Summary Modulation of NF-κB-dependent responses is critical to the success of attaching/effacing (A/E) human pathogenic E. coli (EPEC and EHEC) and the natural mouse pathogen Citrobacter rodentium. NleB, a highly conserved type III secretion system effector of A/E pathogens, suppresses NF-κB activation, but the underlying mechanisms are unknown. We identified the mammalian glycolysis enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an NleB interacting protein. Further, we discovered that GAPDH interacts with the TNF receptor associated factor 2 (TRAF2), a protein required for TNF-α-mediated NF-κB activation, and regulates TRAF2 polyubiquitination. During infection, NleB functions as a translocated N-acetyl-D-glucosamine (O-GlcNAc) transferase that modifies GAPDH. NleB-mediated GAPDH O-GlcNAcylation disrupts the TRAF2-GAPDH interaction to suppress TRAF2 polyubiquitination and NF-κB activation. Eliminating NleB O-GlcNAcylation activity attenuates C. rodentium colonization of mice. These data identify GAPDH as a TRAF2 signaling cofactor and reveal a virulence strategy employed by A/E pathogens to inhibit NF-κB dependent host innate immune responses.

ABSTRACTThe human pathogens enterohemorrhagic and enteropathogenicEscherichia coli(EHEC and EPEC), as well as the related mouse pathogenCitrobacter rodentium, utilize a type III secretion system (T3SS) to inject multiple effector proteins into host cells. TheE. coliO157:H7 strain EDL933 carries two copies of non-locus of enterocyte effacement (LEE)-encoded protein H, designated NleH1 and NleH2, both of which bind to the human ribosomal protein S3 (RPS3), a subunit of NF-κB transcriptional complexes. In this study, we describe significant functional differences between NleH1 and NleH2 in their ability to regulate the host NF-κB pathway. We show that the EHEC and EPEC NleH effectors are functionally equivalent in their ability to affect RPS3 nuclear translocation. NleH1, but not NleH2, inhibited NF-κB activity without altering the kinetics of IκBα phosphorylation/degradation. We also determined that the class I PSD-95/Disc Large/ZO-1 (PDZ)-binding domain of NleH was important for its activity in the NF-κB pathway. In addition to binding RPS3, we found that NleH1 and NleH2 are able to bind to each otherin vitroandin vivo, suggesting an additional mechanism by which theE. coliNleH effectors may regulate the extent and duration of NF-κB activation after their T3SS-dependent translocation. We also performed mouse infection experiments and established that mouse mortality andCitrobactercolonization were reduced in mice infected with ΔnleH. Complementing ΔnleHwith NleH1 restoredCitrobactervirulence and colonization to wild-type levels, whereas complementing with NleH2 reduced them. Taken together, our data show that NleH1 and NleH2 have pronounced functional differences in their ability to alter host transcriptional responses to bacterial infection.

The human pathogens enterohemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC), as well as the mouse pathogen Citrobacter rodentium encode type III secretion system (T3SS) effector proteins to promote their survival in the infected host. The mechanisms of action and the host targets of T3SS effectors are under active investigation because of their importance to bacterial virulence. The non-locus of enterocyte effacement (LEE)-encoded protein F, NleF, contributes to E. coli and C. rodentium colonization of piglets and mice, respectively. Here we sought to characterize the host binding partners of NleF. Using a yeast two-hybrid screen, we identified Tmp21, a type-I integral membrane protein and COPI-vesicle receptor involved in trans-Golgi network function, as an NleF-binding partner. We confirmed this interaction using immunoprecipitation and bimolecular fluorescence complementation (BiFC). We expressed a temperature-sensitive vesicular stomatitis virus glycoprotein (tsVSVG) to monitor protein trafficking and determined that NleF slows the intracellular trafficking of tsVSVG from the endoplasmic reticulum to the Golgi.

Purpose To test the effects of varying vitrification protocols on the cell cycle status and chromosomal integrity in cumulusenclosed GV stage rat oocytes. Methods Vitrified and thawed rat oocytes were labeled with fluorescent markers for chromatin, cell cycle activation, and factin and analyzed by conventional and laser scanning confocal microscopy. Results In all vitrification groups, significant alterations in cumulus cell connectivity, cell cycle status, and cytoplasmic actin integrity were observed following warming compared to fresh control oocytes. Based on the protein phosphorylation marker MPM-2, it is clear that warmed oocytes rapidly enter M-phase but are unable to maintain chromosome integrity as a result of multiple chromatin fusions. A prominent reduction in f-actin is evident in both the ooplasm and at the cortex of vitrified oocytes. Finally, an irreversible but irregular retraction of TZPs occurs on the majority of oocytes subjected to any of the vitrification protocols. Conclusions These findings draw attention to undesirable consequences of immature oocyte vitrification that compromise cell cycle status and chromatin and cytoskeleton integrity that may not be evident until after fertilization.

An important adjunct to the field of fertility preservation is cryobiology. At present, the long-term storage of oocytes, embryos or ovarian tissues relies upon cryopreservation technologies that fall into roughly two different modalities: traditional slow freeze (SF) or rapid cooling, often invoking the process of vitrification. Unlike most cells in the body, female germ cells or oocytes present unique biophysical constraints as either isolated entities or within the context of ovarian follicles. Especially relevant is the fact that the oocyte nucleus, often referred to as the germinal vesicle, is highly hydrated and presents a voluminous non-chromatin occupied space that undergoes significant alterations in chromatin organization during its development. While the impact of cryopreservation on the integrity of the oocyte plasma membrane, organelles, and spindle cytoskeleton have been the focus of most studies to date, the short-term and long-term consequences of chilling and cryoprotectants on the chromosomal and genomic integrity has received much less attention. This chapter reviews the topic of genomic integrity at the level of the oocyte and provides guidelines for the design and implementation of strategies that will permit objective assessment of current and future protocols applied in the field of fertility preservation.