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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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Related in: MedlinePlus

Number of recSodalisCFU (DNA equivalent) in abdomen and thorax of streptozotocin-treated and non-treated male flies injected with 5x104CFU ofSod_pFliCpelBNb46fliC. Prior to injection, treated flies were given three blood meals supplemented with 20 μg/ml streptozotocin during the first week after emergence, while non-treated flies received normal blood meals. qPCR on the pFliCpelBNb46FliC plasmid was used as a means to estimate the number of recSodalis. The bars represent the mean recSodalis CFU (DNA equivalent) ± SD present in abdomen and thorax tissues of at least 5 individual flies from each treatment group at the time of sampling. The number of CFU (DNA equivalent) is represented in log scale on the y-axis. P values were calculated using the Mann–Whitney U test (*p <0.05, **p <0.01).
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Fig3: Number of recSodalisCFU (DNA equivalent) in abdomen and thorax of streptozotocin-treated and non-treated male flies injected with 5x104CFU ofSod_pFliCpelBNb46fliC. Prior to injection, treated flies were given three blood meals supplemented with 20 μg/ml streptozotocin during the first week after emergence, while non-treated flies received normal blood meals. qPCR on the pFliCpelBNb46FliC plasmid was used as a means to estimate the number of recSodalis. The bars represent the mean recSodalis CFU (DNA equivalent) ± SD present in abdomen and thorax tissues of at least 5 individual flies from each treatment group at the time of sampling. The number of CFU (DNA equivalent) is represented in log scale on the y-axis. P values were calculated using the Mann–Whitney U test (*p <0.05, **p <0.01).

Mentions: We explored the capability of recSodalis to colonize the tsetse fly after introduction through intrathoracic injection. We first evaluated the necessity of the prior reduction of the WT Sodalis population in tsetse for recSodalis to establish and colonize its host. In female flies that received 3 streptozotocin supplemented blood meals, the WT Sodalis population was reduced by 95% and 88% in abdomen and thorax tissues respectively, compared to flies fed on normal blood. This treatment did not affect the obligatory Wigglesworthia symbiont population that mainly resides in the tsetse fly abdomen (Additional file 1: Figure S1). Next, the in vivo persistence and growth of the recombinant bacteria in streptozotocin-treated and non-treated flies injected intrathoracically with 5×104 CFU Sod_FliCpelBNb46fliC was evaluated using qPCR (Figure 3). In streptozotocin-treated flies, recSodalis was able to proliferate inside its host, reaching densities of 10- and 20-fold the initial injected dose in respectively abdomen and thorax, whereas in non-treated flies the injected recSodalis population was not able to expand and remained present at its initial density.Figure 3


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)

Number of recSodalisCFU (DNA equivalent) in abdomen and thorax of streptozotocin-treated and non-treated male flies injected with 5x104CFU ofSod_pFliCpelBNb46fliC. Prior to injection, treated flies were given three blood meals supplemented with 20 μg/ml streptozotocin during the first week after emergence, while non-treated flies received normal blood meals. qPCR on the pFliCpelBNb46FliC plasmid was used as a means to estimate the number of recSodalis. The bars represent the mean recSodalis CFU (DNA equivalent) ± SD present in abdomen and thorax tissues of at least 5 individual flies from each treatment group at the time of sampling. The number of CFU (DNA equivalent) is represented in log scale on the y-axis. P values were calculated using the Mann–Whitney U test (*p <0.05, **p <0.01).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig3: Number of recSodalisCFU (DNA equivalent) in abdomen and thorax of streptozotocin-treated and non-treated male flies injected with 5x104CFU ofSod_pFliCpelBNb46fliC. Prior to injection, treated flies were given three blood meals supplemented with 20 μg/ml streptozotocin during the first week after emergence, while non-treated flies received normal blood meals. qPCR on the pFliCpelBNb46FliC plasmid was used as a means to estimate the number of recSodalis. The bars represent the mean recSodalis CFU (DNA equivalent) ± SD present in abdomen and thorax tissues of at least 5 individual flies from each treatment group at the time of sampling. The number of CFU (DNA equivalent) is represented in log scale on the y-axis. P values were calculated using the Mann–Whitney U test (*p <0.05, **p <0.01).
Mentions: We explored the capability of recSodalis to colonize the tsetse fly after introduction through intrathoracic injection. We first evaluated the necessity of the prior reduction of the WT Sodalis population in tsetse for recSodalis to establish and colonize its host. In female flies that received 3 streptozotocin supplemented blood meals, the WT Sodalis population was reduced by 95% and 88% in abdomen and thorax tissues respectively, compared to flies fed on normal blood. This treatment did not affect the obligatory Wigglesworthia symbiont population that mainly resides in the tsetse fly abdomen (Additional file 1: Figure S1). Next, the in vivo persistence and growth of the recombinant bacteria in streptozotocin-treated and non-treated flies injected intrathoracically with 5×104 CFU Sod_FliCpelBNb46fliC was evaluated using qPCR (Figure 3). In streptozotocin-treated flies, recSodalis was able to proliferate inside its host, reaching densities of 10- and 20-fold the initial injected dose in respectively abdomen and thorax, whereas in non-treated flies the injected recSodalis population was not able to expand and remained present at its initial density.Figure 3

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