A comparative proteomic approach to identify and validate African trypanosome proteins at the host-parasite interface
Lead Research Organisation:
University of Cambridge
Department Name: Pathology
Abstract
Parasitic diseases afflict many of the world?s population, and the majority of people on our planet at at risk from one or several such agents. The diseases these parasites cause range from malaria to gut, skin and organ infections. In Africa, the trypanosome parasite is responsible for much death and morbidity - infection is brought about when an infected tsetse fly takes a blood meal. Without treatment the infected person will progress through general ill health symptoms to multiple organ malfunction, coma and death. Related trypanosomes also cause disease in Asia, South America, the Middle East and Southern Europe.
Due to a remarkable system where the African trypanosome changes its coat at regular intervals, the host immune system is unable to control the parasite, which results in efficient infection and invariably death if untreated. Vaccines are unlikely due to the varying coat, and the drugs in use at over 50 years old, frequently toxic and failing due to emergence of resistance. Urgent new research is needed to identify potential new therapeutic options and diagnostics.
One aspect of the parasite that is unique is a region of the surface where all molecules that form the coat emerge when they are made, called the flagellar pocket. As the surface is the boundary between host and parasite, understanding how this is maintained is of major scientific interest as well as of practical utility in uncovering new ways in which the trypanosome may be vulnerable to drug treatments.
We wish to find out what proteins are present in the flagellar pocket, with the expectation that an understanding of the molecules that are present will provide insights into how the pocket performs its job. There has so far been limited success with defining the flagellar pocket, and we propose a method that requires considerable computer power and wide-raging analysis as a solution to the problem. Most importantly, following on from the identification of candidate flagellar pocket proteins, we will look to see which expected proteins have been identified, and also, in combination with computer predictions, will localize a range of unknown proteins using the jellyfish green fluorescent protein, or GFP. This will allow us to assess how many of the proteins really are parts of the flagellar pocket, and hence begin to understand how this organelle contributes to the ability of the trypanosome to cause disease.
Due to a remarkable system where the African trypanosome changes its coat at regular intervals, the host immune system is unable to control the parasite, which results in efficient infection and invariably death if untreated. Vaccines are unlikely due to the varying coat, and the drugs in use at over 50 years old, frequently toxic and failing due to emergence of resistance. Urgent new research is needed to identify potential new therapeutic options and diagnostics.
One aspect of the parasite that is unique is a region of the surface where all molecules that form the coat emerge when they are made, called the flagellar pocket. As the surface is the boundary between host and parasite, understanding how this is maintained is of major scientific interest as well as of practical utility in uncovering new ways in which the trypanosome may be vulnerable to drug treatments.
We wish to find out what proteins are present in the flagellar pocket, with the expectation that an understanding of the molecules that are present will provide insights into how the pocket performs its job. There has so far been limited success with defining the flagellar pocket, and we propose a method that requires considerable computer power and wide-raging analysis as a solution to the problem. Most importantly, following on from the identification of candidate flagellar pocket proteins, we will look to see which expected proteins have been identified, and also, in combination with computer predictions, will localize a range of unknown proteins using the jellyfish green fluorescent protein, or GFP. This will allow us to assess how many of the proteins really are parts of the flagellar pocket, and hence begin to understand how this organelle contributes to the ability of the trypanosome to cause disease.
Technical Summary
African trypanosomes, Trypanosoma brucei spp., are the causative agents of sleeping sickness, which kills ~50 000 people annually in sub-Saharan Africa. N?gana, the related cattle disease, has major detrimental economic impact in the same areas. Expression of a series of immunologically-distinct surface variant surface glycoproteins (VSG) enables the parasite to avoid clearance by the adaptive immune response. Extensive antigenic variation of VSG essentially eliminates the prospect of a vaccine for sleeping sickness, and the need for novel drug therapies remains critical. Trypanosomes must maintain the VSG coat free of many invariant surface receptor proteins while still performing essential biological functions, such as nutrient uptake and cell signalling. This is achieved by specialization of the plasma membrane at the base of the flagellum, the flagellar pocket, that is the sole site for endo- and exocytosis and has been implicated in protein trafficking, control of cell division and pathogenicity. Importantly, disruption of flagellar pocket function by loss of associated factors is lethal to the parasite, highlighting the potential of this organelle as a therapeutic target with essential functions. However, the mechanisms by which it functions remain poorly understood.
We wish to define the molecular composition of this structure to understand parasitism in trypanosomes, to expose new and exploitable vulnerabilities, and to bring novel insight into the host-parasite interaction and parasite biology. We propose to use a coincidence-based mass-spectrometric strategy that seeks to identify the polypeptides present in a covalently-labelled surface protein fraction, those associated with the endosomal apparatus and also analysis of a flagellar pocket-enriched fraction. The identified polypeptides will be parsed using informatic criteria for inclusion or exclusion from the proteome, such as functional annotation, signal sequence, domain architecture, GPI-addition signal, transmembrane domain, expression profile etc. Coincidence, i.e. recovery in more than one proteome and passing criteria will result in inclusion as a candidate for genomic in vivo tagging with GFP for robust validation as a flagellar pocket factor. We previously applied such a strategy to an extensive proteome consisting of over 800 polypeptides, to identify 22 nucleoporins.
We wish to define the molecular composition of this structure to understand parasitism in trypanosomes, to expose new and exploitable vulnerabilities, and to bring novel insight into the host-parasite interaction and parasite biology. We propose to use a coincidence-based mass-spectrometric strategy that seeks to identify the polypeptides present in a covalently-labelled surface protein fraction, those associated with the endosomal apparatus and also analysis of a flagellar pocket-enriched fraction. The identified polypeptides will be parsed using informatic criteria for inclusion or exclusion from the proteome, such as functional annotation, signal sequence, domain architecture, GPI-addition signal, transmembrane domain, expression profile etc. Coincidence, i.e. recovery in more than one proteome and passing criteria will result in inclusion as a candidate for genomic in vivo tagging with GFP for robust validation as a flagellar pocket factor. We previously applied such a strategy to an extensive proteome consisting of over 800 polypeptides, to identify 22 nucleoporins.
