.

Nolan LM, Cavaliere R, Turnbull L, Whitchurch CB - BMC Microbiol. (2015)

Examination of twitching motility-mediated biofilm expansion in response to eATP by wildtypeP. aeruginosastrains and mutants ofcyaA,cyaB,cpdAor the Chp chemosensory system. The twitching motility response of P. aeruginosa strains to eATP at the interstitial space between a solidified nutrient media-coated microscope slide and a coverslip after incubation for 15 h at 37°C. (A) Phase-contrast microscopy of interstitial biofilms of wildtype P. aeruginosa strains PAK, PAO1, PA14 and PA103 in the presence of a H2O-saturated disc (upper images) or ATP-saturated disc (lower images). Scalebar is 50 μm and the arrows indicate the direction of expansion towards the disc. Images are representative of three independent experiments. (B) Surface areas of interstitial biofilms formed in the absence of eATP (black bars) or a constant concentration of 7.5 mM eATP (grey bars); (C) the distances expanded towards a H2O-saturated disc (black bars) or ATP-saturated disc (grey bars). The data are represented as the mean ± SD for three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4355966&req=5

Fig4: Examination of twitching motility-mediated biofilm expansion in response to eATP by wildtypeP. aeruginosastrains and mutants ofcyaA,cyaB,cpdAor the Chp chemosensory system. The twitching motility response of P. aeruginosa strains to eATP at the interstitial space between a solidified nutrient media-coated microscope slide and a coverslip after incubation for 15 h at 37°C. (A) Phase-contrast microscopy of interstitial biofilms of wildtype P. aeruginosa strains PAK, PAO1, PA14 and PA103 in the presence of a H2O-saturated disc (upper images) or ATP-saturated disc (lower images). Scalebar is 50 μm and the arrows indicate the direction of expansion towards the disc. Images are representative of three independent experiments. (B) Surface areas of interstitial biofilms formed in the absence of eATP (black bars) or a constant concentration of 7.5 mM eATP (grey bars); (C) the distances expanded towards a H2O-saturated disc (black bars) or ATP-saturated disc (grey bars). The data are represented as the mean ± SD for three independent experiments.
Mentions: To determine if the observed inhibition by eATP of twitching motility-mediated biofilm expansion by P. aeruginosa strain PAK is likely to be conserved in other P. aeruginosa strains, we examined the influence of eATP gradients on interstitial biofilm expansion by the wild-type P. aeruginosa strains PAO1, PA14 and PA103. These assays showed all strains were similarly inhibited by gradients of eATP (Figure 4A).Figure 4

Bottom Line: We have determined that eATP does not inhibit expression of the tfp major subunit, PilA, but reduces the levels of surface assembled tfp.Endogenous eATP produced by P. aeruginosa serves as a signalling molecule to co-ordinate complex multicellular behaviours of this pathogen.Our findings have significant implications in the development and pathogenesis of P. aeruginosa biofilm infections.

View Article: PubMed Central - PubMed

Affiliation: The ithree institute, University of Technology Sydney, PO Box 123, Broadway, NSW, 2007, Australia. laura.nolan25@gmail.com.

ABSTRACT

Background: Pseudomonas aeruginosa is an opportunistic pathogen that exploits damaged epithelia to cause infection. Type IV pili (tfp) are polarly located filamentous structures which are the major adhesins for attachment of P. aeruginosa to epithelial cells. The extension and retraction of tfp powers a mode of surface translocation termed twitching motility that is involved in biofilm development and also mediates the active expansion of biofilms across surfaces. Extracellular adenosine triphosphate (eATP) is a key "danger" signalling molecule that is released by damaged epithelial cells to alert the immune system to the potential presence of pathogens. As P. aeruginosa has a propensity for infecting damaged epithelial tissues we have explored the influence of eATP on tfp biogenesis and twitching motility-mediated biofilm expansion by P. aeruginosa.

Results: In this study we have found that eATP inhibits P. aeruginosa twitching motility-mediated expansion of interstitial biofilms at levels that are not inhibitory to growth. We have determined that eATP does not inhibit expression of the tfp major subunit, PilA, but reduces the levels of surface assembled tfp. We have also determined that the active twitching zone of expanding P. aeruginosa interstitial biofilms contain large quantities of eATP which may serve as a signalling molecule to co-ordinate cell movements in the expanding biofilm. The inhibition of twitching motility-mediated interstitial biofilm expansion requires eATP hydrolysis and does not appear to be mediated by the Chp chemosensory system.

Conclusions: Endogenous eATP produced by P. aeruginosa serves as a signalling molecule to co-ordinate complex multicellular behaviours of this pathogen. Given the propensity for P. aeruginosa to infect damaged epithelial tissues, our observations suggest that eATP released by damaged cells may provide a cue to reduce twitching motility of P. aeruginosa in order to establish infection at the site of damage. Furthermore, eATP produced by P. aeruginosa biofilms and by damaged epithelial cells may play a role in P. aeruginosa pathogenesis by inducing inflammatory damage and fibrosis. Our findings have significant implications in the development and pathogenesis of P. aeruginosa biofilm infections.

Show MeSH
Related in: MedlinePlus