Purification, characterization, and cloning of a novel pro-inflammatory secreted protein from Staphylococcus aureus.

IMPORTANCE: Staphylococcus aureus causes a myriad of human diseases, ranging from relatively mild soft tissue infections to highly fatal pneumonia, sepsis, and toxic shock syndrome. The organisms primarily cause diseases across mucosal and skin barriers. In order to facilitate penetration of barriers, S. aureus causes harmful inflammation by inducing chemokines from epithelial cells. We report the cloning and characterization of a novel secreted S. aureus protein that induces chemokine production from epithelial cells as its major demonstrable function. This secreted protein possibly helps S. aureus and its secreted proteins to penetrate host barriers.

Glycerol monolaurate inhibits Francisella novicida growth and is produced intracellularly in an ISG15-dependent manner.

Glycerol Monolaurate (GML) is a naturally occurring fatty acid monoester with antimicrobial properties. Francisella tularensis is an agent of bioterrorism known for its unique lipopolysaccharide structure and low immunogenicity. Here we assessed whether exogenous GML would inhibit the growth of Francisella novicida . GML potently impeded Francisella growth and survival in vitro . To appraise the metabolic response to infection, we used GC-MS to survey the metabolome, and surprisingly, observed intracellular GML production following Francisella infection. Notably, the ubiquitin-like protein ISG15 was necessary for increased GML levels induced by bacterial infection, and enhanced ISG15 conjugation correlated with GML levels following serum starvation.

Contrasting roles of MERS-CoV and SARS-CoV-2 internal proteins in pathogenesis in mice.

Betacoronaviruses encode an internal (I) gene via an alternative reading frame within the nucleocapsid gene, called ORF8b for Middle-East respiratory syndrome coronavirus (MERS-CoV) and ORF9b for severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2. Previous reports suggested that proteins 8b and 9b are involved in evading multiple innate immune signaling pathways. However, their roles in mediating pathogenesis in infected animals have not been determined. In this study, we abrogated the expression of protein 8b in MERS-CoV and protein 9b in SARS-CoV-2. Using mouse models of MERS-CoV and SARS-CoV-2 infection, we found that MERS-CoV lacking protein 8b expression was more virulent, while SARS-CoV-2 lacking protein 9b expression was attenuated compared with the respective wild-type viruses. Upon further analysis, we detected increased levels of type I interferon and enhanced infiltration of immune cells to the lungs of mice infected with MERS-CoV lacking protein 8b expression. These data suggest that the I protein of MERS-CoV plays a role in limiting pathogenesis while that of SARS-CoV-2 enhances disease severity. IMPORTANCE The function of betacoronavirus internal protein has been relatively understudied. The earliest report on the internal protein of mouse hepatitis virus suggested that the internal protein is a structural protein without significant functions in virus replication and virulence. However, the internal proteins of severe acute respiratory syndrome coronavirus (SARS-CoV), Middle-East respiratory syndrome coronavirus, and SARS-CoV-2 have been shown to evade immune responses. Despite the reported functions of the internal protein in these highly pathogenic human coronaviruses, its role in mediating pathogenesis in experimentally infected animals has not been characterized. Our data indicated that despite the similar genomic location and expression strategy of these internal proteins, their effects on virulence are vastly different and virus specific, highlighting the complexity between host-virus interaction and disease outcome.

ISG15 Is Required for the Dissemination of Vaccinia Virus Extracellular Virions.

Viruses have developed many different strategies to counteract immune responses, and Vaccinia 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 of Isg15-/- 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, compared to Isg15+/+ cells. Transmission electron microscopy revealed the accumulation of intracellular virus particles and a decrease in extracellular virus particles in the absence of interferon-stimulated gene 15 (ISG15), a finding 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. Lastly, 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, which are essential for EV formation. Altogether, our results indicate that ISG15 is an important host factor in the regulation of VACV dissemination. IMPORTANCE Viral 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.

Characterization of the Tau Interactome in Human Brain Reveals Isoform-Dependent Interaction with 14-3-3 Family Proteins.

Despite exhibiting tau phosphorylation similar to Alzheimer's disease (AD), the human fetal brain is remarkably resilient to tau aggregation and toxicity. To identify potential mechanisms for this resilience, we used co-immunoprecipitation (co-IP) with mass spectrometry to characterize the tau interactome in human fetal, adult, and Alzheimer's disease brains. We found significant differences between the tau interactome in fetal and AD brain tissue, with little difference between adult and AD, although these findings are limited by the low throughput and small sample size of these experiments. Differentially interacting proteins were enriched for 14-3-3 domains, and we found that the 14-3-3-ß, η, and γ isoforms interacted with phosphorylated tau in Alzheimer's disease but not the fetal brain. Since long isoform (4R) tau is only seen in the adult brain and this is one of the major differences between fetal and AD tau, we tested the ability of our strongest hit (14-3-3-ß) to interact with 3R and 4R tau using co-immunoprecipitation, mass photometry, and nuclear magnetic resonance (NMR). We found that 14-3-3-ß interacts preferentially with phosphorylated 4R tau, forming a complex consisting of two 14-3-3-ß molecules to one tau. By NMR, we mapped 14-3-3 binding regions on tau that span the second microtubule binding repeat, which is unique to 4R tau. Our findings suggest that there are isoform-driven differences between the phospho-tau interactome in fetal and Alzheimer's disease brain, including differences in interaction with the critical 14-3-3 family of protein chaperones, which may explain, in part, the resilience of fetal brain to tau toxicity.

Identification of the Extracytoplasmic Function σ Factor σP Regulon in Bacillus thuringiensis.

Bacillus thuringiensis and other members of the Bacillus cereus family are resistant to many ß-lactams. Resistance is dependent upon the extracytoplasmic function sigma factor σP. We used label-free quantitative proteomics to identify proteins whose expression was dependent upon σP. We compared the protein profiles of strains which either lacked σP or overexpressed σP. We identified 8 members of the σP regulon which included four ß-lactamases as well as three penicillin-binding proteins (PBPs). Using transcriptional reporters, we confirmed that these genes are induced by ß-lactams in a σP-dependent manner. These genes were deleted individually or in various combinations to determine their role in resistance to a subset of ß-lactams, including ampicillin, methicillin, cephalexin, and cephalothin. We found that different combinations of ß-lactamases and PBPs are involved in resistance to different ß-lactams. Our data show that B. thuringiensis utilizes a suite of enzymes to protect itself from ß-lactam antibiotics. IMPORTANCE Antimicrobial resistance is major concern for public health. ß-Lactams remain an important treatment option for many diseases. However, the spread of ß-lactam resistance continues to rise. Many pathogens acquire antibiotic resistance from environmental bacteria. Thus, understanding ß-lactam resistance in environmental strains may provide insights into additional mechanisms of antibiotic resistance. Here, we describe how a single regulatory system, σP, in B. thuringiensis controls expression of multiple genes involved in resistance to ß-lactams. Our findings indicate that some of these genes are partially redundant. Our data also suggest that the large number of genes controlled by σP results in increased resistance to a wider range of ß-lactam classes than any single gene could provide.

Listeriolysin S: A bacteriocin from Listeria monocytogenes that induces membrane permeabilization in a contact-dependent manner.

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.

Ring finger protein 213 assembles into a sensor for ISGylated proteins with antimicrobial activity.

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.

Identification of the MuRF1 Skeletal Muscle Ubiquitylome Through Quantitative Proteomics.

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.

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.

The in vivo ISGylome links ISG15 to metabolic pathways and autophagy upon Listeria monocytogenes infection.

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.

Author Correction: N-terminomics identifies Prli42 as a membrane miniprotein conserved in Firmicutes and critical for stressosome activation in Listeria monocytogenes.

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 ).

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.

Listeria monocytogenes: towards a complete picture of its physiology and pathogenesis.

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.

N-terminomics identifies Prli42 as a membrane miniprotein conserved in Firmicutes and critical for stressosome activation in Listeria monocytogenes.

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.

Cytosolic Innate Immune Sensing and Signaling upon Infection.

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.

ISG15 counteracts Listeria monocytogenes infection.

ISG15 is an interferon-stimulated, linear di-ubiquitin-like protein, with anti-viral activity. The role of ISG15 during bacterial infection remains elusive. We show that ISG15 expression in nonphagocytic cells is dramatically induced upon Listeria infection. Surprisingly this induction can be type I interferon independent and depends on the cytosolic surveillance pathway, which senses bacterial DNA and signals through STING, TBK1, IRF3 and IRF7. Most importantly, we observed that ISG15 expression restricts Listeria infection in vitro and in vivo. We made use of stable isotope labeling in tissue culture (SILAC) to identify ISGylated proteins that could be responsible for the protective effect. Strikingly, infection or overexpression of ISG15 leads to ISGylation of ER and Golgi proteins, which correlates with increased secretion of cytokines known to counteract infection. Together, our data reveal a previously uncharacterized ISG15-dependent restriction of Listeria infection, reinforcing the view that ISG15 is a key component of the innate immune response.

Human intracellular ISG15 prevents interferon-α/ß over-amplification and auto-inflammation.

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

Mapping of SUMO sites and analysis of SUMOylation changes induced by external stimuli.

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.

The New Microbiology: a conference at the Institut de France.

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 Académie 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 "Académie des sciences", David Holden and Richard Moxon from the "Royal Society", and Jörg Hacker and Jürgen 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-François Bach, Secrétaire Perpétuel of the Académie des sciences.