title: Unzipping the barriers: Determining the role of the peritrophic matrix in Trypanosoma brucei migration through the midgut of Glossina morsitans morsitans
Creators: name: Rose, C
Publisher: name: University of Liverpool Repository
Year: 2016

description: Tsetse flies serve as biological vectors for several species of African trypanosomes. These parasites undergo a life-cycle stage in both mammals and in the insect. Within the fly, it is proposed that for successful survival, proliferation and establishment of a midgut infection, trypanosomes must cross the tsetse fly peritrophic matrix (PM). Although having been well documented, the mechanism(s) of how trypanosomes are able to cross the PM is not well understood and the crossing event has never been visualised. The peritrophic matrix is an acellular secretion that lines the guts of most insects and is mainly composed of chitin and glycoproteins. The functions of the PM involve facilitating digestion, and epithelial cell protection against ingested pathogens, toxins and against mechanical damage. In order to better understand the nature of the tsetse PM, dissected PMs from lab-reared Glossina morsitans morsitans were solubilised under stringent conditions and their protein composition determined using a mass spectrometry-based proteomics approach. It was found that the tsetse PM is comprised of just short of 300 proteins, including several proteins from the tsetse endosymbiont, Sodalis glossinidius. Tsetse PM proteins were classified into functional groups as peritrophins, enzymes, immune related proteins and other proteins. This study also includes the first report of three tsetse PM peritrophins (GMOY002708, GMOY007191 and GMOY011810), increasing the known repertoire of peritrophins from two to five. Peritrophins were then further classified into four main groups comprising simple, binary, complex and repetitive based on their structural organisation. Comparisons of these peritrophins in Glossina morsitans morsitans were carried out against orthologous proteins of several major Glossina vectors and two comparative non blood-feeders, Musca domestica and Drosophila melanogaster. The vast number of peritrophins in M. domestica and D. melanogaster compared to those in Glossina, and given the feeding habits of these flies, suggested that the PM peritrophins have a role in the protection of the midgut against ingested pathogens. Additionally, peritrophin silencing by RNAi provided information regarding the expression and regulation of peritrophins. Multiple microscopy techniques showed the presence of trypanosomes within the layers of the PM which has previously been shown before. However, there was no evidence of iii trypanosomes crossing the mature PM in the anterior midgut as judged by tissue tomography analysis and 3D reconstruction. Subsequent analysis of the tsetse proventriculus (PV; site of PM secretion) revealed that trypanosomes could be seen in this tissue both between and either side of the PM from as soon as 5 days after receiving a trypanosome-infected bloodmeal. This suggests that the immature PM within the PV provides a probable point of entry for parasites after differentiation into the procyclic life stage. Moreover, the opportunity for breaching of the PM must occur quite quickly after bloodmeal ingestion as refractory flies had already cleared the infection by 5 days post infection. Overall, the results from this thesis suggest that the tsetse PM: a) is structurally composed of a family of peritrophins that differ in complexity and confer resistance to degradation by trypanosomes in the midgut b) functions as a way to clear parasite infections by engulfing them before elimination in the hindgut; and c) the lifecycle of T. brucei may occur entirely (or in parallel with a developmental cycle in the ectoperitrophic space) within the PV of the fly where they are proposed to complete maturation before migration to the salivary glands.