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A Yersinia pestis tat mutant is attenuated in bubonic and small-aerosol pneumonic challenge models of infection but not as attenuated by intranasal challenge.

Bozue J, Cote CK, Chance T, Kugelman J, Kern SJ, Kijek TK, Jenkins A, Mou S, Moody K, Fritz D, Robinson CG, Bell T, Worsham P - PLoS ONE (2014)

Bottom Line: Deletion of the tatA gene resulted in several consequences for the mutant as compared to wild-type.In contrast, when mice were challenged intranasally with the mutant, very little difference in the LD50 was observed between wild-type and mutant strains.Collectively, these findings demonstrate an essential role for the Tat pathway in the virulence of Y. pestis in bubonic and small-aerosol pneumonic infection but less important role for intranasal challenge.

View Article: PubMed Central - PubMed

Affiliation: Bacteriology Division, The United States Army of Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland, United States of America.

ABSTRACT
Bacterial proteins destined for the Tat pathway are folded before crossing the inner membrane and are typically identified by an N-terminal signal peptide containing a twin arginine motif. Translocation by the Tat pathway is dependent on the products of genes which encode proteins possessing the binding site of the signal peptide and mediating the actual translocation event. In the fully virulent CO92 strain of Yersinia pestis, the tatA gene was deleted. The mutant was assayed for loss of virulence through various in vitro and in vivo assays. Deletion of the tatA gene resulted in several consequences for the mutant as compared to wild-type. Cell morphology of the mutant bacteria was altered and demonstrated a more elongated form. In addition, while cultures of the mutant strain were able to produce a biofilm, we observed a loss of adhesion of the mutant biofilm structure compared to the biofilm produced by the wild-type strain. Immuno-electron microscopy revealed a partial disruption of the F1 antigen on the surface of the mutant. The virulence of the ΔtatA mutant was assessed in various murine models of plague. The mutant was severely attenuated in the bubonic model with full virulence restored by complementation with the native gene. After small-particle aerosol challenge in a pneumonic model of infection, the mutant was also shown to be attenuated. In contrast, when mice were challenged intranasally with the mutant, very little difference in the LD50 was observed between wild-type and mutant strains. However, an increased time-to-death and delay in bacterial dissemination was observed in mice infected with the ΔtatA mutant as compared to the parent strain. Collectively, these findings demonstrate an essential role for the Tat pathway in the virulence of Y. pestis in bubonic and small-aerosol pneumonic infection but less important role for intranasal challenge.

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Analysis of Y. pestis proteins.A) Strains (wild-type, lanes 1 and 4; ΔtatA, lanes 2 and 5, and C12, lanes 3 and 6) were grown in HI broth at 28°C or 37°C containing CaCl2, as indicated. Proteins were extracted and ran on a SDS-PAGE gel at equal concentrations. The arrow indicates a protein band that is synthesized at 37°C for the CO92 and to a lesser extent the ΔtatA mutant but not in C12, a F1-antigen mutant strain. Molecular masses are indicated on the left in KDa. Strains were grown in HI broth at 37°C containing CaCl2 and proteins extracted for Western analysis. Equal concentrations of proteins from Y. pestis strains were blotted with a monoclonal antibody to either the F1 antigen (B) or GroEL (C). Lanes: 1, wild-type; 2, ΔtatA; 3, C12; and 4, recombinant protein (F1 antigen or GroEL, respectively). Molecular masses are indicated on the left in KDa. D) Strains were grown in HI broth at 37°C in the presence of MOX to create low calcium conditions. Equal concentrations of proteins from Y. pestis strains were blotted with an antibody to LcrV Lanes: 1, CO92 wild-type; 2, ΔtatA; 3, Y. pestis pLcr-; and 4, rLcrV protein. Molecular masses are indicated on the left in KDa.
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pone-0104524-g004: Analysis of Y. pestis proteins.A) Strains (wild-type, lanes 1 and 4; ΔtatA, lanes 2 and 5, and C12, lanes 3 and 6) were grown in HI broth at 28°C or 37°C containing CaCl2, as indicated. Proteins were extracted and ran on a SDS-PAGE gel at equal concentrations. The arrow indicates a protein band that is synthesized at 37°C for the CO92 and to a lesser extent the ΔtatA mutant but not in C12, a F1-antigen mutant strain. Molecular masses are indicated on the left in KDa. Strains were grown in HI broth at 37°C containing CaCl2 and proteins extracted for Western analysis. Equal concentrations of proteins from Y. pestis strains were blotted with a monoclonal antibody to either the F1 antigen (B) or GroEL (C). Lanes: 1, wild-type; 2, ΔtatA; 3, C12; and 4, recombinant protein (F1 antigen or GroEL, respectively). Molecular masses are indicated on the left in KDa. D) Strains were grown in HI broth at 37°C in the presence of MOX to create low calcium conditions. Equal concentrations of proteins from Y. pestis strains were blotted with an antibody to LcrV Lanes: 1, CO92 wild-type; 2, ΔtatA; 3, Y. pestis pLcr-; and 4, rLcrV protein. Molecular masses are indicated on the left in KDa.

Mentions: As the deletion of the tatA gene would affect translocated proteins, we examined proteins extracted from both the cell pellet and supernatant fractions of mutant and wild-type bacteria grown in HI broth at 28°C or 37°C containing CaCl2. As shown in Fig. 4A, the proteins collected from pellets of Y. pestis grown at 28°C were separated on a 10% Bis-Tris gels, but no obvious differences were observed in the protein profile between wild-type, ΔtatA, or C12 (F1 antigen/caf1 mutant) strains. However, when the protein pellets were examined from Y. pestis grown at 37°C under high calcium conditions, an intense band at <19 kDa appeared for wild-type CO92 which was not present for the 28°C samples. For proteins extracted for the ΔtatA pellet, this band was also present but not as intense as observed for the wild-type strain. We presumed that this band corresponded to the F1 antigen protein encoded by the caf1 gene which is expressed by Y. pestis at 37°C and has a molecular mass of 15.5 kDa [48]–[50]. Further demonstrating that this band corresponded to the F1 antigen, it was absent from the protein profile of the C12 strain of Y. pestis (Fig. 4A).


A Yersinia pestis tat mutant is attenuated in bubonic and small-aerosol pneumonic challenge models of infection but not as attenuated by intranasal challenge.

Bozue J, Cote CK, Chance T, Kugelman J, Kern SJ, Kijek TK, Jenkins A, Mou S, Moody K, Fritz D, Robinson CG, Bell T, Worsham P - PLoS ONE (2014)

Analysis of Y. pestis proteins.A) Strains (wild-type, lanes 1 and 4; ΔtatA, lanes 2 and 5, and C12, lanes 3 and 6) were grown in HI broth at 28°C or 37°C containing CaCl2, as indicated. Proteins were extracted and ran on a SDS-PAGE gel at equal concentrations. The arrow indicates a protein band that is synthesized at 37°C for the CO92 and to a lesser extent the ΔtatA mutant but not in C12, a F1-antigen mutant strain. Molecular masses are indicated on the left in KDa. Strains were grown in HI broth at 37°C containing CaCl2 and proteins extracted for Western analysis. Equal concentrations of proteins from Y. pestis strains were blotted with a monoclonal antibody to either the F1 antigen (B) or GroEL (C). Lanes: 1, wild-type; 2, ΔtatA; 3, C12; and 4, recombinant protein (F1 antigen or GroEL, respectively). Molecular masses are indicated on the left in KDa. D) Strains were grown in HI broth at 37°C in the presence of MOX to create low calcium conditions. Equal concentrations of proteins from Y. pestis strains were blotted with an antibody to LcrV Lanes: 1, CO92 wild-type; 2, ΔtatA; 3, Y. pestis pLcr-; and 4, rLcrV protein. Molecular masses are indicated on the left in KDa.
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pone-0104524-g004: Analysis of Y. pestis proteins.A) Strains (wild-type, lanes 1 and 4; ΔtatA, lanes 2 and 5, and C12, lanes 3 and 6) were grown in HI broth at 28°C or 37°C containing CaCl2, as indicated. Proteins were extracted and ran on a SDS-PAGE gel at equal concentrations. The arrow indicates a protein band that is synthesized at 37°C for the CO92 and to a lesser extent the ΔtatA mutant but not in C12, a F1-antigen mutant strain. Molecular masses are indicated on the left in KDa. Strains were grown in HI broth at 37°C containing CaCl2 and proteins extracted for Western analysis. Equal concentrations of proteins from Y. pestis strains were blotted with a monoclonal antibody to either the F1 antigen (B) or GroEL (C). Lanes: 1, wild-type; 2, ΔtatA; 3, C12; and 4, recombinant protein (F1 antigen or GroEL, respectively). Molecular masses are indicated on the left in KDa. D) Strains were grown in HI broth at 37°C in the presence of MOX to create low calcium conditions. Equal concentrations of proteins from Y. pestis strains were blotted with an antibody to LcrV Lanes: 1, CO92 wild-type; 2, ΔtatA; 3, Y. pestis pLcr-; and 4, rLcrV protein. Molecular masses are indicated on the left in KDa.
Mentions: As the deletion of the tatA gene would affect translocated proteins, we examined proteins extracted from both the cell pellet and supernatant fractions of mutant and wild-type bacteria grown in HI broth at 28°C or 37°C containing CaCl2. As shown in Fig. 4A, the proteins collected from pellets of Y. pestis grown at 28°C were separated on a 10% Bis-Tris gels, but no obvious differences were observed in the protein profile between wild-type, ΔtatA, or C12 (F1 antigen/caf1 mutant) strains. However, when the protein pellets were examined from Y. pestis grown at 37°C under high calcium conditions, an intense band at <19 kDa appeared for wild-type CO92 which was not present for the 28°C samples. For proteins extracted for the ΔtatA pellet, this band was also present but not as intense as observed for the wild-type strain. We presumed that this band corresponded to the F1 antigen protein encoded by the caf1 gene which is expressed by Y. pestis at 37°C and has a molecular mass of 15.5 kDa [48]–[50]. Further demonstrating that this band corresponded to the F1 antigen, it was absent from the protein profile of the C12 strain of Y. pestis (Fig. 4A).

Bottom Line: Deletion of the tatA gene resulted in several consequences for the mutant as compared to wild-type.In contrast, when mice were challenged intranasally with the mutant, very little difference in the LD50 was observed between wild-type and mutant strains.Collectively, these findings demonstrate an essential role for the Tat pathway in the virulence of Y. pestis in bubonic and small-aerosol pneumonic infection but less important role for intranasal challenge.

View Article: PubMed Central - PubMed

Affiliation: Bacteriology Division, The United States Army of Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland, United States of America.

ABSTRACT
Bacterial proteins destined for the Tat pathway are folded before crossing the inner membrane and are typically identified by an N-terminal signal peptide containing a twin arginine motif. Translocation by the Tat pathway is dependent on the products of genes which encode proteins possessing the binding site of the signal peptide and mediating the actual translocation event. In the fully virulent CO92 strain of Yersinia pestis, the tatA gene was deleted. The mutant was assayed for loss of virulence through various in vitro and in vivo assays. Deletion of the tatA gene resulted in several consequences for the mutant as compared to wild-type. Cell morphology of the mutant bacteria was altered and demonstrated a more elongated form. In addition, while cultures of the mutant strain were able to produce a biofilm, we observed a loss of adhesion of the mutant biofilm structure compared to the biofilm produced by the wild-type strain. Immuno-electron microscopy revealed a partial disruption of the F1 antigen on the surface of the mutant. The virulence of the ΔtatA mutant was assessed in various murine models of plague. The mutant was severely attenuated in the bubonic model with full virulence restored by complementation with the native gene. After small-particle aerosol challenge in a pneumonic model of infection, the mutant was also shown to be attenuated. In contrast, when mice were challenged intranasally with the mutant, very little difference in the LD50 was observed between wild-type and mutant strains. However, an increased time-to-death and delay in bacterial dissemination was observed in mice infected with the ΔtatA mutant as compared to the parent strain. Collectively, these findings demonstrate an essential role for the Tat pathway in the virulence of Y. pestis in bubonic and small-aerosol pneumonic infection but less important role for intranasal challenge.

Show MeSH
Related in: MedlinePlus