Internalization signals in synaptotagmin VII utilizing two independent pathways are masked by intramolecular inhibitions

Dasgupta, Shoumita; Kelly, Regis B.

doi:10.1242/jcs.00290

Cited by 12 publications

(8 citation statements)

References 47 publications

(49 reference statements)

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“…To verify the lack of localization to lysosomes, we used keratinocytes transfected with a plasmid encoding the lysosomal protein SytVII fused to GFP. In CHO cells and NRK cells, SytVII is primarily expressed in the lysosomal membrane (29)(30)(31), whereas in PC-12 pheochromocytoma cells and rat neurons, SytVII expression is restricted to the plasma membrane, as is the case with other synaptotagmins involved in synaptic vesicle trafficking (32,33). In OKP7 keratinocytes, we found that SytVII-GFP was distributed both to lysosomes and the plasma membrane (Fig.…”

Section: Slo Prevents Internalization Of Gas By Oropharyngeal Keratinmentioning

confidence: 69%

Cytolysin-dependent evasion of lysosomal killing

Håkansson

Bentley

Shakhnovic

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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Local host defenses limit proliferation and systemic spread of pathogenic bacteria from sites of mucosal colonization. For pathogens such as streptococci that fail to grow intracellularly, internalization and killing by epithelial cells contribute to the control of bacterial growth and dissemination. Here, we show that group A Streptococcus (GAS), the agent of streptococcal sore throat and invasive soft tissue infections, evades internalization and intracellular killing by pharyngeal epithelial cells. Production of the cholesterol-binding cytotoxin streptolysin O (SLO) prevented internalization of GAS into lysosomes. In striking contrast, GAS rendered defective in production of SLO were internalized directly or rapidly transported into lysosomes, where they were killed by a pH-dependent mechanism. Because SLO is the prototype of cholesterol-dependent cytolysins produced by many Grampositive bacteria, cytolysin-mediated evasion of lysosomal killing may be a general mechanism to protect such pathogens from clearance by host epithelial cells.Streptococcus pyogenes ͉ streptolysin O ͉ virulence E pithelial cells of mucosal surfaces constitute an early line of defense against infecting microorganisms. These cells protect the host not only by acting as a physical barrier but also by secreting antimicrobial molecules and by recruiting professional immune cells to the site of infection (1-5). Also, epithelial cells have the capacity to internalize and kill bacteria. Extracellular pathogens such as streptococci, Staphylococcus aureus, and Pseudomonas aeruginosa can be internalized by epithelial cells but do not appear to multiply in the intracellular environment (6-11). Rather, internalized bacteria are killed over time. Therefore, to persist at the mucosal surface, extracellular bacterial pathogens must evade internalization and killing by epithelial cells.In this study, we investigated the effects of streptolysin O (SLO) on the interactions of group A Streptococcus (GAS), the agent of streptococcal sore throat and severe, invasive, or ''flesh-eating'' infections, with human oropharyngeal epithelial cells. SLO is the prototype of a family of cholesterol-binding cytotoxins produced by many pathogenic Gram-positive bacteria including Streptococcus pneumoniae (pneumolysin), Listeria monocytogenes (listeriolysin O), Clostridium perfringens (perfringolysin O), and Bacillus anthracis (anthrolysin) (12-14). The cholesterol-binding cytotoxins share the property of poreforming activity for a broad range of cholesterol-containing cell membranes (12, 15). However, it remains unknown whether they serve a common functional role in pathogen-host biology for the diverse species that produce them. Here, we report that SLO is a critical modulator of GAS internalization, intracellular trafficking, and bacterial killing by human oropharyngeal keratinocytes. We found that SLO prevented direct uptake of GAS into lysosomes and efficient pH-dependent intracellular killing. On the basis of these findings, we propose that SLO enhances GAS surviv...

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Section: Slo Prevents Internalization Of Gas By Oropharyngeal Keratinmentioning

confidence: 69%

Cytolysin-dependent evasion of lysosomal killing

Håkansson

Bentley

Shakhnovic

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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“…a short linker region (16). Syt I, IV, and VII are the most highly conserved (8) and are likely involved in membrane trafficking, particularly, Syt I and Syt VII, which mediate exocytic fusion of synaptic and secretory vesicles in neural and exocrine tissues (9). However, several studies have reported that Syt I is also involved in endocytosis (14,20) and membrane retrieval following kiss-and-run or stimulated exocytosis (2).…”

Section: Discussionmentioning

confidence: 99%

Synaptotagmin I binds intestinal epithelial NHE3 and mediates cAMP- and Ca²⁺-induced endocytosis by recruitment of AP2 and clathrin

Musch

Arvans

Walsh-Reitz

et al. 2007

American Journal of Physiology-Gastrointestinal and Liver Physi

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Apical membrane sodium hydrogen exchanger 3 (NHE3), a major pathway for non-nutrient-dependent intestinal Na(+) absorption, is tightly regulated by second messenger systems that affect its functional activity and membrane trafficking. However, the events and components involved in NHE3 regulation are only partially understood. We report that the adaptor protein synaptotagmin I (Syt I) plays a pivotal role in cAMP- and Ca(2+)-induced cargo recognition of NHE3 and initiation of its endocytosis. Both mouse small intestine (jejunum) and Caco-2BBe Syt I coimmunoprecipitated with NHE3, particularly following increases in cellular cAMP or Ca(2+). Following short interfering RNA (siRNA) suppression of Syt I expression, cAMP- and Ca(2+)-induced inhibition of NHE3 activity were still observed but NHE3 endocytosis was blocked, as assessed by (22)Na influx and apical membrane biotin labeling, respectively. Similar effects on NHE3 inhibition and endocytosis were observed by siRNA suppression of either the mu-subunit of the adaptor protein 2 (AP2) complex or the heavy chain of clathrin. Coimmunoprecipitation analyses of NHE3 with these adaptor proteins revealed that cAMP- and Ca(2+)-induced NHE3-Syt I interaction preceded and was required for recruitment of AP2 and the clathrin complex. Confocal microscopy confirmed both the time sequence and protein associations of these events. We conclude that Syt I plays a pivotal role in mediating cAMP- and Ca(2+)-induced endocytosis of NHE3 (but not in inhibition of activity) through cargo recognition of NHE3 and subsequent recruitment of AP2-clathrin assembly required for membrane endocytosis.

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“…HA-tagged wild-type and AA/ASRR mutant cDNAs of the GABA B receptor subunit GB1 (MargetaMitrovic et al, 2000) were gifts from Dr. L. Y. Jan [University of California at San Francisco (UCSF)]. A pIRES2 plasmid expressing HA-tagged K44E mutant dynamin I in tandem with enhanced green fluorescent protein (Schmidlin et al, 2001;Dasgupta and Kelly, 2003) was a gift from Dr. R. Kelly (UCSF). BiP cDNA (Werstuck et al, 2001) and p50ATF6 cDNA (Yoshida et al, 2000) were kindly provided by Dr. R. Austin (McMartin University, Hamilton, Canada) and Dr. K. Mori (Kyoto University, Kyoto, Japan), respectively.…”

Section: Methodsmentioning

confidence: 99%

Interaction with the Unfolded Protein Response Reveals a Role for Stargazin in Biosynthetic AMPA Receptor Transport

Vandenberghe

Nicoll²,

Bredt³

2005

J. Neurosci.

109

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The transmembrane protein stargazin enhances levels of functional AMPA receptors at the neuronal plasma membrane and at synapses. To clarify the mechanism for this effect, we studied trafficking of the AMPA receptor subunit glutamate receptor 1 (GluR1) in transfected COS7 cells. GluR1 expressed poorly on the surface of these cells and was primarily retained in the endoplasmic reticulum (ER). Stargazin expression strongly increased the surface fraction of GluR1. This effect was not reduced by a dominant-negative dynamin mutant, suggesting that stargazin does not inhibit AMPA receptor endocytosis. Interestingly, upregulation of ER chaperones as part of the unfolded protein response (UPR) both mimicked and occluded the effect of stargazin, suggesting a role for stargazin in ER processing of AMPA receptors. Consistent with this idea, we detected UPR induction in cerebellar granule cells lacking stargazin. Finally, residual AMPA receptor currents in stargazin-deficient neurons were suppressed by inhibition of the UPR. These findings uncover a role for stargazin in AMPA receptor trafficking through the early compartments of the biosynthetic pathway. Furthermore, they provide evidence for modulation of AMPA receptor trafficking by the UPR.

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Internalization signals in synaptotagmin VII utilizing two independent pathways are masked by intramolecular inhibitions

Cited by 12 publications

References 47 publications

Cytolysin-dependent evasion of lysosomal killing

Cytolysin-dependent evasion of lysosomal killing

Synaptotagmin I binds intestinal epithelial NHE3 and mediates cAMP- and Ca²⁺-induced endocytosis by recruitment of AP2 and clathrin

Interaction with the Unfolded Protein Response Reveals a Role for Stargazin in Biosynthetic AMPA Receptor Transport

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Internalization signals in synaptotagmin VII utilizing two independent pathways are masked by intramolecular inhibitions

Cited by 12 publications

References 47 publications

Cytolysin-dependent evasion of lysosomal killing

Cytolysin-dependent evasion of lysosomal killing

Synaptotagmin I binds intestinal epithelial NHE3 and mediates cAMP- and Ca2+-induced endocytosis by recruitment of AP2 and clathrin

Interaction with the Unfolded Protein Response Reveals a Role for Stargazin in Biosynthetic AMPA Receptor Transport

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Synaptotagmin I binds intestinal epithelial NHE3 and mediates cAMP- and Ca²⁺-induced endocytosis by recruitment of AP2 and clathrin