Pancreatic cancer (PC) is one of the deadliest cancers worldwide. Cancer cells secrete excessive numbers of exosomes that play essential roles in tumorigenesis. Long non-coding RNAs (lncRNAs) are essential non-coding RNAs for cancer progression. However, the role of lncRNA plasmacytoma variant translocation 1 (PVT1) in exosome secretion of PC remains to be comprehensively investigated. Thus, nanoparticle tracking analysis and transmission electron microscopy were performed to determine exosome secretion. Confocal microscopy, western blots, real-time PCR, immunofluorescence, pull-down and RNA immunoprecipitation assays, and rescue experiments were applied to investigate the mechanism underlying the role of PVT1 in exosome secretion. The results showed that PVT1 was upregulated in PC cells, along with increased levels of YKT6 v-SNARE homolog (YKT6), ras-related protein Rab-7 (RAB7), and vesicle-associated membrane protein 3 (VAMP3). Also, PVT1 promoted the transportation of multivesicular bodies (MVBs) towards the plasma membrane. In addition, PVT1 promoted the docking of MVBs by altering RAB7 expression and localization. Moreover, PVT1 promoted the fusion of MVBs with the plasma membrane through regulating YKT6 and VAMP3 colocalization and the palmitoylation of YKT6. Taken together, the results suggest that PVT1 promoted exosome secretion of PC cells and thus, can expand the understanding of PVT1 in tumor biology.
Infections by many bacterial pathogens rely on their ability to degrade host glycans by producing glycoside hydrolases (GHs). Here, we discovered a conserved multifunctional GH, SsGalNagA, containing a unique combination of two family 32 carbohydrate-binding modules (CBM), a GH16 domain and a GH20 domain, in the zoonotic pathogen Streptococcus suis 05ZYH33. Enzymatic assays revealed that the SsCBM-GH16 domain displays endo-(β1,4)-galactosidase activity specifically toward the host-derived αGal epitope Gal(α1,3)Gal(β1,4)Glc(NAc)-R, whereas the SsGH20 domain has a wide spectrum of exo-β-N-acetylhexosaminidase activities, including exo-(β1,3)-N-acetylglucosaminidase activity, and employs this activity to act in tandem with SsCBM-GH16 on the αGal-epitope glycan. Further, we found that the CBM32 domain adjacent to the SsGH16 domain is indispensable for SsGH16 catalytic activity. Surface plasmon resonance experiments uncovered that both CBM32 domains specifically bind to αGal-epitope glycan, and together they had a KD of 3.5 mM toward a pentasaccharide αGal-epitope glycan. Cell-binding and αGal epitope-removal assays revealed that SsGalNagA efficiently binds to both swine erythrocytes and tracheal epithelial cells and removes the αGal epitope from these cells, suggesting that SsGalNagA potentially functions in nutrient acquisition or alters host signaling in S. suis. Both binding and removal activities were blocked by an αGal-epitope glycan. SsGalNagA is the first enzyme reported to sequentially act on a glycan containing the αGal epitope. These findings shed detailed light on the evolution of GHs and an important host-pathogen interaction.
Streptococcus suis is an important zoonotic pathogen, however, an efficient markerless genetic manipulation system is still lacking for further physiological and pathological studies on this bacterium. Several techniques have been developed for markerless genetic manipulation of S. suis utilizing either a temperature-sensitive vector or a counterselectable markers (CSMs), however, at present, the efficiency of these techniques is not very satisfactory. In this study, we developed a strategy for markerless genetic manipulation of S. suis employing a CSM based on a conditionally lethal mutant allele of pheS, which encodes the α-subunit of phenylalanyl-tRNA synthetase (PheS). This mutant pheS, mPheS, was constructed by introducing site-directed mutations for a T261S/A315G double-substitution and a number of silent mutations to decrease its similarity with the endogenous wild type pheS gene (wtPheS). Additionally, five potentially strong promoters from S. suis were screened for their ability to drive high-level expression of mPheS, thus endowing the carrier strain with sufficient sensitivity to the phenylalanine analog p-chloro-phenylalanine (p-Cl-phe). Insertion of these P-mPheS cassettes into a vector or into the chromosomal locus via a linked erythromycin resistance gene revealed that mPheS allele driven by promoters P0530 and P1503 renders S. suis sensitive to as low as 0.01% (or 0.5 mM) of p-Cl-phe. This offers two potential CSMs for S. suis with p-Cl-phe as a counterselective agent. P1503-mPheS was revealed to be 100% efficient for counter-selection in S. suis by application in a precise gene deletion. Using P1503-mPheS as a CSM, a two-step insertion and excision strategy for markerless genetic manipulation of S. suis were developed, supplying a powerful tool for markerless genetic manipulation of S. suis.
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