Pseudomonas aeruginosa flagellum is critical for invasion, cutaneous persistence and induction of inflammatory response of skin epidermis

Figures & data

Figure 1. Cytokine and AMP mRNA expression in response to keratinocyte infection by each kind of bacterial strain. IL-1β (A), IL-6 (B), IL-23α (C), IL-32 (D), TSLP (E), TNFα (F), hBD2 (G) and S100A7 (H) mRNA expression by keratinocytes infected for 6 h with wild-type PAK strain, PAK ∆fliC (Fla⁻), PAK∆xcpQ (T2SS ⁻ ), PAK∆pscF (T3SS ⁻ ), PAK ∆fliC∆xcpQ∆pscF (Fla⁻ /T2SS ⁻ /T3SS ⁻ ) strains or wild-type PAK strain in presence of the anti-TLR5 monoclonal antibody. mRNA expression levels are expressed as the fold increase above unstimulated cultures. Data are represented as mean + SEM of five independent experiments. *p < 0.05, **p < 0.01 and *** p < 0.001.

Figure 2. Chemokine mRNA expression and secretion in response to keratinocyte infection by each kind of bacterial strain and inflammatory response to flagellin stimulation. CXCL1, CXCL8, CCL20 mRNA expression (A to C) and secretion (D to F) by keratinocytes infected for 6 h with wild-type PAK strain, PAK ∆fliC (Fla⁻), PAK ∆xcpQ (T2SS ⁻ ), PAK ∆pscF (T3SS ⁻ ), PAK ∆fliC∆xcpQ∆pscF (Fla⁻ /T2SS ⁻ /T3SS ⁻ ) strains or wild-type PAK strain in presence of the anti-TLR5 monoclonal antibody. mRNA expression levels are expressed as the fold increase above unstimulated cultures. Protein concentrations (pg/mL) were measured in culture supernatants by ELISA assays. G: Cytokine, chemokine and AMP mRNA induction after 6 h of keratinocyte stimulation with purified flagellin from P. aeruginosa (1 µg/mL). Data are represented as mean + SEM of five independent experiments. *p < 0.05, **p < 0.01 and *** p < 0.001.

Figure 3. Cytokine and AMP mRNA expression in response to RHE infection by each kind of bacterial strain. IL-1β (A), IL-6 (B), IL-23α (C), IL-32 (D), TSLP (E), TNFα (F), hBD2 (G), S100A7 (H) mRNA expression in fold increase by RHE infected for 16 h with wild-type PAK strain, PAK ∆fliC (Fla⁻), PAK ∆xcpQ (T2SS ⁻ ), PAK ∆pscF (T3SS ⁻ ), PAK ∆fliC∆xcpQ∆pscF (Fla⁻ /T2SS ⁻ /T3SS ⁻ ) strains. mRNA expression levels are expressed as the fold increase above unstimulated cultures. Data are represented as mean + SEM of three independent experiments. *p < 0.05 and **p < 0.01.

Figure 4. Chemokine mRNA expression in response to RHE infection by each kind of bacterial strain and inflammatory response to flagellin stimulation. CXCL1, CXCL8, CCL20 mRNA expression (A to C) and secretion (D to F) by RHE infected with wild-type PAK strain, PAK ∆fliC (Fla⁻), PAK ∆xcpQ (T2SS ⁻ ), PAK ∆pscF (T3SS ⁻ ) or PAK ∆fliC∆xcpQ∆pscF (Fla⁻ /T2SS ⁻ /T3SS ⁻ ) strains. G: Cytokine, chemokine and AMP mRNA induction after 16 h of RHE stimulation with purified flagellin from P. aeruginosa (1µg/mL) in culture medium. mRNA expression levels are expressed as the fold increase above unstimulated cultures. Protein concentrations (pg/mL) were measured in culture supernatants by ELISA assays. Data are represented as mean + SEM of five independent experiments. *p < 0.05, **p < 0.01 and *** p < 0.001.

Figure 5. A to H: Level of S100A7 tissue production (red) assessed by immunohistochemistry on RHE infected with the different PAK strains of P. aeruginosa for 24 h. Nuclei were counter-stained using a hematoxylin solution (blue). A: control (sterile PBS), B: wild-type PAK strain, C: PAK ∆fliC (Fla⁻), D: PAK ∆xcpQ (T2SS ⁻ ), E: PAK ∆pscF (T3SS ⁻ ), F: PAK ∆fliC∆xcpQ∆pscF (Fla⁻ /T2SS ⁻ /T3SS ⁻ ), G: ultrapure flagellin deposited in culture medium, H: ultra-pure flagellin deposited on the upper layer of RHE. I to P: Immunofluorescence assays (green) using a polyclonal anti-P. aeruginosa antibody on RHE infected with the different PAK strains. Nuclei were counter-stained using a solution of propidium iodide red-fluorescent staining. I: control (sterile PBS), J: wild-type PAK strain, K: PAK ∆fliC (Fla⁻), L: PAK ∆xcpQ (T2SS ⁻ ), M: PAK ∆pscF (T3SS ⁻ ), N: PAK ∆fliC∆xcpQ∆pscF (Fla⁻ /T2SS ⁻ /T3SS ⁻ ), O: ultrapure flagellin deposited in culture medium, P: ultra-pure flagellin deposited on the upper layer of RHE.

Figure 6. Flagellum promotes P. aeruginosa persistence in cutaneous infection. (A) Balb/c mice were inoculated with 1.10⁷ CFU of either PAK-lux (●) or PAK ∆fliC-lux (□) strains. Infection was assessed by quantifying luminescence emission on living animals. Data represent the mean ± SEM (n = 4–5 mice/group). *p < 0.05. (B) Pictures of luminescence imaging on living animals recorded immediately after infection (D0) and after 4 days (D4) post-inoculation.

Table 1. Strains and plasmids used in this study.

Strains and plasmids	Characteristics	Source or reference
P. aeruginosa strains
PAK	P. aeruginosa strain K, wild-type	[25]
PAK ∆xcpQ	In-frame partial deletion of xcpQ in strain PAK: defective for type II secretion system (T2SS^−)	[11]
PAK ∆pscF	In-frame partial deletion of pscF in strain PAK: defective for type III secretion system (T3SS^−)	[11]
PAK ∆fliC	In-frame partial deletion of fliC in strain PAK; non-motile (Fla^−)	[11]
PAK ∆fliC∆xcpQ∆pscF	In-frame partial deletion of xcpQ and pscF in strain PAK∆fliC: defective for both type II (T2SS^−) and type III secretion system (T3SS^−); non-motile (Fla^−)	[11]
PAK-lux	PAK strain in which luxCDABE gene expression cassette from plasmid pUC18T-miniTn7T-lux was inserted into the chromosomal att site	This work
PAK ∆fliC-lux	PAK-∆fliC strain in which luxCDABE gene expression cassette from plasmid pUC18T-miniTn7T-lux was inserted into the chromosomal att site	This work
Plasmids^a
pUC18T-miniTn7T-lux	KC848884; suicide vector for shuttling single copies of genes directly to the chromosome via a mini-Tn7 element; aacC1 gene encoding gentamicin resistance marker on Tn7 element; contains oriT for mobilization; P1 integron promoter driven expression of luxCDABE; Amp^r Gm^r	[65]
pTNS3	Helper plasmid encoding the Tn7 site-specific transposition pathway; Amp^r	[62]

^aAmp^r and Gm^r are resistant to ampicillin and gentamycin, respectively.

Greene CM , Carroll TP , Smith SGJ , et al. TLR-induced inflammation in cystic fibrosis and non-cystic fibrosis airway epithelial cells. J Immunol. 2005;174(3):1638–1646.

 PubMed Web of Science ®Google Scholar

Kita K , Sueyoshi N , Okino N , et al. Activation of bacterial ceramidase by anionic glycerophospholipids: possible involvement in ceramide hydrolysis on atopic skin by Pseudomonas ceramidase. Biochem J. 2002;362(Pt 3):619–626.

 PubMed Web of Science ®Google Scholar

Damron FH , McKenney ES , Barbier M , et al. Construction of mobilizable mini-Tn7 vectors for bioluminescent detection of gram-negative bacteria and single-copy promoter lux reporter analysis. Appl Environ Microbiol. 2013;79(13):4149–4153.

 PubMed Web of Science ®Google Scholar

Choi K-H , Schweizer HP . mini-Tn7 insertion in bacteria with single attTn7 sites: example Pseudomonas aeruginosa . Nat Protoc. 2006;1(1):153–161.

 PubMed Web of Science ®Google Scholar