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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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DNA sequencing analysis of the pdpC gene.(A) Mixtures of pdpC1 and pdpC2 DNAs amplified from the SCHU P0 (upper panel), P5 (middle panel), and P9 (lower panel) genomes were directly sequenced using Sanger’s method. The sequencing electropherograms of a region of pdpC from positions 2,031 to 2,056 are shown. (B) pdpC1 (left panel) and pdpC2 (right panel) were individually amplified by the long PCR method, and the amplicons were sequenced directly. The A tract found in the electropherograms is indicated as A8 or A9, as appropriate. (C) Deduced encoded amino acid sequences of PdpC2 from positions 661–720 obtained from SCHU P0, P5, and P9 were aligned. The asterisks and hyphens indicate the identical and untranslated amino acids, respectively. (D) Schematic representation of the structures of the pdpC1 and pdpC2 of Francisella tularensis SCHU S4, SCHU P0, SCHU P5, and SCHU P9. The translated and untranslated regions are indicated by black and white, respectively. The stop codons created by frameshift are indicated by arrowheads. (E) SCHU P0, P5, and P9 were grown in CDM and the lysates were subjected to western blot analyses using anti-PdpC polyclonal antibody [20]. Intact PdpC was observed only in SCHU P9. Bands corresponding to the expected sizes of the intact or truncated PdpCs are indicated by gray and black arrowheads, respectively.
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pone-0089075-g003: DNA sequencing analysis of the pdpC gene.(A) Mixtures of pdpC1 and pdpC2 DNAs amplified from the SCHU P0 (upper panel), P5 (middle panel), and P9 (lower panel) genomes were directly sequenced using Sanger’s method. The sequencing electropherograms of a region of pdpC from positions 2,031 to 2,056 are shown. (B) pdpC1 (left panel) and pdpC2 (right panel) were individually amplified by the long PCR method, and the amplicons were sequenced directly. The A tract found in the electropherograms is indicated as A8 or A9, as appropriate. (C) Deduced encoded amino acid sequences of PdpC2 from positions 661–720 obtained from SCHU P0, P5, and P9 were aligned. The asterisks and hyphens indicate the identical and untranslated amino acids, respectively. (D) Schematic representation of the structures of the pdpC1 and pdpC2 of Francisella tularensis SCHU S4, SCHU P0, SCHU P5, and SCHU P9. The translated and untranslated regions are indicated by black and white, respectively. The stop codons created by frameshift are indicated by arrowheads. (E) SCHU P0, P5, and P9 were grown in CDM and the lysates were subjected to western blot analyses using anti-PdpC polyclonal antibody [20]. Intact PdpC was observed only in SCHU P9. Bands corresponding to the expected sizes of the intact or truncated PdpCs are indicated by gray and black arrowheads, respectively.

Mentions: We found 35 mutations in the SCHU P0 genome compared with the SCHU S4 genome. Because nucleotide substitutions found at 29 positions in the SCHU P0 sequence were shared by the SCHU P5 and P9 strains, these substitutions may not contribute to the increased virulence of SCHU P9 (Table S3). Among six other mutations, a nucleotide deletion in the SCHU P0 pdpC was also found in SCHU P5 (Table S4). In the pdpC of SCHU P0 and P5, an adenine (A) residue was missing from a 9-A residue stretch observed in SCHU S4. However, the pdpC of SCHU P9 appeared to comprise two different molecular species with an 8-A and a 9-A stretch, respectively. The read depths of these two species were 218 and 304, indicating that the molar ratio of these two species was nearly 1∶1 in SCHU P9 genome. This was confirmed by sequencing PCR amplicons of the pdpC using Sanger’s method (Fig. 3A). The data clearly show eight consecutive A residues followed by a thymidine (T) residue in the pdpC of SCHU P0 and P5 (Fig. 3A, upper and middle electropherograms), whereas a mixture of A and T was found at the same position in the SCHU P9 sequence (Fig. 3A, lower electropherogram).


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)

DNA sequencing analysis of the pdpC gene.(A) Mixtures of pdpC1 and pdpC2 DNAs amplified from the SCHU P0 (upper panel), P5 (middle panel), and P9 (lower panel) genomes were directly sequenced using Sanger’s method. The sequencing electropherograms of a region of pdpC from positions 2,031 to 2,056 are shown. (B) pdpC1 (left panel) and pdpC2 (right panel) were individually amplified by the long PCR method, and the amplicons were sequenced directly. The A tract found in the electropherograms is indicated as A8 or A9, as appropriate. (C) Deduced encoded amino acid sequences of PdpC2 from positions 661–720 obtained from SCHU P0, P5, and P9 were aligned. The asterisks and hyphens indicate the identical and untranslated amino acids, respectively. (D) Schematic representation of the structures of the pdpC1 and pdpC2 of Francisella tularensis SCHU S4, SCHU P0, SCHU P5, and SCHU P9. The translated and untranslated regions are indicated by black and white, respectively. The stop codons created by frameshift are indicated by arrowheads. (E) SCHU P0, P5, and P9 were grown in CDM and the lysates were subjected to western blot analyses using anti-PdpC polyclonal antibody [20]. Intact PdpC was observed only in SCHU P9. Bands corresponding to the expected sizes of the intact or truncated PdpCs are indicated by gray and black arrowheads, respectively.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0089075-g003: DNA sequencing analysis of the pdpC gene.(A) Mixtures of pdpC1 and pdpC2 DNAs amplified from the SCHU P0 (upper panel), P5 (middle panel), and P9 (lower panel) genomes were directly sequenced using Sanger’s method. The sequencing electropherograms of a region of pdpC from positions 2,031 to 2,056 are shown. (B) pdpC1 (left panel) and pdpC2 (right panel) were individually amplified by the long PCR method, and the amplicons were sequenced directly. The A tract found in the electropherograms is indicated as A8 or A9, as appropriate. (C) Deduced encoded amino acid sequences of PdpC2 from positions 661–720 obtained from SCHU P0, P5, and P9 were aligned. The asterisks and hyphens indicate the identical and untranslated amino acids, respectively. (D) Schematic representation of the structures of the pdpC1 and pdpC2 of Francisella tularensis SCHU S4, SCHU P0, SCHU P5, and SCHU P9. The translated and untranslated regions are indicated by black and white, respectively. The stop codons created by frameshift are indicated by arrowheads. (E) SCHU P0, P5, and P9 were grown in CDM and the lysates were subjected to western blot analyses using anti-PdpC polyclonal antibody [20]. Intact PdpC was observed only in SCHU P9. Bands corresponding to the expected sizes of the intact or truncated PdpCs are indicated by gray and black arrowheads, respectively.
Mentions: We found 35 mutations in the SCHU P0 genome compared with the SCHU S4 genome. Because nucleotide substitutions found at 29 positions in the SCHU P0 sequence were shared by the SCHU P5 and P9 strains, these substitutions may not contribute to the increased virulence of SCHU P9 (Table S3). Among six other mutations, a nucleotide deletion in the SCHU P0 pdpC was also found in SCHU P5 (Table S4). In the pdpC of SCHU P0 and P5, an adenine (A) residue was missing from a 9-A residue stretch observed in SCHU S4. However, the pdpC of SCHU P9 appeared to comprise two different molecular species with an 8-A and a 9-A stretch, respectively. The read depths of these two species were 218 and 304, indicating that the molar ratio of these two species was nearly 1∶1 in SCHU P9 genome. This was confirmed by sequencing PCR amplicons of the pdpC using Sanger’s method (Fig. 3A). The data clearly show eight consecutive A residues followed by a thymidine (T) residue in the pdpC of SCHU P0 and P5 (Fig. 3A, upper and middle electropherograms), whereas a mixture of A and T was found at the same position in the SCHU P9 sequence (Fig. 3A, lower electropherogram).

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