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Yersinia pestis endowed with increased cytotoxicity is avirulent in a bubonic plague model and induces rapid protection against pneumonic plague.

Zauberman A, Tidhar A, Levy Y, Bar-Haim E, Halperin G, Flashner Y, Cohen S, Shafferman A, Mamroud E - PLoS ONE (2009)

Bottom Line: Following subcutaneous infection, this strain had reduced ability to colonize internal organs, was unable to induce septicemia and exhibited at least a 10(7)-fold reduction in virulence.Yet, upon intravenous or intranasal infection, it was still as virulent as the wild-type strain.These observations have novel implications for the development of vaccines/therapies against Y. pestis and shed new light on the virulence strategies of Y. pestis in nature.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Genetics, Israel Institute for Biological Research, Ness-Ziona, Israel.

ABSTRACT
An important virulence strategy evolved by bacterial pathogens to overcome host defenses is the modulation of host cell death. Previous observations have indicated that Yersinia pestis, the causative agent of plague disease, exhibits restricted capacity to induce cell death in macrophages due to ineffective translocation of the type III secretion effector YopJ, as opposed to the readily translocated YopP, the YopJ homologue of the enteropathogen Yersinia enterocolitica Oratio8. This led us to suggest that reduced cytotoxic potency may allow pathogen propagation within a shielded niche, leading to increased virulence. To test the relationship between cytotoxic potential and virulence, we replaced Y. pestis YopJ with YopP. The YopP-expressing Y. pestis strain exhibited high cytotoxic activity against macrophages in vitro. Following subcutaneous infection, this strain had reduced ability to colonize internal organs, was unable to induce septicemia and exhibited at least a 10(7)-fold reduction in virulence. Yet, upon intravenous or intranasal infection, it was still as virulent as the wild-type strain. The subcutaneous administration of the cytotoxic Y. pestis strain appears to activate a rapid and potent systemic, CTL-independent, immunoprotective response, allowing the organism to overcome simultaneous coinfection with 10,000 LD(50) of virulent Y. pestis. Moreover, three days after subcutaneous administration of this strain, animals were also protected against septicemic or primary pneumonic plague. Our findings indicate that an inverse relationship exists between the cytotoxic potential of Y. pestis and its virulence following subcutaneous infection. This appears to be associated with the ability of the engineered cytotoxic Y. pestis strain to induce very rapid, effective and long-lasting protection against bubonic and pneumonic plague. These observations have novel implications for the development of vaccines/therapies against Y. pestis and shed new light on the virulence strategies of Y. pestis in nature.

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Expression of YopP in Y. pestis enhances its cytotoxicity in macrophages and reduces its virulence.(A) Assessment of bacterial YopJ/P expression. Y. pestis recombinant strains were grown for 3 hrs at 37°C. Western blot analysis was conducted with anti-YopJ/P antibodies (upper lane) or anti-YopE antibodies (lower lane). Endogenous YopJ expression in both wild-type Kim53 and Kim53pGFP strains could be detected only under non-standard conditions of considerably longer exposure time (data not shown). (B) Cytotoxic effect of Y. pestis recombinant strains on J774A.1 macrophage cell line. Cells were infected with Kim53pGFP (circle), Kim53ΔJpGFP (X symbol), Kim53ΔJ+J (square) and Kim53ΔJ+P (triangle) at the indicated MOIs for 1 hour. Cell death was determined by LDH release test. (C) Growth curves of Kim53pGFP (circle), Kim53ΔJ+J (square) and Kim53ΔJ+P (triangle). Bacteria were inoculated to OD630 0.005 in heart infusion broth-containing microplate wells and incubated in a plate reader (Sunrise, Tecan) for an additional 22 hrs at 28°C. (D) Attenuation of YopP-expressing Y. pestis recombinant strain. Groups of mice were infected subcutaneously with 1×102 cfu of Kim53 (10 mice per group, white diamond), Kim53pGFP (5 mice per group, black circle) or Kim53ΔJ+J (5 mice per group, white square), or with 1×106 cfu of Kim53ΔJ+P (5 mice per group, white triangle). Mortality rates were followed daily for 14 days after infection. (E) Spleen sections, isolated from mice 48 hrs after s.c. infection with 1×104 cfu of Kim53pGFP or Kim53ΔJ+P, stained with anti-active caspase-3 antibodies (left panel). Scale bar = 50 µm. The arrow head indicates apoptotic cells. Caspase-positive cells in at least 20 random non-overlapping microscopic fields (magnification, ×1000) were counted (three mice per group, right panel).
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pone-0005938-g001: Expression of YopP in Y. pestis enhances its cytotoxicity in macrophages and reduces its virulence.(A) Assessment of bacterial YopJ/P expression. Y. pestis recombinant strains were grown for 3 hrs at 37°C. Western blot analysis was conducted with anti-YopJ/P antibodies (upper lane) or anti-YopE antibodies (lower lane). Endogenous YopJ expression in both wild-type Kim53 and Kim53pGFP strains could be detected only under non-standard conditions of considerably longer exposure time (data not shown). (B) Cytotoxic effect of Y. pestis recombinant strains on J774A.1 macrophage cell line. Cells were infected with Kim53pGFP (circle), Kim53ΔJpGFP (X symbol), Kim53ΔJ+J (square) and Kim53ΔJ+P (triangle) at the indicated MOIs for 1 hour. Cell death was determined by LDH release test. (C) Growth curves of Kim53pGFP (circle), Kim53ΔJ+J (square) and Kim53ΔJ+P (triangle). Bacteria were inoculated to OD630 0.005 in heart infusion broth-containing microplate wells and incubated in a plate reader (Sunrise, Tecan) for an additional 22 hrs at 28°C. (D) Attenuation of YopP-expressing Y. pestis recombinant strain. Groups of mice were infected subcutaneously with 1×102 cfu of Kim53 (10 mice per group, white diamond), Kim53pGFP (5 mice per group, black circle) or Kim53ΔJ+J (5 mice per group, white square), or with 1×106 cfu of Kim53ΔJ+P (5 mice per group, white triangle). Mortality rates were followed daily for 14 days after infection. (E) Spleen sections, isolated from mice 48 hrs after s.c. infection with 1×104 cfu of Kim53pGFP or Kim53ΔJ+P, stained with anti-active caspase-3 antibodies (left panel). Scale bar = 50 µm. The arrow head indicates apoptotic cells. Caspase-positive cells in at least 20 random non-overlapping microscopic fields (magnification, ×1000) were counted (three mice per group, right panel).

Mentions: Expression of YopP, the YopJ homologue in Y. enterocolitica O∶8, in the background of the attenuated Y. pestis EV76 strain was previously shown to render this strain highly cytotoxic to J774A.1 and RAW 264.7 macrophage cells [10]. In order to explore whether enhanced cytotoxic potency would attenuate Y. pestis virulence, in this study, we used the highly virulent yopJ-deleted strain of Y. pestis Kimberley53 (Kim53ΔJ) [25] to generate recombinant strains expressing in trans either YopP (Kim53ΔJ+P) or YopJ (Kim53ΔJ+J) (Table 1). Plasmid construction was based on replacing the green fluorescent protein (GFP) gene from the pUC19 derivative plasmid pGFPuv by yopP or yopJ genes, under the control of the lac promoter. As a control, we used wild-type Kim53 carrying the same expression vector with the gfp gene (Kim53pGFP). As was shown in the past with the attenuated EV76-based strains [10], the newly constructed Kim53-derived strains exhibited expression levels comparable to those of the two homologous effectors (YopJ and YopP), but with pronounced differences in induction of cell death upon infection of macrophages. Expression of YopP conferred high cytotoxic potential, which expression of YopJ did not (Figure 1A, upper lanes and 1B). It should be noted that expression of YopJ/P had no apparent effect on the bacterial growth rate in vitro (Figure 1C).


Yersinia pestis endowed with increased cytotoxicity is avirulent in a bubonic plague model and induces rapid protection against pneumonic plague.

Zauberman A, Tidhar A, Levy Y, Bar-Haim E, Halperin G, Flashner Y, Cohen S, Shafferman A, Mamroud E - PLoS ONE (2009)

Expression of YopP in Y. pestis enhances its cytotoxicity in macrophages and reduces its virulence.(A) Assessment of bacterial YopJ/P expression. Y. pestis recombinant strains were grown for 3 hrs at 37°C. Western blot analysis was conducted with anti-YopJ/P antibodies (upper lane) or anti-YopE antibodies (lower lane). Endogenous YopJ expression in both wild-type Kim53 and Kim53pGFP strains could be detected only under non-standard conditions of considerably longer exposure time (data not shown). (B) Cytotoxic effect of Y. pestis recombinant strains on J774A.1 macrophage cell line. Cells were infected with Kim53pGFP (circle), Kim53ΔJpGFP (X symbol), Kim53ΔJ+J (square) and Kim53ΔJ+P (triangle) at the indicated MOIs for 1 hour. Cell death was determined by LDH release test. (C) Growth curves of Kim53pGFP (circle), Kim53ΔJ+J (square) and Kim53ΔJ+P (triangle). Bacteria were inoculated to OD630 0.005 in heart infusion broth-containing microplate wells and incubated in a plate reader (Sunrise, Tecan) for an additional 22 hrs at 28°C. (D) Attenuation of YopP-expressing Y. pestis recombinant strain. Groups of mice were infected subcutaneously with 1×102 cfu of Kim53 (10 mice per group, white diamond), Kim53pGFP (5 mice per group, black circle) or Kim53ΔJ+J (5 mice per group, white square), or with 1×106 cfu of Kim53ΔJ+P (5 mice per group, white triangle). Mortality rates were followed daily for 14 days after infection. (E) Spleen sections, isolated from mice 48 hrs after s.c. infection with 1×104 cfu of Kim53pGFP or Kim53ΔJ+P, stained with anti-active caspase-3 antibodies (left panel). Scale bar = 50 µm. The arrow head indicates apoptotic cells. Caspase-positive cells in at least 20 random non-overlapping microscopic fields (magnification, ×1000) were counted (three mice per group, right panel).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2691952&req=5

pone-0005938-g001: Expression of YopP in Y. pestis enhances its cytotoxicity in macrophages and reduces its virulence.(A) Assessment of bacterial YopJ/P expression. Y. pestis recombinant strains were grown for 3 hrs at 37°C. Western blot analysis was conducted with anti-YopJ/P antibodies (upper lane) or anti-YopE antibodies (lower lane). Endogenous YopJ expression in both wild-type Kim53 and Kim53pGFP strains could be detected only under non-standard conditions of considerably longer exposure time (data not shown). (B) Cytotoxic effect of Y. pestis recombinant strains on J774A.1 macrophage cell line. Cells were infected with Kim53pGFP (circle), Kim53ΔJpGFP (X symbol), Kim53ΔJ+J (square) and Kim53ΔJ+P (triangle) at the indicated MOIs for 1 hour. Cell death was determined by LDH release test. (C) Growth curves of Kim53pGFP (circle), Kim53ΔJ+J (square) and Kim53ΔJ+P (triangle). Bacteria were inoculated to OD630 0.005 in heart infusion broth-containing microplate wells and incubated in a plate reader (Sunrise, Tecan) for an additional 22 hrs at 28°C. (D) Attenuation of YopP-expressing Y. pestis recombinant strain. Groups of mice were infected subcutaneously with 1×102 cfu of Kim53 (10 mice per group, white diamond), Kim53pGFP (5 mice per group, black circle) or Kim53ΔJ+J (5 mice per group, white square), or with 1×106 cfu of Kim53ΔJ+P (5 mice per group, white triangle). Mortality rates were followed daily for 14 days after infection. (E) Spleen sections, isolated from mice 48 hrs after s.c. infection with 1×104 cfu of Kim53pGFP or Kim53ΔJ+P, stained with anti-active caspase-3 antibodies (left panel). Scale bar = 50 µm. The arrow head indicates apoptotic cells. Caspase-positive cells in at least 20 random non-overlapping microscopic fields (magnification, ×1000) were counted (three mice per group, right panel).
Mentions: Expression of YopP, the YopJ homologue in Y. enterocolitica O∶8, in the background of the attenuated Y. pestis EV76 strain was previously shown to render this strain highly cytotoxic to J774A.1 and RAW 264.7 macrophage cells [10]. In order to explore whether enhanced cytotoxic potency would attenuate Y. pestis virulence, in this study, we used the highly virulent yopJ-deleted strain of Y. pestis Kimberley53 (Kim53ΔJ) [25] to generate recombinant strains expressing in trans either YopP (Kim53ΔJ+P) or YopJ (Kim53ΔJ+J) (Table 1). Plasmid construction was based on replacing the green fluorescent protein (GFP) gene from the pUC19 derivative plasmid pGFPuv by yopP or yopJ genes, under the control of the lac promoter. As a control, we used wild-type Kim53 carrying the same expression vector with the gfp gene (Kim53pGFP). As was shown in the past with the attenuated EV76-based strains [10], the newly constructed Kim53-derived strains exhibited expression levels comparable to those of the two homologous effectors (YopJ and YopP), but with pronounced differences in induction of cell death upon infection of macrophages. Expression of YopP conferred high cytotoxic potential, which expression of YopJ did not (Figure 1A, upper lanes and 1B). It should be noted that expression of YopJ/P had no apparent effect on the bacterial growth rate in vitro (Figure 1C).

Bottom Line: Following subcutaneous infection, this strain had reduced ability to colonize internal organs, was unable to induce septicemia and exhibited at least a 10(7)-fold reduction in virulence.Yet, upon intravenous or intranasal infection, it was still as virulent as the wild-type strain.These observations have novel implications for the development of vaccines/therapies against Y. pestis and shed new light on the virulence strategies of Y. pestis in nature.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Genetics, Israel Institute for Biological Research, Ness-Ziona, Israel.

ABSTRACT
An important virulence strategy evolved by bacterial pathogens to overcome host defenses is the modulation of host cell death. Previous observations have indicated that Yersinia pestis, the causative agent of plague disease, exhibits restricted capacity to induce cell death in macrophages due to ineffective translocation of the type III secretion effector YopJ, as opposed to the readily translocated YopP, the YopJ homologue of the enteropathogen Yersinia enterocolitica Oratio8. This led us to suggest that reduced cytotoxic potency may allow pathogen propagation within a shielded niche, leading to increased virulence. To test the relationship between cytotoxic potential and virulence, we replaced Y. pestis YopJ with YopP. The YopP-expressing Y. pestis strain exhibited high cytotoxic activity against macrophages in vitro. Following subcutaneous infection, this strain had reduced ability to colonize internal organs, was unable to induce septicemia and exhibited at least a 10(7)-fold reduction in virulence. Yet, upon intravenous or intranasal infection, it was still as virulent as the wild-type strain. The subcutaneous administration of the cytotoxic Y. pestis strain appears to activate a rapid and potent systemic, CTL-independent, immunoprotective response, allowing the organism to overcome simultaneous coinfection with 10,000 LD(50) of virulent Y. pestis. Moreover, three days after subcutaneous administration of this strain, animals were also protected against septicemic or primary pneumonic plague. Our findings indicate that an inverse relationship exists between the cytotoxic potential of Y. pestis and its virulence following subcutaneous infection. This appears to be associated with the ability of the engineered cytotoxic Y. pestis strain to induce very rapid, effective and long-lasting protection against bubonic and pneumonic plague. These observations have novel implications for the development of vaccines/therapies against Y. pestis and shed new light on the virulence strategies of Y. pestis in nature.

Show MeSH
Related in: MedlinePlus