Virulence of an exotoxin A-deficient strain of Pseudomonas aeruginosa toward the silkworm, Bombyx mori

We studied the contribution of exotoxin A to the virulence of Pseudomonas aeruginosa against the silkworm, Bombyx mori. First, an exotoxin A-deficient mutant strain (PAO1toxA) was created, and its virulence compared with that of the parental PAO1 strain. In a short-term mortality assay, the mutant harboring pBBR1MCS2 did not kill B. mori until 120 h after inoculation and complementation of the corresponding gene in trans restored the strain’s virulence. Next, to ascertain whether or not it lost all virulence, PAO1toxA (pBBR1MCS2, pGFP) was used in a long-term mortality assay. B. mori inoculated with the mutant strain did not die until early in the 5th instar (240 h after inoculation). However, 50% of the inoculated B. mori died late in the 5th instar or in the early pupal stage (408 h after inoculation). All had died by the pupal stage (600 h after inoculation). The mutant strain was isolated from dead larvae and cocoons. The bacterial population of PAO1toxA in hemolymph reached 4.77 × 107 cfu/ml. These results indicated that exotoxin A acts as a virulence factor in B. mori and that other virulence factor(s) are involved during the late stages of infection.

► We constructed an exotoxin A-deficient mutant of Pseudomonas aeruginosa. ► In a short-term mortality assay, the mutant did not kill Bombyx mori until 120 h. ► This result indicated that exotoxin A acts as a virulence factor in B. mori. ► However, all had died by the pupal stage in a long-term mortality assay. ► Other virulence factor(s) are involved during the late stages of infection.