Rai, Rajeev (2016) Mechanisms underlying Leishmania parasites survival and dissemination at late stage of infection in human macrophages. PhD thesis, University of Greenwich.

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Abstract

Leishmania parasites are the causative agent of a wide range of human diseases known as leishmaniasis. The progression of disease pathogenesis is dependent on the ability of intracellular parasites to disseminate between human macrophages. It is assumed that parasites could exit macrophages following cell lysis. However, there is still a major gap in knowledge in understanding the exact mechanism pertaining to this critical step. This has been partially caused by the lack of reliable experimental models to study this stage of host-parasite interaction. This research aims to elucidate the mechanism behind cell-tocell spreading of Leishmania in vitro.

A link between host cell apoptosis and Leishmania propagation was initially established by determining the effect of apoptotic induction on infection over a period of 96 hours. The result showed that 60-90% of cells were inoculated with L. aethiopica and L. mexicana within 72 and 96 hours infection. Although, the concentration of viable cells was greatly reduced as compared to non-induced samples, a heavily infected cell population was obtained with this approach. Hence, this population of L. aethiopica and L. mexicana infected cells at 72 hours was used to infect differentiated THP-1 macrophages and validated as a model for infection spreading. Amastigotes spreading was detected within 12 hours of coculture as confirmed by flow cytometry and live cell imaging techniques. Interestingly, the live imaging revealed inter-cellular extrusion of L. aethiopica and L. mexicana from infected to a recipient cells without cell lysis. To address whether parasite extrusion is linked to apoptotic induction, this model was used to detect expression of several apoptotic markers. The results showed that L. aethiopica (but not L. mexicana) dissemination was correlated with host apoptotic induction, as there was a significant increase (p<0.05) in phosphatidyl serine (PS) externalization and active caspase-3 expression. In addition, western blot analysis demonstrated that L. aethiopica spread was associated with downregulation of three major apoptotic signaling pathways: AKT, NF-κB and PKC-δ. Further comparative proteomic analysis of this model supported these findings and showed that during L. aethiopica spreading, a number of host physiological processes were subverted. Specifically caspase-3 and caspase-9 activation strengthens the involvement of mitochondria in initiation of apoptosis. Interestingly, chitinase-3-like protein (inducer of IL-13 production) and butyrophilin (inhibitor of IFN-γ and IL-12) were markedly increased confirming the downregulation of inflammatory response. Finally, elevated increase in glutathione reductase and metallothionein-2 proteins, which are involved in scavenging free radicals, suggests that anti-microbial activity is also downregulated during L. aethiopica spreading.

In conclusion, by developing and validating for the first time a credible in vitro spreading model in human macrophages, this research has clarified the mechanism of Leishmania spreading. L. ethiopica promotes host cell apoptosis in order to disseminate from cell-to-cell without activating inflammatory and anti-microbial responses. In particular, apoptotic induction takes place via downregulation of AKT and NF-κB, release of cytochrome C and consequent activation of caspase-3 and PS exhibition, independently from PKC-δ. This model clearly fits with the silent spread of parasites that takes place in the first weeks (to months) of infection in mice model. Interestingly, the fact that L. mexicana utilizes a non-apoptotic strategy to spread suggests that alternative mechanisms of parasitic extrusion also exist underlying the necessity of species specific investigations. Understanding the molecular basis of Leishmania spreading contributes to filling a current gap in knowledge in the biology of Leishmania infection strategy of this very successful parasite. Moreover, it has the potential to open up a novel set of targets for drugs and vaccine development against this increasingly widespread neglected disease.

Item Type:	Thesis (PhD)
Uncontrolled Keywords:	Leishmania parasites; molecular interaction; macrophage; parasite culture; Leishmania infection; host cell apoptosis;
Subjects:	Q Science > QD Chemistry
Faculty / School / Research Centre / Research Group:	Faculty of Engineering & Science Faculty of Engineering & Science > School of Science (SCI)
Last Modified:	21 Nov 2017 17:17
URI:	http://gala.gre.ac.uk/id/eprint/18109

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