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SummaryThe ubiquitin-like protein, ISG15, can act as a cytokine or can covalently modify host and pathogen-derived proteins. The consequences of ISG15 modification on substrate fate remain unknown. Here we reveal that ISGylation of the Arp2/3 complex slows actin filament formation and stabilizes Arp2/3 dependent structures including cortical actin and lamella. When properly controlled, this serves as an antibacterial and antiviral host defense strategy to directly restrict actin-mediated pathogen spread. However,Listeria monocytogenestakes advantage in models of dysregulated ISGylation, leading to increased mortality due to augmented spread. The underlying molecular mechanism responsible for the ISG15-dependent impact on actin-based motility is due to failed bacterial separation after division. This promotes spread by enabling the formation of multi-headed bacterial “bazookas” with stabilized comet tails that can disseminate deeper into tissues. A bacterial mutant that cannot recruit Arp2/3 or a non-ISGylatable mutant of Arp3 is sufficient to rescue slowed comet tail speed and restrict spread. Importantly, ISG15-deficient neonatal mice have aberrant epidermal epithelia characterized by keratinocytes with diffuse cortical actin, which could underlie observed defects in wound healing in human patients who lack ISG15. Ultimately, our discovery links host innate immune responses to cytoskeletal dynamics with therapeutic implications for viral infection and metastasis.

Viruses have developed many different strategies to counteract immune responses, andVaccinia virus(VACV) is one of a kind in this aspect. To ensure an efficient infection, VACV undergoes a complex morphogenetic process resulting in the production of two types of infective virions: intracellular mature virus (MV) and extracellular enveloped virus (EV), whose spread depends on different dissemination mechanisms. MVs disseminate after cell lysis, whereas EVs are released or propelled in actin tails from living cells. Here we show that ISG15 participates in the control of VACV dissemination. Infection ofIsg15-/-mouse embryonic fibroblasts with VACV International Health Department-J (IHD-J) strain resulted in decreased EV production, concomitant with reduced induction of actin tails and the abolition of comet-shaped plaque formation, comparing withIsg15+/+cells. Transmission electron microscopy revealed accumulation of intracellular and a decrease in extracellular virus particles in the absence of Interferon Stimulated Gene 15 (ISG15), consistent with altered virus egress. Immunoblot and quantitative proteomic analysis of sucrose gradient-purified virions from both genotypes reported differences in protein levels and composition of viral proteins present on virions, suggesting an ISG15-mediated control of viral proteome. Last, the generation of a recombinant IHD-J expressing V5-tagged ISG15 (IHD-J-ISG15) allowed us to identify several viral proteins as potential ISG15 targets, highlighting the proteins A34 and A36, essential for EV formation. Altogether, our results indicate that ISG15 is an important host factor in the regulation of VACV dissemination.Author SummaryViral infections are a constant battle between the virus and the host. While the host’s only goal is victory, the main purpose of the virus is to spread and conquer new territories at the expense of the host’s resources. Along millions of years of incessant encounters, Poxviruses have developed a unique strategy consisting in the production two specialized “troops”: intracellular mature virions (MVs) and extracellular virions (EVs). MVs mediate transmission between hosts, and EVs ensure advance on the battlefield mediating the long-range dissemination.The mechanism by which the virus ‘decides’ to shed from the primary site of infection and its significant impact in viral transmission is not yet fully established. Here, we demonstrate that this process is finely regulated by ISG15/ISGylation, an interferon-induced ubiquitin-like protein with broad antiviral activity. Studying the mechanism that viruses use during infection could result in new ways of understanding our perpetual war against disease and how we might win the next great battle.

Significance Listeria monocytogenes (Lm) is a bacterial pathogen that causes listeriosis, a foodborne disease characterized by gastroenteritis, meningitis, bacteremia, and abortions in pregnant women. The most severe human listeriosis outbreaks are associated with a subset of Lm hypervirulent clones that encode the bacteriocin Listeriolysin S (LLS), which modifies the gut microbiota and allows efficient Lm gut colonization and invasion of deeper organs. Our present work identifies the killing mechanism displayed by LLS to outcompete gut commensal bacteria, demonstrating that it induces membrane permeabilization and membrane depolarization of target bacteria. Moreover, we show that LLS is a thiazole/oxazole–modified microcin that displays a contact-dependent inhibition mechanism., Listeriolysin S (LLS) is a thiazole/oxazole–modified microcin (TOMM) produced by hypervirulent clones of Listeria monocytogenes. LLS targets specific gram-positive bacteria and modulates the host intestinal microbiota composition. To characterize the mechanism of LLS transfer to target bacteria and its bactericidal function, we first investigated its subcellular distribution in LLS-producer bacteria. Using subcellular fractionation assays, transmission electron microscopy, and single-molecule superresolution microscopy, we identified that LLS remains associated with the bacterial cell membrane and cytoplasm and is not secreted to the bacterial extracellular space. Only living LLS-producer bacteria (and not purified LLS-positive bacterial membranes) display bactericidal activity. Applying transwell coculture systems and microfluidic-coupled microscopy, we determined that LLS requires direct contact between LLS-producer and -target bacteria in order to display bactericidal activity, and thus behaves as a contact-dependent bacteriocin. Contact-dependent exposure to LLS leads to permeabilization/depolarization of the target bacterial cell membrane and adenosine triphosphate (ATP) release. Additionally, we show that lipoteichoic acids (LTAs) can interact with LLS and that LTA decorations influence bacterial susceptibility to LLS. Overall, our results suggest that LLS is a TOMM that displays a contact-dependent inhibition mechanism.

MuRF1 (TRIM63) is a muscle-specific E3 ubiquitin ligase and component of the ubiquitin proteasome system. MuRF1 is transcriptionally upregulated under conditions that cause muscle loss, in both rodents and humans, and is a recognized marker of muscle atrophy. In this study, we used in vivo electroporation to determine whether MuRF1 overexpression alone can cause muscle atrophy and, in combination with ubiquitin proteomics, identify the endogenous MuRF1 substrates in skeletal muscle. Overexpression of MuRF1 in adult mice increases ubiquitination of myofibrillar and sarcoplasmic proteins, increases expression of genes associated with neuromuscular junction instability, and causes muscle atrophy. A total of 169 ubiquitination sites on 56 proteins were found to be regulated by MuRF1. MuRF1-mediated ubiquitination targeted both thick and thin filament contractile proteins, as well as, glycolytic enzymes, deubiquitinases, p62, and VCP. These data reveal a potential role for MuRF1 in not only the breakdown of the sarcomere but also the regulation of metabolism and other proteolytic pathways in skeletal muscle.

ISG15 is an interferon-stimulated, ubiquitin-like protein that can conjugate to substrate proteins (ISGylation) to counteract microbial infection, but the underlying mechanisms remain elusive. Here, we use a virus-like particle trapping technology to identify ISG15-binding proteins and discover Ring Finger Protein 213 (RNF213) as an ISG15 interactor and cellular sensor of ISGylated proteins. RNF213 is a poorly characterized, interferon-induced megaprotein that is frequently mutated in Moyamoya disease, a rare cerebrovascular disorder. We report that interferon induces ISGylation and oligomerization of RNF213 on lipid droplets, where it acts as a sensor for ISGylated proteins. We show that RNF213 has broad antimicrobial activity in vitro and in vivo, counteracting infection with Listeria monocytogenes, herpes simplex virus 1, human respiratory syncytial virus and coxsackievirus B3, and we observe a striking co-localization of RNF213 with intracellular bacteria. Together, our findings provide molecular insights into the ISGylation pathway and reveal RNF213 as a key antimicrobial effector., During microbial infection, proteins are modified by the ubiquitin-like protein ISG15. Here, the authors uncover RNF213 as a sensor for ISGylated proteins on the surface of lipid droplets, showing that RNF213 has antiviral properties but also directly targets intracellular bacteria in infected cells.

Protein modification with ISG15 acts cooperatively with IFIT proteins and preserves glucose homeostasis., 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.

It is well documented that influenza A viruses selectively package 8 distinct viral ribonucleoprotein complexes (vRNPs) into each virion; however, the role of host factors in genome assembly is not completely understood. To evaluate the significance of cellular factors in genome assembly, we generated a reporter virus carrying a tetracysteine tag in the NP gene (NP-Tc virus) and assessed the dynamics of vRNP localization with cellular components by fluorescence microscopy. At early time points, vRNP complexes were preferentially exported to the MTOC; subsequently, vRNPs associated on vesicles positive for cellular factor Rab11a and formed distinct vRNP bundles that trafficked to the plasma membrane on microtubule networks. In Rab11a deficient cells, however, vRNP bundles were smaller in the cytoplasm with less co-localization between different vRNP segments. Furthermore, Rab11a deficiency increased the production of non-infectious particles with higher RNA copy number to PFU ratios, indicative of defects in specific genome assembly. These results indicate that Rab11a+ vesicles serve as hubs for the congregation of vRNP complexes and enable specific genome assembly through vRNP:vRNP interactions, revealing the importance of Rab11a as a critical host factor for influenza A virus genome assembly., Author summary The influenza A virus (IAV) genome is composed of 8 distinct RNA segments. It has remained unclear how these 8 individual RNA segments are assembled together to form infectious virus particles. Our study shows that Rab11a+ vesicles serve as platforms for the congregation and assembly of 8 individual viral RNA segments needed to form infectious virus particles. However, in cells lacking Rab11a, viral RNA segments fail to congregate together, resulting in increased production of defective virus particles, likely due to misassembling of viral RNA segments. Thus, our study reveals the important role for Rab11a in influenza virus genome assembly and production of infectious virus particles.

ISG15 is an interferon-stimulated, ubiquitin-like protein, with anti-viral and anti-bacterial activity. Here, we map the endogenous in vivo ISGylome in the liver following Listeria monocytogenes infection by combining murine models of reduced or enhanced ISGylation with quantitative proteomics. Our method identifies 930 ISG15 sites in 434 proteins and also detects changes in the host ubiquitylome. The ISGylated targets are enriched in proteins which alter cellular metabolic processes, including upstream modulators of the catabolic and antibacterial pathway of autophagy. Computational analysis of substrate structures reveals that a number of ISG15 modifications occur at catalytic sites or dimerization interfaces of enzymes. Finally, we demonstrate that animals and cells with enhanced ISGylation have increased basal and infection-induced autophagy through the modification of mTOR, WIPI2, AMBRA1, and RAB7. Taken together, these findings ascribe a role of ISGylation to temporally reprogram organismal metabolism following infection through direct modification of a subset of enzymes in the liver., ISG15 is a ubiquitin-like modifier that can be upregulated in response to bacterial infections. Here, the authors use proteomics to identify endogenous ISGylation substrates in the liver of Listeria monocytogenes infected mice and show that ISGylation alters basal and infection-induced autophagy.

International audience; 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 (lmo1656) 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 colocal-izes 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 mam-malian host. The foodborne pathogen Listeria monocytogenes (Lm) 5 can cross several physiological barriers and infect multiple cell types. The pathogenic potential of Lm relies on the ability of this bacterium to cross multiple physiological barriers as well as its ability to enter and replicate within a wide variety of host cell types (for recent reviews, see Refs. 1 and 2). Upon binding to host cell surface receptors, Lm induces its internalization into both professional phagocytes and nonphagocytic cells (for a recent review, see Ref. 2). From there, Lm escapes into the cyto-sol by rupturing its vacuole. Lm is able to evade host cell immune responses (for a recent review, see Ref. 3) and subvert the host cell actin cytoskeleton to drive intra-and intercellular motility (for recent reviews, see Refs. 4 –6). Secreted and surface-exposed Lm proteins can encounter host components and serve as virulence factors. For example, the secreted pore-forming toxin listeriolysin O (LLO) is one of the most well-characterized and potent virulence factors of Lm (for a review, see Ref. 7). Secretion of LLO occurs prior to Lm entry into the host cell. It inserts into the host plasma membrane and makes large pores. The resulting ion flux drives a diverse array of responses within the cell from global deSUMOylation (8) to mitochondrial fragmentation (9). Upon entry, Lm can escape into the host cytosol by lysing the phagosomal membrane through the combined actions of secreted LLO and phospholipases A and B (PlcA and PlcB) (10 –12). Recent work has uncovered novel secreted Lm virulence factors and their binding partners in the host cell. The secreted protein Listeria nuclear targeted protein A (LntA) targets the host epigenetic regulator BAHD1, altering host cell transcription (13). The small secreted protein internalin C (InlC) sequesters Tuba, a Cdc42 guanine exchange factor, to induce relaxation of membrane cortical tension, thereby facilitating increased bacterial cell-to-cell spread (14, 15). InlC also directly binds to host IB kinase , interfering with host innate immunity (16). The recent plethora of genomics data and the rise of bioin-formatics pipelines have enabled the rapid comparison of mul

International audience; Listeria monocytogenes is a food-borne pathogen responsible for a disease called listeriosis, which is potentially lethal in immunocompromised individuals. This bacterium, first used as a model to study cell-mediated immunity, has emerged over the past 20 years as a paradigm in infection biology, cell biology and fundamental microbiology. In this Review, we highlight recent advances in the understanding of human listeriosis and L. monocytogenes biology. We describe unsuspected modes of hijacking host cell biology, ranging from changes in organelle morphology to direct effects on host transcription via a new class of bacterial effectors called nucleomodulins. We then discuss advances in understanding infection in vivo, including the discovery of tissue-specific virulence factors and the 'arms race' among bacteria competing for a niche in the microbiota. Finally, we describe the complexity of bacterial regulation and physiology, incorporating new insights into the mechanisms of action of a series of riboregulators that are critical for efficient metabolic regulation, antibiotic resistance and interspecies competition.

International audience; To adapt to changing environments, bacteria have evolved numerous pathways that activate stress response genes. In Gram-positive bacteria, the stressosome, a cytoplasmic complex, relays external cues and activates the sigma B regulon. The stressosome is structurally well-characterized in Bacillus, but how it senses stress remains elusive. Here, we report a genome-wide N-terminomic approach in Listeria that strikingly led to the discovery of 19 internal translation initiation sites and 6 miniproteins, among which one, Prli42, is conserved in Firmicutes. Prli42 is membrane-anchored and interacts with orthologues of Bacillus stressosome components. We reconstituted the Listeria stressosome in vitro and visualized its supramolecular structure by electron microscopy. Analysis of a series of Prli42 mutants demonstrated that Prli42 is important for sigma B activation, bacterial growth following oxidative stress and for survival in macrophages. Taken together, our N-terminonic approach unveiled Prli42 as a long-sought link between stress and the stressosome.

International audience; In May 2012, three European Academies held a conference on the present and future of microbiology. The conference, entitled "The New Microbiology", was a joint effort of the French Academie des sciences, of the German National Academy of Sciences Leopoldina and of the British Royal Society. The organizers - Pascale Cossart and Philippe Sansonetti from the "Academie des sciences", David Holden and Richard Moxon from the "Royal Society", and Jorg Hacker and Jurgen Hesseman from the "Leopoldina Nationale Akademie der Wissenschaften" - wanted to highlight the current renaissance in the field of microbiology mostly due to the advent of technological developments and allowing for single-cell analysis, rapid and inexpensive genome-wide comparisons, sophisticated microscopy and quantitative large-scale studies of RNA regulation and proteomics. The conference took place in the historical Palais de l'Institut de France in Paris with the strong support of Jean-Francois Bach, Secretaire Perpetuel of the Academie des sciences.

SummaryATG12, an ubiquitin-like modifier required for macroautophagy, has a single known conjugation target, another autophagy regulator called ATG5. Here, we identify ATG3 as a substrate for ATG12 conjugation. ATG3 is the E2-like enzyme necessary for ATG8/LC3 lipidation during autophagy. ATG12-ATG3 complex formation requires ATG7 as the E1 enzyme and ATG3 autocatalytic activity as the E2, resulting in the covalent linkage of ATG12 onto a single lysine on ATG3. Surprisingly, disrupting ATG12 conjugation to ATG3 does not affect starvation-induced autophagy. Rather, the lack of ATG12-ATG3 complex formation produces an expansion in mitochondrial mass and inhibits cell death mediated by mitochondrial pathways. Overall, these results unveil a role for ATG12-ATG3 in mitochondrial homeostasis and implicate the ATG12 conjugation system in cellular functions distinct from the early steps of autophagosome formation.

This Article contains a URL for a publically available whole-genome browser ( http://nterm.listeriomics.pasteur.fr ). However, due to technical constraint, this website has been replaced with an alternative ( https://listeriomics.pasteur.fr ).

International audience; Cytosolic sensing of pathogens is essential to a productive immune response. Recent reports have emphasized the importance of signaling platforms emanating from organelles and cytosolic sensors, particularly during the response to intracellular pathogens. Here, we highlight recent discoveries identifying the key mediators of nucleic acid and cyclic nucleotide sensing and discuss their importance in host defense. This review will also cover strategies evolved by pathogens to manipulate these pathways.

Intracellular ISG15 is an interferon (IFN)-alpha/beta-inducible ubiquitin-like modifier which can covalently bind other proteins in a process called ISGylation; it is an effector of IFN-alpha/beta-dependent antiviral immunity in mice(1-4). We previously published a study describing humans with inherited ISG15deficiency but without unusually severe viral diseases(5). We showed that these patients were prone to mycobacterial disease and that human ISG15 was non-redundant as an extracellular IFN-gamma-inducing molecule. We show here that ISG15-deficient patients also display unanticipated cellular, immunological and clinical signs of enhanced IFN-alpha/beta immunity, reminiscent of the Mendelian autoinflammatory interferonopathies Aicardi-Goutieres syndrome and spondyloenchondrodysplasia(6-9). We further show that an absence of intracellular ISG15 in the patients' cells prevents the accumulation of USP18(10,11), a potent negative regulator of IFN-alpha/beta signalling, resulting in the enhancement and amplification of IFN-alpha/beta responses. Human ISG15, therefore, is not only redundant for antiviral immunity, but is a key negative regulator of IFN-alpha/beta immunity. In humans, intracellular ISG15 is IFN-alpha/beta-inducible not to serve as a substrate for ISGylation-dependent antiviral immunity, but to ensure USP18-dependent regulation of IFN-alpha/beta and prevention of IFN-alpha/beta-dependent autoinflammation.

International audience; SUMOylation is an essential ubiquitin-like modification involved in important biological processes in eukaryotic cells. Identification of small ubiquitin-related modifier (SUMO)-conjugated residues in proteins is critical for understanding the role of SUMOylation but remains experimentally challenging. We have set up a powerful and high-throughput method combining quantitative proteomics and peptide immunocapture to map SUMOylation sites and have analyzed changes in SUMOylation in response to stimuli. With this technique we identified 295 SUMO1 and 167 SUMO2 sites on endogenous substrates of human cells. We further used this strategy to characterize changes in SUMOylation induced by listeriolysin O, a bacterial toxin that impairs the host cell SUMOylation machinery, and identified several classes of host proteins specifically deSUMOylated in response to this toxin. Our approach constitutes an unprecedented tool, broadly applicable to various SUMO-regulated cellular processes in health and disease.

Ubiquitin-like (UBL) protein conjugations, such as ubiquitination and sumoylation, alter protein fate, localization, function and transcriptional activity. Two UBL protein conjugation systems, the ATG12 and ATG8 systems, are required for macroautophagy. The early steps of autophagosome formation require that these two conjugation systems covalently attach: (1) the UBL ATG12 to the target protein ATG5 (Fig. 1A); and (2) the UBL ATG8 to the lipid phosphotidylethanolamine (PE) (Fig. 1B). Interestingly, since the initial discovery of the ATG12 conjugation system, ATG5 has remained as the only known target of ATG12. Recently, we demonstrated that ATG12 can modify multiple protein targets in mammalian cells, and identified ATG3, the E2-like enzyme that conjugates ATG8 to PE, as a second ATG12 conjugation target. Figure 1 ATG12 conjugation to ATG3. (A) ATG 12 conjugation system. The E1 enzyme ATG7 activates the C-terminal glycine of ATG12. Subsequently, ATG12 is transferred to the E2 conjugating enzyme ATG10, resulting in the formation of the ATG12-ATG5 complex (top). ... Although ATG12 and ATG3 have been previously reported to physically interact, to our knowledge, we provide the first evidence for a covalent complex between these two essential components of the autophagy conjugation machineries. Similar to ATG12-ATG5, formation of the ATG12-ATG3 complex requires activation of ATG12 by the E1-like enzyme ATG7. Subsequently, ATG3 autocatalyzes the conjugation of ATG12 onto a single conserved lysine residue (K243) of this E2-like enzyme (Fig. 1A, see gray box). Based on immunoprecipitation studies, both free ATG3 and the ATG12-ATG3 complex are concomitantly detected in mammalian cells; notably, only a fraction of the total pool of endogenous ATG3 is in a complex with ATG12. This contrasts with ATG5, which appears to be completely conjugated to ATG12. To gain insight into the cellular function of the ATG12-ATG3 complex, we complemented atg3-deficient mouse fibroblasts with either wild-type ATG3 (WTATG3) or a mutant version of ATG3 that is unable to be conjugated to ATG12 due to loss of a single lysine required for isopeptide bond linkage (ATG3KR). Because ATG12, in complex with ATG5, and ATG3 both play critical roles in the biogenesis and elongation of the autophagosomal membrane, we hypothesized that the ATG12-ATG3 complex would likewise regulate the early steps of autophagy. However, upon disrupting ATG12 conjugation to ATG3, we observed no effects on the lipidation of LC3 or other mammalian ATG8 orthologues. We also found no significant changes in nonselective autophagy in response to multiple stresses, including nutrient starvation, rapamycin-mediated mTOR inhibition and carbonyl cyanide m-chlorophenylhydrazone (CCCP)-induced mitochondrial depolarization. Although nonselective autophagy appears intact, our results indicate that the disruption of ATG12 conjugation to ATG3 in cells does have profound effects on mitochondria. ATG3KR cells possess increased mitochondrial mass relative to WTATG3 cells, both at baseline and in response to mitochondrial depolarization with CCCP. Upon mitochondrial depolarization, mitochondria targeting to autophagosomes in ATG3KR cells is significantly reduced relative to WTATG3 cells, suggesting a defect in mitochondrial autophagy or mitophagy. To date, the prevailing view has been that specific adapter molecules target mitochondria for autophagic degradation in mammalian cells. For example, the two related BH3 family proteins, BNIP3 and NIX/BNIP3L, promote mitochondrial clearance during reticulocyte development, whereas the PINK1-Parkin pathway directs mitophagy in response to depolarization. In contrast, the ATGs comprising the conjugation systems have not been previously implicated in the selective targeting of mitochondria; rather, they are proposed to contribute more generally by forming the autophagosome. Our results now broach the hypothesis that this newly identified complex between two conjugation ATGs mediates a unique function for selectively targeting mitochondria for autophagic degradation. In addition, we discovered that the mitochondrial network of ATG3KR cells is highly fragmented. Mitochondrial fragmentation is similarly present in atg3-deficient cells, whereas cells reconstituted with WTATG3 exhibit tubular mitochondrial networks. Mitochondria undergo extensive remodeling through continuous cycles of mitochondrial fusion and fission; appropriate balance of these two opposing processes is necessary for maintenance of overall mitochondrial structure and function. Mitochondrial fragmentation often indicates an imbalance in mitochondrial fission and fusion; in support of this prediction, cells lacking the ATG12-ATG3 complex display reduced mitochondrial fusion activity. Nonetheless, further studies are required to clarify if ATG12-ATG3 serves as a novel link between autophagy and the mitochondrial fusion machinery. Importantly, one can alternatively hypothesize that the effects of ATG12-ATG3 on mitochondrial fusion are secondary to its effects on mitophagy, since previous work indicates that fragmented mitochondria are preferentially degraded by mitophagy. In other words, due to reduced mitophagy, disruption of ATG12 conjugation to ATG3 results in the build-up of fusion defective mitochondria. Finally, ATG12 conjugation to ATG3 sensitizes cells to apoptosis mediated by mitochondrial pathways, but has no effect on death-receptor mediated apoptosis. The resistance to intrinsic apoptosis in ATG3KR cells correlates with an increase in the basal protein levels of antiapoptotic Bcl-2 proteins, namely Bcl-xL. Accordingly, specific antagonism of antiapoptotic Bcl-2 family members with obatoclax, a pharmacological BH3 mimetic, causes equivalent levels of cell death between WTATG3 and ATG3KR cells. An important question for future study is whether these increases in Bcl-XL also contribute functionally to the changes in mitochondrial mass and morphology found in ATG3KR cells. Dissecting the exact mechanisms through which the ATG12-ATG3 complex mediates these diverse effects on mitochondrial homeostasis, morphology and cell death remains an important topic for future investigation. Since a fraction of total ATG3 is in complex with ATG12, this motivates the hypothesis that ATG12 conjugation to ATG3 promotes the relocation of this complex to mitochondria. Alternatively, ATG12 conjugation may change ATG3 substrate specificity, alter its enzymatic activity, or facilitate new protein-protein interactions. Importantly, our results support the idea that the effects of the ATG12-ATG3 complex on mitochondrial homeostasis and cell death are unique functions that can be completely separated from the established roles of each individual ATG in autophagosome formation. Although this is a novel finding for ATG12 and ATG3, other studies demonstrate that ATGs may control cellular functions distinct from autophagy. For example, during phagocytosis, LC3 becomes recruited to, and incorporated into, the plasma membrane by a macroautophagy-independent mechanism that requires both ATG5 and Beclin 1. Hence, we propose that ATG12, like ubiquitin and SUMO, may serve as a broadbased protein modification that mediates diverse cellular functions in addition to autophagy.

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.

Nature 517, 89–93 (2015); doi:10.1038/nature13801In this Letter, author J.-L.C. was inadvertently missing one of their affiliations; they should also have been associated with affiliation number 2 (St. Giles Laboratory of Human Genetics of Infectious Diseases, Rockefeller Branch, The Rockefeller University, New York, New York 10065, USA). This has been corrected in the online versions of the paper.

In the last decades, Listeria has become a key model organism for the study of host-pathogen interactions, noncoding RNA regulation, and bacterial adaptation to stress. To study these mechanisms, several genomics, transcriptomics, and proteomics data sets have been produced. We have developed Listeriomics, an interactive web platform to browse and correlate these heterogeneous sources of information. Our website will allow listeriologists and microbiologists to decipher key regulation mechanism by using a systems biology approach., As for many model organisms, the amount of Listeria omics data produced has recently increased exponentially. There are now >80 published complete Listeria genomes, around 350 different transcriptomic data sets, and 25 proteomic data sets available. The analysis of these data sets through a systems biology approach and the generation of tools for biologists to browse these various data are a challenge for bioinformaticians. We have developed a web-based platform, named Listeriomics, that integrates different tools for omics data analyses, i.e., (i) an interactive genome viewer to display gene expression arrays, tiling arrays, and sequencing data sets along with proteomics and genomics data sets; (ii) an expression and protein atlas that connects every gene, small RNA, antisense RNA, or protein with the most relevant omics data; (iii) a specific tool for exploring protein conservation through the Listeria phylogenomic tree; and (iv) a coexpression network tool for the discovery of potential new regulations. Our platform integrates all the complete Listeria species genomes, transcriptomes, and proteomes published to date. This website allows navigation among all these data sets with enriched metadata in a user-friendly format and can be used as a central database for systems biology analysis. IMPORTANCE In the last decades, Listeria has become a key model organism for the study of host-pathogen interactions, noncoding RNA regulation, and bacterial adaptation to stress. To study these mechanisms, several genomics, transcriptomics, and proteomics data sets have been produced. We have developed Listeriomics, an interactive web platform to browse and correlate these heterogeneous sources of information. Our website will allow listeriologists and microbiologists to decipher key regulation mechanism by using a systems biology approach.

Listeria monocytogenes (Lm) is a ubiquitous bacterium able to survive and thrive within the environment and readily colonizes a wide range of substrates, often as a biofilm. It is also a facultative intracellular pathogen, which actively invades diverse hosts and induces listeriosis. So far, these two complementary facets of Lm biology have been studied independently. Here we demonstrate that the major Lm virulence determinant ActA, a PrfA-regulated gene product enabling actin polymerization and thereby promoting its intracellular motility and cell-to-cell spread, is critical for bacterial aggregation and biofilm formation. We show that ActA mediates Lm aggregation via direct ActA-ActA interactions and that the ActA C-terminal region, which is not involved in actin polymerization, is essential for aggregation in vitro. In mice permissive to orally-acquired listeriosis, ActA-mediated Lm aggregation is not observed in infected tissues but occurs in the gut lumen. Strikingly, ActA-dependent aggregating bacteria exhibit an increased ability to persist within the cecum and colon lumen of mice, and are shed in the feces three order of magnitude more efficiently and for twice as long than bacteria unable to aggregate. In conclusion, this study identifies a novel function for ActA and illustrates that in addition to contributing to its dissemination within the host, ActA plays a key role in Lm persistence within the host and in transmission from the host back to the environment., Author Summary Listeria monocytogenes (Lm) is a ubiquitous bacterium that survives and thrives within the environment, and a facultative intracellular pathogen that induces listeriosis. So far, these two complementary facets of Lm biology have been studied independently. Here we identify ActA, which is a major Lm virulence determinant mediating actin-based motility, as critical for bacterial aggregation and biofilm formation. ActA promotes Lm aggregation via direct ActA-ActA interaction and ActA C-terminal region, which is not involved in actin polymerization, is essential for aggregation. Whereas ActA-mediated Lm aggregation is not observed in infected tissues, it occurs in the gut lumen. Strikingly, ActA-dependent aggregating bacteria exhibit an increased ability to persist within the gut lumen, and are shed in the feces three order of magnitude more and for twice as long than bacteria unable to aggregate. This study identifies a novel function for ActA, which plays a key role in Lm persistence within the host and transmission.