African sleeping sickness or African Trypanosomiasis is a disease caused by the trypanosoma parasite which is biologically transmitted by the tsetse fly (Glossina sp.). The insect is essential for the completion of the parasite’s life cycle. Individuals with the disease exhibit symptoms in two stages. First stage being the haemolymphatic phase and the second stage being the neurological phase. The disease is known as the sleeping sickness due to the second phase which includes symptoms such as confusion, lack of coordination and disrupted sleep cycle.
Sodalis glossinidius is a gram-negative endosymbiotic bacteria living in the alimentary canal of the tsetse fly. These bacteria help the tsetse fly to acquire nutrients that are not synthesized by the fly or not present in their diet. The striking feature of S. glossinidius is that it is one of the few endosymbiotic bacteria that could be cultured and modified genetically.
A group of scientists from Belgium have utilized this feature to genetically modify the endosymbiote to express proteins that can control the sleeping sickness parasite. This is known as paratransgenesis. A transgene is introduced into S. glossinidius where the gene products interfere with the development of the pathogen inside the tsetse host and at the same time preserving host and the symbiote’s viability and fitness.
Nanobodies® (Nbs) are the smallest known antigen binding fragments that target specific antigenic epitopes that are less efficiently targeted by antibodies. Nbs have several superior properties such as small size and stability and therefore are perfect candidates to be used in paratransgenesis. A trypanosome surface glycoprotein specific Nb_An33 Nanobody® was used for targeting the trypanosoma parasite.
Expression plasmids were constructed using the Nbs gene (Nb_An33) with a lac promoter and two genes for the secretion signal for extracellular release of Nb_An33 Nanobody®. The Nb_An33 secreting strains exhibited growth comparable to the wild type. Expressed Nanobody concentrations were determined using ELISA. Binding ability of the extracellularly expressed Nb_An33 with trypanasoma parasite was determined using flow cytometry and fluorescence microscopy.
These results indicate that S. glossinidius could be manipulated to be a paratransgenic organism and the use of Nbs effectors in biological control of other vector borne diseases.
Source : Microbial cell factories