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Title: Identification and characterisation of a set of differentially expressed genes from Glossina morsitans morsitans refractory to a trypanosome infection
Author: Ejeh, Nicholas
ISNI:       0000 0004 6057 9245
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2015
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Tsetse flies (Diptera: Glossinidae) are the vectors of African trypanosomiasis, a parasitic disease of humans and other animals that can be fatal if untreated. Trypanosomes undergo a complex life cycle in the fly involving a series of morphological and biochemical changes that culminate in the development of mammalian infective metacyclics in the salivary glands (Trypanosoma brucei) or the mouthparts (T. congolense). Once in the fly’s midgut, the stumpy forms, which are thought to be pre-adapted for life in the tsetse midgut, differentiate into the procyclic form. However, the majority of flies can effectively get rid of parasites before they develop or establish an infection in the midgut. One would expect that as a result of the high disease burden in Africa, there would be a corresponding large number of flies that are also infected, yet paradoxically, just very few number of flies ( < 5%) can actually transmit the parasites to susceptible mammalian hosts. This means that tsetse flies are generally resistant to trypanosome infection, but the molecular mechanisms underlying this phenomenon are yet to be elucidated. The reasons for this natural refractoriness to a trypanosome infection are considered to be multifactorial and include the age of the fly, maturation of the fly immune system and peritrophic matrix among others, but so far few tsetse genes have been associated with this intrinsic phenotype. In this thesis, I have identified and partially characterised several midgut genes from the tsetse vector, Glossina morsitans, that have been identified as differentially expressed (DE) in flies refractory to a trypanosome infection. A bioinformatics analysis of the 454 transcriptome data shows a total of 55 genes were DE in flies that were challenged with trypanosome. Four differentially regulated midgut genes (i.e. Chitinase (CHIT; GMOY000153), O-GlcNAc transferase (OGT; GMOY002400), secreted Phospholipase A2 (sPLA2; GMOY009713) and serine proteinase inhibitor (SPI; GMOY006016)) were further analysed in silico as representative of different metabolic pathways. RNAi knockdown of midgut-expressed CHIT, OGT, sPLA2 and SPI genes significantly increased susceptibility to T. brucei infection in young files, suggesting that these genes are involved in the immune response to trypanosome infection in G. m. morsitans. However, reversion of the infection phenotype after RNAi silencing was not higher than ~40%, indicating that several proteins/pathways may be implicated in preventing establishment of a trypanosome infection in the tsetse midgut. The potential role of the midgut-expressed sPLA2 activity as a trypanocidal molecule was also studied. Over a period of two weeks, it was found that expression of midgut sPLA2, in flies receiving blood meals infected with bloodstream forms (BSF), is suppressed at the early stage of trypanosome infection, but its rises sharply after 14 days post-infection (dpi). The level of sPLA2 expression in response to infection with procyclic forms (PCF) did not differ from the expression seen when flies were challenged with BSFs, ruling out the possibility of sPLA2 expression being in response to the presence of mature PCFs. However, expression of sPLA2 was augmented at 14 dpi in flies challenged with a higher parasite load, but not with either dead parasites or bacteria, suggesting that its expression is somewhat dependent on the density of live parasite density. Surprisingly, when flies are fed with live Staphylococcus aureus, but not Escherichia coli there was a strong induction of sPLA2 as early as 5 dpi, suggesting that its expression may be linked or modulated by the Toll pathway in response to certain pathogens. Furthermore, in vitro killing assays using recombinant sPLA2 showed that procyclic trypanosomes were lysed after 24 hours when co-cultured with at least 50 g/ml of active enzyme. Thus, it is hypothesised that at 14 dpi, when the trypanosome infection is well established in both the midgut and proventriculus of the fly, a release of PLA2 may help control parasite infection, but is unable to clear it. The data obtained from this thesis will offer new insights on the molecular dialogue that ensues between trypanosomes and tsetse following the ingestion of trypanosomes by tsetse in a blood meal.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available