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. 2009 Jul;117(7):537-46.
doi: 10.1111/j.1600-0463.2009.02466.x.

Inactivation of the rhlA Gene in Pseudomonas Aeruginosa Prevents Rhamnolipid Production, Disabling the Protection Against Polymorphonuclear Leukocytes

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Free PMC article

Inactivation of the rhlA Gene in Pseudomonas Aeruginosa Prevents Rhamnolipid Production, Disabling the Protection Against Polymorphonuclear Leukocytes

Maria Van Gennip et al. APMIS. .
Free PMC article

Abstract

Many of the virulence factors produced by the opportunistic human pathogen Pseudomonas aeruginosa are quorum-sensing (QS) regulated. Among these are rhamnolipids, which have been shown to cause lysis of several cellular components of the human immune system, e.g. monocyte-derived macrophages and polymorphonuclear leukocytes (PMNs). We have previously shown that rhamnolipids produced by P. aeruginosa cause necrotic death of PMNs in vitro. This raises the possibility that rhamnolipids may function as a 'biofilm shield'in vivo, which contributes significantly to the increased tolerance of P. aeruginosa biofilms to PMNs. In the present study, we demonstrate the importance of the production of rhamnolipids in the establishment and persistence of P. aeruginosa infections, using an in vitro biofilm system, an intraperitoneal foreign-body model and a pulmonary model of P. aeruginosa infections in mice. Our experimental data showed that a P. aeruginosa strain, unable to produce any detectable rhamnolipids due to an inactivating mutation in the single QS-controlled rhlA gene, did not induce necrosis of PMNs in vitro and exhibited increased clearance compared with its wild-type counterpart in vivo. Conclusively, the results support our model that rhamnolipids are key protective agents of P. aeruginosa against PMNs.

Figures

Fig. 1
Fig. 1
Rhamnolipid production by wild-type Pseudomonas aeruginosa, its corresponding rhlA mutant and the rhlA+ complemented rhlA strain. Production of rhamnolipids was measured in planktonic grown shaking cultures (black) and a stationary grown biofilm after 24 h (white). The average concentration (±SEM) of rhamnolipids from three experiments is shown.
Fig. 2
Fig. 2
Pseudomonas aeruginosa biofilms at day 5 (green) exposed to polymorphonuclear leukocytes (PMNs) for 120 min at 37°C and stained with the DNA stain propidium iodide (red). Wild-type P. aeruginosa biofilms with extensive PMN necrosis [necrotic PMNs (red color) are indicated by a ring and an arrow in (B), and dead bacteria are indicated in magnification in (A) by a triangle and an arrow]. (C, D) rhlA mutant biofilms mainly with intact PMNs as indicated by an arrow a and ring in (D). (A, C) 3D images; (B, D) top views. The biofilms were visualized by combined fluorescence and light microscopy. Scale bars (50 μm) are shown in (B) and (D). All images were obtained with a ×40/dry objective.
Fig. 3
Fig. 3
(A) Clearance of wild-type Pseudomonas aeruginosa and the rhlA mutant in the foreign-body infection model. Implants were removed 3 days post-insertion and the CFU/implant was determined. There was a significant difference in CFU recovered on comparing mice infected with wild-type P. aeruginosa with mice infected with the rhlA mutant (p<0.0001). The median CFU/implant measured on control implants, not inserted into the mice, was adjusted to an OD600nm of 0.1, correlating to 6.4×105–6.9×105 CFU/implant. The median CFU/implant on control implants not inserted into the mice, from pooled data from two experiments, was arbitrarily assigned the value of 100% and used to normalize the respective CFUs obtained 3 days after infection. RhlA, rhlA mutant (n=24); WT, wild type (n=22). (B) Clearance of wild-type P. aeruginosa and the rhlA mutant in a pulmonary infection model 3 days post-infection. There was a significant difference in the recovery of bacteria on comparing mice infected with the wild-type P. aeruginosa with mice infected with the rhlA mutant (p<0.005). Four mice were euthanized immediately after the challenge to establish the inocula. The median CFU/lung, obtained from pooled data from two experiments, from mice infected with the wild-type P. aeruginosa was 5.6×106 CFU/lung, while mice infected with the rhlA mutant had a median of 1.8×106 CFU/lung. The respective medians were arbitrarily assigned the value of 100% and used to normalize the CFUs obtained 3 days after infection. rhlA, rhlA mutant (n=28); WT, wild type (n=20). Squares and triangles represent CFU/lung in individual mice; scale bars represent the median.

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