Shigella bacteria invade macrophages and epithe-lial cells and following internalization lyse the phagosome and escape to the cytoplasm. Galectin-3, an abundant protein in macrophages and epithelial cells, belongs to a family of beta-galactoside-binding proteins, the galectins, with many proposed functions in immune response, development, differentiation, cancer and infection. Galectins are synthesized as cytosolic proteins and following non-classical secretion bind extra-cellular beta-galactosides. Here we analysed the localization of galectin-3 following entry of Shigella into the cytosol and detected a striking phenomenon. Very shortly after bacterial invasion, intracellular galectin-3 accumulated in structures in vicinity to internalized bacteria. By using immuno-electron microscopy analysis we identified galectin-3 in membranes localized in the phagosome and in tubules and vesicles that derive from the endocytic pathway. We also demonstrated that the binding of galectin-3 to host N-acetyllactosamine-containing glycans, was required for forming the structures. Accumulation of the structures was a type three secretion system-dependent process. More specifically, existence of structures was strictly dependent upon lysis of the phagocytic vacuole and could be shown also by Gram-positive Listeria and Salmonella sifA mutant. We suggest that galectin-3-containing structures may serve as a potential novel tool to spot vacuole lysis.

Intracellular pathogens like Shigella flexneri enter host cells by phagocytosis. Once inside, the pathogen breaks the vacuolar membrane for cytosolic access. The fate and function of the vacuolar membrane remnants are not clear. Examining Shigella-infected nonmyeloid cells, we observed that proteins associated with vacuolar membrane remnants are polyubiquinated, recruit the autophagy marker LC3 and adaptor p62, and are targeted to autophagic degradation. Further, inflammasome components and caspase-1 were localized to these membranes and correlated with dampened inflammatory response and necrotic cell death. In Atg4B mutant cells in which autophagosome maturation is blocked, polyubiquitinated proteins and P62 accumulated on membrane remnants, and as in autophagy-deficient Atg5(-/-) cells, the early inflammatory and cytokine response was exacerbated. Our results suggest that host membranes, after rupture by an invading cytoplasm-targeted bacterium, contribute to the cellular responses to infection by acting as a signaling node, with autophagy playing a central role in regulating these responses.

Internal initiation of translation, whereby ribosomes are directed to internal AUG codon independently of the 5 end of the mRNA, has been observed rarely in higher eucaryotes and has not been demonstrated in living yeast. We report here that starved yeast cells are capable of initiating translation of a dicistronic message internally. The studied element that functions as an internal ribosome entry site (IRES) is hardly functional or not functional at all in logarithmically growing cells. Moreover, during the logarithmic growth phase, this element seems to inhibit translation reinitiation when placed as an intercistronic spacer or to inhibit translation when placed in the 5-untranslated region of a monocistronic message. Inhibition of translation is likely due to the putative strong secondary structure of the IRES that interferes with the cap-dependent scanning process. When cells exit the logarithmic growth phase, or when artificially starved for carbon source, translation of the IRES-containing messages is substantially induced. Our findings imply that the capacity to translate internally is a characteristic of starved rather than vegetatively growing yeast cells.The ribosome scanning model has been originally proposed by Kozak (1) to explain how the translation process is initiated. Numerous studies have corroborated the model whereby the initiation complex is assembled near or at the 5Ј end of the mRNA, facilitated by the interaction of the cap structure with the eucaryotic initiation factor 4E, and starts scanning the mRNA until the first AUG is encountered (for recent review see Ref. 2). An alternative mode of selecting an initiation codon, whereby ribosomes are directed to an internal AUG by an internal ribosome entry sequence (IRES), 1 has also been demonstrated. Well documented cases of internal initiation events are those of the uncapped picornaviral mRNAs (3). IRESes have also been found in the 5Ј-untranslated region (5Ј-UTR) of several cellular mRNAs (4 -13). During evolution, IRESes have been utilized as targets for translation regulation during normal differentiation and development. For example, an IRES was shown to play a role in the translation of platelet-derived growth factor 2 mRNA that increases after megacaryocitic cells undergo terminal differentiation (10). IRESes have also been found to mediate the differential translation of Antenapedia and Ultrabithorax during Drosophila melanogaster development (6,12). Surprisingly, no IRES has been shown to function in yeasts, despite the observations that the yeast cell-free system is capable of recognizing plant viral IRESes (14) as well as natural yeast leader sequences (15). Attempts to promote internal initiation in living yeast cells by using IRESes of poliovirus (16) and encephalomyocarditis virus (17) have thus far failed. These studies were done with optimally growing cells. In their natural environment, however, yeast occasionally encounter starvation and enter into a distinct quiescent state called stationary phase (SP) (reviewed in Refs. ...

SummaryShigella bacteria invade macrophages and epithelial cells and following internalization lyse the phagosome and escape to the cytoplasm. Galectin-3, an abundant protein in macrophages and epithelial cells, belongs to a family of betagalactoside-binding proteins, the galectins, with many proposed functions in immune response, development, differentiation, cancer and infection. Galectins are synthesized as cytosolic proteins and following non-classical secretion bind extracellular beta-galactosides. Here we analysed the localization of galectin-3 following entry of Shigella into the cytosol and detected a striking phenomenon. Very shortly after bacterial invasion, intracellular galectin-3 accumulated in structures in vicinity to internalized bacteria. By using immuno-electron microscopy analysis we identified galectin-3 in membranes localized in the phagosome and in tubules and vesicles that derive from the endocytic pathway. We also demonstrated that the binding of galectin-3 to host N-acetyllactosamine-containing glycans, was required for forming the structures. Accumulation of the structures was a type three secretion system-dependent process. More specifically, existence of structures was strictly dependent upon lysis of the phagocytic vacuole and could be shown also by Gram-positive Listeria and Salmonella sifA mutant. We suggest that galectin-3-containing structures may serve as a potential novel tool to spot vacuole lysis.

No abstract

The eukaryotic translation initiation factor 4E (eIF4E) interacts with the mRNA 5 cap structure (m 7 GpppX) and is essential for the appropriate translation of the vast majority of eukaryotic mRNAs. Most studies of the yeast Saccharomyces cerevisiae CDC33 gene product, eIF4E, have been carried out with logarithmically growing cells, and little is known about its role in starved, nonproliferating cells that enter the stationary phase (SP). It has previously been found that the rate of translation in SP cells is more than 2 orders of magnitude lower than it is in dividing yeast cells. Here we show that this low rate of translation is essential for maintaining the viability of starved yeast cells that enter SP. Specifically, starved cells whose eIF4A is inactive or treated with cycloheximide rapidly lose viability. Moreover, after heat inactivation of the cdc33 temperature-sensitive product, the synthesis of most proteins is abolished and only a small group of proteins is still produced. Unexpectedly, starved cdc33 mutant cells whose eIF4E is inactive and which therefore fail to synthesize the bulk of their proteins remain viable for long periods of time, indistinguishable from their isogenic wild-type counterparts. Taken together, our results indicate that eIF4E-independent translation is necessary and sufficient for survival of yeast cells during long periods of starvation.