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Engineering attenuated virulence of a Theileria annulata-infected macrophage.

Echebli N, Mhadhbi M, Chaussepied M, Vayssettes C, Di Santo JP, Darghouth MA, Langsley G - PLoS Negl Trop Dis (2014)

Bottom Line: The attenuation process is empirical and occurs only after many months, sometimes years, of in vitro culture of virulent clinical isolates.During this extensive culturing, attenuated lines lose their vaccine potential.Specific ablation of a single infected host cell virulence trait (c-Jun) induced a complete failure of Theileria annulata-transformed macrophages to disseminate, whereas virulent macrophages disseminated to the kidneys, spleen, and lungs of Rag2/γC mice.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Biologie Cellulaire Comparative des Apicomplexes, Faculté de Médicine, Université Paris Descartes - Sorbonne Paris Cité, Paris, France; Inserm U1016, Cnrs UMR8104, Cochin Institute, Paris, France.

ABSTRACT
Live attenuated vaccines are used to combat tropical theileriosis in North Africa, the Middle East, India, and China. The attenuation process is empirical and occurs only after many months, sometimes years, of in vitro culture of virulent clinical isolates. During this extensive culturing, attenuated lines lose their vaccine potential. To circumvent this we engineered the rapid ablation of the host cell transcription factor c-Jun, and within only 3 weeks the line engineered for loss of c-Jun activation displayed in vitro correlates of attenuation such as loss of adhesion, reduced MMP9 gelatinase activity, and diminished capacity to traverse Matrigel. Specific ablation of a single infected host cell virulence trait (c-Jun) induced a complete failure of Theileria annulata-transformed macrophages to disseminate, whereas virulent macrophages disseminated to the kidneys, spleen, and lungs of Rag2/γC mice. Thus, in this heterologous mouse model loss of c-Jun expression led to ablation of dissemination of T. annulata-infected and transformed macrophages. The generation of Theileria-infected macrophages genetically engineered for ablation of a specific host cell virulence trait now makes possible experimental vaccination of calves to address how loss of macrophage dissemination impacts the disease pathology of tropical theileriosis.

No MeSH data available.


Related in: MedlinePlus

Dissemination of Jed 4 macrophages in Rag2/γC mice.A (Virulent line) & B (Engineered attenuated line), The Ts4 micro-satellite locus was amplified from DNA extracted from the lungs (tracks 1–7), kidneys (tracks 8–11) and spleens (tracks 12–18) of 18 mice injected with virulent Jed 4 macrophages. Apart from a Ts4 specific band in the positive control no parasite DNA was amplified from extracts of lungs (tracks 1–8), kidneys (tracks 9–11) and spleens (tracks 12–18) of 18 mice injected with V-Delta 169 macrophages. (+) = Positive control DNA, (−) = negative control water, MW = molecular-weight size marker (200 bp ladder).
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pntd-0003183-g004: Dissemination of Jed 4 macrophages in Rag2/γC mice.A (Virulent line) & B (Engineered attenuated line), The Ts4 micro-satellite locus was amplified from DNA extracted from the lungs (tracks 1–7), kidneys (tracks 8–11) and spleens (tracks 12–18) of 18 mice injected with virulent Jed 4 macrophages. Apart from a Ts4 specific band in the positive control no parasite DNA was amplified from extracts of lungs (tracks 1–8), kidneys (tracks 9–11) and spleens (tracks 12–18) of 18 mice injected with V-Delta 169 macrophages. (+) = Positive control DNA, (−) = negative control water, MW = molecular-weight size marker (200 bp ladder).

Mentions: It is likely that the reduced dissemination of live attenuated vaccines, as estimated in heterologous mouse models, allows them to confer protection of cattle against theileriosis without killing the vaccinated animal [35]. Moreover, reduced dissemination of live attenuated vaccines has been ascribed to loss of MMP9 activity [36]. Above, we showed that the engineered attenuated Jed 4 line has not only reduced MMP9 activity, increased timp3 levels and ablated ADAM19 expression compared to control virulent Jed 4 (V: Jed4p18) macrophages, so we tested whether this translated into a failure to disseminate by their subcutaneous injection into Rag2/γC mice [12]. Dissemination of virulent Theileria-transformed Jed 4 macrophages (V: Jed4p18) gave rise to metastases in kidneys, spleen and lungs, and the presence of T. annulata in these tumours was confirmed by PCR detection of 4 different microsatellite parasite markers (Ts4, Ts6, Ts8 and Ts25) in tissue extracts of the 3 different organs and shown in Figure 4 is the result with Ts4. In contrast, we failed to detect tumours (or parasite DNA) in the same organs of mice injected with the engineered attenuated line. Thus, in this heterologous mouse model for Theileria-transformed macrophage dissemination ectopic expression of Flag-Δ169-c-Jun that diminishes MMP9/ADAM19 and increases TIMP3 expression led to ablation of tumour dissemination by the engineered attenuated line.


Engineering attenuated virulence of a Theileria annulata-infected macrophage.

Echebli N, Mhadhbi M, Chaussepied M, Vayssettes C, Di Santo JP, Darghouth MA, Langsley G - PLoS Negl Trop Dis (2014)

Dissemination of Jed 4 macrophages in Rag2/γC mice.A (Virulent line) & B (Engineered attenuated line), The Ts4 micro-satellite locus was amplified from DNA extracted from the lungs (tracks 1–7), kidneys (tracks 8–11) and spleens (tracks 12–18) of 18 mice injected with virulent Jed 4 macrophages. Apart from a Ts4 specific band in the positive control no parasite DNA was amplified from extracts of lungs (tracks 1–8), kidneys (tracks 9–11) and spleens (tracks 12–18) of 18 mice injected with V-Delta 169 macrophages. (+) = Positive control DNA, (−) = negative control water, MW = molecular-weight size marker (200 bp ladder).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4222746&req=5

pntd-0003183-g004: Dissemination of Jed 4 macrophages in Rag2/γC mice.A (Virulent line) & B (Engineered attenuated line), The Ts4 micro-satellite locus was amplified from DNA extracted from the lungs (tracks 1–7), kidneys (tracks 8–11) and spleens (tracks 12–18) of 18 mice injected with virulent Jed 4 macrophages. Apart from a Ts4 specific band in the positive control no parasite DNA was amplified from extracts of lungs (tracks 1–8), kidneys (tracks 9–11) and spleens (tracks 12–18) of 18 mice injected with V-Delta 169 macrophages. (+) = Positive control DNA, (−) = negative control water, MW = molecular-weight size marker (200 bp ladder).
Mentions: It is likely that the reduced dissemination of live attenuated vaccines, as estimated in heterologous mouse models, allows them to confer protection of cattle against theileriosis without killing the vaccinated animal [35]. Moreover, reduced dissemination of live attenuated vaccines has been ascribed to loss of MMP9 activity [36]. Above, we showed that the engineered attenuated Jed 4 line has not only reduced MMP9 activity, increased timp3 levels and ablated ADAM19 expression compared to control virulent Jed 4 (V: Jed4p18) macrophages, so we tested whether this translated into a failure to disseminate by their subcutaneous injection into Rag2/γC mice [12]. Dissemination of virulent Theileria-transformed Jed 4 macrophages (V: Jed4p18) gave rise to metastases in kidneys, spleen and lungs, and the presence of T. annulata in these tumours was confirmed by PCR detection of 4 different microsatellite parasite markers (Ts4, Ts6, Ts8 and Ts25) in tissue extracts of the 3 different organs and shown in Figure 4 is the result with Ts4. In contrast, we failed to detect tumours (or parasite DNA) in the same organs of mice injected with the engineered attenuated line. Thus, in this heterologous mouse model for Theileria-transformed macrophage dissemination ectopic expression of Flag-Δ169-c-Jun that diminishes MMP9/ADAM19 and increases TIMP3 expression led to ablation of tumour dissemination by the engineered attenuated line.

Bottom Line: The attenuation process is empirical and occurs only after many months, sometimes years, of in vitro culture of virulent clinical isolates.During this extensive culturing, attenuated lines lose their vaccine potential.Specific ablation of a single infected host cell virulence trait (c-Jun) induced a complete failure of Theileria annulata-transformed macrophages to disseminate, whereas virulent macrophages disseminated to the kidneys, spleen, and lungs of Rag2/γC mice.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Biologie Cellulaire Comparative des Apicomplexes, Faculté de Médicine, Université Paris Descartes - Sorbonne Paris Cité, Paris, France; Inserm U1016, Cnrs UMR8104, Cochin Institute, Paris, France.

ABSTRACT
Live attenuated vaccines are used to combat tropical theileriosis in North Africa, the Middle East, India, and China. The attenuation process is empirical and occurs only after many months, sometimes years, of in vitro culture of virulent clinical isolates. During this extensive culturing, attenuated lines lose their vaccine potential. To circumvent this we engineered the rapid ablation of the host cell transcription factor c-Jun, and within only 3 weeks the line engineered for loss of c-Jun activation displayed in vitro correlates of attenuation such as loss of adhesion, reduced MMP9 gelatinase activity, and diminished capacity to traverse Matrigel. Specific ablation of a single infected host cell virulence trait (c-Jun) induced a complete failure of Theileria annulata-transformed macrophages to disseminate, whereas virulent macrophages disseminated to the kidneys, spleen, and lungs of Rag2/γC mice. Thus, in this heterologous mouse model loss of c-Jun expression led to ablation of dissemination of T. annulata-infected and transformed macrophages. The generation of Theileria-infected macrophages genetically engineered for ablation of a specific host cell virulence trait now makes possible experimental vaccination of calves to address how loss of macrophage dissemination impacts the disease pathology of tropical theileriosis.

No MeSH data available.


Related in: MedlinePlus