Cip29 And Wdr1 Brucella Invasion And Macrophage Function In A Preliminary Study

Brucella are intramacrophage pathogens that induce chronic infections called brucellosis in a wide range of mammals, including domestic animals and humans, afflicting over 500 000 people per year. Brucellosis has a major impact on the health of both man and livestock in many regions of the world with the concomitant economic consequences. In contrast to other intracellular pathogens, Brucella do not produce exotoxins, antiphagocytic capsules, resistance forms or fimbriae, invasive proteases, toxic lipopolysaccharide, virulence plasmids and lysogenic phages and do not show antigenic variation. The key aspect of the virulence of Brucella is their ability to proliferate within professional phagocytic host cells[1,2]. Macrophages can kill most pathogens and induce specific and non-specific immune response as the immunity-regulating cells and effector cells. The strategies of Brucella escaping from the killing of macrophages and surviving in them are: (1) rapid escape from the phagosome to gain entry into the cytosol; (2) stall of phagosome maturation at various stages preceding fusion with lysosomes; (3) inhibit the excretion of cytokine by macrophages; (4)inhibit the macrophages apoptosis; (5) inhibit the antigen present ability of macrophages[3-6]. Therefore, the macrophage response to infection has important consequences for both the survival of phagocytosed bacteria and the further development of host immunity.However, very little is known about the macrophage cell signaling pathways initiated upon infection and the virulence strategy that Brucella use to counteract these responses and secure their survival. To discover the mechanisms of Brucella survival in macrophage and interactions between Brucella and host, using 2D electrophoretic technique combined with mass spectroscopy, we found 38 different-expression proteins of THP-1 cells after infection with Brucella in our previous research. According to literatures, two of the different-expression proteins-CIP29 and WDR1 were selected to study their funtions in THP-1 cells and Brucella infection in this paper.Up to present, very little is known about CIP29 and WDR1. It is predicted that CIP29 has the ability of binding nucleotides, contribute to the regulation of transcription and post-translation, involve in metabolism and apoptosis of cells, so it has a complicated role in controlling the fate of cells. WDR1 contains nine WD-repeat structural domains, and it can facilitate actin turnover and regulate cells morphous. In our previous study, the expression of CIP29 and WDR1 in cells infection with Brucella was changed obviously, so in this paper we will interfere in CIP29 and WDR1 expression to study their functions in macrophage and Burcella infection.The study was carried out in two parts. On the first part, preliminarily study functions of CIP29 and its effect to the number of Brucella during infection in THP-1 cells.This part including the following:(1)obtein CIP29 gene from THP-1 cells by RT-PCR. (2) construct recombinant plasmids pET28a-CIP29, express and purify recombinant protien CIP29 and prepare its antibodies.(3) construct recombinant plasmids pEGFP-CIP29, then transfect to cells to up-regulate CIP29 expression. (4)detect the apoptosis of cells and TNF-αconcentration in supernatant. (5) observation on the numbers of Brucella in THP-1 cells. On the second part, preliminarily study functions of WDR1 and its effect to the number of Brucella during infection in THP-1 cells. This part including the following: (1)obtein WDR1 gene from THP-1 cells by RT-PCR. (2) construct recombinant plasmids pET28a-WDR1, express and purify WDR1 and prepare its antibodies.(3) design and transfect siRNA to cells to down-regulate WDR1 expression in cells. (4) observation on the numbers of Brucella in THP-1 cells. (5) detect TNF-αconcentration in supernatant and phagocytosis activity of macrophage.Results: Successfully obteined CIP29 and WDR1 genes from THP-1 cells, constructed recombinant plasmids, and prepared their antibodies. Up-regulation of CIP29 could induce apoptosis of THP-1 cells with statistical significance, but it had no effect to TNF-αsecretion and a little effect to infection of Brucella in THP-1 cells without statistical significance. Down-regulation of WDR1 had no effect to TNF-αsecretion and the numbers of Brucella during infection in THP-1 cells, but WDR1 had obviously effect to phagocytosis activity of THP-1 cells with statistical significance.Conclusion: Defence of Brucella infection is a complicated process with many proteins coordination in macrophages. Research on functions of proteins by interventing in their expression is only a simple method, due to different proteins with different functions, it is necessary use diverse methods to study their specific roles . In this study, CIP29 and WDR1 have no obviouse functions during macrophages infected with Brucella, the reason may be owe to the limitation of technology used, or because the complicated process during infection which make it impossible to display the functions of a single protein, this will be verified in the further study. This study indicated that single protein of THP-1 cells had little influence on Brucella infection, and host defense of Brucella infection need multiple proteins cooperation together.