Differential ubiquitination as an effective strategy employed by Blood-Brain Barrier for prevention of bacterial transcytosis

ABSTRACT The protective mechanisms of blood-brain barrier (BBB) prohibiting entry of blood borne pathogens and toxins into the central nervous system (CNS) is critical for maintenance of homeostasis in the brain. These include various forms of intracellular defence mechanisms which are vital to block bacterial transcytosis, the major route of trafficking adopted by meningeal pathogens to transit into the CNS. However, mechanistic details of the defence mechanisms and their exploitation to prevent bacterial meningitis remain unexplored. In this study, we established that brain endothelium driven ubiquitination acts as a major intracellular defence mechanism for clearance of S. pneumoniae, a critical neurotropic pathogen, during its transit through BBB. Our findings suggest that brain endothelium employs differential ubiquitination with either K48 or K63-Ub chain topologies as an effective strategy to target SPN towards diverse killing pathways. While K63-Ub decoration triggers autophagic killing, K48-Ub directs pneumococcus exclusively to the proteasome machinery. Indeed, time lapse fluorescence imaging involving proteasomal marker LMP2 revealed that in BBB, majority of the ubiquitinated SPN were cleared by proteasome. Fittingly, pharmacological inhibition of proteasome and autophagy pathway not only led to exclusive accumulation of K48-Ub and K63-Ub marked SPN, respectively, but also triggered significant increment in intracellular SPN burden. Moreover, genetic impairment of formation of either K48 or K63-Ub chain topology demonstrated that though both chain types play important roles in disposal of intracellular SPN, K48-Ub chains and subsequent proteasomal degradation has more pronounced contribution towards ubiquitinated SPN killing in brain endothelium. Collectively, these observations for the first time illustrated a pivotal role of differential ubiquitination in orchestrating a symphony of intracellular defence mechanisms blocking pathogen trafficking into the brain which could be further exploited to prevent bacterial CNS infections. IMPORTANCE Among the different cellular barriers present in the human body, Blood-Brain Barrier (BBB) is unique as it not only provides structural integrity but also protects the central nervous system (CNS) from pathogen invasion. In recent past, ubiquitination, which is known to be involved in protein quality control and cellular homeostasis, has been proven to be critically involved in pathogen clearance. In this study, employing S. pneumoniae as a model CNS pathogen, we wanted to decipher the critical contribution of ubiquitination in protective mechanism of BBB while tackling bacterial entry into the CNS. Our results suggest, that BBB deploys differential ubiquitination as an effective strategy to prevent neurotropic bacterial trafficking into the brain. By portraying a comprehensive picture of ubiquitin coat on SPN, we figured out that different ubiquitin chain topologies formed on the pneumococcus dictated the selection of downstream degradative pathways, namely, autophagy and proteasomal machinery. Amongst these, contribution of proteasomal system in clearance of pneumococcus was found to be more pronounced. Overall our study revealed how BBB deploys differential ubiquitination as a strategy to trigger autophagy and proteasomal system, which work in tandem to ensure brain’s identity as an immunologically sterile site.