Modelling tuberculous meningitis in zebrafish usingMycobacterium marinum

Abstract

Tuberculous meningitis (TBM) is one of the most severe extrapulmonary manifestations of tuberculosis, with a high morbidity and mortality. Characteristic pathological features of TBM are Rich foci, i.e. brain- and spinal-cord-specific granulomas formed after hematogenous spread of pulmonary tuberculosis. Little is known about the early pathogenesis of TBM and the role of Rich foci. We have adapted the zebrafish model of Mycobacterium marinum infection (zebrafish–M. marinum model) to study TBM. First, we analyzed whether TBM occurs in adult zebrafish and showed that intraperitoneal infection resulted in granuloma formation in the meninges in 20% of the cases, with occasional brain parenchyma involvement. In zebrafish embryos, bacterial infiltration and clustering of infected phagocytes was observed after infection at three different inoculation sites: parenchyma, hindbrain ventricle and caudal vein. Infection via the bloodstream resulted in the formation of early granulomas in brain tissue in 70% of the cases. In these zebrafish embryos, infiltrates were located in the proximity of blood vessels. Interestingly, no differences were observed when embryos were infected before or after early formation of the blood-brain barrier (BBB), indicating that bacteria are able to cross this barrier with relatively high efficiency. In agreement with this observation, infected zebrafish larvae also showed infiltration of the brain tissue. Upon infection of embryos with an M. marinum ESX-1 mutant, only small clusters and scattered isolated phagocytes with high bacterial loads were present in the brain tissue. In conclusion, our adapted zebrafish–M. marinum infection model for studying granuloma formation in the brain will allow for the detailed analysis of both bacterial and host factors involved in TBM. It will help solve longstanding questions on the role of Rich foci and potentially contribute to the development of better diagnostic tools and therapeutics.

High content analysis of granuloma histology and neutrophilic inflammation in adult zebrafish infected with Mycobacterium marinum

“…Optional: decapitate zebrafish using two tweezers to guillotine the head off at the gills. Although observed by other groups using the M. marinum E11 strain, we have never observed M. marinum M strain dissemination to the head following intraperitoneal injection (van Leeuwen et al, 2014). Decapitation may also aid fitting particularly large zebrafish into a cryomold.…”

iniBAC induction Is Vitamin B12- and MutAB-dependent in Mycobacterium marinum

“…Zebrafish embryos were injected with 2000 cfus by microinjection in the hindbrain ventricle as previously described (45,46). Infection was monitored at specified times by means of fluorescence microscopy.…”

Mycobacteria employ two different mechanisms to cross the blood-brain barrier

“…Importantly, upon infection with M. marinum, TBM does occur in adult zebrafish, with granuloma formation in the meninges and brain parenchyma in 20% of the cases (van Leeuwen et al, 2014). Therefore, the zebrafish model allows us to specifically address questions regarding mycobacterial invasion into the brain in an in vivo model (Bernut et al, 2014;Tenor, Oehlers, Yang, Tobin, & Perfect, 2015;van Leeuwen et al, 2014) This study provides in vivo evidence that mycobacteria utilise phagocytic cells to cross the BBB. Additionally, by using in vivo macrophage depletion and correlated light and electron microscopy (CLEM), we show that mycobacteria also employ transcellular migration by infecting and damaging brain endothelial cells in an ESX-1-dependent manner.…”

“…Already after 3-days postfertilisation (dpf), the zebrafish BBB functionally prevents exchange of large molecules (Fleming, Diekmann, & Goldsmith, 2013;Xie, Farage, Sugimoto, & Anand-Apte, 2010). Importantly, upon infection with M. marinum, TBM does occur in adult zebrafish, with granuloma formation in the meninges and brain parenchyma in 20% of the cases (van Leeuwen et al, 2014). Therefore, the zebrafish model allows us to specifically address questions regarding mycobacterial invasion into the brain in an in vivo model (Bernut et al, 2014;Tenor, Oehlers, Yang, Tobin, & Perfect, 2015;van Leeuwen et al, 2014) This study provides in vivo evidence that mycobacteria utilise phagocytic cells to cross the BBB.…”