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. 2009 Dec 23;4(12):e8439.
doi: 10.1371/journal.pone.0008439.

Pseudomonas aeruginosa exploits lipid A and muropeptides modification as a strategy to lower innate immunity during cystic fibrosis lung infection

Cristina Cigana  1 , Laura Curcurù, Maria Rosaria Leone, Teresa Ieranò, Nicola Ivan Lorè, Irene Bianconi, Alba Silipo, Flora Cozzolino, Rosa Lanzetta, Antonio Molinaro, Maria Lina Bernardini, Alessandra Bragonzi
Affiliations

Pseudomonas aeruginosa exploits lipid A and muropeptides modification as a strategy to lower innate immunity during cystic fibrosis lung infection

Cristina Cigana et al. PLoS One. .

Abstract

Pseudomonas aeruginosa can establish life-long airways chronic infection in patients with cystic fibrosis (CF) with pathogenic variants distinguished from initially acquired strain. Here, we analysed chemical and biological activity of P. aeruginosa Pathogen-Associated Molecular Patterns (PAMPs) in clonal strains, including mucoid and non-mucoid phenotypes, isolated during a period of up to 7.5 years from a CF patient. Chemical structure by MS spectrometry defined lipopolysaccharide (LPS) lipid A and peptidoglycan (PGN) muropeptides with specific structural modifications temporally associated with CF lung infection. Gene sequence analysis revealed novel mutation in pagL, which supported lipid A changes. Both LPS and PGN had different potencies when activating host innate immunity via binding TLR4 and Nod1. Significantly higher NF-kB activation, IL-8 expression and production were detected in HEK293hTLR4/MD2-CD14 and HEK293hNod1 after stimulation with LPS and PGN respectively, purified from early P. aeruginosa strain as compared to late strains. Similar results were obtained in macrophages-like cells THP-1, epithelial cells of CF origin IB3-1 and their isogenic cells C38, corrected by insertion of cystic fibrosis transmembrane conductance regulator (CFTR). In murine model, altered LPS structure of P. aeruginosa late strains induces lower leukocyte recruitment in bronchoalveolar lavage and MIP-2, KC and IL-1beta cytokine levels in lung homogenates when compared with early strain. Histopathological analysis of lung tissue sections confirmed differences between LPS from early and late P. aeruginosa. Finally, in this study for the first time we unveil how P. aeruginosa has evolved the capacity to evade immune system detection, thus promoting survival and establishing favourable conditions for chronic persistence. Our findings provide relevant information with respect to chronic infections in CF.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Chemical structure of P. aeruginosa lipid A.
MALDI MS spectra of lipid A blend obtained by acid hydrolysis of LPS from P. aeruginosa clinical strains isolated at the onset of chronic colonization (AA2) (A) and after years of chronic infection (AA43 and AA44) from a CF patient (B and C). A difference of 238 Da corresponds to a 16∶0 fatty acid residue whereas 170 corresponds to a 10∶0 (3-OH) residue and 80 Da are indicative of a phosphate group. The 16 Da difference is relative to the presence of a Hydroxy group at C-2 of the secondary 12∶0 fatty acids. The non indicated ion peaks are relative to the species already indicated and bearing sodium or potassium counter-ions.
Figure 2
Figure 2. Stimulation of HEK 293-hTLR4/MD2-CD14 with LPS derived from the three clinical isolates of P. aeruginosa AA2, AA43 and AA44.
A) Fold of activation of NF-kB after 4 h of stimulation with different concentrations of LPS; commercial LPS of P. aeruginosa was used as a control. B) IL-8 mRNA induction after stimulation with 100 ng/mL of LPS for 4 h. C) IL-8 secretion after stimulation with 100 ng/mL of LPS for 24 h. Commercial LPS of PAO1 was used as a control. *p<0.05, **p<0.01, ***p<0.001 in the Student's t-test.
Figure 3
Figure 3. Response of IB3-1, C38 and THP-1 cells after stimulation with LPS derived from the three clinical isolates of P. aeruginosa AA2, AA43 and AA44.
A) Fold of induction of IL-8 and C) TNF-α mRNA in IB3-1 and C38 cells after stimulation with 100 ng/mL of LPS for 4 h. Commercial LPS of PAO1 was used as control. The values represent the expression levels relative to untreated IB3-1 (means±SD). B) IL-8 secretion from IB3-1 and C38 cells after stimulation with 100 ng/mL of LPS for 24 h. LPS of PAO1 was used as control. D) TNF-α secretion from THP-1 after stimulation for 6 h with 100 ng/mL of LPS derived from the clinical isolates AA2, AA43 and AA44. LPSs from P. aeruginosa serotype 1022 and from E. coli serotype OIII:B4 (Sigma) were used as controls. *p<0.05, **p<0.01, ***p<0.001 in the Student's t-test.
Figure 4
Figure 4. Neutrophils recruitment and cytokines release in murine lungs after 16 h treatment with LPS derived from clinical P. aeruginosa strains.
C57Bl/6 mouse were exposed for 16 h to 10 mg/mouse of LPS derived from a P. aeruginosa reference strain (PAO1) and three clinical isolates (AA2, AA43 and AA44). A) Total cells were recovered from the BALF and quantified. B, C and D) MIP-2, IL-1β and KC secretions were quantified by ELISA in lung homogenates. *p<0.05, **p<0.01 in the Student's t-test.
Figure 5
Figure 5. Murine lung histology after treatment with LPS derived from clinical P. aeruginosa strains.
Mice exposed for 16 h to 10 mg/mouse of LPS derived from three P. aeruginosa clinical isolates (AA2, AA43 and AA44). After H&E staining, extensive recruitment of inflammatory cells is visible in the bronchial lumen (asterisk) of animals treated with LPS of AA2 strain (A and B), whereas treatment of mice with LPS of AA43 (C and D) and AA44 (E and F) showed limited accumulation.
Figure 6
Figure 6. Stimulation of HEK 293-Nod1/psiNod1 and HEK 293-Nod2 with PGN derived from the three clinical isolates of P. aeruginosa AA2, AA43 and AA44.
A, D) Fold of activation of NF-kB after 18 h of stimulation with different concentration of PGN (0,5 µg/mL; 1 µg/mL; 10 µg/mL); triDAP and MDP were used as controls. B, E) IL-8 mRNA induction after stimulation with 1 µg/mL of PGN for 18 h. C, F) IL-8 secretion after stimulation with 1 µg/mL of PGN for 24 h. *p<0.05; **p<0.01 in the Student's t-test.
Figure 7
Figure 7. Response of IB3-1 and C38 cells after stimulation with PGN derived from the three clinical isolates of P. aeruginosa AA2, AA43 and AA44.
A) Fold of induction of TNF-α and (B) IL-8 mRNA after stimulation with 1 µg/mL of PGN for 4 h. The values represent the expression levels relative to untreated IB3-1 (means±SD). C) IL-8 secretion after stimulation with 1 µg/mL of PGN for 24 h. *p<0.05, **p<0.01, ***p<0.001 in the Student's t-test.
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