DiMeN Doctoral Training Partnership: Live imaging of leukocyte signalling dynamics during tuberculosis infection in an in vivo zebrafish model

The innate immune response is the body’s first line of defense against injury and invading pathogens. We are interested in the cellular response to infection, involving the white blood cells (leukocytes) of the host. Our disease of interest is Tuberculosis, a bacterial infection on the rise due to the increasing prevalence of multi-drug resistance. Mycobacterium tuberculosis is able to highjack leukocytes, escaping their antimicrobial mechanisms thereby surviving and proliferating allowing the formation of granulomas, protective structures in which the bacterium can survive for decades. To investigate the complex molecular and cellular processes leading to granuloma formation our model of choice is the zebrafish embryo infected with Mycobacterium marinum (Mm, fish TB). Zebrafish embryos have an innate immune response comparable to humans, and are transparent, allowing detailed spatial and temporal analysis of the immune response during infection.
We focus on hypoxia inducible factor alpha (Hif-α), a transcription factor that is upregulated when cells are experiencing low levels of oxygen. Using fluorescent transgenic lines and antibody labelling we have previously shown that infected leukocytes transiently upregulate Hif- leading to an upregulation of nitric oxide (NO), a potent antimicrobial (Elks et al., 2013. http://goo.gl/CxdYld). However, this response is transient, with both Hif- and NO levels decreasing via unknown mechanisms, allowing permissive conditions for granuloma formation (Elks et al., 2014. http://goo.gl/zRt9ey). Understanding the mechanisms by which Hif-α is downregulated, and developing ways to sustain Hif- levels in infected leukocytes may therefore represent an exciting host-derived therapeutic opportunity, one that would not drive resistance to antimycobacterial agents.
The aim of this study will be to characterise the dynamics of the Hif- and NO response to infection to understand the mechanisms by which these antimicrobial mechanisms are initially upregulated, but then counteracted by Mm.
Transgenic fluorescent reporter zebrafish lines of Hif- and NO signalling will be used in order to follow, in realtime, their activation in leukocytes after Mm infection. We have previously shown that Hif- and NO are downregulated by the time granulomas form, and hypothesise that this is a Mm driven process to allow permissive conditions for granuloma formation. To determine the bacterial mechanisms involved the transgenic lines will be infected with, live Mm, heatkilled Mm, and a panel of attenuated Mm mutants with mutations in genes activated when in the intracellular environment. To further define the molecular mechanisms underlying the dynamics of Hif- turnover, a 3-6 month period will be spent in the lab of the second supervisor, Dr Violaine See (University of Liverpool), a specialist in HIF- imaging in living cells. Mammalian fibroblast-like (COS7 cells) and macrophage-like cell lines (RAW 264.7) will be transfected with HIF-GFP fusion constructs and reporters of HIF activity to visualise the stability of signal over time once infected with Mm (wildtype and mutants). Cell line models will allow the dynamics of Hif- turnover after infection to be quantified using a previously established two-component mathematical model (Bagnall et al., 2014).
The host immune pathways silenced by Mm will be determined in the in vitro and in vivo models using qPCR in combination with existing fluorescent zebrafish transgenic lines of important cytokines (eg., tnfalpha:GFP). Hif-1 will be manipulated and RNAseq will be performed to investigate which immune pathways are modulated to improve infection outcomes.
This work will take place in the young and vibrant research group of Dr Philip Elks (http://elkslab.weebly.com) at The Bateson Centre, The University of Sheffield, and the candidate will be well trained in a combination of cutting-edge molecular biology and microscopy techniques. There will be regular input from the second supervisor, Dr Violaine See at the University of Liverpool, a hypoxia/HIF expert, with regular meetings and additional short training periods in Liverpool.