Shigella escapes lysosomal degradation through inactivation of Rab31 by IpaH4.5

Introduction. Shigella flexneri is an intracellular bacterial pathogen that utilizes a type III secretion apparatus to inject effector proteins into host cells. Hypothesis/Gap Statement. The T3SS effector IpaH4.5 is important for the virulence of Shigella . Aim. This study aimed to elucidate the molecular mechanism and host target of the IpaH4.5 as well as its roles in S. flexneri infection. Methodology. The GAP assay was used to identify substrate Rab GTPases of IpaH4.5. A coimmunoprecipitation assay was applied to identify the interaction of Rab GTPases with IpaH4.5. A confocal microscopy analysis was used to assess the effects of IpaH4.5 on mannose 6-phosphate receptor (MPR) trafficking. To identify the effects of IpaH4.5 GAP activity on the activity of lysosomal cathepsin B, the Magic Red-RR assay was used. Finally, the intracellular persistence assay was used to identify IpaH4.5 GAP activity in S. flexneri intracellular growth. Results. We found that the effector IpaH4.5 disrupts MPR trafficking and lysosomal function, thereby counteracting host lysosomal degradation. IpaH4.5 harbours TBC-like dual-finger motifs and exhibits potent RabGAP activities towards Rab31. IpaH4.5 disrupts the transport of the cation-dependent mannose 6-phosphate receptor (CD-MPR) from the Golgi to the endosome by targeting Rab31, thereby attenuating lysosomal function. As a result, the intracellular persistence of S. flexneri requires IpaH4.5 TBC-like GAP activity to mediate bacterial escape from host lysosome-mediated elimination. Conclusion. We identified an unknown function of IpaH4.5 and its potential role in S. flexneri infection.

Tissue plasminogen activator is a ligand of cation-independent mannose 6-phosphate receptor and consists of glycoforms that contain mannose 6-phosphate

Plasmin is the key enzyme in fibrinolysis. Upon interaction with plasminogen activators, the zymogen plasminogen is converted to active plasmin. Some studies indicate plasminogen activation is regulated by cation-independent mannose 6-phosphate receptor (CI-MPR), a protein that facilitates lysosomal enzyme trafficking and insulin-like growth factor 2 downregulation. Plasminogen regulation may be accomplished by CI-MPR binding to plasminogen or urokinase plasminogen activator receptor. We asked whether other members of the plasminogen activation system, such as tissue plasminogen activator (tPA), also interact with CI-MPR. Because tPA is a glycoprotein with three N-linked glycosylation sites, we hypothesized that tPA contains mannose 6-phosphate (M6P) and binds CI-MPR in a M6P-dependent manner. Using surface plasmon resonance, we found that two sources of tPA bound the extracellular region of human and bovine CI-MPR with low-mid nanomolar affinities. Binding was partially inhibited with phosphatase treatment or M6P. Subsequent studies revealed that the five N-terminal domains of CI-MPR were sufficient for tPA binding, and this interaction was also partially mediated by M6P. The three glycosylation sites of tPA were analyzed by mass spectrometry, and glycoforms containing M6P and M6P-N-acetylglucosamine were identified at position N448 of tPA. In summary, we found that tPA contains M6P and is a CI-MPR ligand.

Dual enzyme responsive mannose-6-phosphate based vesicle for controlled lysosomal delivery

Dual enzyme responsive stable biomimetic vesicles composed of mannose-6-phosphate lipid can encapsulate and deliver dual dye/drug and protein/enzyme exclusively to the lysosome in HEK-293 cells.

Chemoenzymatic glycan-selective remodeling of a therapeutic lysosomal enzyme with high-affinity M6P-glycan ligands. Enzyme substrate specificity is the name of the game

Functionalization of therapeutic lysosomal enzymes with mannose-6-phosphate (M6P) glycan ligands represents a major strategy for enhancing the cation-independent M6P receptor (CI-MPR)-mediated cellular uptake, thus improving the overall therapeutic efficacy of...