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Bmf contributes to the onset of anoikis by translocating from cytoskeleton to mitochondria when cells lose attachment to the extracellular matrix. However, the structural details of Bmf cytoskeleton tethering and the control of Bmf release upon loss of anchorage remained unknown. Here we showed that cell detachment induced rapid and sustained activation of p38 MAPK in mammary epithelial cell lines. Inhibition of p38 signaling or Bmf knockdown rescued anoikis. Activated p38 MAPK could directly phosphorylate Bmf at multiple sites including a non-proline-directed site threonine 72 (T72). Crystallographic studies revealed that Bmf T72 directly participated in DLC2 binding and its phosphorylation would block Bmf/DLC2 interaction through steric hindrance. Finally, we showed that phosphomimetic mutation of T72 enhanced Bmf apoptotic activity in vitro and in a knock-in mouse model. This work unraveled a novel regulatory mechanism of Bmf activity during anoikis and provided structural basis for Bmf cytoskeleton tethering and dissociation.

Photocleavable molecules can enable the light-dependent modulation of biomolecular activities with high spatiotemporal precision. We have previously reported a photocleavable protein (PhoCl1) that, uniquely, is a fully genetically encoded photocleavable molecule that can be introduced into cells in the form of its corresponding gene to enable optogenetic control of biomolecular activities. However, the first generation PhoCl1 exhibited a relatively slow rate of dissociation, potentially limiting its utility. Here, we report the X-ray crystal structures of the PhoCl1 green state, red state, and cleaved empty barrel. Molecular dynamics (MD) simulations were performed to provide insight into the precise dissociation mechanism. Using structure-guided engineering and directed evolution, we have developed PhoCl2c with higher contrast ratio and PhoCl2f with faster dissociation. We characterized the performance of these new variants as purified proteins and in cultured cells. Our results demonstrate that PhoCl2 variants exhibit faster and more efficient dissociation, which should enable improved optogenetic manipulations of protein localization and protein–protein interactions in living cells., Photocleavable proteins can enable the light-dependent modulation of biomolecular activities with high spatiotemporal precision.

Motivated by the growing recognition of citrate as a central metabolite in a variety of biological processes associated with healthy and diseased cellular states, we have developed a series of high-performance genetically encoded citrate biosensors suitable for imaging of citrate concentrations in mammalian cells. The design of these biosensors was guided by structural studies of the citrate-responsive sensor histidine kinase and took advantage of the same conformational changes proposed to propagate from the binding domain to the catalytic domain. Following extensive engineering based on a combination of structure guided mutagenesis and directed evolution, we produced an inverse-response biosensor (ΔF/Fmin ≈ 18) designated Citroff1 and a direct-response biosensor (ΔF/Fmin ≈ 9) designated Citron1. We report the X-ray crystal structure of Citron1 and demonstrate the utility of both biosensors for qualitative and quantitative imaging of steady-state and pharmacologically perturbed citrate concentrations in live cells., Development of high-performance genetically encoded biosensors for citrate, based on engineered fluorescent proteins, enables visualization and quantification of intracellular citrate concentrations.

In Acinetobacter baumannii, resistance-nodulation-cell division (RND)-type efflux is a resistance mechanism of great importance since it contributes to reduced susceptibility to multiple antimicrobial compounds. Some mutations within the genes encoding the two-component regulatory system AdeRS appear to play a major role in increased expression of the RND efflux pump AdeABC and, consequently, in reduced antimicrobial susceptibility, as they are commonly observed in multidrug-resistant (MDR) A. baumannii. In the present study, the impact of frequently identified amino acid substitutions, namely, D21V and D26N in AdeR and T156M in AdeS, on adeB expression, efflux activity, and antimicrobial susceptibility was investigated. Reverse transcription-quantitative PCR (qRT-PCR) studies revealed significantly increased adeB expression caused by D26N (AdeR) and T156M (AdeS). In addition, accumulation assays have shown that these mutations induce increased efflux activity. Subsequently, antimicrobial susceptibility testing via agar dilution and broth microdilution confirmed the importance of these substitutions for the MDR phenotype, as the MICs for various antimicrobials of different classes were increased. In contrast, the amino acid substitution D21V in AdeR did not lead to increased adeB expression and did not reduce antimicrobial susceptibility. This study demonstrates the impact of the D26N (AdeR) and T156M (AdeS) amino acid substitutions, highlighting that these regulators represent promising targets for interfering with efflux activity to restore antimicrobial susceptibility. IMPORTANCE The active efflux of antimicrobials by bacteria can lead to antimicrobial resistance and persistence and can affect multiple different classes of antimicrobials. Efflux pumps are tightly regulated, and their overexpression can be mediated by changes in their regulators. Identifying these changes is one step in the direction of resistance prediction, but it also opens the possibility of targeting efflux pump regulation as a strategy to overcome antimicrobial resistance. Here, we have investigated commonly found changes in the regulators of the main efflux pumps in Acinetobacter baumannii.

Potassium ions (K+) play a critical role as an essential electrolyte in all biological systems. Here we report the crystal structure-guided optimization and directed evolution of an improved genetically encoded fluorescent K+ biosensor, GINKO2. GINKO2 is highly sensitive and specific for K+ and enables in vivo detection of K+ dynamics in multiple species.

MYC drives the tumorigenesis of human cancers, including prostate cancer (PrCa), thus deubiquitinase (DUB) that maintains high level of c-Myc oncoprotein is a rational therapeutic target. Several ubiquitin-specific protease (USP) family members of DUB have been reported to deubiquitinate c-Myc, but none of them is the physiological DUB for c-Myc in PrCa. By screening all the DUBs, here we reveal that OTUD6A is exclusively amplified and overexpressed in PrCa but not in other cancers, eliciting a prostatic-specific oncogenic role through deubiquitinating and stabilizing c-Myc oncoprotein. Moreover, genetic ablation of OTUD6A efficiently represses prostatic tumorigenesis of both human PrCa cells and the Hi-Myc transgenic PrCa mice, via reversing the metabolic remodeling caused by c-Myc overexpression in PrCa. These results indicate that OTUD6A is a physiological DUB for c-Myc in PrCa setting and specifically promotes prostatic tumorigenesis through stabilizing c-Myc oncoprotein, suggesting that OTUD6A could be a unique therapeutic target for Myc-driven PrCa.

Staphylococcus aureus ArlRS is a key two-component regulatory system necessary for adhesion, biofilm formation, and virulence. The response regulator ArlR consists of a C-terminal DNA-binding effector domain and an N-terminal receiver domain that is phosphorylated by ArlS, the cognate transmembrane sensor histidine kinase. We demonstrate that the receiver domain of ArlR adopts the canonical α5β5 response regulator assembly, which dimerizes upon activation, using beryllium trifluoride as an aspartate phosphorylation mimic. Activated ArlR recognizes a 20-bp imperfect inverted repeat sequence in the ica operon, which is involved in intercellular adhesion polysaccharide production. Crystal structures of the inactive and activated forms reveal that activation induces a significant conformational change in the β4-α4 and β5-α5-connecting loops, in which the α4 and α5 helices constitute the homodimerization interface. Crystal structures of the DNA-binding ArlR effector domain indicate that it is able to dimerize via a non-canonical β1–β2 hairpin domain swapping, raising the possibility of a new mechanism for signal transduction from the receiver domain to effector domain. Taken together, the current study provides structural insights into the activation of ArlR and its recognition, adding to the diversity of response regulation mechanisms that may inspire novel antimicrobial strategies specifically targeting Staphylococcus.

International audience; The C-type lectin receptor Clec4f has been identified as a specific surface marker for Kupffer cells, although its ortholog is absent in humans and its biological function remains elusive. Here, we report the crystal structure of a truncated mouse trimeric Clec4f. The orientation between the carbohydrate-recognition domain of Clec4f and its neck region differs from other C-type lectins, resulting in an observed distance of 45 Å between the glycan-binding sites within the Clec4f trimer. Interestingly, the trimeric coiled-coil interface within its heptad neck region contains multiple polyglutamine interactions instead of the predominantly hydrophobic leucine zipper found in other C-type lectin receptors. The Clec4f trimeric structure displays unique features regarding its assembly and ligand recognition, shedding light on the evolution and diversity of the C-type lectin family.

Summary Bacterial two-component regulatory systems are ubiquitous environment-sensing signal transducers involved in pathogenesis and antibiotic resistance. The Acinetobacter baumannii two-component regulatory system AdeRS is made up of a sensor histidine kinase AdeS and a cognate response regulator AdeR, which together reduce repression of the multidrug-resistant efflux pump AdeABC. Herein we demonstrate that an N-terminal intrinsically disordered tail in AdeR is important for the upregulation of adeABC expression, although it greatly increases the susceptibility of AdeR to proteasome-mediated degradation. We also show that AdeS assembles into a hexameric state that is necessary for its full histidine kinase activity, which appears to occur via cis autophosphorylation. Taken together, this study demonstrates new structural mechanisms through which two-component systems can transduce environmental signals to impact gene expression and enlightens new potential antimicrobial approach by targeting two-component regulatory systems., Graphical abstract, Highlights • Crystal structure of AdeR dimer with traceable N-terminal intrinsically disordered region. • N-terminal intrinsically disordered region AdeR is involved in proteasome proteolysis. • Crystal structure of AdeS catalytic domain demonstrates cis autophosphorylation. • AdeS can assemble into hexamer and is crucial for its full kinase activity., Molecular Structure ; Microbiology; Protein structure aspects

To enable investigations of the emerging roles of cell-to-cell shuttling of L-lactate, we have developed an intensiometric green fluorescent genetically encoded biosensor for extracellular L-lactate. We demonstrate that this biosensor, designated eLACCO1.1, enables minimally invasive cellular resolution imaging of extracellular L-lactate in cultured mammalian cells and brain tissue.

The photocleavable protein (PhoCl) is a green-to-red photoconvertible fluorescent protein that, when illuminated with violet light, undergoes main chain cleavage followed by spontaneous dissociation of the resulting fragments. The first generation PhoCl (PhoCl1) exhibited a relative slow rate of dissociation, potentially limiting its utilities for optogenetic control of cell physiology. In this work, we report the X-ray crystal structures of the PhoCl1 green state, red state, and cleaved empty barrel. Using structure-guided engineering and directed evolution, we have developed PhoCl2c with higher contrast ratio and PhoCl2f with faster dissociation. We characterized the performance of these new variants as purified proteins and expressed in cultured cells. Our results demonstrate that PhoCl2 variants exhibit faster and more efficient dissociation, which should enable improved optogenetic manipulations of protein localization and protein-protein interactions in living cells.

Summary DUSP6 functions as an important negative feedback component of the MAPK/ERK signaling pathway. Although DUSP6 expression is tightly regulated by ERK1/2 signaling, the molecular mechanism of this regulation remains partially understood. In this work, we show that the transcriptional repressor CIC functions downstream of the ERK1/2 signaling to negatively regulate DUSP6 expression. CIC directly represses DUSP6 transcription by binding to three cis-regulatory elements (CREs) in DUSP6 promoter. p90RSK, a downstream target of ERK1/2, phosphorylates CIC at S173 and S301 sites, which creates a 14-3-3 recognition motif, resulting in 14-3-3-mediated nuclear export of CIC and derepression of DUSP6. Finally, we demonstrate that the oncogenic CIC-DUX4 fusion protein acts as a transcriptional activator of DUSP6 and its nuclear/cytoplasmic distribution remains regulated by ERK1/2 signaling. These results complete an ERK1/2/p90RSK/CIC/DUSP6 negative feedback circuit and elucidate the molecular mechanism of how RTK/MAPK signaling harnesses the transcriptional repressor activity of CIC in mammalian cells., Graphical Abstract, Highlights • CIC represses DUSP6 transcription through direct promoter binding • p90RSK phosphorylates CIC at S173 and S301 sites • S173/S301 phosphorylated CIC binds to 14-3-3 to promote its nuclear export • ERK/p90RSK signaling regulates the subcellular localization of CIC-DUX4 protein, Biochemistry; Structural Biology; Cancer

Polyhydroxyalkanoate (PHA) is a class of microbial synthesized biodegradable and biocompatible aliphatic polymer which has been developed into nanoparticles (NPs) for sustained release of hydrophobic compounds. Taking advantage of the natural PHA binding protein PhaP which could be steadily adsorbed onto PHA NPs through hydrophobic interaction, a tumor targeting system was developed in this study by presenting an epidermal growth factor receptor (EGFR)-targeting peptide (ETP) on the surface of PHA NPs, via PhaP mediated adsorption. To reveal the effects of residual emulsifiers on PhaP mediated ETP modification and optimize the tumor targeting capacity of the system, a novel emulsifier-free PHA NPs (EF-NPs) was fabricated together with other two kinds of conventional emulsifier-required PHA NPs (PVA-NPs and P68-NPs, which were prepared with poly(vinyl alcohol) (PVA) and Pluronic F68 as emulsifiers, respectively). By analyzing the surface hydrophobicity, the amount of adsorbed fusion protein, and the cellular uptake of all kinds of PHA NPs, our results demonstrated that EF-NPs with stronger surface hydrophobicity were the most proper formulation for further PhaP mediated ETP functionalization. The residual PVA and Pluronic F68 affected the modification efficiency and secondary structure of ETP-PhaP fusion protein, and finally obstructed the targeting effect of ETP-PhaP modified PVA-NPs and P68-NPs to EGFR over-expressed tumor cells. The animal experiment further confirmed the effectiveness and feasibility of in vivo application of ETP-PhaP functionalized EF-NPs, indicating that it could be served as a promising tumor targeting system with satisfactory EGFR targeting ability. This PhaP mediated bio-modification process also opens a wide way for developing various PHA-based targeting systems by presenting different tumor or other tissue-specific targeting peptides.

The master regulator CsgD switches planktonic growth to biofilm formation by activating synthesis of curli fimbriae and cellulose in Enterobacteriaceae. CsgD was classified to be the LuxR response regulatory family, while its cognate sensor histidine kinase has not been identified yet. CsgD consists of a C-terminal DNA binding domain and an N-terminal regulatory domain that provokes the upstream signal transduction to further modulate its function. We provide the crystal structure of Salmonella Typhimurium CsgD regulatory domain, which reveals an atypical β5α5 response regulatory receiver domain folding with the α2 helix representing as a disorder loop compared to the LuxR/FixJ canonical response regulator, and the structure indicated a noteworthy α5 helix similar to the non-canonical master regulator VpsT receiver domain α6. CsgD regulatory domain assembles with two dimerization interfaces mainly through α1 and α5, which has shown similarity to the c-di-GMP independent and stabilized dimerization interface of VpsT from Vibrio cholerae respectively. The potential phosphorylation site D59 is directly involved in the interaction of interfaces I and mutagenesis studies indicated that both dimerization interfaces could be crucial for CsgD activity. The structure reveals important molecular details for the dimerization assembly of CsgD and will shed new insight into its regulation mechanism.

This study investigated the emulsification properties of polysaccharides from Dioscorea opposita Thunb. Graded alcohol precipitation was used to extract Dioscorea opposita polysaccharides fractions (4 samples) in different ranges of molecular weight. Sample 3 contained more glucose and protein (80.13% and 0.34%, respectively), and molecular weight was approximately 34,790 Da, distributing narrowly. The droplet sizes and stabilities of emulsions made of gum arabic (GA) and polysaccharide samples at different concentrations and ratios were measured, specifically the emulsions of GA and medium-chain-triglycerides (MCT); polysaccharides and MCT; and polysaccharides, GA and MCT (1:1:1). The results indicated that sample 2 and 3 had emulsifying properties, and the emulsions made with sample 2, GA and MCT (1:1:1) presented the best emulsification properties. Therefore, polysaccharides of Dioscorea opposita could be utilised as a natural emulsifier that can be improved synergistically with other emulsifiers, such as gum arabic.

A major pharmacological barrier to peptide therapeutics is their susceptibility to proteolytic degradation and poor membrane permeability, which, in principle, can be overcome by nanoparticle-based delivery technologies. Proteins, by definition, are nano materials and have been clinically proven as an efficient delivery vehicle for small molecule drugs. Here we describe the design of a protein-based peptide drug carrier derived from the tetramerization domain of the chimeric oncogenic protein Bcr/Abl of chronic myeloid leukemia. A dodecameric peptide inhibitor of the p53-MDM2/MDMX interaction, termed PMI, was grafted to the N-terminal helical region of Bcr/Abl tetramer. To antagonize intracellular MDM2/MDMX for p53 activation, we extended this protein, (PMI)Bcr/Abl, by a C-terminal Arg-repeating hexapeptide to facilitate its cellular uptake. The resultant tetrameric protein (PMI)Bcr/Abl-R6 adopted an alpha-helical conformation in solution and bound to MDM2 at an affinity of 32 nM.(PMI)Bcr/Abl-R6 effectively induced apoptosis of HCT116 p53(+/+) cells in vitro in a p53-dependent manner and potently inhibited tumor growth in a nude mouse xenograft model by stabilizing p53 in vivo. Our protein-based delivery strategy thus provides a clinically viable solution to p53-inspired anticancer therapy and is likely applicable to the development of many other peptide therapeutics to target a great variety of intracellular protein-protein interactions responsible for disease initiation and progression.

Objective Acinetobacter baumannii has emerged as a problematic hospital pathogen and tigecycline is among the few remaining antibiotics retaining activity against multidrug-resistant A. baumannii. This study was aimed to elucidate the tigecycline resistance mechanisms in 28 unique clinical A. baumannii strains from nine provinces in China. Methods Whole genome sequences were obtained via Illumina HiSeq sequencing and regulatory genes of efflux pumps were analyzed. Minimal inhibitory concentrations (MICs) were determined by agar/microbroth dilution according to the guidelines recommended by Clinical and Laboratory Standards Institute (CLSI). Tigecycline susceptibility data was interpreted using breakpoints for Enterobacterales recommended by EUCAST v8.1. Results The majority of isolates belonged to the international clonal lineage IC2 (n = 27, 96.4%). Four isolates were considered tigecycline-intermediate (MIC = 2 mg/L), twenty-four isolates were tigecycline-resistant. The insertion of ISAba1 in adeS was found in six isolates and was the most prevalent insertion element (IS). In four isolates we observed an insertion of ISAba1 in adeN, and two of them had IS26 insertions. Two mutations in adeN (deletion and premature stop codon) were observed only in the MIC = 4 mg/L isolates. Other mutations in adeRS (amino acid insertion/substitutions and premature stop codons) were only detected in the MIC ≥ 8 group. The novel substitutions E219 K in adeR and A130 T in adeS were observed in five and four isolates respectively, suggesting a mutational hotspot. Conclusions This study demonstrates that changes in transcription regulators were important mechanisms in tigecycline resistance in A. baumannii. Also, we identified several chromosomal hotspots that can be used for prediction of tigecycline resistance.

Salmonella Enteritidis has developed the potential to contaminate eggs by surviving in the antimicrobial environment of the hen's egg white. This has led to a worldwide pandemic of foodborne salmonellosis infections in humans due to the consumption of contaminated eggs and egg-derived products. The molecular mechanisms of Salmonella Enteritidis egg white survival are not fully clear. Using in vivo expression technology and promoter-reporter fusions we showed that the promoter of the tolC gene, encoding the TolC outer membrane channel that is used by multidrug efflux pumps to export harmful molecules and to secrete bacterial products, is activated by egg white at the chicken body temperature. Using a Salmonella Enteritidis tolC deletion mutant we showed that TolC has an important role in egg white survival. Chromatographic separation techniques and subsequent testing of antimicrobial activities of separated egg white fractions led to the identification of ovotransferrin as the egg white antimicrobial factor which is capable of inhibiting growth of a tolC deletion strain but not the wild type strain. We provide evidence that TolC protects Salmonella Enteritidis against ovotransferrin-mediated growth inhibition in egg white.

The compound MCI-154 was previously shown to increase the calcium sensitivity of cardiac muscle contraction. Using solution NMR spectroscopy, we demonstrate that MCI-154 interacts with the calcium-sensing subunit of the cardiac troponin complex, cardiac troponin C (cTnC). Surprisingly, however, it binds only to the structural C-terminal domain of cTnC (cCTnC), and not to the regulatory N-terminal domain (cNTnC) that determines the calcium sensitivity of cardiac muscle. Physiologically, cTnC is always bound to cardiac troponin I (cTnI), so we examined its interaction with MCI-154 in the presence of two soluble constructs, cTnI1–77 and cTnI135–209, which contain all of the segments of cTnI known to interact with cTnC. Neither the cTnC-cTnI1–77 complex nor the cTnC-cTnI135–209 complex binds to MCI-154. Since residues 39–60 of cTnI are known to bind tightly to the cCTnC domain to form a structured core that is invariant throughout the cardiac cycle, we conclude that MCI-154 does not bind to cTnC when it is part of the intact cardiac troponin complex. Thus, MCI-154 likely exerts its calcium sensitizing effect by interacting with a target other than cardiac troponin., Highlights • MCI-154 is a small molecule calcium sensitizer in cardiac muscle. • The N-domain of cardiac troponin C controls calcium sensitivity in cardiac muscle. • MCI-154 binds weakly to the promiscuous C-terminal domain of troponin C. • Cardiac troponin C does not bind MCI-154 in the presence of troponin I. • MCI-154 does not exert its calcium sensitizing effect directly through troponin C.

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Objectives To investigate the mechanisms of tigecycline resistance in isogenic Acinetobacter baumannii isolate pairs as well as 65 unique clinical A. baumannii isolates obtained during the MagicBullet clinical trial from Greece, Italy and Spain. Methods A. baumannii isolates were subjected to WGS and the regulatory genes of resistance-nodulation-cell division (RND)-type efflux pumps were analysed. MICs were determined by agar dilution and the expression of RND-type efflux pumps was measured by semi-quantitative RT-PCR. Results In isolate pairs, disruption of adeS or adeN by ISs increased adeB or adeJ expression and conferred increased resistance to at least three antimicrobial classes, respectively. The insertion of ISAba1 in adeN was observed in more than 30% of tested isolates and was the most prevalent IS. Furthermore, the insertion of ISAba125 and ISAba27 into adeN was observed for the first time in A. baumannii isolates. Besides ISs, several different mutations were observed in adeN (e.g. deletions and premature stop codons), all of which led to increased tigecycline MICs. Moreover, several amino acid substitutions were detected in AdeRS, AdeN and AdeL. Of note, the substitutions D21V, G25S and D26N in AdeR were found in multiple sequences and suggest a mutational hotspot. Conclusions This study provides an insight into the different mechanisms associated with tigecycline resistance using a genomic approach and points out the importance of considering adeRS and adeN as markers for tigecycline-resistant A. baumannii isolates.

The master regulator CsgD switches planktonic growth to biofilm formation by activating synthesis of curli fimbriae and cellulose in Enterobacteriaceae. CsgD was classified to be the LuxR response regulatory family, while its cognate sensor histidine kinase has not been identified yet. CsgD consists of a C‐terminal DNA binding domain and an N‐terminal regulatory domain that provokes the upstream signal transduction to further modulate its function. We provide the crystal structure of Salmonella Typhimurium CsgD regulatory domain, which reveals an atypical β5α5 response regulatory receiver domain folding with the α2 helix representing as a disorder loop compared to the LuxR/FixJ canonical response regulator, and the structure indicated a noteworthy α5 helix similar to the non‐canonical master regulator VpsT receiver domain α6. CsgD regulatory domain assembles with two dimerization interfaces mainly through α1 and α5, which has shown similarity to the c‐di‐GMP independent and stabilized dimerization interface of VpsT from Vibrio cholerae respectively. The potential phosphorylation site D59 is directly involved in the interaction of interfaces I and mutagenesis studies indicated that both dimerization interfaces could be crucial for CsgD activity. The structure reveals important molecular details for the dimerization assembly of CsgD and will shed new insight into its regulation mechanism.

Vsig4 is a recently identified immune regulatory protein related to the B7 family with dual functionality: a negative regulator of T cell activation and a receptor for the complement components C3b and C3c. Here we present a structural evaluation of a nanobody, Nb119, against the extracellular IgV domain protein of both mouse and human recombinant Vsig4, which have a high degree of sequence identity. Although mouse and human Vsig4 bind to Nb119 with a 250 times difference in dissociation constants, the interaction results in a highly identical assembly with a RMSD of 0.4Å. The molecular determinants for Vsig4 recognition and cross reactivity unveiled by the atomic structure of Nb119 in complex with mVsig4 and hVsig4 afford new insights useful for the further optimization of the nanobody for potential use in humans. Additionally, structural analysis of the Vsig4-Nb119 complexes indicates that Nb119 occupies the interface on Vsig4 recognized by the macroglobulin-like domains MG4 and MG5 of C3b. Thus an affinity-improved Nb119 may have the potential to influence the activation of both T cells and complement.

Nearly all bacteria exhibit a type of phenotypic growth described as persistence that is thought to underlie antibiotic tolerance and recalcitrant chronic infections. The chromosomally encoded high-persistence (Hip) toxin–antitoxin proteins HipASO and HipBSO from Shewanella oneidensis, a proteobacterium with unusual respiratory capacities, constitute a type II toxin–antitoxin protein module. Here we show that phosphorylated HipASO can engage in an unexpected ternary complex with HipBSO and double-stranded operator DNA that is distinct from the prototypical counterpart complex from Escherichia coli. The structure of HipBSO in complex with operator DNA reveals a flexible C-terminus that is sequestered by HipASO in the ternary complex, indicative of its role in binding HipASO to abolish its function in persistence. The structure of HipASO in complex with a non-hydrolyzable ATP analogue shows that HipASO autophosphorylation is coupled to an unusual conformational change of its phosphorylation loop. However, HipASO is unable to phosphorylate the translation factor Elongation factor Tu, contrary to previous reports, but in agreement with more recent findings. Our studies suggest that the phosphorylation state of HipA is an important factor in persistence and that the structural and mechanistic diversity of HipAB modules as regulatory factors in bacterial persistence is broader than previously thought.

One of the most pertinent recent outcomes of molecular microbiology efforts to understand bacterial behavior is the discovery of a wide range of toxin-antitoxin (TA) systems that are tightly controlling bacterial persistence. While TA systems were originally linked to control over the genetic material, for example plasmid maintenance, it is now clear that they are involved in essential cellular processes like replication, gene expression, and cell wall synthesis. Toxin activity is induced stochastically or after environmental stimuli, resulting in silencing of the above-mentioned biological processes and entry in a dormant state. In this minireview, we highlight the recent developments in research on these intriguing systems with a focus on their role in biofilms and in bacterial virulence. We discuss their potential as targets in antimicrobial drug discovery.

SummaryThe discovery that hematopoietic human colony stimulating factor-1 receptor (CSF-1R) can be activated by two distinct cognate cytokines, colony stimulating factor-1 (CSF-1) and interleukin-34 (IL-34), created puzzling scenarios for the two possible signaling complexes. We here employ a hybrid structural approach based on small-angle X-ray scattering (SAXS) and negative-stain EM to reveal that bivalent binding of human IL-34 to CSF-1R leads to an extracellular assembly hallmarked by striking similarities to the CSF-1:CSF-1R complex, including homotypic receptor-receptor interactions. Thus, IL-34 and CSF-1 have evolved to exploit the geometric requirements of CSF-1R activation. Our models include N-linked oligomannose glycans derived from a systematic approach resulting in the accurate fitting of glycosylated models to the SAXS data. We further show that the C-terminal region of IL-34 is heavily glycosylated and that it can be proteolytically cleaved from the IL-34:hCSF-1R complex, providing insights into its role in the functional nonredundancy of IL-34 and CSF-1.

The type II secretion system is a multiprotein assembly spanning the inner and outer membranes in Gram-negative bacteria. It is found in almost all pathogenic bacteria where it contributes to virulence, host tissue colonization, and infection. The exoproteins are secreted across the outer membrane via a large translocation channel, the secretin, which typically adopts a dodecameric structure. These secretin channels have large periplasmic N-terminal domains that reach out into the periplasm for communication with the inner membrane platform and with a pseudopilus structure that spans the periplasm. Here we report the crystal structure of the N-terminal periplasmic domain of the secretin XcpQ from Pseudomonas aeruginosa, revealing a two-lobe dimeric assembly featuring parallel subunits engaging in well defined interactions at the tips of each lobe. We have employed structure-based engineering of disulfide bridges and native mass spectrometry to show that the periplasmic domain of XcpQ dimerizes in a concentration-dependent manner. Validation of these insights in the context of cellular full-length XcpQ and further evaluation of the functionality of disulfide-linked XcpQ establishes that the basic oligomerization unit of XcpQ is a dimer. This is consistent with the notion that the dodecameric secretin assembles as a hexamer of dimers to ensure correct projection of the N-terminal domains into the periplasm. Therefore, our studies provide a key conceptual advancement in understanding the assembly principles and dynamic function of type II secretion system secretins and challenge recent studies reporting monomers as the basic subunit of the secretin oligomer.

The properties of mucilage obtained from Dioscorea opposita, generated during industrial manufacturing were investigated in this study. Characteristics such as monosaccharide content, amino acid content, molecular weight, and structural features were measured, whereas morphology was observed using a scanning/transmission electron microscope. Additionally, emulsification properties at different concentrations (0.2%, 0.4%, 0.6%, 0.8%, and 1.0%) and under acidic and basic pH (5.0 and 9.0) conditions were studied. The results showed that emulsions prepared from mucilage and medium-chain triglycerides presented more effective emulsifying functions and higher stability, especially at low concentrations. Both, acidic and basic conditions improved the overall emulsification properties, which suggested that the isoelectric point of amino acids may be involved in the emulsification properties. The results of this study show that mucilage from Dioscorea opposita can be considered as a sustainable resource of a natural emulsifier obtained from industrial waste. (C) 2017 Elsevier Ltd. All rights reserved.

Single-photon emission computed tomography combined with micro-CT (SPECT/μCT) imaging using Nanobodies against complement receptor of the Ig superfamily (CRIg), found on tissue macrophages such as synovial macrophages, has promising potential to visualize joint inflammation in experimental arthritis. Here, we further addressed the specificity and assessed the potential for arthritis monitoring. Signals obtained with 99mTc-labelled NbV4m119 Nanobody were compared in joints of wild type (WT) versus CRIg−/− mice with collagen-induced arthritis (CIA) or K/BxN serum transfer-induced arthritis (STIA). In addition, SPECT/μCT imaging was used to investigate arthritis development in STIA and in CIA under dexamethasone treatment. 99mTc-NbV4m119 accumulated in inflamed joints of WT, but not CRIg−/− mice with CIA and STIA. Development and spontaneous recovery of symptoms in STIA was reflected in initially increased and subsequently reduced joint accumulation of 99mTc-NbV4m119. Dexamethasone treatment of CIA mice reduced 99mTc-NbV4m119 accumulation as compared to saline control in most joints except knees. SPECT/μCT imaging with 99mTc-NbV4m119 allows specific assessment of inflammation in different arthritis models and provides complementary information to clinical scoring for quantitatively and non-invasively monitoring the pathological process and the efficacy of arthritis treatment.