Search

Sodalis glossinidius, a secondary bacterial symbiont of the tsetse fly, is currently considered as a potential delivery system for anti-trypanosomal components interfering with African trypanosome transmission (i.e. paratransgenesis). Nanobodies (Nbs) have been proposed as potential candidates to target the parasite during development in the tsetse fly. In this study, we have generated an immune Nb-library and developed a panning strategy to select Nbs against the Trypanosoma brucei brucei procyclic developmental stage present in the tsetse fly midgut. Selected Nbs were expressed, purified, assessed for binding and tested for their impact on the survival and growth of in vitro cultured procyclic T. b. brucei parasites. Next, we engineered S. glossinidius to express the selected Nbs and validated their ability to block T. brucei development in the tsetse fly midgut. Genetically engineered S. glossinidius expressing Nb_88 significantly compromised parasite development in the tsetse fly midgut both at the level of infection rate and parasite load. Interestingly, expression of Nb_19 by S. glossinidius resulted in a significantly enhanced midgut establishment. These data are the first to show in situ delivery by S. glossinidius of effector molecules that can target the trypanosome-tsetse fly crosstalk, interfering with parasite development in the fly. These proof-of-principle data represent a major step forward in the development of a control strategy based on paratransgenic tsetse flies. Finally, S. glossinidius-based Nb delivery can also be applied as a powerful laboratory tool to unravel the molecular determinants of the parasite-vector association.

Tsetse flies (Glossina sp.) are medically and veterinary important vectors of African trypanosomes, protozoan parasites that cause devastating diseases in humans and livestock in sub-Saharan Africa. These flies feed exclusively on vertebrate blood and harbor a limited diversity of obligate and facultative bacterial commensals. They have a well-developed innate immune system that plays a key role in protecting the fly against invading pathogens and in modulating the fly's ability to transmit African trypanosomes. In this review, we briefly summarize our current knowledge on the tsetse fly innate immune system and its interaction with the bacterial commensals and the trypanosome parasite.

Background Tsetse flies (Diptera: Glossinidae) are the cyclical vectors of the causative agents of African Trypanosomosis, which has been identified as a neglected tropical disease in both humans and animals in many regions of sub-Saharan Africa. The sterile insect technique (SIT) has shown to be a powerful method to manage tsetse fly populations when used in the frame of an area-wide integrated pest management (AW-IPM) program. To date, the release of sterile males to manage tsetse fly populations has only been implemented in areas to reduce transmission of animal African Trypanosomosis (AAT). The implementation of the SIT in areas with Human African Trypanosomosis (HAT) would require additional measures to eliminate the potential risk associated with the release of sterile males that require blood meals to survive and hence, might contribute to disease transmission. Paratransgenesis offers the potential to develop tsetse flies that are refractory to trypanosome infection by modifying their associated bacteria (Sodalis glossinidius) here after referred to as Sodalis. Here we assessed the feasibility of combining the paratransgenesis approach with SIT by analyzing the impact of ionizing radiation on the copy number of Sodalis and the vectorial capacity of sterilized tsetse males. Results Adult Glossina morsitans morsitans that emerged from puparia irradiated on day 22 post larviposition did not show a significant decline in Sodalis copy number as compared with non-irradiated flies. Conversely, the Sodalis copy number was significantly reduced in adults that emerged from puparia irradiated on day 29 post larviposition and in adults irradiated on day 7 post emergence. Moreover, irradiating 22-day old puparia reduced the copy number of Wolbachia and Wigglesworthia in emerged adults as compared with non-irradiated controls, but the radiation treatment had no significant impact on the vectorial competence of the flies. Conclusion Although the radiation treatment significantly reduced the copy number of some tsetse fly symbionts, the copy number of Sodalis recovered with time in flies irradiated as 22-day old puparia. This recovery offers the opportunity to combine a paratransgenesis approach – using modified Sodalis to produce males refractory to trypanosome infection – with the release of sterile males to minimize the risk of disease transmission, especially in HAT endemic areas. Moreover, irradiation did not increase the vector competence of the flies for trypanosomes. Electronic supplementary material The online version of this article (10.1186/s12866-018-1283-8) contains supplementary material, which is available to authorized users.

With the absence of effective prophylactic vaccines and drugs against African trypanosomosis, control of this group of zoonotic neglected tropical diseases depends the control of the tsetse fly vector. When applied in an area-wide insect pest management approach, the sterile insect technique (SIT) is effective in eliminating single tsetse species from isolated populations. The need to enhance the effectiveness of SIT led to the concept of investigating tsetse-trypanosome interactions by a consortium of researchers in a five-year (2013–2018) Coordinated Research Project (CRP) organized by the Joint Division of FAO/IAEA. The goal of this CRP was to elucidate tsetse-symbiome-pathogen molecular interactions to improve SIT and SIT-compatible interventions for trypanosomoses control by enhancing vector refractoriness. This would allow extension of SIT into areas with potential disease transmission. This paper highlights the CRP’s major achievements and discusses the science-based perspectives for successful mitigation or eradication of African trypanosomosis.

Sodalis glossinidius, a maternally inherited secondary symbiont of the tsetse fly, is a bacterium in the early/intermediate state of the transition toward symbiosis, representing an important model for investigating establishment and evolution of insect-bacteria symbiosis. The absence of phylogenetic congruence in tsetse-Sodalis coevolution and the existence of Sodalis genotypic diversity in field flies are suggestive for a horizontal transmission route. However, to date no natural mechanism for the horizontal transfer of this symbiont has been identified. Using novel methodologies for the stable fluorescent-labeling and introduction of modified Sodalis in tsetse flies, we unambiguously show that male-borne Sodalis is 1) horizontally transferred to females during mating and 2) subsequently vertically transmitted to the progeny, that is, paternal transmission. This mixed mode of transmission has major consequences regarding Sodalis' genome evolution as it can lead to coinfections creating opportunities for lateral gene transfer which in turn could affect the interaction with the tsetse host.

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. Electronic supplementary material The online version of this article (doi:10.1186/s12934-014-0156-6) contains supplementary material, which is available to authorized users.

Blood feeding arthropods are responsible for the transmission of a large array of medically important infectious agents that include viruses, bacteria, protozoan parasites and helminths. The recent development of transgenic and paratransgenic technologies have enabled supplementing the immune system of these arthropod vectors with anti-pathogen effector molecules in view of compromising their vector competence for these microbial agents. The characteristics of the selected anti-pathogen compound will largely determine the efficacy and specificity of this approach. Low specificity will generally result in bystander effects, likely having a direct or indirect fitness cost for the arthropod. In contrast, the use of highly specific compounds from the adaptive immune system of vertebrates such as antibody derived fragments is more likely to enable highly specific effects without conferring a selective disadvantage to the (para)transgenic arthropods. Here, Nanobodies® are excellent candidates to increase the immune competence of arthropods. Moreover they were shown to exert a novel type of anti-pathogen activity that uniquely depends on their small size.

Sodalis glossinidius, a maternally inherited endosymbiont of the tsetse fly, maintains genes encoding homologues of the PhoP-PhoQ two-component regulatory system. This two-component system has been extensively studied in facultative bacterial pathogens and is known to serve as an environmental magnesium sensor and a regulator of key virulence determinants. In the current study, we show that the inactivation of the response regulator, phoP, renders S. glossinidius sensitive to insect derived cationic antimicrobial peptides (AMPs). The resulting mutant strain displays reduced expression of genes involved in the structural modification of lipid A that facilitates resistance to AMPs. In addition, the inactivation of phoP alters the expression of type-III secretion system (TTSS) genes encoded within three distinct chromosomal regions, indicating that PhoP-PhoQ also serves as a master regulator of TTSS gene expression. In the absence of phoP, S. glossinidius is unable to superinfect either its natural tsetse fly host or a closely related hippoboscid louse fly. Furthermore, we show that the S. glossinidius PhoQ sensor kinase has undergone functional adaptations that result in a substantially diminished ability to sense ancestral signals. The loss of PhoQ's sensory capability is predicted to represent a novel adaptation to the static symbiotic lifestyle, allowing S. glossinidius to constitutively express genes that facilitate resistance to host derived AMPs., Author Summary Mutualistic insect endosymbionts are known to undergo a process of degenerative evolution that streamlines their gene inventory in accordance with the obligate nature of the host associated lifestyle. Here we show that the mutualistic insect endosymbiont Sodalis glossinidius utilizes a two-component regulatory system (PhoP-PhoQ) that evolved to facilitate the regulation of virulence genes in facultative pathogens in accordance with environmental magnesium. While the regulatory targets of PhoP-PhoQ are conserved among S. glossinidius and a wide range of bacterial pathogens, the S. glossinidius PhoP-PhoQ system is unusual because it has a substantially reduced ability to sense magnesium as a repressing signal. This enables S. glossinidius to express genes encoding critical symbiosis determinants within host environments that are rich in both magnesium and antimicrobial peptides. These findings illustrate how a change in the ecology of a host–symbiont association can influence the dynamic functionality of a regulatory system in the symbiont.

Background Sodalis glossinidius, a gram-negative bacterial endosymbiont of the tsetse fly, has been proposed as a potential in vivo drug delivery vehicle to control trypanosome parasite development in the fly, an approach known as paratransgenesis. Despite this interest of S. glossinidius as a paratransgenic platform organism in tsetse flies, few potential effector molecules have been identified so far and to date none of these molecules have been successfully expressed in this bacterium. Results In this study, S. glossinidius was transformed to express a single domain antibody, (Nanobody®) Nb_An33, that efficiently targets conserved cryptic epitopes of the variant surface glycoprotein (VSG) of the parasite Trypanosoma brucei. Next, we analyzed the capability of two predicted secretion signals to direct the extracellular delivery of significant levels of active Nb_An33. We show that the pelB leader peptide was successful in directing the export of fully functional Nb_An33 to the periplasm of S. glossinidius resulting in significant levels of extracellular release. Finally, S. glossinidius expressing pelBNb_An33 exhibited no significant reduction in terms of fitness, determined by in vitro growth kinetics, compared to the wild-type strain. Conclusions These data are the first demonstration of the expression and extracellular release of functional trypanosome-interfering Nanobodies® in S. glossinidius. Furthermore, Sodalis strains that efficiently released the effector protein were not affected in their growth, suggesting that they may be competitive with endogenous microbiota in the midgut environment of the tsetse fly. Collectively, these data reinforce the notion for the potential of S. glossinidius to be developed into a paratransgenic platform organism.