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Role of pathogenicity determinant protein C (PdpC) in determining the virulence of the Francisella tularensis subspecies tularensis SCHU.

Uda A, Sekizuka T, Tanabayashi K, Fujita O, Kuroda M, Hotta A, Sugiura N, Sharma N, Morikawa S, Yamada A - PLoS ONE (2014)

Bottom Line: Moreover, SCHU P9 grew more efficiently in J774.1 murine macrophages compared with that in the less pathogenic SCHU P0 and P5.To validate the pathogenicity of PdpC, both copies of the pdpC gene in SCHU P9 have been inactivated by Targetron mutagenesis.These results demonstrate that PdpC is crucial in determining the virulence of F. tularensis SCHU.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterinary Science, National Institute of Infectious Diseases, Shinjuku, Tokyo, Japan.

ABSTRACT
Francisella tularensis subspecies tularensis, the etiological agent of tularemia, is highly pathogenic to humans and animals. However, the SCHU strain of F. tularensis SCHU P0 maintained by passaging in artificial media has been found to be attenuated. To better understand the molecular mechanisms behind the pathogenicity of F. tularensis SCHU, we attempted to isolate virulent bacteria by serial passages in mice. SCHU P5 obtained after 5th passages in mice remained avirulent, while SCHU P9 obtained after 9th passages was completely virulent in mice. Moreover, SCHU P9 grew more efficiently in J774.1 murine macrophages compared with that in the less pathogenic SCHU P0 and P5. Comparison of the nucleotide sequences of the whole genomes of SCHU P0, P5, and P9 revealed only 1 nucleotide difference among P0, P5 and P9 in 1 of the 2 copies of pathogenicity determinant protein C (pdpC) gene. An adenine residue deletion was observed in the pdpC1 gene of SCHU P0, P5, and P9 and in the pdpC2 gene of SCHU P0, and P5, while P9 was characterized by the wild type pdpC2 gene. Thus, SCHU P0 and P5 expressed only truncated forms of PdpC protein, while SCHU P9 expressed both wild type and truncated versions. To validate the pathogenicity of PdpC, both copies of the pdpC gene in SCHU P9 have been inactivated by Targetron mutagenesis. SCHU P9 mutants with inactivated pdpC gene showed low intracellular growth in J774.1 cells and did not induce severe disease in experimentally infected mice, while virulence of the mutants was restored by complementation with expression of the intact PdpC. These results demonstrate that PdpC is crucial in determining the virulence of F. tularensis SCHU.

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Pathogenicity of Francisella strains in mice.(A) The scheme for SCHU P0, P5, and P9 isolation. Three C57BL/6J mice were inoculated intraperitoneally with 5×106 CFU ml−1 of SCHU P0 suspension and mice were sacrificed 4 days after infection. Three mice spleens obtained from the infected mice were homogenized. One hundred micro litter of 10% spleen homogenate was plated on chocolate II agar, and cultured for 3 days. After cultivation, the bacteria grown confluently on the plate were suspended with 1 ml of chemically defined medium (CDM). Subsequent passages were performed similarly. When mice demonstrated severe clinical signs or >20% weight loss, they were humanely sacrificed by isoflurane inhalation. If the mice did not show apparent clinical signs, they were sacrificed 4 days after infection. The inoculation dose and CFU in spleen at 4 days post-inoculation in each passage were shown in top table. Spleen homogenates prepared at the 5th and 9th passages were cultured on chocolate II agar. Single colonies were then isolated and designated as SCHU P5 and P9, respectively. Bacterial stocks were prepared by cultivating respective strains in CDM at 37°C for 24 h and stored in CDM containing 10% glycerol at −80°C until use. (B–E) Four C57BL/6J mice from each group were intraperitoneally infected with 1×106 (B, D) or 1×103 (C, E) CFU of the SCHU P0 (circle), P5 (diamond), or P9 (square) strains. Their survival rates (B, C) and body weights (D, E) were measured and observed for 14 days. Survival rates and mean ± standard error of the mean (SEM) of percentage body weights are shown in the upper and lower panels, respectively. (F–J) Three C57BL/6J mice in each group were intranasally inoculated with 102 CFU SCHU P9 (circle), 106 CFU SCHU P9 (diamond), and 106 CFU SCHU P5 (square). Their survival rates (F) and body weights (G) were measured and observed for 18 days. Mice were sacrificed at the indicated days post-inoculation, and then the averages ± SEM of bacterial CFU in lungs (H), spleens (I), and livers (J) were shown. The data in CFU of SCHU P9 106 CFU at 4 dpi were defected because all the mice were sacrificed within 3 dpi.
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pone-0089075-g001: Pathogenicity of Francisella strains in mice.(A) The scheme for SCHU P0, P5, and P9 isolation. Three C57BL/6J mice were inoculated intraperitoneally with 5×106 CFU ml−1 of SCHU P0 suspension and mice were sacrificed 4 days after infection. Three mice spleens obtained from the infected mice were homogenized. One hundred micro litter of 10% spleen homogenate was plated on chocolate II agar, and cultured for 3 days. After cultivation, the bacteria grown confluently on the plate were suspended with 1 ml of chemically defined medium (CDM). Subsequent passages were performed similarly. When mice demonstrated severe clinical signs or >20% weight loss, they were humanely sacrificed by isoflurane inhalation. If the mice did not show apparent clinical signs, they were sacrificed 4 days after infection. The inoculation dose and CFU in spleen at 4 days post-inoculation in each passage were shown in top table. Spleen homogenates prepared at the 5th and 9th passages were cultured on chocolate II agar. Single colonies were then isolated and designated as SCHU P5 and P9, respectively. Bacterial stocks were prepared by cultivating respective strains in CDM at 37°C for 24 h and stored in CDM containing 10% glycerol at −80°C until use. (B–E) Four C57BL/6J mice from each group were intraperitoneally infected with 1×106 (B, D) or 1×103 (C, E) CFU of the SCHU P0 (circle), P5 (diamond), or P9 (square) strains. Their survival rates (B, C) and body weights (D, E) were measured and observed for 14 days. Survival rates and mean ± standard error of the mean (SEM) of percentage body weights are shown in the upper and lower panels, respectively. (F–J) Three C57BL/6J mice in each group were intranasally inoculated with 102 CFU SCHU P9 (circle), 106 CFU SCHU P9 (diamond), and 106 CFU SCHU P5 (square). Their survival rates (F) and body weights (G) were measured and observed for 18 days. Mice were sacrificed at the indicated days post-inoculation, and then the averages ± SEM of bacterial CFU in lungs (H), spleens (I), and livers (J) were shown. The data in CFU of SCHU P9 106 CFU at 4 dpi were defected because all the mice were sacrificed within 3 dpi.

Mentions: Figure 1A illustrates the scheme for SCHU P0, P5, and P9 isolation. SCHU P0 was cultured to confluence for 3 days on chocolate II agar (Becton Dickinson and Co., Cockeysville, MD) from its glycerol stock and resuspended in saline. Three C57BL/6J mice were inoculated intraperitoneally with 5×106 CFU ml−1 of SCHU suspension and mice were sacrificed 4 days after infection. Three spleens obtained from the infected mice were homogenized. One hundred microlitter of 10% spleen homogenate was plated on chocolate II agar, and cultured for 3 days. After cultivation, the bacteria grown confluently on the plate were suspended with 1 ml of chemically defined medium (CDM). Subsequent passages were performed similarly. When mice demonstrated severe clinical signs or >20% weight loss, they were humanely sacrificed by isoflurane inhalation. If the mice did not show apparent clinical signs, they were sacrificed 4 days after infection. The inoculation dose and CFU in spleen at 4 days post-inoculation in each passage are shown in figure 1A. Spleen homogenates prepared at the 5th and 9th passages were cultured on chocolate II agar. Single colonies were then isolated and designated as SCHU P5 and P9, respectively. Bacterial stocks were prepared by cultivating respective strains in CDM at 37°C for 24 h and stored in CDM containing 10% glycerol at −80°C until use.


Role of pathogenicity determinant protein C (PdpC) in determining the virulence of the Francisella tularensis subspecies tularensis SCHU.

Uda A, Sekizuka T, Tanabayashi K, Fujita O, Kuroda M, Hotta A, Sugiura N, Sharma N, Morikawa S, Yamada A - PLoS ONE (2014)

Pathogenicity of Francisella strains in mice.(A) The scheme for SCHU P0, P5, and P9 isolation. Three C57BL/6J mice were inoculated intraperitoneally with 5×106 CFU ml−1 of SCHU P0 suspension and mice were sacrificed 4 days after infection. Three mice spleens obtained from the infected mice were homogenized. One hundred micro litter of 10% spleen homogenate was plated on chocolate II agar, and cultured for 3 days. After cultivation, the bacteria grown confluently on the plate were suspended with 1 ml of chemically defined medium (CDM). Subsequent passages were performed similarly. When mice demonstrated severe clinical signs or >20% weight loss, they were humanely sacrificed by isoflurane inhalation. If the mice did not show apparent clinical signs, they were sacrificed 4 days after infection. The inoculation dose and CFU in spleen at 4 days post-inoculation in each passage were shown in top table. Spleen homogenates prepared at the 5th and 9th passages were cultured on chocolate II agar. Single colonies were then isolated and designated as SCHU P5 and P9, respectively. Bacterial stocks were prepared by cultivating respective strains in CDM at 37°C for 24 h and stored in CDM containing 10% glycerol at −80°C until use. (B–E) Four C57BL/6J mice from each group were intraperitoneally infected with 1×106 (B, D) or 1×103 (C, E) CFU of the SCHU P0 (circle), P5 (diamond), or P9 (square) strains. Their survival rates (B, C) and body weights (D, E) were measured and observed for 14 days. Survival rates and mean ± standard error of the mean (SEM) of percentage body weights are shown in the upper and lower panels, respectively. (F–J) Three C57BL/6J mice in each group were intranasally inoculated with 102 CFU SCHU P9 (circle), 106 CFU SCHU P9 (diamond), and 106 CFU SCHU P5 (square). Their survival rates (F) and body weights (G) were measured and observed for 18 days. Mice were sacrificed at the indicated days post-inoculation, and then the averages ± SEM of bacterial CFU in lungs (H), spleens (I), and livers (J) were shown. The data in CFU of SCHU P9 106 CFU at 4 dpi were defected because all the mice were sacrificed within 3 dpi.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3928404&req=5

pone-0089075-g001: Pathogenicity of Francisella strains in mice.(A) The scheme for SCHU P0, P5, and P9 isolation. Three C57BL/6J mice were inoculated intraperitoneally with 5×106 CFU ml−1 of SCHU P0 suspension and mice were sacrificed 4 days after infection. Three mice spleens obtained from the infected mice were homogenized. One hundred micro litter of 10% spleen homogenate was plated on chocolate II agar, and cultured for 3 days. After cultivation, the bacteria grown confluently on the plate were suspended with 1 ml of chemically defined medium (CDM). Subsequent passages were performed similarly. When mice demonstrated severe clinical signs or >20% weight loss, they were humanely sacrificed by isoflurane inhalation. If the mice did not show apparent clinical signs, they were sacrificed 4 days after infection. The inoculation dose and CFU in spleen at 4 days post-inoculation in each passage were shown in top table. Spleen homogenates prepared at the 5th and 9th passages were cultured on chocolate II agar. Single colonies were then isolated and designated as SCHU P5 and P9, respectively. Bacterial stocks were prepared by cultivating respective strains in CDM at 37°C for 24 h and stored in CDM containing 10% glycerol at −80°C until use. (B–E) Four C57BL/6J mice from each group were intraperitoneally infected with 1×106 (B, D) or 1×103 (C, E) CFU of the SCHU P0 (circle), P5 (diamond), or P9 (square) strains. Their survival rates (B, C) and body weights (D, E) were measured and observed for 14 days. Survival rates and mean ± standard error of the mean (SEM) of percentage body weights are shown in the upper and lower panels, respectively. (F–J) Three C57BL/6J mice in each group were intranasally inoculated with 102 CFU SCHU P9 (circle), 106 CFU SCHU P9 (diamond), and 106 CFU SCHU P5 (square). Their survival rates (F) and body weights (G) were measured and observed for 18 days. Mice were sacrificed at the indicated days post-inoculation, and then the averages ± SEM of bacterial CFU in lungs (H), spleens (I), and livers (J) were shown. The data in CFU of SCHU P9 106 CFU at 4 dpi were defected because all the mice were sacrificed within 3 dpi.
Mentions: Figure 1A illustrates the scheme for SCHU P0, P5, and P9 isolation. SCHU P0 was cultured to confluence for 3 days on chocolate II agar (Becton Dickinson and Co., Cockeysville, MD) from its glycerol stock and resuspended in saline. Three C57BL/6J mice were inoculated intraperitoneally with 5×106 CFU ml−1 of SCHU suspension and mice were sacrificed 4 days after infection. Three spleens obtained from the infected mice were homogenized. One hundred microlitter of 10% spleen homogenate was plated on chocolate II agar, and cultured for 3 days. After cultivation, the bacteria grown confluently on the plate were suspended with 1 ml of chemically defined medium (CDM). Subsequent passages were performed similarly. When mice demonstrated severe clinical signs or >20% weight loss, they were humanely sacrificed by isoflurane inhalation. If the mice did not show apparent clinical signs, they were sacrificed 4 days after infection. The inoculation dose and CFU in spleen at 4 days post-inoculation in each passage are shown in figure 1A. Spleen homogenates prepared at the 5th and 9th passages were cultured on chocolate II agar. Single colonies were then isolated and designated as SCHU P5 and P9, respectively. Bacterial stocks were prepared by cultivating respective strains in CDM at 37°C for 24 h and stored in CDM containing 10% glycerol at −80°C until use.

Bottom Line: Moreover, SCHU P9 grew more efficiently in J774.1 murine macrophages compared with that in the less pathogenic SCHU P0 and P5.To validate the pathogenicity of PdpC, both copies of the pdpC gene in SCHU P9 have been inactivated by Targetron mutagenesis.These results demonstrate that PdpC is crucial in determining the virulence of F. tularensis SCHU.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterinary Science, National Institute of Infectious Diseases, Shinjuku, Tokyo, Japan.

ABSTRACT
Francisella tularensis subspecies tularensis, the etiological agent of tularemia, is highly pathogenic to humans and animals. However, the SCHU strain of F. tularensis SCHU P0 maintained by passaging in artificial media has been found to be attenuated. To better understand the molecular mechanisms behind the pathogenicity of F. tularensis SCHU, we attempted to isolate virulent bacteria by serial passages in mice. SCHU P5 obtained after 5th passages in mice remained avirulent, while SCHU P9 obtained after 9th passages was completely virulent in mice. Moreover, SCHU P9 grew more efficiently in J774.1 murine macrophages compared with that in the less pathogenic SCHU P0 and P5. Comparison of the nucleotide sequences of the whole genomes of SCHU P0, P5, and P9 revealed only 1 nucleotide difference among P0, P5 and P9 in 1 of the 2 copies of pathogenicity determinant protein C (pdpC) gene. An adenine residue deletion was observed in the pdpC1 gene of SCHU P0, P5, and P9 and in the pdpC2 gene of SCHU P0, and P5, while P9 was characterized by the wild type pdpC2 gene. Thus, SCHU P0 and P5 expressed only truncated forms of PdpC protein, while SCHU P9 expressed both wild type and truncated versions. To validate the pathogenicity of PdpC, both copies of the pdpC gene in SCHU P9 have been inactivated by Targetron mutagenesis. SCHU P9 mutants with inactivated pdpC gene showed low intracellular growth in J774.1 cells and did not induce severe disease in experimentally infected mice, while virulence of the mutants was restored by complementation with expression of the intact PdpC. These results demonstrate that PdpC is crucial in determining the virulence of F. tularensis SCHU.

Show MeSH
Related in: MedlinePlus