Deborah T. Hung

Targeting virulence: a new paradigm for antimicrobial therapy

Clinically significant antibiotic resistance has evolved against virtually every antibiotic deployed. Yet the development of new classes of antibiotics has lagged far behind our growing need for such drugs. Rather than focusing on therapeutics that target in vitro viability, much like conventional antibiotics, an alternative approach is to target functions essential for infection, such as virulence factors required to cause host damage and disease. This approach has several potential advantages including expanding the repertoire of bacterial targets, preserving the host endogenous microbiome, and exerting less selective pressure, which may result in decreased resistance. We review new approaches to targeting virulence, discuss their advantages and disadvantages, and propose that in addition to targeting virulence, new antimicrobial development strategies should be expanded to include targeting bacterial gene functions that are essential for in vivo viability. We highlight both new advances in identifying these functions and prospects for antimicrobial discovery targeting this unexploited area.

Identification of the immunophilins capable of mediating inhibition of signal transduction by cyclosporin A and FK506: roles of calcineurin binding and cellular location.

The immunosuppressants cyclosporin A (CsA) and FK506 appear to block T-cell function by inhibiting the calcium-regulated phosphatase calcineurin. While multiple distinct intracellular receptors for these drugs (cyclophilins and FKBPs, collectively immunophilins) have been characterized, the functionally active ones have not been discerned. We found that overexpression of cyclophilin A or B or FKBP12 increased T-cell sensitivity to CsA or FK506, respectively, demonstrating that they are able to mediate the inhibitory effects of their respective immunosuppressants in vivo. In contrast, cyclophilin C, FKBP13, and FKBP25 had no effect. Direct comparison of the Ki of each drug-immunophilin complex for calcineurin in vitro revealed that although calcineurin binding was clearly necessary, it was not sufficient to explain the in vivo activity of the immunophilin. Subcellular localization was shown also to play a role, since gene deletions of cyclophilins B and C which changed their intracellular locations altered their activities significantly. Cyclophilin B has been shown previously to be located within calcium-containing intracellular vesicles; its ability to mediate CsA inhibition implies that certain components of the signal transduction machinery are also spatially restricted within the cell.

Characterization of a functional thrombin receptor. Issues and opportunities.

Introduction How does the protease thrombin activate platelets and other cells? This question presents intriguing issues from both basic scientific and clinical perspectives. From a basic view, the fact that thrombin is a protease raises the possibility of a novel proteolytic mechanism of receptor activation. From a clinical view, thrombin regulates hemostasis and thrombosis. Moreover, a host of thrombin actions on cells have been defined in vitro; in vivo these actions may be critical for both normal responses to wounding and for pathological vascular events. An understanding of the mechanism of thrombin-induced cell activation promises reagents for identifying thrombins role in cellular responses in vivo and possibly new therapeutics. In this Perspectives, we review the recent cloning and characterization of a platelet thrombin receptor in our laboratory (1). Identification of the thrombin receptor did reveal a unique proteolytic mechanism of receptor activation, and led to the development of a novel agonist peptide which activates the thrombin receptor independent of thrombin and thrombins protease activity. This agonist peptide is a useful new tool for addressing the role of thrombin receptor activation in cellular responses. It is our hope that having the cloned receptor in hand will also allow the development of thrombin receptor antagonists. Such reagents will define the role of thrombin receptor activation in normal and disease processes in vivo, and may provide the basis for a new class of antithrombotic or antiproliferative pharmaceuticals.

Genomic epidemiology of the Escherichia coli O104:H4 outbreaks in Europe, 2011

The degree to which molecular epidemiology reveals information about the sources and transmission patterns of an outbreak depends on the resolution of the technology used and the samples studied. Isolates of Escherichia coli O104:H4 from the outbreak centered in Germany in May–July 2011, and the much smaller outbreak in southwest France in June 2011, were indistinguishable by standard tests. We report a molecular epidemiological analysis using multiplatform whole-genome sequencing and analysis of multiple isolates from the German and French outbreaks. Isolates from the German outbreak showed remarkably little diversity, with only two single nucleotide polymorphisms (SNPs) found in isolates from four individuals. Surprisingly, we found much greater diversity (19 SNPs) in isolates from seven individuals infected in the French outbreak. The German isolates form a clade within the more diverse French outbreak strains. Moreover, five isolates derived from a single infected individual from the French outbreak had extremely limited diversity. The striking difference in diversity between the German and French outbreak samples is consistent with several hypotheses, including a bottleneck that purged diversity in the German isolates, variation in mutation rates in the two E. coli outbreak populations, or uneven distribution of diversity in the seed populations that led to each outbreak.

Cloned platelet thrombin receptor is necessary for thrombin-induced platelet activation.

Platelet activation by thrombin is critical for hemostasis and thrombosis. Structure-function studies with a recently cloned platelet thrombin receptor suggest that a hirudin-like domain in the receptors extracellular amino terminal extension is a thrombin-binding determinant important for receptor activation. We now report that a peptide antiserum to this domain is a potent and specific antagonist of thrombin-induced platelet activation. This study demonstrates that the cloned platelet thrombin receptor is necessary for platelet activation by thrombin, and provides a strategy for developing blocking monoclonal antibodies of potential therapeutic value.

Understanding and controlling the cell cycle with natural products

Small molecule natural products have aided in the discovery and characterization of many proteins critical to the progression and maintenance of the cell cycle. Identification of the direct target of a natural product gives scientists a tool to control a specific aspect of the cell cycle, thus facilitating the study of the cell-cycle machinery.

(+)-Discodermolide binds to microtubules in stoichiometric ratio to tubulin dimers, blocks taxol binding and results in mitotic arrest

BACKGROUND The marine natural product (+)-discodermolide has potent immunosuppressive activity. It inhibits proliferation of a wide range of human and murine cells, induces cell cycle arrest in the G2 or M phase and was recently shown to stabilize microtubules. Total synthesis of discodermolide has made it possible to generate variants of the compound to study its intracellular function in detail. RESULTS We have determined that (+)-discodermolide arrests MG63 cells at M phase, and has a stabilizing effect on microtubules. In vitro studies show that discodermolide induces polymerization of purified tubulin in the absence of microtubule-associated proteins, and that it binds to tubulin dimers in microtubules at 1:1 stoichiometry. Discodermolide binds taxol-polymerized microtubules at near stoichiometric level, whereas taxol binds discodermolide-induced microtubules poorly. Competition data show that the binding of microtubules by discodermolide and taxol are mutually exclusive; discodermolide binds with higher affinity than taxol. The results of binding assays carried out in vivo or in cell lysates also suggest that the microtubule network is discodermolides cellular target. CONCLUSIONS (+)-Discodermolide causes cell cycle arrest at the metaphase-anaphase transition in mitosis, presumably due to its stabilizing effect on microtubules. In vitro, discodermolide polymerizes purified tubulin potently in the absence of MAPs. It binds microtubules at one molecule per tubulin dimer with a higher affinity than taxol, and the binding of microtubules by discodermolide and taxol are mutually exclusive. In total cell lysates discodermolide displays binding activity that is consistent with its effects on microtubules.

Eradication of bacterial persisters with antibiotic-generated hydroxyl radicals

During Mycobacterium tuberculosis infection, a population of bacteria likely becomes refractory to antibiotic killing in the absence of genotypic resistance, making treatment challenging. We describe an in vitro model capable of yielding a phenotypically antibiotic-tolerant subpopulation of cells, often called persisters, within populations of Mycobacterium smegmatis and M. tuberculosis. We find that persisters are distinct from the larger antibiotic-susceptible population, as a small drop in dissolved oxygen (DO) saturation (20%) allows for their survival in the face of bactericidal antibiotics. In contrast, if high levels of DO are maintained, all cells succumb, sterilizing the culture. With increasing evidence that bactericidal antibiotics induce cell death through the production of reactive oxygen species (ROS), we hypothesized that the drop in DO decreases the concentration of ROS, thereby facilitating persister survival, and maintenance of high DO yields sufficient ROS to kill persisters. Consistent with this hypothesis, the hydroxyl-radical scavenger thiourea, when added to M. smegmatis cultures maintained at high DO levels, rescues the persister population. Conversely, the antibiotic clofazimine, which increases ROS via an NADH-dependent redox cycling pathway, successfully eradicates the persister population. Recent work suggests that environmentally induced antibiotic tolerance of bulk populations may result from enhanced antioxidant capabilities. We now show that the small persister subpopulation within a larger antibiotic-susceptible population also shows differential susceptibility to antibiotic-induced hydroxyl radicals. Furthermore, we show that stimulating ROS production can eradicate persisters, thus providing a potential strategy to managing persistent infections.

Nucleic acid detection with CRISPR-Cas13a/C2c2

Sensitive and specific CRISPR diagnostics Methods are needed that can easily detect nucleic acids that signal the presence of pathogens, even at very low levels. Gootenberg et al. combined the allele-specific sensing ability of CRISPR-Cas13a with recombinase polymerase amplification methods to detect specific RNA and DNA sequences. The method successfully detected attomolar levels of Zika virus, as well as the presence of pathogenic bacteria. It could also be used to perform human genotyping from cell-free DNA. Science, this issue p. 438 An ortholog of CRISPR-Cas13a/C2c2 can be used as a highly sensitive detector of specific RNA and DNA sequences. Rapid, inexpensive, and sensitive nucleic acid detection may aid point-of-care pathogen detection, genotyping, and disease monitoring. The RNA-guided, RNA-targeting clustered regularly interspaced short palindromic repeats (CRISPR) effector Cas13a (previously known as C2c2) exhibits a “collateral effect” of promiscuous ribonuclease activity upon target recognition. We combine the collateral effect of Cas13a with isothermal amplification to establish a CRISPR-based diagnostic (CRISPR-Dx), providing rapid DNA or RNA detection with attomolar sensitivity and single-base mismatch specificity. We use this Cas13a-based molecular detection platform, termed Specific High-Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK), to detect specific strains of Zika and Dengue virus, distinguish pathogenic bacteria, genotype human DNA, and identify mutations in cell-free tumor DNA. Furthermore, SHERLOCK reaction reagents can be lyophilized for cold-chain independence and long-term storage and be readily reconstituted on paper for field applications.

Bile acids stimulate biofilm formation in Vibrio cholerae

Vibrio cholerae is a Gram‐negative bacterium that causes the acute diarrhoeal disease cholera. After the bacterium is ingested, it passes through the digestive tract, encountering various environmental stresses including the acidic milieu of the stomach and the toxic effects of bile in the duodenum. While these stresses serve as part of a host defence system, V. cholerae has evolved resistance mechanisms that allow it to evade these defences and establish infection. We examined the expression profiles of V. cholerae in response to bile or bile acids and found an induction of biofilm genes. We found that V. cholerae shows significantly enhanced biofilm formation in response to bile acids, and that bacteria within the biofilm are more resistant to the toxicity of bile acids compared with planktonic cells. Bile acid induction of biofilms was found to be dependent on the vps genes (Vibriopolysaccharidesynthesis) and their transcriptional activator VpsR, but VpsT is not required. These results contribute to the developing picture of a complex relationship between V. cholerae and its environment within the host during infection.