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All antibiotics have to engage bacterial amphiphilic barriers such as the lipopolysaccharide-rich outer membrane or the phospholipid-based inner membrane in some manner, either by disrupting them outright and/or permeating them and thereby allow the antibiotic to get into bacteria. There is a growing class of cyclic antibiotics, many of which are of bacterial origin, that exhibit activity against Gram-negative bacteria, which constitute an urgent problem in human health. We examine a diverse collection of these cyclic antibiotics, both natural and synthetic, which include bactenecin, polymyxin B, octapeptin, capreomycin, and Kirshenbaum peptoids, in order to identify what they have in common when they interact with bacterial lipid membranes. We find that they virtually all have the ability to induce negative Gaussian curvature (NGC) in bacterial membranes, the type of curvature geometrically required for permeation mechanisms such as pore formation, blebbing, and budding. This is interesting since permeation of membranes is a function usually ascribed to antimicrobial peptides (AMPs) from innate immunity. As prototypical test cases of cyclic antibiotics, we analyzed amino acid sequences of bactenecin, polymyxin B, and capreomycin using our recently developed machine-learning classifier trained on α-helical AMP sequences. Although the original classifier was not trained on cyclic antibiotics, a modified classifier approach correctly predicted that bactenecin and polymyxin B have the ability to induce NGC in membranes, while capreomycin does not. Moreover, the classifier was able to recapitulate empirical structure-activity relationships from alanine scans in polymyxin B surprisingly well. These results suggest that there exists some common ground in the sequence design of hybrid cyclic antibiotics and linear AMPs.

Constitutively active BCR-ABL kinase fusions are causative mutations in the pathogenesis of hematopoietic neoplasias including chronic myelogenous leukemia (CML). Although these fusions have been successfully targeted with kinase inhibitors, drug-resistance and relapse continue to limit long-term survival, highlighting the need for continued innovative drug discovery. We developed a time-resolved Förster resonance energy transfer (TR-FRET) -based assay to identify compounds that disrupt stimulation of the ABL kinase by blocking its ability to bind the positive regulator RIN1. This assay was used in a high throughput screen (HTS) of two small molecule libraries totaling 444,743 compounds. 708 confirmed hits were counter-screened to eliminate off-target inhibitors and reanalyzed to prioritize compounds with IC50 values below 10 μM. The CML cell line K562 was then used to identify five compounds that decrease MAPK1/3 phosphorylation, which we determined to be an indicator of RIN1-dependent ABL signaling. One of these compounds is a thiadiazole, and the other four are structurally related acyl piperidine amides. Notably, these five compounds lower cellular BCR-ABL1 kinase activity by blocking a positive regulatory interaction rather than directly inhibiting ABL catalytic function.

Intracellular acting protein exotoxins produced by bacteria and plants are important molecular determinants that drive numerous human diseases. A subset of these toxins, the cytolethal distending toxins (CDTs), are encoded by several Gram-negative pathogens and have been proposed to enhance virulence by allowing evasion of the immune system. CDTs are trafficked in a retrograde manner from the cell surface through the Golgi apparatus and into the endoplasmic reticulum (ER) before ultimately reaching the host cell nucleus. However, the mechanism by which CDTs exit the ER is not known. Here we show that three central components of the host ER associated degradation (ERAD) machinery, Derlin-2 (Derl2), the E3 ubiquitin-protein ligase Hrd1, and the AAA ATPase p97, are required for intoxication by some CDTs. Complementation of Derl2-deficient cells with Derl2:Derl1 chimeras identified two previously uncharacterized functional domains in Derl2, the N-terminal 88 amino acids and the second ER-luminal loop, as required for intoxication by the CDT encoded by Haemophilus ducreyi (Hd-CDT). In contrast, two motifs required for Derlin-dependent retrotranslocation of ERAD substrates, a conserved WR motif and an SHP box that mediates interaction with the AAA ATPase p97, were found to be dispensable for Hd-CDT intoxication. Interestingly, this previously undescribed mechanism is shared with the plant toxin ricin. These data reveal a requirement for multiple components of the ERAD pathway for CDT intoxication and provide insight into a Derl2-dependent pathway exploited by retrograde trafficking toxins.

Pathogenic microorganisms and toxins have evolved a variety of mechanisms to gain access to the host-cell cytosol and thereby exert virulent effects upon the host. One common mechanism of cellular entry requires trafficking to an acidified endosome, which promotes translocation across the host membrane. To identify small-molecule inhibitors that block this process, a library of 30,000 small molecules was screened for inhibitors of anthrax lethal toxin. Here we report that 4-bromobenzaldehyde N-(2,6-dimethylphenyl)semicarbazone, the most active compound identified in the screen, inhibits intoxication by lethal toxin and blocks the entry of multiple other acid-dependent bacterial toxins and viruses into mammalian cells. This compound, which we named EGA, also delays lysosomal targeting and degradation of the EGF receptor, indicating that it targets host-membrane trafficking. In contrast, EGA does not block endosomal recycling of transferrin, retrograde trafficking of ricin, phagolysosomal trafficking, or phagosome permeabilization by Franciscella tularensis. Furthermore, EGA does not neutralize acidic organelles, demonstrating that its mechanism of action is distinct from pH-raising agents such as ammonium chloride and bafilomycin A1. EGA is a powerful tool for the study of membrane trafficking and represents a class of host-targeted compounds for therapeutic development to treat infectious disease.

Severe chronic postsurgical pain has a prevalence of 4-10% in the surgical population. The underlying nociceptive mechanisms have not been well characterized. Following the late resolution phase of an inflammatory injury, high-dose μ-opioid-receptor inverse agonists reinstate hypersensitivity to nociceptive stimuli. This unmasking of latent pain sensitization has been a consistent finding in rodents while only observed in a limited number of human volunteers. Latent sensitization could be a potential triggering venue in chronic postsurgical pain. The objective of the present trial was in detail to examine the association between injury-induced secondary hyperalgesia and naloxone-induced unmasking of latent sensitization. Healthy volunteers (n = 80) received a cutaneous heat injury (47°C, 420 s, 12.5 cm2). Baseline secondary hyperalgesia areas were assessed 1 h post-injury. Utilizing an enriched enrollment design, subjects with a magnitude of secondary hyperalgesia areas in the upper quartile ('high-sensitizers' [n = 20]) and the lower quartile ('low-sensitizers' [n = 20]) were selected for further study. In four consecutive experimental sessions (Sessions 1 to 4), the subjects at two sessions (Sessions 1 and 3) received a cutaneous heat injury followed 168 h later (Sessions 2 and 4) by a three-step target-controlled intravenous infusion of naloxone (3.25 mg/kg), or normal saline. Assessments of secondary hyperalgesia areas were made immediately before and stepwise during the infusions. Simple univariate statistics revealed no significant differences in secondary hyperalgesia areas between naloxone and placebo treatments (P = 0.215), or between 'high-sensitizers' and 'low-sensitizers' (P = 0.757). In a mixed-effects model, secondary hyperalgesia areas were significantly larger following naloxone as compared to placebo for 'high-sensitizers' (P < 0.001), but not 'low-sensitizers' (P = 0.651). Although we could not unequivocally demonstrate naloxone-induced reinstatement of heat injury-induced hyperalgesia, further studies in clinical postsurgical pain models are warranted.

NOD-like receptors (NLRs) are a group of cytoplasmic molecules that recognize microbial invasion or 'danger signals'. Activation of NLRs can induce rapid caspase-1 dependent cell death termed pyroptosis, or a caspase-1 independent cell death termed pyronecrosis. Bacillus anthracis lethal toxin (LT), is recognized by a subset of alleles of the NLR protein Nlrp1b, resulting in pyroptotic cell death of macrophages and dendritic cells. Here we show that LT induces lysosomal membrane permeabilization (LMP). The presentation of LMP requires expression of an LT-responsive allele of Nlrp1b, and is blocked by proteasome inhibitors and heat shock, both of which prevent LT-mediated pyroptosis. Further the lysosomal protease cathepsin B is released into the cell cytosol and cathepsin inhibitors block LT-mediated cell death. These data reveal a role for lysosomal membrane permeabilization in the cellular response to bacterial pathogens and demonstrate a shared requirement for cytosolic relocalization of cathepsins in pyroptosis and pyronecrosis.

Author(s): Kenneth A. Bradley [1, 2, 3]; Jeremy Mogridge [3, 4, 5]; Michael Mourez [4]; R. John Collier [4]; John A. T. Young (corresponding author) [1] The tripartite toxin secreted [...]

Bacterial species like Escherichia coli, manufacture siderophore molocules like Enterobactin, in response to iron deficiency. EntB, is a component of enterobactin biosynthesis that mediate the production of 2,3 dihydroxybenzoate. Furthermore, Entb acts as an aryl carrier protein which mediates the assembly of enterobactin. EntB also induces enterobactin synthesis by undergoing EntD-mediated phosphopantetheinylation and subsequent acylation of the intermediate product in the presence of EntE.