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Caspase-3 mediates the pathogenic effect of Yersinia pestis YopM in liver of C57BL/6 mice and contributes to YopM's function in spleen.

Ye Z, Gorman AA, Uittenbogaard AM, Myers-Morales T, Kaplan AM, Cohen DA, Straley SC - PLoS ONE (2014)

Bottom Line: The bacteria were found associated with myeloid cells in foci of inflammation and in liver sinusoids.By 2 d post-infection, YopM had no effect on distribution of these cells, but by 3 d cellular decomposition had outstripped acute inflammation in foci due to parent Y. pestis, while foci due to the ΔyopM-1 strain still contained many inflammatory cells.This result identified caspase-3 as a co-factor or effector in YopM's action and supports the hypothesis that in liver YopM's main pathogenic effect is mediated by caspase-3 to cause apoptosis of PMNs.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky, Lexington, KY, United States of America.

ABSTRACT
The virulence protein YopM of the plague bacterium Yersinia pestis has different dominant effects in liver and spleen. Previous studies focused on spleen, where YopM inhibits accumulation of inflammatory dendritic cells. In the present study we focused on liver, where PMN function may be directly undermined by YopM without changes in inflammatory cell numbers in the initial days of infection, and foci of inflammation are easily identified. Mice were infected with parent and ΔyopM-1 Y. pestis KIM5, and effects of YopM were assessed by immunohistochemistry and determinations of bacterial viable numbers in organs. The bacteria were found associated with myeloid cells in foci of inflammation and in liver sinusoids. A new in-vivo phenotype of YopM was revealed: death of inflammatory cells, evidenced by TUNEL staining beginning at d 1 of infection. Based on distributions of Ly6G(+), F4/80(+), and iNOS(+) cells within foci, the cells that were killed could have included both PMNs and macrophages. By 2 d post-infection, YopM had no effect on distribution of these cells, but by 3 d cellular decomposition had outstripped acute inflammation in foci due to parent Y. pestis, while foci due to the ΔyopM-1 strain still contained many inflammatory cells. The destruction depended on the presence of both PMNs in the mice and YopM in the bacteria. In mice that lacked the apoptosis mediator caspase-3 the infection dynamics were novel: the parent Y. pestis was limited in growth comparably to the ΔyopM-1 strain in liver, and in spleen a partial growth limitation for parent Y. pestis was seen. This result identified caspase-3 as a co-factor or effector in YopM's action and supports the hypothesis that in liver YopM's main pathogenic effect is mediated by caspase-3 to cause apoptosis of PMNs.

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PMNs were critical to control growth of 28/37°C-pregrown ΔyopM-1 Y. pestis in liver but not spleen.B6 mice were ablated for PMNs (open symbols) by treatment with anti-Ly6G antibody on days -1 and +1 of infection; mock-treated mice (closed symbols) received nonspecific rat IgG. The mice were infected with 400 28/37°C-grown parent (squares) or ΔyopM-1 (circles) Y. pestis, and viable numbers (CFU) were determined on d 1, 2, and 3 p.i. Group labels: MM, mock-treated mice infected with ΔyopM-1 Y. pestis; AM, Ablated mice infected with ΔyopM-1 Y. pestis; MP, mock-treated mice infected with the parent strain; AP, ablated mice infected with the parent strain. The data were obtained in two experiments, each with 2 to 3 mice per group, for 4 to 6 mice total for each category. Each symbol gives the bacterial burden of one mouse; the horizontal lines mark the geometric means of the pooled data for each category. Statistically significant differences by Student's t test occurred on d 3 p.i. as indicated: *, P<0.05; **, P<0.01.
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pone-0110956-g001: PMNs were critical to control growth of 28/37°C-pregrown ΔyopM-1 Y. pestis in liver but not spleen.B6 mice were ablated for PMNs (open symbols) by treatment with anti-Ly6G antibody on days -1 and +1 of infection; mock-treated mice (closed symbols) received nonspecific rat IgG. The mice were infected with 400 28/37°C-grown parent (squares) or ΔyopM-1 (circles) Y. pestis, and viable numbers (CFU) were determined on d 1, 2, and 3 p.i. Group labels: MM, mock-treated mice infected with ΔyopM-1 Y. pestis; AM, Ablated mice infected with ΔyopM-1 Y. pestis; MP, mock-treated mice infected with the parent strain; AP, ablated mice infected with the parent strain. The data were obtained in two experiments, each with 2 to 3 mice per group, for 4 to 6 mice total for each category. Each symbol gives the bacterial burden of one mouse; the horizontal lines mark the geometric means of the pooled data for each category. Statistically significant differences by Student's t test occurred on d 3 p.i. as indicated: *, P<0.05; **, P<0.01.

Mentions: We had shown that PMNs are critical for control over growth of 28°C-grown ΔyopM-1 Y. pestis in liver [19]. In this study we verified that this also holds when the bacteria were grown at 28/37°C (Figure 1). In one of the experiments of Fig. 1, we evaluated the histology in each of the 3 mice per treatment group on each of the three days. Figure 2 illustrates typical lesions for d 3 p.i. in mice with bacterial burdens near to the means of the pooled data of Figure 1. In mock-treated mice, parent Y. pestis formed compact coagulatively necrotic lesions with only bits of inflammatory cells remaining (Fig. 1A), while foci due to the ΔyopM-1 strain in mock-treated mice were larger and retained many intact cells (panel C). In mice ablated for PMNs (panels B and D), the lesions due to both strains were similar to ones due to ΔyopM-1 Y. pestis in mock-treated mice. These findings are consistent with the critical involvement of PMNs in cellular destruction as previously found in pneumonic plague, where PMNs were shown to be responsible for the architectural damage and major symptoms of disease [21]. Further, it was striking that the extensive destruction of inflammatory cells was seen only when the bacteria expressed YopM and the mice had PMNs (Fig. 2 panel A), even though similarly high bacterial numbers were present in livers of the PMN-ablated mice infected with parent Y. pestis (panel B).


Caspase-3 mediates the pathogenic effect of Yersinia pestis YopM in liver of C57BL/6 mice and contributes to YopM's function in spleen.

Ye Z, Gorman AA, Uittenbogaard AM, Myers-Morales T, Kaplan AM, Cohen DA, Straley SC - PLoS ONE (2014)

PMNs were critical to control growth of 28/37°C-pregrown ΔyopM-1 Y. pestis in liver but not spleen.B6 mice were ablated for PMNs (open symbols) by treatment with anti-Ly6G antibody on days -1 and +1 of infection; mock-treated mice (closed symbols) received nonspecific rat IgG. The mice were infected with 400 28/37°C-grown parent (squares) or ΔyopM-1 (circles) Y. pestis, and viable numbers (CFU) were determined on d 1, 2, and 3 p.i. Group labels: MM, mock-treated mice infected with ΔyopM-1 Y. pestis; AM, Ablated mice infected with ΔyopM-1 Y. pestis; MP, mock-treated mice infected with the parent strain; AP, ablated mice infected with the parent strain. The data were obtained in two experiments, each with 2 to 3 mice per group, for 4 to 6 mice total for each category. Each symbol gives the bacterial burden of one mouse; the horizontal lines mark the geometric means of the pooled data for each category. Statistically significant differences by Student's t test occurred on d 3 p.i. as indicated: *, P<0.05; **, P<0.01.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4220956&req=5

pone-0110956-g001: PMNs were critical to control growth of 28/37°C-pregrown ΔyopM-1 Y. pestis in liver but not spleen.B6 mice were ablated for PMNs (open symbols) by treatment with anti-Ly6G antibody on days -1 and +1 of infection; mock-treated mice (closed symbols) received nonspecific rat IgG. The mice were infected with 400 28/37°C-grown parent (squares) or ΔyopM-1 (circles) Y. pestis, and viable numbers (CFU) were determined on d 1, 2, and 3 p.i. Group labels: MM, mock-treated mice infected with ΔyopM-1 Y. pestis; AM, Ablated mice infected with ΔyopM-1 Y. pestis; MP, mock-treated mice infected with the parent strain; AP, ablated mice infected with the parent strain. The data were obtained in two experiments, each with 2 to 3 mice per group, for 4 to 6 mice total for each category. Each symbol gives the bacterial burden of one mouse; the horizontal lines mark the geometric means of the pooled data for each category. Statistically significant differences by Student's t test occurred on d 3 p.i. as indicated: *, P<0.05; **, P<0.01.
Mentions: We had shown that PMNs are critical for control over growth of 28°C-grown ΔyopM-1 Y. pestis in liver [19]. In this study we verified that this also holds when the bacteria were grown at 28/37°C (Figure 1). In one of the experiments of Fig. 1, we evaluated the histology in each of the 3 mice per treatment group on each of the three days. Figure 2 illustrates typical lesions for d 3 p.i. in mice with bacterial burdens near to the means of the pooled data of Figure 1. In mock-treated mice, parent Y. pestis formed compact coagulatively necrotic lesions with only bits of inflammatory cells remaining (Fig. 1A), while foci due to the ΔyopM-1 strain in mock-treated mice were larger and retained many intact cells (panel C). In mice ablated for PMNs (panels B and D), the lesions due to both strains were similar to ones due to ΔyopM-1 Y. pestis in mock-treated mice. These findings are consistent with the critical involvement of PMNs in cellular destruction as previously found in pneumonic plague, where PMNs were shown to be responsible for the architectural damage and major symptoms of disease [21]. Further, it was striking that the extensive destruction of inflammatory cells was seen only when the bacteria expressed YopM and the mice had PMNs (Fig. 2 panel A), even though similarly high bacterial numbers were present in livers of the PMN-ablated mice infected with parent Y. pestis (panel B).

Bottom Line: The bacteria were found associated with myeloid cells in foci of inflammation and in liver sinusoids.By 2 d post-infection, YopM had no effect on distribution of these cells, but by 3 d cellular decomposition had outstripped acute inflammation in foci due to parent Y. pestis, while foci due to the ΔyopM-1 strain still contained many inflammatory cells.This result identified caspase-3 as a co-factor or effector in YopM's action and supports the hypothesis that in liver YopM's main pathogenic effect is mediated by caspase-3 to cause apoptosis of PMNs.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky, Lexington, KY, United States of America.

ABSTRACT
The virulence protein YopM of the plague bacterium Yersinia pestis has different dominant effects in liver and spleen. Previous studies focused on spleen, where YopM inhibits accumulation of inflammatory dendritic cells. In the present study we focused on liver, where PMN function may be directly undermined by YopM without changes in inflammatory cell numbers in the initial days of infection, and foci of inflammation are easily identified. Mice were infected with parent and ΔyopM-1 Y. pestis KIM5, and effects of YopM were assessed by immunohistochemistry and determinations of bacterial viable numbers in organs. The bacteria were found associated with myeloid cells in foci of inflammation and in liver sinusoids. A new in-vivo phenotype of YopM was revealed: death of inflammatory cells, evidenced by TUNEL staining beginning at d 1 of infection. Based on distributions of Ly6G(+), F4/80(+), and iNOS(+) cells within foci, the cells that were killed could have included both PMNs and macrophages. By 2 d post-infection, YopM had no effect on distribution of these cells, but by 3 d cellular decomposition had outstripped acute inflammation in foci due to parent Y. pestis, while foci due to the ΔyopM-1 strain still contained many inflammatory cells. The destruction depended on the presence of both PMNs in the mice and YopM in the bacteria. In mice that lacked the apoptosis mediator caspase-3 the infection dynamics were novel: the parent Y. pestis was limited in growth comparably to the ΔyopM-1 strain in liver, and in spleen a partial growth limitation for parent Y. pestis was seen. This result identified caspase-3 as a co-factor or effector in YopM's action and supports the hypothesis that in liver YopM's main pathogenic effect is mediated by caspase-3 to cause apoptosis of PMNs.

Show MeSH
Related in: MedlinePlus