Effect of superoxide dismutase gene inactivation on virulence of Pseudomonas aeruginosa PAO1 toward the silkworm, Bombyx mori

Abstract

To investigate the role of superoxide dismutase (SOD) in virulence against the silkworm, Bombyx mori, mutants of Pseudomonas aeruginosa PAO1 lacking manganese-SOD (PAO1sodM), iron-SOD (PAO1sodB), or both (PAO1sodMB) were generated. The mutants were injected into the hemocoel of B. mori. The virulence decreased in the order PAO1=PAO1sodM>PAO1sodB>PAO1sodMB. In particular, PAO1sodMB was avirulent at a dose of 10(5) cells or less. The sod double mutant PAO1sodMB was then complemented with either pSodM or pSodB in trans. In both the complemented strains, the virulence was partially restored. Of the two plasmids, pSodB contributed more to the virulence of P. aeruginosa against B. mori. The results of growth in B. mori hemolymph broth and microscopic analysis suggested that a longer lag phase and superoxide sensitivity correlated with decreased virulence in sod mutants. In conclusion, the SODs are required for full virulence of P. aeruginosa against B. mori and Fe-SOD is more important than Mn-SOD in the infection process.

FIG. 1.

Mortality assay of the sod mutants (A) and the complemented strains (B) of P. aeruginosa PAO1 in B. mori. Each strain was precultured in LB (a) or LPS (b). P. aeruginosa PAO1 (circles), PAO1sodM (triangles), PAO1sodB (squares), and PAO1sodMB (inverted triangles) were injected into the fourth instar of B. mori larvae at various doses (A). Similarly, the complemented strain was inoculated (B). PAO1(pBBR1MCS2), the sod mutants carrying pBBR1MCS2, the sod mutants carrying pSodM, and the sod mutant carrying pSodB are represented as circles, triangles, squares, and inverted triangles, respectively. In the inoculation test using the complemented strains, the dose was 10⁵ cells. Five larvae were injected per dilution. Each data point is an average of three replicates, and the error bar indicates the standard error of the mean.

FIG. 2.

Growth of P. aeruginosa strains in BMH broth. The strains were grown overnight in LB broth at 30°C. Three microliters of the LB culture was subcultured into 3 ml of BMH broth and incubated at 25°C until an OD₆₆₀ of 0.1 was reached. Then, a 30-μl aliquot of preculture was inoculated into 3 ml of fresh BMH broth and shaken at 70 rpm under aerobic conditions at 25°C. The OD₆₆₀ was automatically recorded with an Advantec TVS062CA biophotorecorder (Advantec Toyo Co. Ltd., Tokyo, Japan). Three experiments were repeated to ensure reproducibility and gave equivalent results. Each plot represents the average of triplicates.

FIG. 3.

Light microscopic observation of the hemolymph from B. mori inoculated with P. aeruginosa strains. From each strain 10⁵ cells were injected into the fourth-instar larvae. The hemolymph was collected every 6 h after inoculation. The smear of collected hemolymph was stained with Giemsa solution. A single asterisk means that bacterial cells were not observed. A double asterisk means that the bacterial density was low. A triple asterisk means that the bacterial density was high. On the plate, some of the bacterial cells are shown with arrowheads. Bars, 20 μm.

FIG. 4.

Growth of P. aeruginosa PAO1(pBBR1MCS2) in B. mori. Cells (10⁵) were injected into the fourth-instar larvae. A hemolymph sample of a single larva was collected every 3 h after inoculation. Three samples were independently obtained from different larvae. The number of viable cells in the hemolymph was estimated by a plate count procedure. Each plotted point represents the average of triplicates. Error bars indicates standard errors.