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Multidrug efflux systems play an important role in the invasiveness of Pseudomonas aeruginosa.

Hirakata Y, Srikumar R, Poole K, Gotoh N, Suematsu T, Kohno S, Kamihira S, Hancock RE, Speert DP - J. Exp. Med. (2002)

Bottom Line: All efflux mutants except a deltamexCD-oprJ deletion demonstrated significantly reduced invasiveness compared with WT.Invasiveness was restored to the deltamexAB-oprM mutant by complementation with mexAB-oprM or by addition of culture supernatant from MDCK cells infected with WT.We conclude that the P. aeruginosa MexAB-OprM efflux system exports virulence determinants that contribute to bacterial virulence.

View Article: PubMed Central - PubMed

Affiliation: Division of Infectious and Immunological Diseases, Department of Pediatrics, University of British Columbia, Vancouver, British Columbia, V5Z 4H4 Canada. hirakata@net.nagasaki-u.ac.jp

ABSTRACT
Pseudomonas aeruginosa is an important opportunistic human pathogen. Certain strains can transmigrate across epithelial cells, and their invasive phenotype is correlated with capacity to cause invasive human disease and fatal septicemia in mice. Four multidrug efflux systems have been described in P. aeruginosa, however, their contribution to virulence is unclear. To clarify the role of efflux systems in invasiveness, P. aeruginosa PAO1 wild-type (WT) and its efflux mutants were evaluated in a Madin-Darby canine kidney (MDCK) epithelial cell monolayer system and in a murine model of endogenous septicemia. All efflux mutants except a deltamexCD-oprJ deletion demonstrated significantly reduced invasiveness compared with WT. In particular, a deltamexAB-oprM deletion strain was compromised in its capacity to invade or transmigrate across MDCK cells, and could not kill mice, in contrast to WT which was highly invasive (P < 0.0006) and caused fatal infection (P < 0.0001). The other mutants, including deltamexB and deltamexXY mutants, were intermediate between WT and the deltamexAB-oprM mutant in invasiveness and murine virulence. Invasiveness was restored to the deltamexAB-oprM mutant by complementation with mexAB-oprM or by addition of culture supernatant from MDCK cells infected with WT. We conclude that the P. aeruginosa MexAB-OprM efflux system exports virulence determinants that contribute to bacterial virulence.

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Penetration of P. aeruginosa strain K767 (WT) and its efflux mutants. Bacteria were inoculated at 3.5 × 106 CFU per well to the apical surfaces of MDCK cell monolayers. (A) The assay was performed in triplicate, and results are expressed as mean ± SD. (B) The values are expressed as percentages of values obtained with strain K767 at 3 and 6 h.
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fig1: Penetration of P. aeruginosa strain K767 (WT) and its efflux mutants. Bacteria were inoculated at 3.5 × 106 CFU per well to the apical surfaces of MDCK cell monolayers. (A) The assay was performed in triplicate, and results are expressed as mean ± SD. (B) The values are expressed as percentages of values obtained with strain K767 at 3 and 6 h.

Mentions: Both the parent WT strain K767 and strain K1521 (ΔmexCD-oprJ) penetrated MDCK monolayers by 3 h, whereas K1119 (ΔmexAB-oprM) was not detected in the basolateral medium until 6 h after infection (Fig. 1 A). At all time points, the penetration of both K767 (WT) and K1521 (ΔmexCD-oprJ) was significantly greater than that for K1119 (ΔmexAB-oprM) (P < 0.0006, ANOVA). Compared with the WT, relative capacities of the mutants to penetrate monolayers at 3 and 6 h after infection are summarized in Fig. 1 B. Strains K1523 (ΔmexB) and K1525 (ΔmexXY) were also compromised for invasion, but to lesser extents than the mutant with total deletion of mexAB-oprM (K1119). The invasive capacities of strains OCR1 (nalB) and K1536 (nfxB), hyperexpressing efflux systems, were also intermediate between strains K767 (WT) and K1119 (ΔmexAB-oprM).


Multidrug efflux systems play an important role in the invasiveness of Pseudomonas aeruginosa.

Hirakata Y, Srikumar R, Poole K, Gotoh N, Suematsu T, Kohno S, Kamihira S, Hancock RE, Speert DP - J. Exp. Med. (2002)

Penetration of P. aeruginosa strain K767 (WT) and its efflux mutants. Bacteria were inoculated at 3.5 × 106 CFU per well to the apical surfaces of MDCK cell monolayers. (A) The assay was performed in triplicate, and results are expressed as mean ± SD. (B) The values are expressed as percentages of values obtained with strain K767 at 3 and 6 h.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2194012&req=5

fig1: Penetration of P. aeruginosa strain K767 (WT) and its efflux mutants. Bacteria were inoculated at 3.5 × 106 CFU per well to the apical surfaces of MDCK cell monolayers. (A) The assay was performed in triplicate, and results are expressed as mean ± SD. (B) The values are expressed as percentages of values obtained with strain K767 at 3 and 6 h.
Mentions: Both the parent WT strain K767 and strain K1521 (ΔmexCD-oprJ) penetrated MDCK monolayers by 3 h, whereas K1119 (ΔmexAB-oprM) was not detected in the basolateral medium until 6 h after infection (Fig. 1 A). At all time points, the penetration of both K767 (WT) and K1521 (ΔmexCD-oprJ) was significantly greater than that for K1119 (ΔmexAB-oprM) (P < 0.0006, ANOVA). Compared with the WT, relative capacities of the mutants to penetrate monolayers at 3 and 6 h after infection are summarized in Fig. 1 B. Strains K1523 (ΔmexB) and K1525 (ΔmexXY) were also compromised for invasion, but to lesser extents than the mutant with total deletion of mexAB-oprM (K1119). The invasive capacities of strains OCR1 (nalB) and K1536 (nfxB), hyperexpressing efflux systems, were also intermediate between strains K767 (WT) and K1119 (ΔmexAB-oprM).

Bottom Line: All efflux mutants except a deltamexCD-oprJ deletion demonstrated significantly reduced invasiveness compared with WT.Invasiveness was restored to the deltamexAB-oprM mutant by complementation with mexAB-oprM or by addition of culture supernatant from MDCK cells infected with WT.We conclude that the P. aeruginosa MexAB-OprM efflux system exports virulence determinants that contribute to bacterial virulence.

View Article: PubMed Central - PubMed

Affiliation: Division of Infectious and Immunological Diseases, Department of Pediatrics, University of British Columbia, Vancouver, British Columbia, V5Z 4H4 Canada. hirakata@net.nagasaki-u.ac.jp

ABSTRACT
Pseudomonas aeruginosa is an important opportunistic human pathogen. Certain strains can transmigrate across epithelial cells, and their invasive phenotype is correlated with capacity to cause invasive human disease and fatal septicemia in mice. Four multidrug efflux systems have been described in P. aeruginosa, however, their contribution to virulence is unclear. To clarify the role of efflux systems in invasiveness, P. aeruginosa PAO1 wild-type (WT) and its efflux mutants were evaluated in a Madin-Darby canine kidney (MDCK) epithelial cell monolayer system and in a murine model of endogenous septicemia. All efflux mutants except a deltamexCD-oprJ deletion demonstrated significantly reduced invasiveness compared with WT. In particular, a deltamexAB-oprM deletion strain was compromised in its capacity to invade or transmigrate across MDCK cells, and could not kill mice, in contrast to WT which was highly invasive (P < 0.0006) and caused fatal infection (P < 0.0001). The other mutants, including deltamexB and deltamexXY mutants, were intermediate between WT and the deltamexAB-oprM mutant in invasiveness and murine virulence. Invasiveness was restored to the deltamexAB-oprM mutant by complementation with mexAB-oprM or by addition of culture supernatant from MDCK cells infected with WT. We conclude that the P. aeruginosa MexAB-OprM efflux system exports virulence determinants that contribute to bacterial virulence.

Show MeSH
Related in: MedlinePlus