Limits...
Homogentisate 1-2-Dioxygenase Downregulation in the Chronic Persistence of Pseudomonas aeruginosa Australian Epidemic Strain-1 in the CF Lung.

Harmer CJ, Wynn M, Pinto R, Cordwell S, Rose BR, Harbour C, Triccas JA, Manos J - PLoS ONE (2015)

Bottom Line: In C. elegans, hmgA deactivation resulted in significantly increased PA14 virulence while hmgA downregulation reduced AES-1M virulence.AES-1M was significantly more persistent in mouse lung and showed a significant increase in TNF-α (p<0.0001), sustained even with no detectable bacteria.PA14ΔhmgA did not show increased TNF-α.

View Article: PubMed Central - PubMed

Affiliation: Bacterial Pathogens in Cystic Fibrosis Group, Department of Infectious Diseases and Immunology, Central Clinical School, The University of Sydney, Sydney, Australia; School of Molecular Biosciences, The University of Sydney, Sydney, Australia.

ABSTRACT
Some Pseudomonas aeruginosa strains including Australian Epidemic Strain-1 (AES-1 or AUS-01) cause persistent chronic infection in cystic fibrosis (CF) patients, with greater morbidity and mortality. Factors conferring persistence are largely unknown. Previously we analysed the transcriptomes of AES-1 grown in Luria broth, nematode growth medium for Caenorhabditis elegans assay (both aerobic) and artificial sputum medium (mainly hypoxic). Transcriptional comparisons included chronic AES-1 strains against PAO1 and acute AES-1 (AES-1R) against its chronic isogen (AES-1M), isolated 10.5 years apart from a CF patient and not eradicated in the meantime. Prominent amongst genes downregulated in AES-1M in all comparisons was homogentisate-1-2-dioxygenase (hmgA); an oxygen-dependent gene known to be mutationally deactivated in many chronic infection strains of P. aeruginosa. To investigate if hmgA downregulation and deactivation gave similar virulence persistence profiles, a hmgA mutant made in UCBPP-PA14 utilising RedS-recombinase and AES-1M were assessed in the C. elegans virulence assay, and the C57BL/6 mouse for pulmonary colonisation and TNF-α response. In C. elegans, hmgA deactivation resulted in significantly increased PA14 virulence while hmgA downregulation reduced AES-1M virulence. AES-1M was significantly more persistent in mouse lung and showed a significant increase in TNF-α (p<0.0001), sustained even with no detectable bacteria. PA14ΔhmgA did not show increased TNF-α. This study suggests that hmgA may have a role in P. aeruginosa persistence in chronic infection and the results provide a starting point for clarifying the role of hmgA in chronic AES-1.

No MeSH data available.


Related in: MedlinePlus

Mouse TNF-α response to lung infection with AES-1R, AES-1M, PA14 and PA14ΔhmgA, and neutrophil populations in PA14 and PA14ΔhmgA-infected mice.The TNF-α profile of P. aeruginosa-infected mice was determined in lung homogenates at 3-, 24- and 72-hrs post-infection by ELISA. A. AES-1M elicited a rapid and significantly greater TNF-α response compared to AES-1R at 3 and 24-hr, and both responses were still at >400pg/ml at 72hr despite the absence of bacteria in lungs. B. Both PA14 and PA14hmgA elicited a rapid TNF-α response compared to control uninfected mice by three hrs post-infection however there was no significant difference between them. The significances of differences between strains were determined by ANOVA * p<0.0001 vs. AES-1R. C. Mice were infected with 106 CFU of either wildtype PA14 or PA14ΔhmgA and lungs were harvested at 72-hrs post-infection. Single cells suspensions were stained and analysed by FACS. Neutrophil populations were identified based on their CD11b+Ly6G+ phenotype. Total cell numbers in the lung were determined based on the number of stained cells. Infection with PA14ΔhmgA was marked by a significant increase in the number of neutrophils in the lung compared to mice infected with PA14. The significances of differences between strains were determined by ANOVA * p<0.05 vs. PA14 WT.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4529145&req=5

pone.0134229.g003: Mouse TNF-α response to lung infection with AES-1R, AES-1M, PA14 and PA14ΔhmgA, and neutrophil populations in PA14 and PA14ΔhmgA-infected mice.The TNF-α profile of P. aeruginosa-infected mice was determined in lung homogenates at 3-, 24- and 72-hrs post-infection by ELISA. A. AES-1M elicited a rapid and significantly greater TNF-α response compared to AES-1R at 3 and 24-hr, and both responses were still at >400pg/ml at 72hr despite the absence of bacteria in lungs. B. Both PA14 and PA14hmgA elicited a rapid TNF-α response compared to control uninfected mice by three hrs post-infection however there was no significant difference between them. The significances of differences between strains were determined by ANOVA * p<0.0001 vs. AES-1R. C. Mice were infected with 106 CFU of either wildtype PA14 or PA14ΔhmgA and lungs were harvested at 72-hrs post-infection. Single cells suspensions were stained and analysed by FACS. Neutrophil populations were identified based on their CD11b+Ly6G+ phenotype. Total cell numbers in the lung were determined based on the number of stained cells. Infection with PA14ΔhmgA was marked by a significant increase in the number of neutrophils in the lung compared to mice infected with PA14. The significances of differences between strains were determined by ANOVA * p<0.05 vs. PA14 WT.

Mentions: We compared the TNF-α response in the mouse lung for AES-1R and AES-1M and separately, PA14/ and PA14-ΔhmgA using a TNF-α ELISA. Infection with AES-1M elicited a significantly higher TNF-α response than infection with AES-1R at both 3 and 24 hrs post infection (p<0.001). Even with no detectable lung bacteria at 72 hr, both AES-1R and AES-1M mice still displayed a considerable TNF-α response (ca. 400–500 pg/ml) (Fig 3A). The TNF-α response against PA14 wild type and PA14ΔhmgA (Fig 3B) was rapid by three-hrs post-infection, but there was no statistically significant difference in TNF-α response between PA14ΔhmgA and PA14 wild type at any time point.


Homogentisate 1-2-Dioxygenase Downregulation in the Chronic Persistence of Pseudomonas aeruginosa Australian Epidemic Strain-1 in the CF Lung.

Harmer CJ, Wynn M, Pinto R, Cordwell S, Rose BR, Harbour C, Triccas JA, Manos J - PLoS ONE (2015)

Mouse TNF-α response to lung infection with AES-1R, AES-1M, PA14 and PA14ΔhmgA, and neutrophil populations in PA14 and PA14ΔhmgA-infected mice.The TNF-α profile of P. aeruginosa-infected mice was determined in lung homogenates at 3-, 24- and 72-hrs post-infection by ELISA. A. AES-1M elicited a rapid and significantly greater TNF-α response compared to AES-1R at 3 and 24-hr, and both responses were still at >400pg/ml at 72hr despite the absence of bacteria in lungs. B. Both PA14 and PA14hmgA elicited a rapid TNF-α response compared to control uninfected mice by three hrs post-infection however there was no significant difference between them. The significances of differences between strains were determined by ANOVA * p<0.0001 vs. AES-1R. C. Mice were infected with 106 CFU of either wildtype PA14 or PA14ΔhmgA and lungs were harvested at 72-hrs post-infection. Single cells suspensions were stained and analysed by FACS. Neutrophil populations were identified based on their CD11b+Ly6G+ phenotype. Total cell numbers in the lung were determined based on the number of stained cells. Infection with PA14ΔhmgA was marked by a significant increase in the number of neutrophils in the lung compared to mice infected with PA14. The significances of differences between strains were determined by ANOVA * p<0.05 vs. PA14 WT.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4529145&req=5

pone.0134229.g003: Mouse TNF-α response to lung infection with AES-1R, AES-1M, PA14 and PA14ΔhmgA, and neutrophil populations in PA14 and PA14ΔhmgA-infected mice.The TNF-α profile of P. aeruginosa-infected mice was determined in lung homogenates at 3-, 24- and 72-hrs post-infection by ELISA. A. AES-1M elicited a rapid and significantly greater TNF-α response compared to AES-1R at 3 and 24-hr, and both responses were still at >400pg/ml at 72hr despite the absence of bacteria in lungs. B. Both PA14 and PA14hmgA elicited a rapid TNF-α response compared to control uninfected mice by three hrs post-infection however there was no significant difference between them. The significances of differences between strains were determined by ANOVA * p<0.0001 vs. AES-1R. C. Mice were infected with 106 CFU of either wildtype PA14 or PA14ΔhmgA and lungs were harvested at 72-hrs post-infection. Single cells suspensions were stained and analysed by FACS. Neutrophil populations were identified based on their CD11b+Ly6G+ phenotype. Total cell numbers in the lung were determined based on the number of stained cells. Infection with PA14ΔhmgA was marked by a significant increase in the number of neutrophils in the lung compared to mice infected with PA14. The significances of differences between strains were determined by ANOVA * p<0.05 vs. PA14 WT.
Mentions: We compared the TNF-α response in the mouse lung for AES-1R and AES-1M and separately, PA14/ and PA14-ΔhmgA using a TNF-α ELISA. Infection with AES-1M elicited a significantly higher TNF-α response than infection with AES-1R at both 3 and 24 hrs post infection (p<0.001). Even with no detectable lung bacteria at 72 hr, both AES-1R and AES-1M mice still displayed a considerable TNF-α response (ca. 400–500 pg/ml) (Fig 3A). The TNF-α response against PA14 wild type and PA14ΔhmgA (Fig 3B) was rapid by three-hrs post-infection, but there was no statistically significant difference in TNF-α response between PA14ΔhmgA and PA14 wild type at any time point.

Bottom Line: In C. elegans, hmgA deactivation resulted in significantly increased PA14 virulence while hmgA downregulation reduced AES-1M virulence.AES-1M was significantly more persistent in mouse lung and showed a significant increase in TNF-α (p<0.0001), sustained even with no detectable bacteria.PA14ΔhmgA did not show increased TNF-α.

View Article: PubMed Central - PubMed

Affiliation: Bacterial Pathogens in Cystic Fibrosis Group, Department of Infectious Diseases and Immunology, Central Clinical School, The University of Sydney, Sydney, Australia; School of Molecular Biosciences, The University of Sydney, Sydney, Australia.

ABSTRACT
Some Pseudomonas aeruginosa strains including Australian Epidemic Strain-1 (AES-1 or AUS-01) cause persistent chronic infection in cystic fibrosis (CF) patients, with greater morbidity and mortality. Factors conferring persistence are largely unknown. Previously we analysed the transcriptomes of AES-1 grown in Luria broth, nematode growth medium for Caenorhabditis elegans assay (both aerobic) and artificial sputum medium (mainly hypoxic). Transcriptional comparisons included chronic AES-1 strains against PAO1 and acute AES-1 (AES-1R) against its chronic isogen (AES-1M), isolated 10.5 years apart from a CF patient and not eradicated in the meantime. Prominent amongst genes downregulated in AES-1M in all comparisons was homogentisate-1-2-dioxygenase (hmgA); an oxygen-dependent gene known to be mutationally deactivated in many chronic infection strains of P. aeruginosa. To investigate if hmgA downregulation and deactivation gave similar virulence persistence profiles, a hmgA mutant made in UCBPP-PA14 utilising RedS-recombinase and AES-1M were assessed in the C. elegans virulence assay, and the C57BL/6 mouse for pulmonary colonisation and TNF-α response. In C. elegans, hmgA deactivation resulted in significantly increased PA14 virulence while hmgA downregulation reduced AES-1M virulence. AES-1M was significantly more persistent in mouse lung and showed a significant increase in TNF-α (p<0.0001), sustained even with no detectable bacteria. PA14ΔhmgA did not show increased TNF-α. This study suggests that hmgA may have a role in P. aeruginosa persistence in chronic infection and the results provide a starting point for clarifying the role of hmgA in chronic AES-1.

No MeSH data available.


Related in: MedlinePlus