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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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Transmission of recSodalisto the F1generation was evaluated. Recombinant and total Sodalis numbers were estimated as described above in abdomen and thorax of the F1 progeny (freshly emerged) produced by streptozotocin-treated female flies injected with 5 × 104 CFU of Sod_pFliCpelBNb46fliC. Data points represent the mean recSodalis and total Sodalis CFU (DNA equivalent) ± SD present in the respective tissues of at least 5 individual flies at the time of sampling. The number of CFU (DNA equivalent) is represented in log scale on the y-axis.
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Fig6: Transmission of recSodalisto the F1generation was evaluated. Recombinant and total Sodalis numbers were estimated as described above in abdomen and thorax of the F1 progeny (freshly emerged) produced by streptozotocin-treated female flies injected with 5 × 104 CFU of Sod_pFliCpelBNb46fliC. Data points represent the mean recSodalis and total Sodalis CFU (DNA equivalent) ± SD present in the respective tissues of at least 5 individual flies at the time of sampling. The number of CFU (DNA equivalent) is represented in log scale on the y-axis.

Mentions: The in vivo persistence of Sod_FliCpelBNb46fliC was evaluated by qPCR based estimation of the amount of recSodalis CFU in abdomen and thorax tissues of streptozotocin-treated male flies injected with 1 × 107 recombinant CFU over a 28 day period (Figure 5A). Sod_FliCpelBNb46fliC was able to remain present at high densities in abdomen and thorax tissues of experimental flies throughout the course of the 28-day observation period. In flies injected with Sod_FliCpelBNb46fliC, the entire Sodalis population in abdomen and thorax remained recombinant. Next, we evaluated recSodalis densities in the haemolymph and midgut tissues of flies injected with 1 × 107 recombinant CFU (Figure 5B). Sod_FliCpelBNb46fliC was able to reach the fly midgut where it persisted at densities between 5 × 104 and 1 × 105 CFU (DNA equivalent) throughout the 21-day observation period. In these flies the obligatory Wigglesworthia symbiont population was not affected by the presence of Sod_FliCpelBNb46fliC (Additional file 1: Figure S2) nor did we observe any effect upon the fecundity of female recSodalis colonized flies. Transmission dynamics of the recombinant bacteria to the F1 progeny was evaluated by qPCR. Sod_FliCpelBNb46fliC was transmitted to the F1 generation, although non-plasmid containing Sodalis were dominant in these flies. Indeed, Sod_FliCpelBNb46fliC constituted only 7 and 5% of the entire Sodalis population in respectively abdomen and thorax (Figure 6).Figure 5


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)

Transmission of recSodalisto the F1generation was evaluated. Recombinant and total Sodalis numbers were estimated as described above in abdomen and thorax of the F1 progeny (freshly emerged) produced by streptozotocin-treated female flies injected with 5 × 104 CFU of Sod_pFliCpelBNb46fliC. Data points represent the mean recSodalis and total Sodalis CFU (DNA equivalent) ± SD present in the respective tissues of at least 5 individual flies at the time of sampling. The number of CFU (DNA equivalent) is represented in log scale on the y-axis.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig6: Transmission of recSodalisto the F1generation was evaluated. Recombinant and total Sodalis numbers were estimated as described above in abdomen and thorax of the F1 progeny (freshly emerged) produced by streptozotocin-treated female flies injected with 5 × 104 CFU of Sod_pFliCpelBNb46fliC. Data points represent the mean recSodalis and total Sodalis CFU (DNA equivalent) ± SD present in the respective tissues of at least 5 individual flies at the time of sampling. The number of CFU (DNA equivalent) is represented in log scale on the y-axis.
Mentions: The in vivo persistence of Sod_FliCpelBNb46fliC was evaluated by qPCR based estimation of the amount of recSodalis CFU in abdomen and thorax tissues of streptozotocin-treated male flies injected with 1 × 107 recombinant CFU over a 28 day period (Figure 5A). Sod_FliCpelBNb46fliC was able to remain present at high densities in abdomen and thorax tissues of experimental flies throughout the course of the 28-day observation period. In flies injected with Sod_FliCpelBNb46fliC, the entire Sodalis population in abdomen and thorax remained recombinant. Next, we evaluated recSodalis densities in the haemolymph and midgut tissues of flies injected with 1 × 107 recombinant CFU (Figure 5B). Sod_FliCpelBNb46fliC was able to reach the fly midgut where it persisted at densities between 5 × 104 and 1 × 105 CFU (DNA equivalent) throughout the 21-day observation period. In these flies the obligatory Wigglesworthia symbiont population was not affected by the presence of Sod_FliCpelBNb46fliC (Additional file 1: Figure S2) nor did we observe any effect upon the fecundity of female recSodalis colonized flies. Transmission dynamics of the recombinant bacteria to the F1 progeny was evaluated by qPCR. Sod_FliCpelBNb46fliC was transmitted to the F1 generation, although non-plasmid containing Sodalis were dominant in these flies. Indeed, Sod_FliCpelBNb46fliC constituted only 7 and 5% of the entire Sodalis population in respectively abdomen and thorax (Figure 6).Figure 5

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