Effect of Vitamin A on Listeria monocytogenes Infection in a Silkworm Model

Abstract

Insect infection models have been used increasingly to study various pathogenic agents in evaluations of pathogenicity and drug efficacy. In this study, we demonstrated that larvae of the silkworm Bombyx mori are useful for studying Listeria monocytogenes infections in insects. Infection with the L. monocytogenes wild-type strain induced silkworm death. Infection by a listeriolysin O (LLO) deletion mutant also induced silkworm death, but the bacterial numbers in silkworms were lower than those of the wild-type strain. Intracellular growth was observed when the silkworm ovary-derived cell line BmN4 was infected with the wild-type strain. Explosive replication was not observed in BmN4 cells infected with the LLO mutant and the bacterial numbers of the LLO mutant were lower than those of the wild-type strain. Pretreatment with vitamin A did not affect silkworm mortality after bacterial infection, but the efficiency of infecting the hemocytes and BmN4 cells was decreased with vitamin A treatment. Our results indicate that silkworm larvae are a useful insect infection model for L. monocytogenes and that vitamin A has protective effects against bacterial infection in silkworms.

Fig 1. Infection of silkworms with L. monocytogenes.

(A) Survival rate of silkworms. Fifteen insects per group were infected and monitored to determine the time required to kill 50% of the silkworms. Approximately 10⁴ bacteria belonging to the wild-type strain (WT, circles), LLO deletion mutant (Δhly, triangles), and LLO-complemented strain (Δhly::hly, squares) were sufficient to kill 50% (LD50) of the silkworms within 4 and 5 days. Significant differences were accepted at P < 0.05 and they are indicated by asterisks (*). (B) The bacterial numbers of each strain detected in silkworms at 48 h post inoculation. The data represent averages based on triplicate samples from three identical experiments and the error bars represent the standard error of the mean (SEM) (n = 9). Significant differences were accepted at P < 0.05 and they are indicated by asterisks (*). (C) Hemocytes (red) containing L. monocytogenes strains (green, arrowheads) were observed by confocal laser scanning microscopy at 1 h and 24 h post inoculation. Scale bar represents 10 μm. (D) Immunoblot analysis of LLO expression. Samples were prepared from the wild-type strain, LLO deletion mutant, and LLO-complemented strain cultivated at 37°C or 25°C in BHI broth.

Fig 2. Intracellular replication of L. monocytogenes in BmN4 cells.

(A) L. monocytogenes replicates intracellularly in BmN4 cells. The silkworm ovary-derived cell line BmN4 was infected with the wild-type strain (WT), LLO deletion mutant (Δhly), and LLO-complemented strain (Δhly::hly) at an MOI of 10. Bacterial invasion and intracellular replication by each strain were measured at 3 h and 24 h after infection. The data represent averages based on triplicate samples from three identical experiments and the error bars represent the standard deviations. Significant differences were accepted at P < 0.05 and they are indicated by asterisks (*). (B) BmN4 cells (red) containing L. monocytogenes strains (green, white arrowheads) were observed by confocal laser scanning microscopy at 3 h and 24 h post inoculation. Scale bar represents 20 μm. Explosive bacterial replication is indicated by open arrowheads. The experiment was replicated three times independently.

Fig 3. Infection of L. monocytogenes to silkworm pretreated with vitamin A.

(A) Survival of infected silkworms after previous supplementation with vitamin A. Approximately 10⁴ bacteria of the wild-type strain were used to infect silkworms pretreated with (open circles) or without vitamin A (black circles). Groups of five insects per group were checked daily for survival. Survival was observed until 8 days post infection. (B) Bacterial numbers of the wild-type strain in silkworms pretreated with (black bars) or without vitamin A (open bars) until 48 h post inoculation. The data represent averages based on triplicate samples from three identical experiments and the error bars represent the SEM (n = 9). Significant differences were accepted at P < 0.05 and they are indicated by asterisks (*). (C) Hemocytes (red) containing L. monocytogenes strains (green, arrowheads) isolated from silkworm pretreated with or without vitamin A were observed by confocal laser scanning microscopy at 3 h and 24 h post inoculation. Scale bar represents 10 μm.

Fig 4. L. monocytogenes infection of BmN4 cells after pretreatment with vitamin A.

(A) Pretreatment with vitamin A inhibited L. monocytogenes infection in BmN4 cells. BmN4 was infected with the wild-type strain at a multiplicity of infection of 10. Bacterial invasion and intracellular replication were measured at 3 h and 24 h after infection in BmN4 cells at the indicated vitamin A concentrations (mg/ml). The data represent averages based on triplicate samples from three identical experiments and the error bars represent the standard deviations. Significant differences were accepted at P < 0.05 and they are indicated by asterisks (*). (B) BmN4 cells (red) containing L. monocytogenes strains (green, white arrowheads) were observed by confocal laser scanning microscopy at 3 h and 24 h post inoculation. BmN4 cells were pretreated with (11.25 mg/ml) or without vitamin A. Scale bar represents 20 μm. Explosive bacterial replication is indicated by open arrowheads. Filopodia-like structure are indicated by arrows. The experiment was replicated three times independently.