A Bacterial Effector Reveals the V-ATPase-ATG16L1 Axis that Initiates Xenophagy.

Antibacterial autophagy (xenophagy) is an important host defense, but how it is initiated is unclear. Here, we performed a bacterial transposon screen and identified a T3SS effector SopF that potently blocked Salmonella autophagy. SopF was a general xenophagy inhibitor without affecting canonical autophagy. S. Typhimurium ΔsopF resembled S. flexneri ΔvirAΔicsB with the majority of intracellular bacteria targeted by autophagy, permitting a CRISPR screen that identified host V-ATPase as an essential factor. Upon bacteria-caused vacuolar damage, the V-ATPase recruited ATG16L1 onto bacteria-containing vacuole, which was blocked by SopF. Mammalian ATG16L1 bears a WD40 domain required for interacting with the V-ATPase. Inhibiting autophagy by SopF promoted S. Typhimurium proliferation in vivo. SopF targeted Gln124 of ATP6V0C in the V-ATPase for ADP-ribosylation. Mutation of Gln124 also blocked xenophagy, but not canonical autophagy. Thus, the discovery of SopF reveals the V-ATPase-ATG16L1 axis that critically mediates autophagic recognition of intracellular pathogen.

Mitochondrial NAD+ Controls Nuclear ARTD1-Induced ADP-Ribosylation.

In addition to its role as an electron transporter, mitochondrial nicotinamide adenine dinucleotide (NAD+) is an important co-factor for enzymatic reactions, including ADP-ribosylation. Although mitochondria harbor the most intra-cellular NAD+, mitochondrial ADP-ribosylation remains poorly understood. Here we provide evidence for mitochondrial ADP-ribosylation, which was identified using various methodologies including immunofluorescence, western blot, and mass spectrometry. We show that mitochondrial ADP-ribosylation reversibly increases in response to respiratory chain inhibition. Conversely, H2O2-induced oxidative stress reciprocally induces nuclear and reduces mitochondrial ADP-ribosylation. Elevated mitochondrial ADP-ribosylation, in turn, dampens H2O2-triggered nuclear ADP-ribosylation and increases MMS-induced ARTD1 chromatin retention. Interestingly, co-treatment of cells with the mitochondrial uncoupler FCCP decreases PARP inhibitor efficacy. Together, our results suggest that mitochondrial ADP-ribosylation is a dynamic cellular process that impacts nuclear ADP-ribosylation and provide evidence for a NAD+-mediated mitochondrial-nuclear crosstalk.

PARP7-mediated ADP-ribosylation of FRA1 promotes cancer cell growth by repressing IRF1- and IRF3-dependent apoptosis.

PARP7 was reported to promote tumor growth in a cell-autonomous manner and by repressing the antitumor immune response. Nevertheless, the molecular mechanism of how PARP7-mediated ADP-ribosylation exerts these effects in cancer cells remains elusive. Here, we identified PARP7 as a nuclear and cysteine-specific mono-ADP-ribosyltransferase that modifies targets critical for regulating transcription, including the AP-1 transcription factor FRA1. Loss of FRA1 ADP-ribosylation via PARP7 inhibition by RBN-2397 or mutation of the ADP-ribosylation site C97 increased FRA1 degradation by the proteasome via PSMC3. The reduction in FRA1 protein levels promoted IRF1- and IRF3-dependent cytokine as well as proapoptotic gene expression, culminating in CASP8-mediated apoptosis. Furthermore, high PARP7 expression was indicative of the PARP7 inhibitor response in FRA1-positive lung and breast cancer cells. Collectively, our findings highlight the connected roles of PARP7 and FRA1 and emphasize the clinical potential of PARP7 inhibitors for FRA1-driven cancers.

Persistent anthrax as a major driver of wildlife mortality in a tropical rainforest.

Anthrax is a globally important animal disease and zoonosis. Despite this, our current knowledge of anthrax ecology is largely limited to arid ecosystems, where outbreaks are most commonly reported. Here we show that the dynamics of an anthrax-causing agent, Bacillus cereus biovar anthracis, in a tropical rainforest have severe consequences for local wildlife communities. Using data and samples collected over three decades, we show that rainforest anthrax is a persistent and widespread cause of death for a broad range of mammalian hosts. We predict that this pathogen will accelerate the decline and possibly result in the extirpation of local chimpanzee (Pan troglodytes verus) populations. We present the epidemiology of a cryptic pathogen and show that its presence has important implications for conservation.

From Coffee Waste to Active Ingredient for Cosmetic Applications.

Coffee silverskin (CS) is the thin epidermis covering and protecting the coffee bean and it represents the main by-product of the coffee roasting process. CS has recently gained attention due to its high content in bioactive molecules and the growing interest in valuable reutilization of waste products. Drawing inspiration from its biological function, here its potential in cosmetic applications was investigated. CS was recovered from one of the largest coffee roasters located in Switzerland and processed through supercritical CO2 extraction, thereby generating coffee silverskin extract. Chemical profiling of this extract revealed the presence of potent molecules, among which cafestol and kahweol fatty acid esters, as well as acylglycerols, ß-sitosterol and caffeine. The CS extract was then dissolved in organic shea butter, yielding the cosmetic active ingredient SLVR'Coffee™. In vitro gene expression studies performed on keratinocytes showed an upregulation of genes involved in oxidative stress responses and skin-barrier functionality upon treatment with the coffee silverskin extract. In vivo, our active protected the skin against Sodium Lauryl Sulfate (SLS)-induced irritation and accelerated its recovery. Furthermore, this active extract improved measured as well as perceived skin hydration in female volunteers, making it an innovative, bioinspired ingredient that comforts the skin and benefits the environment.

Evidence against Zika virus infection of pets and peri-domestic animals in Latin America and Africa.

Decades after its discovery in East Africa, Zika virus (ZIKV) emerged in Brazil in 2013 and infected millions of people during intense urban transmission. Whether vertebrates other than humans are involved in ZIKV transmission cycles remained unclear. Here, we investigate the role of different animals as ZIKV reservoirs by testing 1723 sera of pets, peri-domestic animals and African non-human primates (NHP) sampled during 2013-2018 in Brazil and 2006-2016 in Côte d'Ivoire. Exhaustive neutralization testing substantiated co-circulation of multiple flaviviruses and failed to confirm ZIKV infection in pets or peri-domestic animals in Côte d'Ivoire (n=259) and Brazil (n=1416). In contrast, ZIKV seroprevalence was 22.2% (2/9, 95% CI, 2.8-60.1) in West African chimpanzees (Pan troglodytes verus) and 11.1% (1/9, 95% CI, 0.3-48.3) in king colobus (Colobus polycomos). Our results indicate that while NHP may represent ZIKV reservoirs in Africa, pets or peri-domestic animals likely do not play a role in ZIKV transmission cycles.

Establishment of a Mass-Spectrometry-Based Method for the Identification of the In Vivo Whole Blood and Plasma ADP-Ribosylomes.

Blood and plasma proteins are heavily investigated as biomarkers for different diseases. However, the post-translational modification states of these proteins are rarely analyzed since blood contains many enzymes that rapidly remove these modifications after sampling. In contrast to the well-described role of protein ADP-ribosylation in cells and organs, its role in blood remains mostly uncharacterized. Here, we discovered that plasma phosphodiesterases and/or ADP-ribosylhydrolases rapidly demodify in vitro ADP-ribosylated proteins. Thus, to identify the in vivo whole blood and plasma ADP-ribosylomes, we established a mass-spectrometry-based workflow that was applied to blood samples collected from LPS-treated pigs (Sus scrofa domesticus), which serves as a model for human systemic inflammatory response syndrome. These analyses identified 60 ADP-ribosylated proteins, 17 of which were ADP-ribosylated plasma proteins. This new protocol provides an important step forward for the rapidly developing field of ADP-ribosylation and defines the blood and plasma ADP-ribosylomes under both healthy and disease conditions.

Research Trends and Future Perspectives in Marine Biomimicking Robotics.

Mechatronic and soft robotics are taking inspiration from the animal kingdom to create new high-performance robots. Here, we focused on marine biomimetic research and used innovative bibliographic statistics tools, to highlight established and emerging knowledge domains. A total of 6980 scientific publications retrieved from the Scopus database (1950-2020), evidencing a sharp research increase in 2003-2004. Clustering analysis of countries collaborations showed two major Asian-North America and European clusters. Three significant areas appeared: (i) energy provision, whose advancement mainly relies on microbial fuel cells, (ii) biomaterials for not yet fully operational soft-robotic solutions; and finally (iii), design and control, chiefly oriented to locomotor designs. In this scenario, marine biomimicking robotics still lacks solutions for the long-lasting energy provision, which presently hinders operation autonomy. In the research environment, identifying natural processes by which living organisms obtain energy is thus urgent to sustain energy-demanding tasks while, at the same time, the natural designs must increasingly inform to optimize energy consumption.

Comprehensive ADP-ribosylome analysis identifies tyrosine as an ADP-ribose acceptor site.

Despite recent mass spectrometry (MS)-based breakthroughs, comprehensive ADP-ribose (ADPr)-acceptor amino acid identification and ADPr-site localization remain challenging. Here, we report the establishment of an unbiased, multistep ADP-ribosylome data analysis workflow that led to the identification of tyrosine as a novel ARTD1/PARP1-dependent in vivo ADPr-acceptor amino acid. MS analyses of in vitro ADP-ribosylated proteins confirmed tyrosine as an ADPr-acceptor amino acid in RPS3A (Y155) and HPF1 (Y238) and demonstrated that trans-modification of RPS3A is dependent on HPF1. We provide an ADPr-site Localization Spectra Database (ADPr-LSD), which contains 288 high-quality ADPr-modified peptide spectra, to serve as ADPr spectral references for correct ADPr-site localizations.

Detection of possible spillover of a novel hantavirus in a Natal mastomys from Guinea.

To date, only two rodent-borne hantaviruses have been detected in sub-Saharan Africa. Here, we report the detection of a yet unknown hantavirus in a Natal mastomys (Mastomys natalensis) in Méliandou, Guinea, in 2014. The phylogenetic placement of this virus suggests that it might represent a cross-order spillover event from an unknown bat or eulipotyphlan host.

Absence of frequent herpesvirus transmission in a nonhuman primate predator-prey system in the wild.

Emergence of viruses into the human population by transmission from nonhuman primates (NHPs) represents a serious potential threat to human health that is primarily associated with the increased bushmeat trade. Transmission of RNA viruses across primate species appears to be relatively frequent. In contrast, DNA viruses appear to be largely host specific, suggesting low transmission potential. Herein, we use a primate predator-prey system to study the risk of herpesvirus transmission between different primate species in the wild. The system was comprised of western chimpanzees (Pan troglodytes verus) and their primary (western red colobus, Piliocolobus badius badius) and secondary (black-and-white colobus, Colobus polykomos) prey monkey species. NHP species were frequently observed to be coinfected with multiple beta- and gammaherpesviruses (including new cytomegalo- and rhadinoviruses). However, despite frequent exposure of chimpanzees to blood, organs, and bones of their herpesvirus-infected monkey prey, there was no evidence for cross-species herpesvirus transmission. These findings suggest that interspecies transmission of NHP beta- and gammaherpesviruses is, at most, a rare event in the wild.

Gas-Phase Fragmentation of ADP-Ribosylated Peptides: Arginine-Specific Side-Chain Losses and Their Implication in Database Searches.

ADP-ribosylation is a reversible post-translational modification of proteins that has been linked to many biological processes. The identification of ADP-ribosylated proteins and particularly of their acceptor amino acids remains a major challenge. The attachment sites of the modification are difficult to localize by mass spectrometry (MS) because of the labile nature of the linkage and the complex fragmentation pattern of the ADP-ribose in MS/MS experiments. In this study we performed a comprehensive analysis of higher-energy collisional dissociation (HCD) spectra acquired from ADP-ribosylated peptides which were modified on arginine, serine, glutamic acid, aspartic acid, tyrosine, or lysine residues. In addition to the fragmentation of the peptide backbone, various cleavages of the ADP-ribosylated amino acid side chains were investigated. We focused on gas-phase fragmentations that were specific either to ADP-ribosylated arginine or to ADP-ribosylated serine and other O-linked ADP-ribosylations. The O-glycosidic linkage between ADP-ribose and serine, glutamic acid, or aspartic acid was the major cleavage site, making localization of these modification sites difficult. In contrast, the bond between ADP-ribose and arginine was relatively stable. The main cleavage site was the inner bond of the guanidine group, which resulted in the formation of ADP-ribosylated carbodiimide and of ornithine in place of modified arginine. Taking peptide fragment ions resulting from this specific cleavage into account, a considerably larger number of peptides containing ADP-ribosylated arginine were identified in database searches. Furthermore, the presence of diagnostic ions and of losses of fragments from peptide ions allowed us, in most cases, to distinguish between ADP-ribosylated arginine and serine residues.

Implementation of Automated Blood Culture With Quality Assurance in a Resource-Limited Setting.

Background: Blood cultures (BC) have a high clinical relevance and are a priority specimen for surveillance of antimicrobial resistance. Manual BC are still most frequently used in resource-limited settings. Data on automated BC performance in Africa are scarce. We implemented automated BC at a surveillance site of the African Network for improved Diagnostics, Epidemiology and Management of Common Infectious Agents (ANDEMIA). Methods: Between June 2017 and January 2018, pairs of automated BC (BacT/ALERT®FA Plus) and manual BC (brain-heart infusion broth) were compared at a University hospital in Bouaké, Côte d'Ivoire. BC were inoculated each with a target blood volume of 10 ml from the same venipuncture. Automated BC were incubated for up to 5 days, manual BC for up to 10 days. Terminal subcultures were performed for manual BC only. The two systems were compared regarding yield, contamination, and turnaround time. For quality assurance, isolates were retested in a German routine microbiological laboratory. Results: BC sampling was increased from on average 24 BC to 63 BC per month. A total of 337 matched pairs of BC were included. Automated BC was positive in 36.5%, manual BC in 24.0% (p-value < 0.01), proportion of contamination was 47.9 and 43.8%, respectively (p-value = 1.0). Turnaround time of positive BC was shortened by 2.5 days with automated compared to manual BC (p < 0.01). Most common detected pathogens in both systems were Klebsiella spp. (26.0%) and Staphylococcus aureus (18.2%). Most contaminants were members of the skin flora. Retesting of 162 isolates was concordant in 79.6% on family level. Conclusions: Implementing automated BC in a resource-limited setting is possible and improves microbiological diagnostic performance. Automated BC increased yield and shortened turnaround times. Regular training and mentorship of clinicians has to be intensified to increase number and quality of BC. Pre-analytical training to improve diagnostic stewardship is essential when implementing a new microbiological method. Retesting highlighted that manual identification and antimicrobial susceptibility testing can be of good quality and sustainable. The implementation of automated tools should be decided individually according to economic considerations, number of samples, stable supply chain of consumables, and technical sustainability.

Engineering Af1521 improves ADP-ribose binding and identification of ADP-ribosylated proteins.

Protein ADP-ribosylation is a reversible post-translational modification that regulates important cellular functions. The identification of modified proteins has proven challenging and has mainly been achieved via enrichment methodologies. Random mutagenesis was used here to develop an engineered Af1521 ADP-ribose binding macro domain protein with 1000-fold increased affinity towards ADP-ribose. The crystal structure reveals that two point mutations K35E and Y145R form a salt bridge within the ADP-ribose binding domain. This forces the proximal ribose to rotate within the binding pocket and, as a consequence, improves engineered Af1521 ADPr-binding affinity. Its use in our proteomic ADP-ribosylome workflow increases the ADP-ribosylated protein identification rates and yields greater ADP-ribosylome coverage. Furthermore, generation of an engineered Af1521 Fc fusion protein confirms the improved detection of cellular ADP-ribosylation by immunoblot and immunofluorescence. Thus, this engineered isoform of Af1521 can also serve as a valuable tool for the analysis of cellular ADP-ribosylation under in vivo conditions.

Author Correction: Evidence for Human Streptococcus pneumoniae in wild and captive chimpanzees: A potential threat to wild populations.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

PARP1 Hinders Histone H2B Occupancy at the NFATc1 Promoter to Restrain Osteoclast Differentiation.

Induction of nuclear factor of activated T cell cytoplasmic 1 (NFATc1) by macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL) is essential for macrophage differentiation into osteoclasts (OCs), but the underlying mechanisms remain unclear. The ability of poly(ADP-ribose) polymerase 1 (PARP1) to poly-ADP-ribosylate NFATc1 in T cells prompted us to investigate the PARP1 and NFATc1 interaction during osteoclastogenesis. However, extensive studies failed to directly link PARP1 to NFATc1. A combination of transcriptomics and proteomics studies was then used to identify PARP1 targets under these conditions. These unbiased approaches in conjunction with site-directed mutagenesis studies revealed that PARP1 inhibited NFATc1 expression and OC formation by ADP-ribosylating histone H2B at serine 7 and decreasing the occupancy of this histone variant at the NFATc1 promoter. The anti-osteoclastogenic function of PARP1 was confirmed in vivo in several mouse models of PARP1 loss-of-function or gain-of-function, including a novel model in which PARP1 was conditionally ablated in myeloid cells. Thus, PARP1 ADP-ribosylates H2B to negatively regulate NFATc1 expression and OC differentiation. © 2019 American Society for Bone and Mineral Research.

Geographically structured genomic diversity of non-human primate-infecting Treponema pallidum subsp. pertenue.

Many non-human primate species in sub-Saharan Africa are infected with Treponema pallidum subsp. pertenue, the bacterium causing yaws in humans. In humans, yaws is often characterized by lesions of the extremities and face, while T. pallidum subsp. pallidum causes venereal syphilis and is typically characterized by primary lesions on the genital, anal or oral mucosae. It remains unclear whether other T. pallidum subspecies found in humans also occur in non-human primates and how the genomic diversity of non-human primate T. pallidum subsp. pertenue lineages is distributed across hosts and space. We observed orofacial and genital lesions in sooty mangabeys (Cercocebus atys) in Taï National Park, Côte d'Ivoire and collected swabs and biopsies from symptomatic animals. We also collected non-human primate bones from 8 species in Taï National Park and 16 species from 11 other sites across sub-Saharan Africa. Samples were screened for T. pallidum DNA using polymerase chain reactions (PCRs) and we used in-solution hybridization capture to sequence T. pallidum genomes. We generated three nearly complete T. pallidum genomes from biopsies and swabs and detected treponemal DNA in bones of six non-human primate species in five countries, allowing us to reconstruct three partial genomes. Phylogenomic analyses revealed that both orofacial and genital lesions in sooty mangabeys from Taï National Park were caused by T. pallidum subsp. pertenue. We showed that T. pallidum subsp. pertenue has infected non-human primates in Taï National Park for at least 28 years and has been present in two non-human primate species that had not been described as T. pallidum subsp. pertenue hosts in this ecosystem, western chimpanzees (Pan troglodytes verus) and western red colobus (Piliocolobus badius), complementing clinical evidence that started accumulating in Taï National Park in 2014. More broadly, simian T. pallidum subsp. pertenue strains did not form monophyletic clades based on host species or the symptoms caused, but rather clustered based on geography. Geographical clustering of T. pallidum subsp. pertenue genomes might be compatible with cross-species transmission of T. pallidum subsp. pertenue within ecosystems or environmental exposure, leading to the acquisition of closely related strains. Finally, we found no evidence for mutations that confer antimicrobial resistance.

Outcomes of contemporary interventional therapy of ST elevation infarction in patients older than 75 years.

BACKGROUND: Data on contemporary real-world outcomes of interventional revascularization in patients > or = 75 y of age with ST elevation infarction (STEMI) are limited. METHODS: We analyzed all 504 consecutive patients who underwent angiography for acute STEMI between 1999 and 2005 at our center, and followed them up over one year. Outcomes in patients > or = 75 y of age were compared with younger patients. RESULTS: Patients > or = 75 y of age (n = 115) were majority females (55% versus 21%, p < 0.001), more cases of diabetes (42% versus 27%, p = 0.004), hypertension (78% versus 65%, p = 0.03) and a history of coronary events (25% versus 15%, p = 0.002). Younger patients were more often smokers (63% versus 30%, p < 0.001). After coronary angiography patients > or = 75 y of age underwent less frequent intervention (PCI; 84% versus 93%, p = 0.01). However, if PCI was performed, technical success rates were similar to younger patients (84% versus 86%). The 30-d mortality was 13% versus 6.4% (p = 0.03), but after successful PCI, the 30-d mortality rate was not significantly higher in old patients (7.4% versus 3.9%, p = 0.23). Bleeding complications were very low in both age groups if the radial access route was chosen. Multivariate predictors of 30-d mortality were cardiogenic shock/survived cardiac arrest, ejection fraction < 30%, conservative treatment or unsuccessful PCI (OR 3.01), renal insufficiency, diabetes, and age. One-y mortality was higher in the elderly (24.3% versus 9.9%, p < 0.001). Among 30-d-survivors, only multivessel disease and age were multivariate predictors of 1-y mortality. CONCLUSION: Patients > or = 75 y of age benefit from PCI in STEMI, and failed or unattempted PCI worsens prognosis in the old as well as in younger patients.

Clinically Relevant ESBL-Producing K. pneumoniae ST307 and E. coli ST38 in an Urban West African Rat Population.

High-risk ESBL-producing Enterobacteriaceae (ESBL-E) have been described in wild birds and rodents worldwide. Rats are of special interest not only due to their indicator role for environmental pollution with multi-resistant bacteria but also as possible infection source. Data on the presence of high-risk ESBL-E in urban wildlife from Africa remain scarce, however. Twenty-nine animals from three different rat (Rattus) species were captured in the city of Conakry (Guinea, West Africa) in 2015. Rectal swabs were analyzed for ESBL-E using selective media. Species typing and phenotypic antimicrobial resistance analysis to broad-spectrum beta-lactams and other classes of antimicrobials was performed for Enterobacteriaceae-like isolates using the VITEK®2 system (BioMérieux, Germany). Confirmed ESBL-producing E. coli and K. pneumoniae were whole-genome sequenced and resistance genes, phylogenetic background and genes related to bacterial fitness and virulence were analyzed. In total, six of twenty-nine rats (20%) carried ESBL-E (K. pneumoniae and E. coli). All ESBL-producers were multi-drug resistant with blaCTX-M-15 as the dominating ESBL-type. Interestingly, ESBL-associated clonal lineages E. coli ST38 and K. pneumoniae ST307 were found. The ESBL-plasmid in K. pneumoniae ST307 revealed high sequence similarities to pKPN3-307_TypeC, a >200 kbp IncFII plasmid originating from a human clinical ST307 isolate. This was in contrast to the core genome: the rat isolate was distantly related to the human clinical ST307 isolate (27 SNPs/Mbp). In addition, we identified π-fimbrial, capsule 2, and glycogen synthesis clusters in the rodent ST307 isolate, whose involvement in the adaptation to survival outside the host and in human urinary tracts has been suggested. Our results demonstrate the presence of clinically relevant, ESBL-producing K. pneumoniae ST307 and E. coli ST38 clonal lineages in an urban West African rat population. The human community is likely the initial source of ESBL-E however, rats might function as infection source and transmission hub, accelerated by frequent interactions at a human-wildlife interface.