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The α-proteobacterium Sinorhizobium meliloti establishes a chronic intracellular infection during the symbiosis with its legume hosts. Within specialized host cells, S. meliloti differentiates into highly polyploid, enlarged nitrogen-fixing bacteroids. This differentiation is driven by host cells through the production of defensin-like peptides called “nodule-specific cysteine-rich” (NCR) peptides. Recent research has shown that synthesized NCR peptides exhibit antimicrobial activity at high concentrations but cause bacterial endoreduplication at sublethal concentrations. We leveraged synchronized S. meliloti populations to determine how treatment with a sublethal NCR peptide affects the cell cycle and physiology of bacteria at the molecular level. We found that at sublethal levels a representative NCR peptide specifically blocks cell division and antagonizes Z-ring function. Gene-expression profiling revealed that the cell division block was produced, in part, through the substantial transcriptional response elicited by sublethal NCR treatment that affected ∼15% of the genome. Expression of critical cell-cycle regulators, including ctrA , and cell division genes, including genes required for Z-ring function, were greatly attenuated in NCR-treated cells. In addition, our experiments identified important symbiosis functions and stress responses that are induced by sublethal levels of NCR peptides and other antimicrobial peptides. Several of these stress-response pathways also are found in related α-proteobacterial pathogens and might be used by S. meliloti to sense host cues during infection. Our data suggest a model in which, in addition to provoking stress responses, NCR peptides target intracellular regulatory pathways to drive S. meliloti endoreduplication and differentiation during symbiosis.

Sinorhizobium meliloti differentiates into persisting, nitrogen-fixing bacteroids within root nodules of the legume Medicago truncatula. Nodule-specific cysteine-rich antimicrobial peptides (NCR AMPs) and the bacterial BacA protein are essential for bacteroid development. However, the bacterial factors central to the NCR AMP response and the in planta role of BacA are unknown. We investigated the hypothesis that BacA is critical for the bacterial response towards NCR AMPs. We found that BacA was not essential for NCR AMPs to induce features of S. meliloti bacteroids in vitro. Instead, BacA was critical to reduce the amount of NCR AMP-induced membrane permeabilization and bacterial killing in vitro. Within M. truncatula, both wild-type and BacA-deficient mutant bacteria were challenged with NCR AMPs, but this resulted in persistence of the wild-type bacteria and rapid cell death of the mutant bacteria. In contrast, BacA was dispensable for bacterial survival in an M. truncatula dnf1 mutant defective in NCR AMP transport to the bacterial compartment. Therefore, BacA is critical for the legume symbiosis by protecting S. meliloti against the bactericidal effects of NCR AMPs. Host AMPs are ubiquitous in nature and BacA proteins are essential for other chronic host infections by symbiotic and pathogenic bacteria. Hence, our findings suggest that BacA-mediated protection of bacteria against host AMPs is a critical stage in the establishment of different prolonged host infections.

A metabotropic glutamate receptor coupled to phospholipase D (PLD‐mGluR) was discovered in the hippocampus over three decades ago. Its pharmacology and direct linkage to PLD activation are well established and indicate it is a highly atypical glutamate receptor. A receptor with the same pharmacology is present in spindle primary sensory terminals where its blockade can totally abolish, and its activation can double, the normal stretch‐evoked firing. We report here the first identification of this PLD‐mGluR protein, by capitalizing on its expression in primary mechanosensory terminals, developing an enriched source, pharmacological profiling to identify an optimal ligand, and then functionalizing it as a molecular tool. Evidence from immunofluorescence, western and far‐western blotting indicates PLD‐mGluR is homomeric GluK2, since GluK2 is the only glutamate receptor protein/receptor subunit present in spindle mechanosensory terminals. Its expression was also found in the lanceolate palisade ending of hair follicle, also known to contain the PLD‐mGluR. Finally, in a mouse model with ionotropic function ablated in the GluK2 subunit, spindle glutamatergic responses were still present, confirming it acts purely metabotropically. We conclude the PLD‐mGluR is a homomeric GluK2 kainate receptor signalling purely metabotropically and it is common to other, perhaps all, primary mechanosensory endings. What is the central question of this study? The metabotropic glutamate receptor coupled to phospholipase D (PLD‐mGluR) is a glutamate receptor previously only characterized pharmacologically but essential for maintaining stretch responsiveness in muscle spindle mechanosensory primary endings: what is the PLD‐mGluR protein? What is the main finding and its importance? PLD‐mGluR was identified as a homomeric GluK2 receptor signalling metabotropically. This identifies PLD‐mGluR 30 years after its discovery. This is important because: PLD‐mGluR is essential for muscle spindle stretch sensitivity; it is the first native kainate receptor shown to signal solely metabotropically; and, as it is the only GluR expressed in spindle mechanosensory endings, muscle spindles make a good functional assay of the native receptor.

The discovery of liquid water at several locations in the solar system raises the possibility that microbial life may have evolved outside Earth and as such could be accidently introduced into the Earth’s ecosystem. Unusual sugars or amino acids, like non-proteinogenic isovaline and α-aminoisobutyric acid that are vanishingly rare or absent from life forms on Earth, have been found in high abundance on non-terrestrial carbonaceous meteorites. It is therefore conceivable that exo-microorganisms might contain proteins that include these rare amino acids. We therefore asked whether the mammalian immune system would be able to recognize and induce appropriate immune responses to putative proteinaceous antigens that include these rare amino acids. To address this, we synthesised peptide antigens based on a backbone of ovalbumin and introduced isovaline and α-aminoisobutyric acid residues and demonstrated that these peptides can promote naïve OT-I cell activation and proliferation, but did so less efficiently than the canonical peptides. This is relevant to the biosecurity of missions that may retrieve samples from exoplanets and moons that have conditions that may be permissive for life, suggesting that accidental contamination and exposure to exo-microorganisms with such distinct proteomes might pose an immunological challenge.

There is an urgent need to develop effective therapies and treatment strategies to treat hypoxic tumors, which have a very poor prognosis and do not respond well to existing therapies. A novel hypoxia-targeting agent, KEMTUB012-NI2, was synthesized by conjugating a 2-nitroimidazole hypoxia-targeting moiety to a synthetic tubulysin, a very potent antimitotic. Its hypoxic selectivity and mode of action were studied in breast cancer cell lines. KEMTUB012-NI2 exhibited a similar selectivity for hypoxic cells to that of tirapazamine, a well-established hypoxia-targeting agent, but was >1,000 times more potent in cell cytotoxicity assays. The hypoxia-targeting mechanism for both KEMTUB012-NI2 and tirapazamine was selective and mediated by one-electron reductases. However, while cytochrome p450 reductase (POR) downregulation could inhibit tirapazamine cytotoxicity, it actually sensitized hypoxic cells to KEMTUB012-NI2. KEMTUB012-NI2 is a potent new agent that can selectively target hypoxic cancer cells. The hypoxia selectivity of KEMTUB012-NI2 and tirapazamine appears to be differentially activated by reductases. Since reductases are heterogeneously expressed in tumors, the different activation mechanisms will allow these agents to complement each other. Combining POR downregulation with KEMTUB012-NI2 treatment could be a new treatment strategy that maximizes efficacy toward hypoxic tumor cells while limiting systemic toxicity.

The CB1 receptor represents a promising target for the treatment of several disorders including pain-related disease states. However, therapeutic applications of Δ(9)-tetrahydrocannabinol and other CB1 orthosteric receptor agonists remain limited because of psychoactive side effects. Positive allosteric modulators (PAMs) offer an alternative approach to enhance CB1 receptor function for therapeutic gain with the promise of reduced side effects. Here we describe the development of the novel synthetic CB1 PAM, 6-methyl-3-(2-nitro-1-(thiophen-2-yl)ethyl)-2-phenyl-1H-indole (ZCZ011), which augments the in vitro and in vivo pharmacological actions of the CB1 orthosteric agonists CP55,940 and N-arachidonoylethanolamine (AEA). ZCZ011 potentiated binding of [(3)H]CP55,940 to the CB1 receptor as well as enhancing AEA-stimulated [(35)S]GTPγS binding in mouse brain membranes and β-arrestin recruitment and ERK phosphorylation in hCB1 cells. In the whole animal, ZCZ011 is brain penetrant, increased the potency of these orthosteric agonists in mouse behavioral assays indicative of cannabimimetic activity, including antinociception, hypothermia, catalepsy, locomotor activity, and in the drug discrimination paradigm. Administration of ZCZ011 alone was devoid of activity in these assays and did not produce a conditioned place preference or aversion, but elicited CB1 receptor-mediated antinociceptive effects in the chronic constriction nerve injury model of neuropathic pain and carrageenan model of inflammatory pain. These data suggest that ZCZ011 acts as a CB1 PAM and provide the first proof of principle that CB1 PAMs offer a promising strategy to treat neuropathic and inflammatory pain with minimal or no cannabimimetic side effects.

PurposeMolecular imaging of αvβ3 integrin has exhibited real potential to guide the appropriate use of anti-angiogenic therapies. However, an incomplete understanding of the factors that influence binding of αvβ3 integrin-specific radiotracers currently limits their use for assessing response to therapy in cancer patients. This study identifies two fundamental factors that modulate uptake of these radiotracers.ProceduresExperiments were performed in prostate cancer (PC3) and glioblastoma (U87MG) cells, which differentially express αvβ3 integrin. αvβ3 integrin-specific radiotracers were used to investigate the effect of manipulating αvβ3 integrin expression or activation in cellular binding assays. β3 integrin and αvβ3 integrin expression were measured by western blotting and flow cytometry, respectively. The effect of select pharmacological inhibitors on αvβ3 integrin activation and expression was also determined.ResultsRadiotracer binding was proportional to αvβ3 integrin expression when it was decreased (β3 knock-down cells) or increased, either using pharmacological inhibitors of cell signalling or by culturing cells for different times. Studies with both small molecule and arginine–glycine–aspartic acid (RGD)-based radiotracers revealed increased radiotracer binding after activation of αvβ3 integrin with Mn2+ or talin head domain. Moreover, inhibition of fundamental signalling pathways (mitogen-activated protein kinase kinase (MEK), Src and VEGFR2) decreased radiotracer binding, reflecting reduced αvβ3 integrin activity.ConclusionBinding of small molecule ligands and radiolabelled RGD peptides is modulated by expression and activation status of αvβ3 integrin. αvβ3 integrin-specific radiotracers can provide otherwise inaccessible information of the effect of signalling pathways on αvβ3 integrin. This has significant implications for assessing response to anti-angiogenic therapies in clinical studies.

Purpose Tubulysins are natural tetrapeptides that inhibit tubulin polymerisation. Tubulysins are very potent inhibitors of mammalian cancer cell growth, but restricted availability has limited their characterisation and development as anti-cancer compounds. KEMTUB10 was recently developed as a synthetic analogue of natural tubulysins. Methods The cell cytotoxicity of KEMTUB10 was studied in cell lines that represent the main breast cancer sub-types. The KEMTUB10 pro-apoptotic mechanism of action was studied in MCF7 and MDAMB231 cells. Results KEMTUB10 exerts a potent cytotoxic effect in cells representing the main breast cancer sub-types. KEMTUB10 blocks cells in the G2/M phase of the cell cycle and is a strong stimulator of apoptosis/cell death. KEMTUB10-induced apoptosis involves p53 and Bim, and to some extent Bcl-2 phosphorylation. Conclusions KEMTUB10 is a promising new anti-mitotic that exerts a potent cytotoxic effect in breast cancer cells, blocks cells in the G2/M phase of the cell cycle and stimulates apoptosis/cell death. KEMTUB10 has a distinct mode of action to taxol, appears to be sensitive to different molecular factors in cells and is likely subject to different mechanisms of acquired resistance. KEMTUB10 has the potential to be an important addition to the anti-cancer therapeutic armoury.

Our resistance to infection is critically dependent upon the ability of pattern recognition receptors to recognise microbial invasion and induce protective immune responses. One such family of receptors are the C-type lectins, which play central roles in antifungal immunity1. These receptors activate key effector mechanisms upon recognition of conserved fungal cell wall carbohydrates. However, several other immunologically active fungal ligands have been described, including melanin2,3, whose mechanisms of recognition remain largely undefined. Here we identify a C-type lectin receptor, Melanin sensing C-type Lectin receptor (MelLec), that plays an essential role in antifungal immunity through recognition of the naphthalene-diol unit of 1,8-dihydroxynaphthalene (DHN)-melanin. MelLec recognises melanin in conidial spores of Aspergillus fumigatus, as well as other DHN-melanised fungi and is ubiquitously expressed by CD31+ endothelial cells in mice. MelLec is also expressed by a sub-population of these cells in mice that co-express EpCAM and which were detected only in the lung and liver. In mouse models, MelLec was required for protection against disseminated infection with A. fumigatus. In humans, MelLec is also expressed by myeloid cells, and we identified a single nucleotide polymorphism of this receptor that negatively affected myeloid inflammatory responses and significantly increased susceptibility of stem-cell transplant recipients to disseminated Aspergillus infections. Thus MelLec is a receptor recognising an immunologically active component commonly found on fungi and plays an essential role in protective antifungal immunity in both mice and humans.

Resistance to infection is critically dependent on the ability of pattern recognition receptors to recognize microbial invasion and induce protective immune responses. One such family of receptors are the C-type lectins, which are central to antifungal immunity. These receptors activate key effector mechanisms upon recognition of conserved fungal cell-wall carbohydrates. However, several other immunologically active fungal ligands have been described; these include melanin, for which the mechanism of recognition is hitherto undefined. Here we identify a C-type lectin receptor, melanin-sensing C-type lectin receptor (MelLec), that has an essential role in antifungal immunity through recognition of the naphthalene-diol unit of 1,8-dihydroxynaphthalene (DHN)-melanin. MelLec recognizes melanin in conidial spores of Aspergillus fumigatus as well as in other DHN-melanized fungi. MelLec is ubiquitously expressed by CD31+ endothelial cells in mice, and is also expressed by a sub-population of these cells that co-express epithelial cell adhesion molecule and are detected only in the lung and the liver. In mouse models, MelLec was required for protection against disseminated infection with A. fumigatus. In humans, MelLec is also expressed by myeloid cells, and we identified a single nucleotide polymorphism of this receptor that negatively affected myeloid inflammatory responses and significantly increased the susceptibility of stem-cell transplant recipients to disseminated Aspergillus infections. MelLec therefore recognizes an immunologically active component commonly found on fungi and has an essential role in protective antifungal immunity in both mice and humans.