Study On The Role And Mechanism Of Adenosine Synthase In The Translocation Of Meningitis-causing Bacteria Across The Blood-brain Barrier

Despite advances in antibiotic therapies and vaccinations,the meningitis burden remains high and progress lags substantially behind that of other vaccine preventable diseases.Globally in 2016,incident cases of meningitis were about 3 million,causing hundreds of thousands of deaths.Bacterial meningitis is considered the most severe form of this disease and can rapidly become fatal or lead to severe disability in those who survive.Bacterial meningitis is mostly caused by extracellular pathogens,and most cases are complicated by bacteremia.This study focuses on a key issue in the pathogenesis of blood-borne bacterial meningitis,that is,how bacteria break through the barrier between the blood and the central nervous system(blood-CNS barriers)and enter the brain.It is generally accepted that bacteria can cross blood-CNS barriers through three general routes: transcellular,paracellular,and Trojan-horse mechanisms.These routes are not mutually exclusive,and studies have shown that pathogenic bacteria such as Neisseria meningitidis may invade the CNS from the blood circulation system through different pathways.As a critical natural barrier between the blood and the CNS,the blood-brain barrier(BBB)not only blocks the invasion of pathogens,but also hinders the entry of therapeutic drugs into the brain.Among them,some studies have shown that extracellular adenosine can change the permeability of the BBB by activating the A1 and A2A adenosine receptors present on the surface of brain microvascular endothelial cells,and this mechanism can be applied to drug delivery across the BBB.On the other hand,our previous work identified a 5’-nucleotidase from the cell wall protein of Streptococcus suis serotype 2(S.suis 2)which hydrolyzes AMP,ADP,and ATP to produce adenosine.S.suis 2 is known to cause meningitis and streptococcal toxic shock-like syndrome in humans,and is a major cause of meningitis in several regions of Asia.Despite these clues,any functional contributions of these bacterial adenosine synthase on the ability of pathogenic bacteria to promote their translocation across BBB remain unknown.In the first part of this study,we aimed to explore the function of adenosine synthase Ssads in the process of S.suis crossing BBB and the specific cellular and molecular mechanisms.Using an optimized murine model of S.suis meningitis,we found that mice infected with ssads gene-deficient S.suis(S.suis Δssads)had a lower bacterial load in the brain at 72h post-infection than those infected with wild-type S.suis(S.suis WT),whereas no significant difference was observed in bacterial loads in the blood at 5h and 72h after infection between the two strains.Histologic examination of brain tissue from mice at 72 hours of infection revealed that the ssads gene deficiency reduced the ability of S.suis to cause thickening,hemorrhage and infiltration of inflammatory cells in the brain tissue of mice.Upon an in vitro blood-brain barrier model constructed with a human brain microvascular endothelial cell line(HCMEC/D3),we found that Ssads-mediated adenosine production promoted the passage of S.suis through the HCMEC/D3 monolayer,but not significantly affected the adhesion and invasion rate of S.suis to HCMEC/D3 cells.We used A1,A2 A,A2B adenosine receptor(AR)antagonists for screening experiments and found that A1,A2A receptor signals are of vital importance in the process of S.suis penetrating the BBB in vitro.An extensive assortment of intracellular c AMP assay as well as A1 AR agonist and competitive c AMP antagonist experiments supported that activation of the A1 adenosine receptor signaling cascade in hosts promote S.suis penetration across BBB in vitro.Furthermore,we used monolayers prepared from CRISPR-edited HCMEC/D3 cells,A1 AR agonist/antagonist treated mice,and newly CRISPR knockout mice to show that Ssads enzymatic activity and A1 AR signaling work in concert to promote S.suis penetration across BBB.In addition,using primary brain microvascular endothelial cells isolated from mouse brain tissue for fluorescence confocal experiments,we found that adenosine synthase mediated adenosine production or A1 AR signal may promote cytoskeletal rearrangement and changes of junction protein in brain microvascular endothelial cells the during S.suis infection.Preliminary Western blot experiments revealed the effect of adenosine involve the phosphorylation of ERK1/2 protein and p38 protein in MAPK signaling pathway.Together,these data in this part show that S.suis can cross BBB via the production of extracellular adenosine mediated by Ssads.In the next part of this study,we explore whether other pathogenic bacteria harboring adenosine synthase activity can also use extracellular adenosine to promote their translocation across BBB.We collected information on Gram-positive bacteria and Gram-negative bacteria with adenosine synthase gene based on sequence alignment and literature search.The phylogenetic tree including the 22 identified or predicted5’-nucleotidase sequences was constructed with MEGA software(7.0)by using the Maximum Likelihood method and “WAG+G+I+F” model.Using the phosphate detection kit,we confirmed that several meningitis-causing pathogens,including Group B Streptococcus(GBS),Listeria monocytogenes,Staphylococcus aureus,and Staphylococcus epidermidis,have exposed adenosine synthase activity,but Streptococcus pneumoniae exhibited no enzymatic activity.We next evaluated the translocation ability of GBS,S.aureus,and S.epidermidis across HCMEC/D3 monolayers in the presence or absence of a 5’-nucleotidase inhibitor APCP.All three bacteria could cross the monolayers,and we found that the addition of APCP significantly reduced their translocation ability.Additionally,our laboratory isolated and identified a GBS with capsular serum type 3 and MLST type 17,and we further explored the in vivo role of local adenosine synthesis during GBS entry into murine brains.Using a well-defined and widely used mouse model of GBS hematogenous meningitis,we found that APCP treatment to interrupt adenosine production significantly reduced brain bacterial loads at 72h post-infection.Thus,our results support that that local adenosine synthesis by meningeal species may alter BBB permeability and facilitate bacterial translocation into the CNS.