Efficacy of FimA antibody and clindamycin in silkworm larvae stimulated with Porphyromonas gulae

Abstract

Objective: Porphyromonas gulae, a major periodontal pathogen in animals, possesses fimbriae that have been classified into three genotypes (A, B, C) based on the diversity of fimA genes encoding fimbrillin protein (FimA). P. gulae strains with type C fimbriae were previously shown to be more virulent than other types. In this study, we further examined the host toxicity mediated by P. gulae fimbriae by constructing recombinant FimA (rFimA) expression vectors for each genotype and raised antibodies to the purified proteins. Methods and Results: All larvae died within 204 h following infection with P. gulae type C at the low-dose infection, whereas type A and B did not. Among fimA types, the survival rates of the larvae injected with rFimA type C were remarkably decreased, while the survival rates of the larvae injected with rFimA type A and type B were greater than 50%. Clindamycin treatment inhibited the growth of type C strains in a dose-dependent manner, resulting in an increased rate of silkworm survival. Finally, type C rFimA-specific antiserum prolonged the survival of silkworm larvae stimulated by infection with P. gulae type C strain or injection of rFimA type C protein. Conclusion: These results suggested that type C fimbriae have high potential for enhancement of bacterial pathogenesis, and that both clindamycin and anti-type C rFimA-specific antibodies are potent inhibitors of type C fimbriae-induced toxicity. This is the first report to establish a silkworm infection model using P. gulae for toxicity assessment.

Keywords: Fimbriae; Porphyromonas gulae; genotypes; silkworm larvae.

Figure 1.

Expression of recombinant FimA in Escherichia coli BL21. (a) Coomassie blue staining of purified GST-rFimA fusion proteins separated by SDS-PAGE. Lane 1, rFimA type A, Lane 2, rFimA type B, Lane 3, rFimA type C. (b) Western blot analysis using each anti-rFimA antibody. Lane 1, rFimA type A; Lane 2, rFimA type B; Lane 3, rFimA type C. M: molecular marker

Figure 2.

Virulence of P. gulae infection in silkworm larvae. larvae were injected with 10 ml suspension of P. gulaeat. low dose (A; 1 × 10⁷ CFU) or high dose (B; 5 × 10⁷ CFU) and incubated at 37°C. The survival rate was recorded at the time points indicated. PBS was used as a negative control. Data are representative of three independent experiments

Figure 3.

Effect of P. gulae rFimA protein injection on silkworm larvae. A total of 50 µl (5 µg) of each rFimA protein was injected into larvae and incubated at 37°C. The survival rate was recorded at the time points indicated. PBS was used as a negative control. Data are representative of three independent experiments. Survival rates in the silkworm larvae in each group were evaluated with a Kaplan–Meier plot, which was analyzed by a log-rank test (*P < 0.001)

Figure 4.

Inhibitory effects of antibiotics on the growth of P. gulae. P. gulae (5 × 10⁷ CFU) was cultured in trypticase soy broth TSB and an antibiotic at the concentrations indicated for 24 h. after incubation, bacterial growth was measured using a microplate reader. Data are expressed as the relative ratio of treated/untreated cultures and represent means ± SD from three independent experiments analyzed with a t-test. *P < 0.05; **P < 0.01; ***P < 0.001

Figure 5.

Effect of clindamycin on P. gulae D049 (type C) infection of silkworm larvae. Larvae (n = 10) were injected with a 50-µl suspension of P. gulae (5 × 10⁷ CFU) and a 50-μl clindamycin solution (0.4 μg/ml) and incubated at 37°C. The survival was recorded for the time indicated. PBS was used as a negative control. Data are representative of three independent experiments. Survival rates in the silkworm larvae in each group were evaluated with a Kaplan-Meier plot, which was analyzed by a log-rank test. *P < 0.001

Figure 6.

Effect of clindamycin on infection of silkworm larvae with different P. gulae strains. Larvae (n = 10) were injected with a 50-μl of P. gulae suspensions (5 × 10⁷ CFU) (a) strain ATCC51700 (type A), (b) strain D040 (type B), or (c) strain D049 (type C), and a 50-μl solution of 0.4 μg/ml clindamycin, and incubated at 37°C. The survival rate was recorded for the time points indicated. PBS was used as a negative control. Data are representative of three independent experiments. Survival rates in the silkworm larvae in each group were evaluated with a Kaplan-Meier plot, which was analyzed by a log-rank test. *P < 0.05; **P < 0.01

Figure 7.

Effects of rFimA type C antisera on survival of silkworm larvae injected with rFimA proteins. Silkworm larvae (n = 10) were injected with 50 μl of each rFimA protein (5 μg each of type A, B and C) and anti-rFimA type C antisera, and incubated at 37°C. The survival rate was recorded for the time points indicated. Data are representative of three independent experiments. Survival rates in the silkworm larvae in each group were evaluated with a Kaplan-Meier plot, which was analyzed by a log-rank test. *P < 0.05; **P < 0.01

Figure 8.

Effects of antisera raised against each rFimA protein type on silkworm larvae injected with the corresponding rFimA protein. Larvae (n = 10) were injected with 5 μg in 50 μl of type A (a), type B (b) or type C (c) rFimA protein, with without the corresponding anti-rFimA antisera (indicated concentrations), and incubated at 37°C. The survival rate was recorded for the time points indicated. PBS was used as a negative control. Data are representative of three independent experiments. Survival rates in the silkworm larvae in each group were evaluated with a Kaplan-Meier plot, which was analyzed by a log-rank test. *P < 0.05; **P < 0.01

Figure 9.

Effect of antisera raised against each rFimA protein type on silkworm larvae infected with P. gulae strains. Larvae (n = 10) were injected with a 50 μl suspension of P. gulae (5 × 10⁷ CFU) type A strain ATCC51700 (a), type B strain D040 (b), or type C strain (c), and were incubated at 37°C. The survival rate was recorded at the time points indicated. PBS was used as a negative control. Data are representative of three independent experiments. Survival rates in the silkworm larvae in each group were evaluated with a Kaplan-Meier plot, which was analyzed by a log-rank test. *P < 0.001