Pseudomonas aeruginosa exploits a PIP3-dependent pathway to transform apical into basolateral membrane

Pseudomonas aeruginosa, an important human pathogen, preferentially binds and enters injured cells from the basolateral (BL) surface. We previously demonstrated that activation of phosphatidylinositol 3-kinase (PI3K) and Akt are necessary and sufficient for P. aeruginosa entry from the apical (AP) surface and that AP addition of phosphatidylinositol 3,4,5-trisphosphate (PIP3) is sufficient to convert AP into BL membrane (Kierbel, A., A. Gassama-Diagne, K. Mostov, and J.N. Engel. 2005. Mol. Biol. Cell. 16:2577–2585; Gassama-Diagne, A., W. Yu, M. ter Beest, F. Martin-Belmonte, A. Kierbel, J. Engel, and K. Mostov. 2006. Nat. Cell Biol. 8:963–970). We now show that P. aeruginosa subverts this pathway to gain entry from the AP surface. In polarized monolayers, P. aeruginosa binds near cell–cell junctions without compromising them where it activates and recruits PI3K to the AP surface. Membrane protrusions enriched for PIP3 and actin accumulate at the AP surface at the site of bacterial binding. These protrusions lack AP membrane markers and are comprised of BL membrane constituents, which are trafficked there by transcytosis. The end result is that this bacterium transforms AP into BL membrane, creating a local microenvironment that facilitates its colonization and entry into the mucosal barrier

Protein modification with ISG15 blocks coxsackievirus pathology by antiviral and metabolic reprogramming

Protein modification with ISG15 (ISGylation) represents a major type I IFN–induced antimicrobial system. Common mechanisms of action and species-specific aspects of ISGylation, however, are still ill defined and controversial. We used a multiphasic coxsackievirus B3 (CV) infection model with a first wave resulting in hepatic injury of the liver, followed by a second wave culminating in cardiac damage. This study shows that ISGylation sets nonhematopoietic cells into a resistant state, being indispensable for CV control, which is accomplished by synergistic activity of ISG15 on antiviral IFIT1/3 proteins. Concurrent with altered energy demands, ISG15 also adapts liver metabolism during infection. Shotgun proteomics, in combination with metabolic network modeling, revealed that ISG15 increases the oxidative capacity and promotes gluconeogenesis in liver cells. Cells lacking the activity of the ISG15-specific protease USP18 exhibit increased resistance to clinically relevant CV strains, therefore suggesting that stabilizing ISGylation by inhibiting USP18 could be exploited for CV-associated human pathologies

Short-term consumption of a high-fat diet increases host susceptibility to Listeria monocytogenes infection

peer-reviewedBackground A westernized diet comprising a high caloric intake from animal fats is known to influence the development of pathological inflammatory conditions. However, there has been relatively little focus upon the implications of such diets for the progression of infectious disease. Here, we investigated the influence of a high-fat (HF) diet upon parameters that influence Listeria monocytogenes infection in mice. Results We determined that short-term administration of a HF diet increases the number of goblet cells, a known binding site for the pathogen, in the gut and also induces profound changes to the microbiota and promotes a pro-inflammatory gene expression profile in the host. Host physiological changes were concordant with significantly increased susceptibility to oral L. monocytogenes infection in mice fed a HF diet relative to low fat (LF)- or chow-fed animals. Prior to Listeria infection, short-term consumption of HF diet elevated levels of Firmicutes including Coprococcus, Butyricicoccus, Turicibacter and Clostridium XIVa species. During active infection with L. monocytogenes, microbiota changes were further exaggerated but host inflammatory responses were significantly downregulated relative to Listeria-infected LF- or chow-fed groups, suggestive of a profound tempering of the host response influenced by infection in the context of a HF diet. The effects of diet were seen beyond the gut, as a HF diet also increased the sensitivity of mice to systemic infection and altered gene expression profiles in the liver. Conclusions We adopted a systems approach to identify the effects of HF diet upon L. monocytogenes infection through analysis of host responses and microbiota changes (both pre- and post-infection). Overall, the results indicate that short-term consumption of a westernized diet has the capacity to significantly alter host susceptibility to L. monocytogenes infection concomitant with changes to the host physiological landscape. The findings suggest that diet should be a consideration when developing models that reflect human infectious disease.This research was funded by the European Union’s Horizon 2020 Research and Innovation Program under the Marie Skłodowska-Curie grant agreement No. 641984, through funding of the List_MAPS consortium. We also acknowledge funding and support from Science Foundation Ireland (SFI) in the form of a center grant (APC Microbiome Ireland grant SFI/12/RC/2273)

Assessment of the Red Cell Proteome of Young Patients with Unexplained Hemolytic Anemia by Two-Dimensional Differential In-Gel Electrophoresis (DIGE)

Erythrocyte cytosolic protein expression profiles of children with unexplained hemolytic anemia were compared with profiles of close relatives and controls by two-dimensional differential in-gel electrophoresis (2D-DIGE). The severity of anemia in the patients varied from compensated (i.e., no medical intervention required) to chronic transfusion dependence. Common characteristics of all patients included chronic elevation of reticulocyte count and a negative workup for anemia focusing on hemoglobinopathies, morphologic abnormalities that would suggest a membrane defect, immune-mediated red cell destruction, and evaluation of the most common red cell enzyme defects, glucose-6-phosphate dehydrogenase and pyruvate kinase deficiency. Based upon this initial workup and presentation during infancy or early childhood, four patients classified as hereditary nonspherocytic hemolytic anemia (HNSHA) of unknown etiology were selected for proteomic analysis. DIGE analysis of red cell cytosolic proteins clearly discriminated each anemic patient from both familial and unrelated controls, revealing both patient-specific and shared patterns of differential protein expression. Changes in expression pattern shared among the four patients were identified in several protein classes including chaperons, cytoskeletal and proteasome proteins. Elevated expression in patient samples of some proteins correlated with high reticulocyte count, likely identifying a subset of proteins that are normally lost during erythroid maturation, including proteins involved in mitochondrial metabolism and protein synthesis. Proteins identified with patient-specific decreased expression included components of the glutathione synthetic pathway, antioxidant pathways, and proteins involved in signal transduction and nucleotide metabolism. Among the more than 200 proteins identified in this study are 21 proteins not previously described as part of the erythrocyte proteome. These results demonstrate the feasibility of applying a global proteomic approach to aid characterization of red cells from patients with hereditary anemia of unknown cause, including the identification of differentially expressed proteins as potential candidates with a role in disease pathogenesis

Autophagy Protein Atg3 is Essential for Maintaining Mitochondrial Integrity and for Normal Intracellular Development of Toxoplasma gondii Tachyzoites

Autophagy is a cellular process that is highly conserved among eukaryotes and permits the degradation of cellular material. Autophagy is involved in multiple survival-promoting processes. It not only facilitates the maintenance of cell homeostasis by degrading long-lived proteins and damaged organelles, but it also plays a role in cell differentiation and cell development. Equally important is its function for survival in stress-related conditions such as recycling of proteins and organelles during nutrient starvation. Protozoan parasites have complex life cycles and face dramatically changing environmental conditions; whether autophagy represents a critical coping mechanism throughout these changes remains poorly documented. To investigate this in Toxoplasma gondii, we have used TgAtg8 as an autophagosome marker and showed that autophagy and the associated cellular machinery are present and functional in the parasite. In extracellular T. gondii tachyzoites, autophagosomes were induced in response to amino acid starvation, but they could also be observed in culture during the normal intracellular development of the parasites. Moreover, we generated a conditional T. gondii mutant lacking the orthologue of Atg3, a key autophagy protein. TgAtg3-depleted parasites were unable to regulate the conjugation of TgAtg8 to the autophagosomal membrane. The mutant parasites also exhibited a pronounced fragmentation of their mitochondrion and a drastic growth phenotype. Overall, our results show that TgAtg3-dependent autophagy might be regulating mitochondrial homeostasis during cell division and is essential for the normal development of T. gondii tachyzoites

ATG12 Conjugation to ATG3 Modulates Mitochondrial Homeostasis and Cell Death

Autophagy is an intracellular response to starvation, which is conserved in eukaryotes from unicellular to metazoan organisms. While autophagy sustains an individual cell from death by a variety of cellular stresses, evidence suggests that it is also critically important on an organismal level for the prevention of cancer, neurodegenerative and autoimmune diseases. In light of the therapeutic implications of autophagy, much remains to be understood about the function of individual autophagy genes. Many of the autophagy genes participate in two ubiquitin-like conjugation systems. The first mediates conjugation of ATG12 to ATG5 and the second mediates lipidation of LC3/ATG8 by phosphatidylethanolamine. We hypothesized that ATG12 conjugates to other substrates in addition to ATG5, as is the case for most ubiquitin-like proteins (ubls). By increasing the cellular pool of ATG12, we were able to identify and characterize a novel covalent complex between ATG12 and ATG3. A small subset of the cellular pool of ATG3 is in complex with ATG12 at baseline conditions in mammalian cells. Formation of the ATG12-ATG3 complex requires activation by ATG7, the autophagy E1-like enzyme. ATG12 subsequently becomes auto-conjugated in cis by ATG3. Unlike ATG12-ATG5 complex formation, ATG12-ATG3 conjugation does not require the E2-like enzyme ATG10. The c-terminal glycine of ATG12 specifically binds to K243 of ATG3.The ATG12-ATG5 complex and ATG3 each modulate steps in early autophagosome expansion and closure. Therefore, we initially expected that the ATG12-ATG3 complex would affect autophagy. To our surprise, disruption of the ATG12-ATG3 complex had no effect on autophagy induced by starvation, rapamycin or chemical uncoupling of the mitochondria. Instead disruption of the ATG12-ATG3 complex leads to a highly fragmented mitochondrial network that is incapable of fusion, increased in mass and defective in mitochondrial autophagy. These phenotypes also correlate with resistance to mitochondrial cell death. In summary, we have demonstrated evidence for a previously unknown conjugate between ATG12 and ATG3. Conjugation between ATG12 and ATG3 endows each protein with an entirely separate function from either protein's known role in autophagosome biogenesis. Our work suggests that ATG12 may be a more broad-based protein modification than previously anticipated

EASY READING MNEMOSYNE ROAD ATLAS

From its very beginnings, my artistic practice has revolved around space, broadly conceived. Phenomenology leant me its language to explain the heuristic marvel of perspective, of the translation of dimensional space into flat, planar information, of the imposition and projection of this practiced thing back onto embodied experience. My protracted, if variable ventures into phenomenology have been crucial to the language of my creative practice, but a syntactical side stepping of that field’s seemingly inborn theoretical dilemmas would be made in bad faith[1]. That phenomenology seems to have been doomed from its very outset to be unfinished, unfinishable indicates the difficulty, the futility of parsing out a subject like meaningful experience of the world. Meaning itself (and its myriad sensate, experiential roots) stubbornly resists categorical partitioning and reduction, a circumstance which bumps up against another to form a paradox -- making meaning of the world is itself is fundamentally a kind of categorical reduction, a thing is a hole in a thing it is not[2]. The environ, the life-world is something that we cannot help but understand through categorical pairs, Foucault’s inviolable oppositions: private | public; leisurely | utilitarian; intimate | social; itinerant | anchored, categories “nurtured by the hidden presence of the sacred.”[3] I cannot accept[4] phenomenology, and likewise cannot circumvent it. I cannot untangle the intersections of subject and life-world much less its intersections with other subjects and their life-worlds; I cannot discern if the home or the road came first[5], or if meaning in phenomenal experience precedes the imagined place or the cinematic image repertoire. I cannot even begin to neatly pull experience and meaning apart into its constitutive parts (though I find myself always trying). And so, in lieu of a salient, tight theoretical opus of my practice and its engagement with space, I will instead recount several passages through it, trips. What follows is EASY READING MNESMOSYNE ROAD ATLAS, a kind of travelogue. The trips included are interrelated; their meanings echo and invert each other. They are real passages, they are remembered, they are imagined, they are virtual, they are dimensional. They are mine, they are taken by others before me, they are implied, they are imaginary, historical, retold. The trips outlined here are not, quite obviously, an exhaustive account of kinds of human passages, of course, just as this is not an exhaustive account of ways of being-in-the-world[6]. It is rather a sampling of passages germane to my work, my dread, passages through places bearing significant meaning to me, places I assign meaning to, places I have lived in and likewise imagined and re-membered for myself constantly, within and outside my studio. I do not endorse all these trips – in fact, I object to, and do not find any personal resonance in the tenor of some of them, but have nevertheless included them as important counterweights for my trips, for my work, for my Mnemosyne Atlas. [1] Real understood in various, often conflicting ways w/ reference to the life-world and elsewhere [2] Robert Smithson, 1968 [3] Michel Foucault, “Of Other Spaces”, 2. [4] Fred Moten, In The Break: The Aesthetics of the Black Radical Tradition (Minneapolis: University of Minnesota Press, 2003), 203. [5] JB Jackson poses this question in “Roads Belong in the Landscape”, in A Sense of Place, A Sense of Time, JB Jackson (New Haven: Yale University Press, 1994.), 189-205. [6] The whole purpose of this structure is to bypass, to avoid the impossible, if quite tempting impulse to work out an exhaustive theoretical framewor

Lmo1656 is a secreted virulence factor of Listeria monocytogenes that interacts with the sorting nexin 6-BAR complex

Listeria monocytogenes (Lm) is a facultative intracellular bacterial pathogen and the causative agent of listeriosis, a rare but fatal disease. During infection, Lm can traverse several physiological barriers; it can cross the intestine and placenta barrier and, in immunocompromised individuals, the blood-brain barrier. With the recent plethora of sequenced genomes available for Lm, it is clear that the complete repertoire of genes used by Lm to interact with its host remains to be fully explored. Recently, we focused on secreted Lm proteins because they are likely to interact with host cell components. Here, we investigated a putatively secreted protein of Lm, Lmo1656, that is present in most sequenced strains of Lm but absent in the nonpathogenic species Listeria innocua. lmo1656 gene is predicted to encode a small, positively charged protein. We show that Lmo1656 is secreted by Lm. Furthermore, deletion of the lmo1656 gene (Δlmo1656) attenuates virulence in mice infected orally but not intravenously, suggesting that Lmo1656 plays a role during oral listeriosis. We identified sorting nexin 6 (SNX6), an endosomal sorting component and BAR domain-containing protein, as a host cell interactor of Lmol656. SNX6 colocalizes with WT Lm during the early steps of infection. This colocalization depends on Lmo1656, and RNAi of SNX6 impairs infection in infected tissue culture cells, suggesting that SNX6 is utilized by Lm during infection. Our results reveal that Lmo1656 is a novel secreted virulence factor of Lm that facilitates recruitment of a specific member of the sorting nexin family in the mammalian host