First Draft of Introduction and Ideas for the project

The role of complement properdin upon infection with Mycobacterium marinum

Introduction

The complement system is a part of the innate immune response upon infection with a pathogen or a foreign particle. The complement activation consists of three main pathways: the classical pathway, the mannose-binding lectin pathway and the alternative pathway. Activation of the complement via the classical pathway involves the formation of antibody-antigen complexes. This recruits the C1 component which cleaves the C2 and C3 components forming a protease known as C3 convertase. The MBLP is very much similar to the CP, however, it is initiated when mannose-binding lectin binds to the mannose on the surface of pathogens.

The AP, on the contrary, is initiated by spontaneous cleavage of C3 in the fluid phase that leads to the formation of C3b segment. This segment associates with factor B in the environment which is cleaved by factor D, forming the AP C3 convertase, C3bBb. The C3 convertase from the three pathways cause the C3 cleavage which produces a small C3a fragment and a large C3b fragment. The latter has three possible functions: it can activate the alternative pathway causing amplification of C3b production, it can attach to the pathogen cell surface and act as opsonins or it can bind to the C3 convertase and form C5 convertase. Formation of C5 convertase causes the cleavage of C5 and the recruitment of C6, C7, C8 and poly-C9 components, forming a membrane attack comples (MAC) and ultimately resulting in cell lysis.

The role of properdin in the complement system was first suggested by Pillemar and collaborators (1954). It was suggested that properdin binds to a target protein and activates the complement system. After initial refutation, the work of Pillemar was vindicated (Lepow, 1980) as the AP was established (Medicus et al., 1976; Pangburn and Muller-Eberhard, 1986). When the alternative pathway was firmly established, the C3 convertase, C3bBb, was shown to be a short-lived complex (Medicus et al., 1976; Pangburn and Muller-Eberhard, 1986) which was stabilized by association with properdin by 5- fold to 10-fold (Fearon et al., 1975). Hence properdin was accepted as a positive regulator of the complement system.

In a recent research by Spitzer and colleagues (2007), surface plasmon resonance (SPR) methodology was used to conclude that bound to a target surface; complement activation can be initiated by properdin using the proteins found in serum. Furthermore, it has been also shown that properdin can recognize a variety of dangerous, nonself targets (for example, zymosan, Neisseria, and LPS-coated microtiter wells), and promotes the initiation of the C3 convertase of the AP (Spitzer et al., 2007; Kimura et al., 2008). These studies led to further studies that demonstrate the involvement of properdin in recognition and removal of altered or dangerous self cells, for example, apoptotic T cells (for review see Kemper et al., 2010).

In this study, the role played by properdin during Mycobacterium marinum infection will be studied using properdin deficient mice.  The first stage will be to compare the abundance of associated and/or internalized M. marinum in non-adherent cells in wild-type and properdin deficient mice using flow cytometry . This can be carried out by labeling M. marinum with a fluorescent dye like calcein (Barker et al., 1997).

This will then be followed by comparing the effects of hypoxia on leukocytes prepared from both wildtype and properdin deficient mice. Cells such as splenic macrophages, bone marrow derived dendritic cells and mast cells will be infected with M. marinum in hypoxic and normoxic conditions. These cells will then be lysed post-infection and the intracellular viable count (CFU) will be determined. The supernatants of the cells will be used to analyze TNF-α bioactivity using L929 indicator cells. In presence of TNF-α, L929 cells undergo cell death by either apoptosis or necrosis (Humphreys and Wilson, 1999).  Finally, gene expression will be analyzed to assess the time dependent host response in macrophage cell lines J774 and RAW.


References 

• Barker LP, George KM, Falkow S, Small, PLC. 1997. Differential Trafficking of Live and Dead . Mycobacterium marinum Organisms in Macrophages. Infection and Immunity 65: 1497-1504.
• Fearon DT, Austen KF. 1975. Properdin: binding to C3b and stabilization of the C3b-dependent C3 convertase. J. Exp. Med.142:856–63
•  Humphreys DT, Wilson MR. 1999. Modes of L929 Cell Death Induced by TNF-α and other Cytotoxic Agents.  Cytokine 11:773-782.
• Kemper, C., Atkinson, J. P., Hourcade, D.E. 2010. Properdin: Emerging roles of a Pattern-Recognition Molecule. Annu. Rev. Immunol. 28:131-55
• Kimura Y, Miwa T, Zhou L, Song WC. 2008. Activator-specific requirement of properdin in the initiation and amplification of the alternative pathway complement. Blood 11:732–40 
• Lepow IH. 1980. Presidential address to American Association of Immunologists in Anaheim, California, April 16, 1980. Louis Pillemer, Properdin, and scientific controversy. J. Immunol.125:471–75
• Medicus RG, Gotze O, Muller-Eberhard HJ. 1976. Alternative pathway of complement: recruitment of precursor properdin by the labile C3/C5 convertase and the potentiation of the pathway. J. Exp. Med. 144:1076–93
• Pangburn MK, Muller-Eberhard HJ. 1986. The C3 convertase of the alternative pathway of human complement. Enzymic properties of the bimolecular proteinase. Biochem. J. 235:723–30
• Pillemer L, Blum L, Lepow IH, Ross OA, Todd EW, Wardlaw AC. 1954. The properdin system and immunity. I. Demonstration and isolation of a new serum protein, properdin, and its role in immune phenomena. Science 120:279–85 
• Spitzer D, Mitchell LM, Atkinson JP, Hourcade DE. 2007. Properdin can initiate complement activation by binding specific target surfaces and providing a platform for de novo convertase assembly.J. Immunol. 179:2600–8