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Delivery of a functional anti-trypanosome Nanobody in different tsetse fly tissues via a bacterial symbiont, Sodalis glossinidius.

De Vooght L, Caljon G, De Ridder K, Van Den Abbeele J - Microb. Cell Fact. (2014)

Bottom Line: We show that recSodalis is competitive with WT Sodalis in in vivo conditions and that tsetse flies transiently cleared of their endogenous WT Sodalis population can be successfully repopulated with recSodalis at high densities.Finally, we demonstrated that recSodalis expressed significant levels (ng range) of functional Nb_An46 in different tsetse fly tissues, including the midgut where an important developmental stage of the trypanosome parasite occurs.We demonstrated the proof-of-concept that the Sodalis symbiont can be genetically engineered to express and release significant amounts of functional anti-trypanosome Nbs in different tissues of the tsetse fly.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences, Unit of Veterinary Protozoology, Institute of Tropical Medicine Antwerp (ITM), Antwerp, Belgium. ldevooght@itg.be.

ABSTRACT

Background: Sodalis glossinidius, a vertically transmitted microbial symbiont of the tsetse fly, is currently considered as a potential delivery system for anti-trypanosomal components that reduce or eliminate the capability of the tsetse fly host to transmit parasitic trypanosomes, an approach also known as paratransgenesis. An essential step in developing paratransgenic tsetse is the stable colonization of adult flies and their progeny with recombinant Sodalis bacteria, expressing trypanocidal effector molecules in tissues where the parasite resides.

Results: In this study, Sodalis was tested for its ability to deliver functional anti-trypanosome nanobodies (Nbs) in Glossina morsitans morsitans. We characterized the in vitro and in vivo stability of recombinant Sodalis (recSodalis) expressing a potent trypanolytic nanobody, i.e. Nb_An46. We show that recSodalis is competitive with WT Sodalis in in vivo conditions and that tsetse flies transiently cleared of their endogenous WT Sodalis population can be successfully repopulated with recSodalis at high densities. In addition, vertical transmission to the offspring was observed. Finally, we demonstrated that recSodalis expressed significant levels (ng range) of functional Nb_An46 in different tsetse fly tissues, including the midgut where an important developmental stage of the trypanosome parasite occurs.

Conclusions: We demonstrated the proof-of-concept that the Sodalis symbiont can be genetically engineered to express and release significant amounts of functional anti-trypanosome Nbs in different tissues of the tsetse fly. The application of this innovative concept of using pathogen-targeting nanobodies delivered by insect symbiotic bacteria could be extended to other vector-pathogen systems.

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ELISA based quantitation of the amount of Nb_An46 present in haemolymph, abdomen, thorax and midgut of streptozotocin-treated flies injected with 1×107CFU ofSod_pFliCpelBNb46fliCat selected time points (○:7 dpi, ▽: 14 dpi, □: 7 dpi) using a 6 × His tag specific detection antibody. Values are presented as ng nanobody per whole tissue extract or 1 μl haemolymph.
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Fig7: ELISA based quantitation of the amount of Nb_An46 present in haemolymph, abdomen, thorax and midgut of streptozotocin-treated flies injected with 1×107CFU ofSod_pFliCpelBNb46fliCat selected time points (○:7 dpi, ▽: 14 dpi, □: 7 dpi) using a 6 × His tag specific detection antibody. Values are presented as ng nanobody per whole tissue extract or 1 μl haemolymph.

Mentions: Nb_An46 expression in flies injected with 1 × 107Sod_FliCpelBNb46fliC CFU was quantified using a VSG-binding ELISA. Nanobody concentrations were determined at different time points post-injection in whole abdomen and thorax extracts, haemolymph and midgut (Figure 7). Functional Nb_An46 accumulated in haemolymph and thorax samples of injected flies over time, indicating a continuous transgene expression by recSodalis in these tissues. In the thorax the Nb_An46 concentration increased from 22 ng on day 14 post-injection to 35 ng on day 21. Although significant quantities of active Nb_An46 were detected in abdomen and midgut using a VSG-binding ELISA, the accuracy of this quantitation could have been hampered by the abundant presence of proteolytic enzymes in the tsetse fly gut, probably resulting in fast degradation of the nanobodies in the tissue homogenates and possibly resulting in the underestimation of the active Nb content of the non-digestive part of the tsetse fly gut.Figure 7


Delivery of a functional anti-trypanosome Nanobody in different tsetse fly tissues via a bacterial symbiont, Sodalis glossinidius.

De Vooght L, Caljon G, De Ridder K, Van Den Abbeele J - Microb. Cell Fact. (2014)

ELISA based quantitation of the amount of Nb_An46 present in haemolymph, abdomen, thorax and midgut of streptozotocin-treated flies injected with 1×107CFU ofSod_pFliCpelBNb46fliCat selected time points (○:7 dpi, ▽: 14 dpi, □: 7 dpi) using a 6 × His tag specific detection antibody. Values are presented as ng nanobody per whole tissue extract or 1 μl haemolymph.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4230353&req=5

Fig7: ELISA based quantitation of the amount of Nb_An46 present in haemolymph, abdomen, thorax and midgut of streptozotocin-treated flies injected with 1×107CFU ofSod_pFliCpelBNb46fliCat selected time points (○:7 dpi, ▽: 14 dpi, □: 7 dpi) using a 6 × His tag specific detection antibody. Values are presented as ng nanobody per whole tissue extract or 1 μl haemolymph.
Mentions: Nb_An46 expression in flies injected with 1 × 107Sod_FliCpelBNb46fliC CFU was quantified using a VSG-binding ELISA. Nanobody concentrations were determined at different time points post-injection in whole abdomen and thorax extracts, haemolymph and midgut (Figure 7). Functional Nb_An46 accumulated in haemolymph and thorax samples of injected flies over time, indicating a continuous transgene expression by recSodalis in these tissues. In the thorax the Nb_An46 concentration increased from 22 ng on day 14 post-injection to 35 ng on day 21. Although significant quantities of active Nb_An46 were detected in abdomen and midgut using a VSG-binding ELISA, the accuracy of this quantitation could have been hampered by the abundant presence of proteolytic enzymes in the tsetse fly gut, probably resulting in fast degradation of the nanobodies in the tissue homogenates and possibly resulting in the underestimation of the active Nb content of the non-digestive part of the tsetse fly gut.Figure 7

Bottom Line: We show that recSodalis is competitive with WT Sodalis in in vivo conditions and that tsetse flies transiently cleared of their endogenous WT Sodalis population can be successfully repopulated with recSodalis at high densities.Finally, we demonstrated that recSodalis expressed significant levels (ng range) of functional Nb_An46 in different tsetse fly tissues, including the midgut where an important developmental stage of the trypanosome parasite occurs.We demonstrated the proof-of-concept that the Sodalis symbiont can be genetically engineered to express and release significant amounts of functional anti-trypanosome Nbs in different tissues of the tsetse fly.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences, Unit of Veterinary Protozoology, Institute of Tropical Medicine Antwerp (ITM), Antwerp, Belgium. ldevooght@itg.be.

ABSTRACT

Background: Sodalis glossinidius, a vertically transmitted microbial symbiont of the tsetse fly, is currently considered as a potential delivery system for anti-trypanosomal components that reduce or eliminate the capability of the tsetse fly host to transmit parasitic trypanosomes, an approach also known as paratransgenesis. An essential step in developing paratransgenic tsetse is the stable colonization of adult flies and their progeny with recombinant Sodalis bacteria, expressing trypanocidal effector molecules in tissues where the parasite resides.

Results: In this study, Sodalis was tested for its ability to deliver functional anti-trypanosome nanobodies (Nbs) in Glossina morsitans morsitans. We characterized the in vitro and in vivo stability of recombinant Sodalis (recSodalis) expressing a potent trypanolytic nanobody, i.e. Nb_An46. We show that recSodalis is competitive with WT Sodalis in in vivo conditions and that tsetse flies transiently cleared of their endogenous WT Sodalis population can be successfully repopulated with recSodalis at high densities. In addition, vertical transmission to the offspring was observed. Finally, we demonstrated that recSodalis expressed significant levels (ng range) of functional Nb_An46 in different tsetse fly tissues, including the midgut where an important developmental stage of the trypanosome parasite occurs.

Conclusions: We demonstrated the proof-of-concept that the Sodalis symbiont can be genetically engineered to express and release significant amounts of functional anti-trypanosome Nbs in different tissues of the tsetse fly. The application of this innovative concept of using pathogen-targeting nanobodies delivered by insect symbiotic bacteria could be extended to other vector-pathogen systems.

Show MeSH
Related in: MedlinePlus