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Pathogenic bacteria exploit transferrin receptor transcytosis to penetrate the blood-brain barrier.

ABSTRACT: The human blood-brain barrier (BBB) comprises a single layer of brain microvascular endothelial cells (HBMECs) protecting the brain from bloodborne pathogens. Meningitis is among the most serious diseases, but the mechanisms by which major meningitis-causing bacterial pathogens cross the BBB to reach the brain remain poorly understood. We found that Streptococcus pneumoniae, group B Streptococcus, and neonatal meningitis Escherichia coli commonly exploit a unique vesicle fusion mechanism to hitchhike on transferrin receptor (TfR) transcytosis to cross the BBB and illustrated the details of this process in human BBB model in vitro and mouse model. Toll-like receptor signals emanating from bacteria-containing vesicles (BCVs) trigger K33-linked polyubiquitination at Lys168 and Lys181 of the innate immune regulator TRAF3 and then activate the formation of a protein complex containing the guanine nucleotide exchange factor RCC2, the small GTPase RalA and exocyst subcomplex I (SC I) on BCVs. The distinct function of SEC6 in SC I, interacting directly with RalA on BCVs and the SNARE protein SNAP23 on TfR vesicles, tethers these two vesicles and initiates the fusion. Our results reveal that innate immunity triggers a unique modification of TRAF3 and the formation of the HBMEC-specific protein complex on BCVs to authenticate the precise recognition and selection of TfR vesicles to fuse with and facilitate bacterial penetration of the BBB.

SUBMITTER: Cheng Z

PROVIDER: S-EPMC10523449 | biostudies-literature | 2023 Sep

REPOSITORIES: biostudies-literature

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Pathogenic bacteria exploit transferrin receptor transcytosis to penetrate the blood-brain barrier.

Cheng Zhihui Z Zheng Yangyang Y Yang Wen W Sun Hongmin H Zhou Fangyu F Huang Chuangjie C Zhang Shuwen S Song Yingying Y Liang Qi'an Q Yang Nan N Li Meifang M Liu Bin B Feng Lu L Wang Lei L

Proceedings of the National Academy of Sciences of the United States of America 20230921 39

The human blood-brain barrier (BBB) comprises a single layer of brain microvascular endothelial cells (HBMECs) protecting the brain from bloodborne pathogens. Meningitis is among the most serious diseases, but the mechanisms by which major meningitis-causing bacterial pathogens cross the BBB to reach the brain remain poorly understood. We found that Streptococcus pneumoniae, group B Streptococcus, and neonatal meningitis Escherichia coli commonly exploit a unique vesicle fus ...[more]

PMID: 37733740

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Project description:Delivering biologics to elicit a therapeutic response in the central nervous system (CNS) remains challenging due to the presence of the blood-brain barrier (BBB). Receptor-mediated transcytosis is a strategy to improve brain exposure after systemic drug administration. The availability of a clinically relevant in vitro BBB model is crucial to investigate transcytosis pathways and to predict the penetration of biologics into the CNS. We created a perfused human in vitro BBB model made of induced pluripotent stem cells (iPSC)-derived brain microvascular endothelial cells (BMEC) for studying transferrin receptor-mediated transcytosis. iPSC-derived BMEC were seeded in the top channel of a three-lane microfluidic device (OrganoPlate®). After 2 days in culture, the established cell model exhibited relevant BBB features, including physiological transendothelial electrical resistance in a transwell setting (1500 Ω*cm2), reduced apparent permeability (Papp) to the fluorescence tracer Lucifer yellow (20-fold less than cell-free chips), expression of key BBB markers such as tight junctions proteins, transporters, receptors and functional P-gp efflux pump. Moreover, the model exhibited functional transferrin receptor-mediated uptake and transcytosis. To assess selective transferrin receptor-mediated transcytosis, a mixture of anti-human transferrin receptor (MEM-189) and control (sheep IgG anti-bovine serum albumin) antibodies was perfused in the top channel for 2 h. The Papp of MEM-189 was 11-fold higher than that of the control antibody, demonstrating facilitated receptor-mediated transcytosis. Compared to published work reporting a 2-fold ratio, this result is remarkable and establishes the suitability of our model for exploring receptor-mediated transcytosis and screening of antibodies for putative brain shuttle application. A perfused in vitro human model made of iPSC-derived BMEC with the chief characteristics (barrier tightness, functionality) of the human BBB can be applied to study transferrin receptor (TfR)-mediated transcytosis of therapeutic antibodies. This may bring critical advances in drug shuttle technology. Graphical abstract generated with biorender.com.

Project description:BackgroundThe blood brain barrier limits entry of macromolecular diagnostic and therapeutic cargos. Blood brain barrier transcytosis via receptor mediated transport systems, such as the transferrin receptor, can be used to carry macromolecular cargos with variable efficiency. Transcytosis involves trafficking through acidified intracellular vesicles, but it is not known whether pH-dependent unbinding of transport shuttles can be used to improve blood brain barrier transport efficiency.MethodsA mouse transferrin receptor binding nanobody, NIH-mTfR-M1, was engineered to confer greater unbinding at pH 5.5 vs 7.4 by introducing multiple histidine mutations. The histidine mutant nanobodies were coupled to neurotensin for in vivo functional blood brain barrier transcytosis testing via central neurotensin-mediated hypothermia in wild-type mice. Multi-nanobody constructs including the mutant M1R56H, P96H, Y102H and two copies of the P2X7 receptor-binding 13A7 nanobody were produced to test proof-of-concept macromolecular cargo transport in vivo using quantitatively verified capillary depleted brain lysates and in situ histology.ResultsThe most effective histidine mutant, M1R56H, P96H, Y102H-neurotensin, caused > 8 °C hypothermia after 25 nmol/kg intravenous injection. Levels of the heterotrimeric construct M1R56H, P96H, Y102H-13A7-13A7 in capillary depleted brain lysates peaked at 1 h and were 60% retained at 8 h. A control construct with no brain targets was only 15% retained at 8 h. Addition of the albumin-binding Nb80 nanobody to make M1R56H, P96H, Y102H-13A7-13A7-Nb80 extended blood half-life from 21 min to 2.6 h. At 30-60 min, biotinylated M1R56H, P96H, Y102H-13A7-13A7-Nb80 was visualized in capillaries using in situ histochemistry, whereas at 2-16 h it was detected in diffuse hippocampal and cortical cellular structures. Levels of M1R56H, P96H, Y102H-13A7-13A7-Nb80 reached more than 3.5 percent injected dose/gram of brain tissue after 30 nmol/kg intravenous injection. However, higher injected concentrations did not result in higher brain levels, compatible with saturation and an apparent substrate inhibitory effect.ConclusionThe pH-sensitive mouse transferrin receptor binding nanobody M1R56H, P96H, Y102H may be a useful tool for rapid and efficient modular transport of diagnostic and therapeutic macromolecular cargos across the blood brain barrier in mouse models. Additional development will be required to determine whether this nanobody-based shuttle system will be useful for imaging and fast-acting therapeutic applications.

Project description:The presence of the blood-brain barrier (BBB) creates a nigh-on impenetrable obstacle for large macromolecular therapeutics that need to be delivered to the brain milieu to treat neurological disorders. To overcome this, one of the strategies used is to bypass the barrier with what is referred to as a "Trojan Horse" strategy, where therapeutics are designed to use endogenous receptor-mediated pathways to piggyback their way through the BBB. Even though in vivo methodologies are commonly used to test the efficacy of BBB-penetrating biologics, comparable in vitro BBB models are in high demand, as they benefit from being an isolated cellular system devoid of physiological factors that can on occasion mask the processes behind BBB transport via transcytosis. We have developed an in vitro BBB model (In-Cell BBB-Trans assay) based on the murine cEND cells that help delineate the ability of modified large bivalent IgG antibodies conjugated to the transferrin receptor binder scFv8D3 to cross an endothelial monolayer grown on porous cell culture inserts (PCIs). Following the administration of bivalent antibodies into the endothelial monolayer, a highly sensitive enzyme-linked immunosorbent assay (ELISA) is used to determine the concentration in the apical (blood) and basolateral (brain) chambers of the PCI system, allowing for the evaluation of apical recycling and basolateral transcytosis, respectively. Our results show that antibodies conjugated to scFv8D3 transcytose at considerably higher levels compared to unconjugated antibodies in the In-Cell BBB-Trans assay. Interestingly, we are able to show that these results mimic in vivo brain uptake studies using identical antibodies. In addition, we are able to transversely section PCI cultured cells, allowing for the identification of receptors and proteins that are likely involved in the transcytosis of the antibodies. Furthermore, studies using the In-Cell BBB-Trans assay revealed that transcytosis of the transferrin-receptor-targeting antibodies is dependent on endocytosis. In conclusion, we have designed a simple, reproducible In-Cell BBB-Trans assay based on murine cells that can be used to rapidly determine the BBB-penetrating capabilities of transferrin-receptor-targeting antibodies. We believe that the In-Cell BBB-Trans assay can be used as a powerful, preclinical screening platform for therapeutic neurological pathologies.

Project description:The blood-brain barrier (BBB) is a formidable obstacle to the delivery of therapeutics to the brain. Antibodies that bind transferrin receptor (TfR), which is enriched in brain endothelial cells, have been shown to cross the BBB and are being developed as fusion proteins to deliver therapeutic cargos to brain targets. Various antibodies have been developed for this purpose and their in vivo evaluation demonstrated that either low affinity or monovalent receptor binding re-directs their transcellular trafficking away from lysosomal degradation and toward improved exocytosis on the abluminal side of the BBB. However, these studies have been performed with antibodies that recognize different TfR epitopes and have different binding characteristics, preventing inter-study comparisons. In this study, the efficiency of transcytosis in vitro and intracellular trafficking in endosomal compartments were evaluated in an in vitro BBB model for affinity variants (Kd from 5 to174 nM) of the rat TfR-binding antibody, OX26. Distribution in subcellular fractions of the rat brain endothelial cells was determined using both targeted quantitative proteomics-selected reaction monitoring and fluorescent imaging with markers of early- and late endosomes. The OX26 variants with affinities of 76 and 108 nM showed improved trancytosis (Papp values) across the in vitro BBB model compared with a 5 nM OX26. Although ~40% of the 5 nM OX26 and ~35% of TfR co-localized with late-endosome/lysosome compartment, 76 and 108 nM affinity variants showed lower amounts in lysosomes and a predominant co-localization with early endosome markers. The study links bivalent TfR antibody affinity to mechanisms of sorting and trafficking away from late endosomes and lysosomes, resulting in improvement in their transcytosis efficiency.Open practicesOpen Science: This manuscript was awarded with the Open Materials Badge. For more information see: https://cos.io/our-services/open-science-badges/ Cover Image for this issue: doi: 10.1111/jnc.14193.

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Pathogenic bacteria exploit transferrin receptor transcytosis to penetrate the blood-brain barrier.

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Pathogenic bacteria exploit transferrin receptor transcytosis to penetrate the blood-brain barrier.

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