Limits...
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.

Show MeSH

Related in: MedlinePlus

Localizing the F1 capsule by microscopy.Y. pestis strains were grown at 37°C in HI broth containing CaCl2 and exposed to an antibody against the F1 capsule. IFM samples: A) wild-type, B) ΔtatA mutant, and C) complemented ΔtatA mutant. The images (100×) were captured for all samples at identical camera settings to maintain relative fluorescence. IEM samples: D) wild-type (micron bar = 0.1 µm), E) ΔtatA mutant (micron bar = 0.1 µm), and F) C12, F1 negative (micron bar = 0.5 µm).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4125294&req=5

pone-0104524-g005: Localizing the F1 capsule by microscopy.Y. pestis strains were grown at 37°C in HI broth containing CaCl2 and exposed to an antibody against the F1 capsule. IFM samples: A) wild-type, B) ΔtatA mutant, and C) complemented ΔtatA mutant. The images (100×) were captured for all samples at identical camera settings to maintain relative fluorescence. IEM samples: D) wild-type (micron bar = 0.1 µm), E) ΔtatA mutant (micron bar = 0.1 µm), and F) C12, F1 negative (micron bar = 0.5 µm).

Mentions: To attempt to define this defect in the concentration of the F1 antigen for the ΔtatA mutant, Y. pestis cells were examined by microscopy using both IFM and IEM. As shown in Fig. 5A, when wild-type CO92 was grown under conditions to promote F1 antigen synthesis and visualized by IFM using an antibody specific to the F1 antigen, the cells displayed a tight layer of fluorescence that surrounded the cells and in close association to the membrane. The ΔtatA mutant was grown under identical conditions but was only slightly fluorescent, indicating the presence of low but still detectable levels of F1 antigen. (Fig. 5B) However, fluorescence was not as intense as observed with the parental strain. In addition, the labeling for the mutant appeared to be more diffuse around the cells and not as tightly associated. To determine if this defect was due specifically to the deletion of the tatA gene, we observed the complemented mutant strain by IFM (Fig. 5C). As expected, the fluorescence of the complemented ΔtatA strain was restored to the intense fluorescent labeling that was associated with the wild-type bacterial cells. Also included in this study was the C12 strain which lacks the F1 antigen protein and no fluorescent labeling of this strain was observed (data not shown).


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)

Localizing the F1 capsule by microscopy.Y. pestis strains were grown at 37°C in HI broth containing CaCl2 and exposed to an antibody against the F1 capsule. IFM samples: A) wild-type, B) ΔtatA mutant, and C) complemented ΔtatA mutant. The images (100×) were captured for all samples at identical camera settings to maintain relative fluorescence. IEM samples: D) wild-type (micron bar = 0.1 µm), E) ΔtatA mutant (micron bar = 0.1 µm), and F) C12, F1 negative (micron bar = 0.5 µm).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4125294&req=5

pone-0104524-g005: Localizing the F1 capsule by microscopy.Y. pestis strains were grown at 37°C in HI broth containing CaCl2 and exposed to an antibody against the F1 capsule. IFM samples: A) wild-type, B) ΔtatA mutant, and C) complemented ΔtatA mutant. The images (100×) were captured for all samples at identical camera settings to maintain relative fluorescence. IEM samples: D) wild-type (micron bar = 0.1 µm), E) ΔtatA mutant (micron bar = 0.1 µm), and F) C12, F1 negative (micron bar = 0.5 µm).
Mentions: To attempt to define this defect in the concentration of the F1 antigen for the ΔtatA mutant, Y. pestis cells were examined by microscopy using both IFM and IEM. As shown in Fig. 5A, when wild-type CO92 was grown under conditions to promote F1 antigen synthesis and visualized by IFM using an antibody specific to the F1 antigen, the cells displayed a tight layer of fluorescence that surrounded the cells and in close association to the membrane. The ΔtatA mutant was grown under identical conditions but was only slightly fluorescent, indicating the presence of low but still detectable levels of F1 antigen. (Fig. 5B) However, fluorescence was not as intense as observed with the parental strain. In addition, the labeling for the mutant appeared to be more diffuse around the cells and not as tightly associated. To determine if this defect was due specifically to the deletion of the tatA gene, we observed the complemented mutant strain by IFM (Fig. 5C). As expected, the fluorescence of the complemented ΔtatA strain was restored to the intense fluorescent labeling that was associated with the wild-type bacterial cells. Also included in this study was the C12 strain which lacks the F1 antigen protein and no fluorescent labeling of this strain was observed (data not shown).

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