Investigation of the Hemidesmosome Protein Components and Their Roles in the Attachment Mechanism of Leishmania.

Bakri, Rowaida Ali (2014) Investigation of the Hemidesmosome Protein Components and Their Roles in the Attachment Mechanism of Leishmania. PhD thesis, Lancaster University.

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Abstract

During their life cycles trypanosomatids differentiate into distinct forms within their hosts. The parasites use their flagella to attach and anchor themselves to the surface of the gut wall in their sand fly vectors. On cuticular surfaces the flagellar tip becomes expanded and forms hemidesmosome-like structures, which enables the parasites to attach strongly to the gut wall. This attachment process occurs in all trypanosomatids and is believed to protect the parasites from elimination by the action of digestive system of the gut so they can survive and multiply, and is also proposed to be an important element of the transmission mechanism. The identity of Leishmania hemidesmosomal protein molecules and their function in the attachment mechanism is not known. In order to investigate this, L. mexicana, L. major and L. tarentolae promastigotes were cultured in the presence of various materials in attempts to replicate this attachment phenomenon in vitro. Most of the materials were discontinued due to their poor attachment performance when cultured with the parasites. However, L. mexicana promastigotes were able attach well to Ethylene-vinyl acetate copolymer (EVA) forming expanded flagella as confirmed by scanning electron microscopy. The EVA material generated high quality and quantity of parasites to use for protein investigations. Protein extraction followed by SDS-PAGE gel staining of selected materials that showed good attachment, revealed a wide range of proteins bands (24. 4 kDa - 88 kDa) in each material, except EVA showed only three bands(24. 4 kDa, 31 kDa and 36. 5 kDa). However, these may have been false positive results and most of bands were probably from media supplements, and therefore molecular approaches were applied for their sensitivity and specificity advantages. Total RNA was extracted from attached promastigotes of L. Mexicana that adhered to EVA surface. A cDNA library was conducted and screened by RT-PCR, cDNA probes and Northern blotting. Screening results showed several genes identified as hypothetical proteins and expressed by trypanosomatids or Leishmania species only. To find specific genes highly or only expressed by attached forms, levels of these genes expression were compared between in vitro attached (haptomonad) promastigotes, metacyclic and log-phase promastigotes. Results showed that the three stages expressed some genes at a similar level (LmxM. 17. 0870, LmxM. 09. 1505, LmxM. 17. 0810 and LmxM. 05. 0450), while other genes were more highly expressed by log-phase promastigotes (LmxM. 01. 0620, LmxM. 36. 3780, LmxM. 31. 2500, LmxM. 30. 2270, LmxM. 08. 0410, LmxM. 31. 1090, LmxM. 36. 3620, LmxM. 36. 5850 and LmxM. 05. 0450). Metacyclic promastigotes also showed different expression levels of several genes (LmxM. 11. 0930, LmxM. 31. 0180, LmxM. 36. 5060 and LmxM. 36. 2450). Three putative haptomonad-upregulated genes were identified (LmxM. 18. 1620, LmxM. 32. 0940 and LmxM. 29. 3025) although due to time restrictions it was not possible to determine if these were associated with hemidesmosomes. Further investigations are recommended by using new technical approaches.