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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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Number of recSodalisCFU (DNA equivalent) present in abdomen (A) and thorax (T) tissues of flies injected with respectively 5×104, 5×105, 1×107and 5×107CFU ofSod_pFliCpelBNb46fliC. qPCR on the pFliCpelBNb46FliCplasmid 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 Kruskal–Wallis test followed by Dunn's test for multiple comparison (*p <0.05, **p <0.01).
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Fig4: Number of recSodalisCFU (DNA equivalent) present in abdomen (A) and thorax (T) tissues of flies injected with respectively 5×104, 5×105, 1×107and 5×107CFU ofSod_pFliCpelBNb46fliC. qPCR on the pFliCpelBNb46FliCplasmid 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 Kruskal–Wallis test followed by Dunn's test for multiple comparison (*p <0.05, **p <0.01).

Mentions: We determined the optimal dose for recSodalis injection in terms of host colonization and its viability. For this, streptozotocin-treated male flies were microinjected with either 5 × 104, 5 × 105, 107 and 5 × 107 CFU Sod_FliCpelBNb46fliC and recSodalis densities in abdomen and thorax tissues were determined 7 and 14 days post-injection (dpi) using qPCR (Figure 4). Injection of 5 × 107 CFU resulted in an increased fly mortality (up to 59% mortality 14 dpi), whereas limited mortality (≤25% 14 dpi) was observed within the other injection groups. In these groups, recSodalis was able to repopulate the abdomen and thorax to densities comparable to natural Sodalis levels present in WT flies (on average respectively 2.5 × 106 and 1.5 × 106 CFU), demonstrating that a wide range of recSodalis doses (i.e. 5 × 104 to 1 × 107) are suitable to initiate colonization without affecting fly viability.Figure 4


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) present in abdomen (A) and thorax (T) tissues of flies injected with respectively 5×104, 5×105, 1×107and 5×107CFU ofSod_pFliCpelBNb46fliC. qPCR on the pFliCpelBNb46FliCplasmid 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 Kruskal–Wallis test followed by Dunn's test for multiple comparison (*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

Fig4: Number of recSodalisCFU (DNA equivalent) present in abdomen (A) and thorax (T) tissues of flies injected with respectively 5×104, 5×105, 1×107and 5×107CFU ofSod_pFliCpelBNb46fliC. qPCR on the pFliCpelBNb46FliCplasmid 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 Kruskal–Wallis test followed by Dunn's test for multiple comparison (*p <0.05, **p <0.01).
Mentions: We determined the optimal dose for recSodalis injection in terms of host colonization and its viability. For this, streptozotocin-treated male flies were microinjected with either 5 × 104, 5 × 105, 107 and 5 × 107 CFU Sod_FliCpelBNb46fliC and recSodalis densities in abdomen and thorax tissues were determined 7 and 14 days post-injection (dpi) using qPCR (Figure 4). Injection of 5 × 107 CFU resulted in an increased fly mortality (up to 59% mortality 14 dpi), whereas limited mortality (≤25% 14 dpi) was observed within the other injection groups. In these groups, recSodalis was able to repopulate the abdomen and thorax to densities comparable to natural Sodalis levels present in WT flies (on average respectively 2.5 × 106 and 1.5 × 106 CFU), demonstrating that a wide range of recSodalis doses (i.e. 5 × 104 to 1 × 107) are suitable to initiate colonization without affecting fly viability.Figure 4

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