Deacon, Andrew D. (2017) Expression of green fluorescent protein (GFP) in Leishmania spp. to identify survival strategies the parasite imposes on the human host. PhD thesis, University of Greenwich.

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Abstract

Leishmania protozoa are the etiological agent of human disease known as leishmaniasis. There are three main forms of this disease, whose manifestation depends on a number of factors including the infecting Leishmania species and the immunological status of the individual. Disease development requires extracellular form of the parasite, the promastigotes to be internalised, transform and replicate as intracellular amastigotes within mammalian cells. Once inside host cells parasites they regulate and control immune responses from inside the host cells through the modulation of protein expression including cytokines. The changes involving Leishmania promastigote and host cells is well documented during the early stage of infection. However, little is known on the modulative effect of L. aethiopica and L. mexicana during infection of host cells. It is key to have an altruistic approach to when studying the different Leishmania spp during host infection, as it is a key factor in the outcome of type of leishmaniasis.

The study of infection is complicated by the inherent difficulty of detecting intracellular parasites, as many methods are laborious and time consuming (microscopy with Giemsa staining). Implementation of fluorescent expressing parasites has been successfully used in a number of species to observe and quantify cellular infection. Although successful stable genetically modified Leishmania parasites are not available for L. mexicana and L. aethiopica, this research aims to investigate survival mechanisms such as cytokine and protein expression during the early stages of infection. This requires monitoring infection as the first step, which involved developing for the first time constitutively expressing GFP in L. aethiopica, L. major, L. mexicana and L. tropica. The effect of infection on host macrophages was directly quantified via comparative proteomics which identified significant differences in protein expression not only between uninfected and infected cells at late stages of infection but also between different species. The effect of infection on the ability of controlling host and neighbouring cells was further characterised by looking at cytokine & antimicrobial peptide expression, including TNFα, IL-10, TGFβ, IL-1RA and cathelicidin at various time points during L. aethiopica infection. As cytokine expression is closely linked to manipulation of T-cells, which are also know to be ultimately responsible for disease exacerbation/resolution, the effect of the parasites on such cells was further investigate. A novel role for T cells on infection was explored, specifically their ability to host parasites.

The successful stable integration of GFP expression in four Leishmania spp. was confirmed by PCR, western blot, fluorescent microscopy and flow cytometry (N=3). These parasites allowed the monitoring of infection in macrophages whilst investigating the expression of cytokines, proteins, and novel human cell types. Cathelicidin expression was shown to
increase during the early stages of infection at 8hrs following by a steady decline towards 48hrs post infection. The anti-inflammatory cytokine IL-10 expression was shown to increase from 2-48hrs peaking at the latest point with >3-fold difference post infection. Interestingly, the inflammatory cytokine TNFα showed expression increased from 2-48hrs, peaking with >5-fold at 48hrs post infection. Both these cytokines have been shown to peak at 8 hours followed by a decline, which may indicate subtle differences at a species (Lapara and Kelly 2010). Proteinomic study revealed L. mexicana and L. aethiopica both up-regulated a total of eight proteins with only two that were influenced by both species (N=3). In addition, down regulation was only identified during infection with L. aethiopica and not L. mexicana. However, during upregulation, the proteins identified were found to be involved in endocytosis (actin), immunological responses (MHC I), free radical scavenging and apoptosis (metallothionein). When comparing with similar proteomic studies by Singh et al, all modulation effects identified by L. aethiopica and L. mexicana were not previously shown during L. donovani infection. Infection was tested against human T lymphocytes as they have been implicated in the production of cytokines for the activation of macrophages allowing the elimination of intracellular parasites within macrophages. This novel work has shown L. aethiopica and L. mexicana are capable of infecting Jurkat cells (N=3) and enriched human T lymphocytes extracted from five different individuals. This finding has uncovered that infection is not only includes T cells (including CD4+ subsets) but percentage of infection is dependant on the species of parasites and the donor lymphocytes.

In conclusion, this research showed for the first time that L. aethiopica and L. mexicana modulate a repertoire of protein expression at 24hrs post infection. The changes of cytokines expression highlights differences between different species and the discovery that Leishmania has the ability to infect human T cells may commence the start of the complete understanding of the survival mechanisms imposed by the parasite on the wider cellular network. This will change the approach to studying immunology of leishmaniasis infection and potentially clarifying the role of these cells for which contrasting evidences in human and mice models have caused much debate.

Item Type:	Thesis (PhD)
Uncontrolled Keywords:	Leishmania spp; protein expression; immune system; parasites; infection
Subjects:	Q Science > QD Chemistry R Medicine > RS Pharmacy and materia medica
Faculty / School / Research Centre / Research Group:	Faculty of Engineering & Science Faculty of Engineering & Science > School of Science (SCI)
Last Modified:	24 Apr 2019 11:01
URI:	http://gala.gre.ac.uk/id/eprint/23694

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