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TB host genes

Final Report Summary - TB HOST GENES (TB host genes)

Summary description of the project objectives

The aim of this project TB HOST GENES was to use the zebrafish embryo model for tuberculosis (TB) to increase understanding of host genes involved in susceptibility and resistance to mycobacterial infection. Mycobacterium tuberculosis (Mtb) is the cause of TB in humans. Because of the large number of infected individuals (one third of the world population) and the increasing occurrence of multidrug resistant strains of Mtb, understanding the mechanistic basis of host-pathogen interactions underlying TB has important medical implications. This presents a major challenge, requiring a combination of modern cell biology and functional genomics approaches in animal models that mimic the human disease. Mycobacterium marinum (Mm) is the closest genetic relative of Mtb and a natural pathogen of the zebrafish where it causes tuberculous granulomas similar as in human TB. Infecting the optically transparent zebrafish embryo with fluorescent bacteria allows us to monitor the infection in vivo and follow granuloma formation in real time. The specific objectives of this study were to perform a knockdown screen of candidate host genes involved in Mm infection and to study their role in granuloma formation, the hallmark of TB infection.

Description of the work performed

Prior knowledge of the host laboratory on macrophage-specific and infection-inducible expression of host genes provided an excellent starting point for selecting candidates for a knockdown screen. To achieve knockdown, morpholino antisense oligonucleotides targeting the selected candidate genes were injected into zebrafish embryos and these embryos were subsequently infected with red fluorescent Mm bacteria. To assess bacterial burden in the infected fish, fluorescence images were analysed with pixel quantification software developed in collaboration with the informatics department. Based on the initial screening results, a subset of candidate genes was selected for more detailed analyses and their role in mycobacterial granuloma formation was confirmed with additional morpholinos. High throughput sequencing technology (RNAseq) was used to gain insight into the specific gene expression changes during the infection process and under knockdown conditions of the selected host genes.

Description of the main results

During the granting period, we integrated available transcriptomics datasets of the host laboratory and selected genes with a potential role in the innate immune response during Mm infection. Morpholino knockdown screens showed that more than half of the selected genes affected granuloma formation, suggesting their involvement in controlling Mm infection. While one gene increased resistance to infection under knockdown condition, knockdown of all other genes resulted in increased susceptibility and bigger granulomas. Using leukocyte-specific staining we confirmed that increased infection was not the result of impaired immune cell development, but more likely due to specific gene knockdown effects on the innate immune response. Additional morpholinos against the same target genes were designed to confirm the specificity of the infection phenotypes. We focused our studies mainly on two genes that are expressed in macrophages, the main target cell type of Mm infection. One of these genes was shown to be involved in phagocytosis of Mm bacteria, while the second gene may be directly involved in bacterial killing. RNA deep sequencing analysis of infected embryos showed that increased granuloma formation under knockdown conditions of these genes was associated with increased expression of inflammation markers. In future work, we will extend these analyses to earlier stages of the infection process to gain better insight in their specific roles in activating primary innate host defense pathways involved in TB.

Expected final results and their potential impact and use

In this project, supported by a European Marie-Curie postdoc fellowship, we successfully employed the zebrafish embryo model for TB to identify host genes involved in controlling mycobacterial growth during early stages of infection when granulomas are being formed. It is expected that further in-depth functional studies of these genes, including ongoing RNA deep sequencing analyses, will contribute to a better understanding of the innate immune response to mycobacterial infection and the mechanisms involved in granuloma formation. Granulomas represent the pathological hallmark structures of TB and form a niche for long-term survival of mycobacteria inside their host. A better understanding of the mechanisms involved in granuloma formation may inspire new strategies to prevent their formation or kill bacteria with granulomas. Development of new TB treatment strategies is especially urgent because virulent multi-drug (MDR), extensively-drug (XDR) and even totally-drug (XDR) resistant strains of Mtb are rapidly emerging. Host-targeted treatments are now being considered for innovative therapeutic approaches that may overcome current limitations of antibiotic treatment. This underscores the importance to understand the key regulating host factors involved in TB and to identify potential targets for future drug development. Morpholino knockdown screening in the zebrafish TB model proved an effective way to identify host genes that modify susceptibility and resistance to mycobacterial infection.

Project web site: http://www.science.leidenuniv.nl/index.php/ibl/racz