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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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In vitrocharacteristics of recSodalisexpressing Nb_An46. A) Growth curve analysis of WT Sodalis and Sod_pFliCpelBNb46fliC. The error bars show the ± SD of two biological replicates. Samples were taken every 24 h. B) ELISA-based Nb_An46 quantitation (bar-chart) of the intra- and extracellular nanobody concentration produced by Sod_pFliCpelBNb46fliC at selected time points in relation to bacterial cell density (OD600nm, solid line) using a 6 × His tag specific detection antibody. Values are presented as ng recombinant protein per ml culture medium.
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Fig2: In vitrocharacteristics of recSodalisexpressing Nb_An46. A) Growth curve analysis of WT Sodalis and Sod_pFliCpelBNb46fliC. The error bars show the ± SD of two biological replicates. Samples were taken every 24 h. B) ELISA-based Nb_An46 quantitation (bar-chart) of the intra- and extracellular nanobody concentration produced by Sod_pFliCpelBNb46fliC at selected time points in relation to bacterial cell density (OD600nm, solid line) using a 6 × His tag specific detection antibody. Values are presented as ng recombinant protein per ml culture medium.

Mentions: Prior to the introduction of recSodalis into experimental flies, the Nb expression profile, in vitro growth rate and plasmid stability of recSodalis expressing a FliCpelBNb_An46 fusion protein (Sod:FliCpelBNb46fliC) was established. Extracellular Nb_An46 expression was confirmed by Western blot analysis of supernatant from cultures grown to the beginning of stationary phase (OD600 0.5-0.6) (Figure 1). Nb_An46 expression and release was quantified at different time points during bacterial growth over a 10-day period by measuring the concentration of active Nb in the whole cell lysate and culture medium using a VSG-binding ELISA assay. Functional Nb_An46 was expressed from day 2 onwards and accumulated in the culture medium to a concentration of 88 ng/ml by day 10 (Figure 2B). RecSodalis showed normal growth kinetics (Figure 2A) with cell population doubling times comparable to a WT Sodalis strain i.e., 15.0 hrs and 14.8 hrs respectively. The number of plasmid copies per cell was estimated to be approximately 20 during the lag and exponential phases of Sod:FliCpelBNb46fliC grown in the presence of antibiotic selection. The stability of the FliCpelBNb46fliC plasmid in recSodalis was measured by maintaining the recombinant bacteria in log phase growth for 27 generations in liquid MM medium in the absence of antibiotic selection. Colony counts on antibiotic-selective plates showed that 94% of the Sodalis population remained antibiotic resistant after 27 generations (corresponding to a 2-month test period) (Table 1).Figure 1


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)

In vitrocharacteristics of recSodalisexpressing Nb_An46. A) Growth curve analysis of WT Sodalis and Sod_pFliCpelBNb46fliC. The error bars show the ± SD of two biological replicates. Samples were taken every 24 h. B) ELISA-based Nb_An46 quantitation (bar-chart) of the intra- and extracellular nanobody concentration produced by Sod_pFliCpelBNb46fliC at selected time points in relation to bacterial cell density (OD600nm, solid line) using a 6 × His tag specific detection antibody. Values are presented as ng recombinant protein per ml culture medium.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig2: In vitrocharacteristics of recSodalisexpressing Nb_An46. A) Growth curve analysis of WT Sodalis and Sod_pFliCpelBNb46fliC. The error bars show the ± SD of two biological replicates. Samples were taken every 24 h. B) ELISA-based Nb_An46 quantitation (bar-chart) of the intra- and extracellular nanobody concentration produced by Sod_pFliCpelBNb46fliC at selected time points in relation to bacterial cell density (OD600nm, solid line) using a 6 × His tag specific detection antibody. Values are presented as ng recombinant protein per ml culture medium.
Mentions: Prior to the introduction of recSodalis into experimental flies, the Nb expression profile, in vitro growth rate and plasmid stability of recSodalis expressing a FliCpelBNb_An46 fusion protein (Sod:FliCpelBNb46fliC) was established. Extracellular Nb_An46 expression was confirmed by Western blot analysis of supernatant from cultures grown to the beginning of stationary phase (OD600 0.5-0.6) (Figure 1). Nb_An46 expression and release was quantified at different time points during bacterial growth over a 10-day period by measuring the concentration of active Nb in the whole cell lysate and culture medium using a VSG-binding ELISA assay. Functional Nb_An46 was expressed from day 2 onwards and accumulated in the culture medium to a concentration of 88 ng/ml by day 10 (Figure 2B). RecSodalis showed normal growth kinetics (Figure 2A) with cell population doubling times comparable to a WT Sodalis strain i.e., 15.0 hrs and 14.8 hrs respectively. The number of plasmid copies per cell was estimated to be approximately 20 during the lag and exponential phases of Sod:FliCpelBNb46fliC grown in the presence of antibiotic selection. The stability of the FliCpelBNb46fliC plasmid in recSodalis was measured by maintaining the recombinant bacteria in log phase growth for 27 generations in liquid MM medium in the absence of antibiotic selection. Colony counts on antibiotic-selective plates showed that 94% of the Sodalis population remained antibiotic resistant after 27 generations (corresponding to a 2-month test period) (Table 1).Figure 1

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