Antimicrobial resistance (AMR) nanomachines: mechanisms for fluoroquinolone and glycopeptide recognition, efflux and/or deactivation

In this review, we discuss mechanisms of resistance identified in bacterial agents Staphylococcus aureus and the enterococci towards two priority classes of antibiotics—the fluoroquinolones and the glycopeptides. Members of both classes interact with a number of components in the cells of these bacteria, so the cellular targets are also considered. Fluoroquinolone resistance mechanisms include efflux pumps (MepA, NorA, NorB, NorC, MdeA, LmrS or SdrM in S. aureus and EfmA or EfrAB in the enterococci) for removal of fluoroquinolone from the intracellular environment of bacterial cells and/or protection of the gyrase and topoisomerase IV target sites in Enterococcus faecalis by Qnr-like proteins. Expression of efflux systems is regulated by GntR-like (S. aureus NorG), MarR-like (MgrA, MepR) regulators or a two-component signal transduction system (TCS) (S. aureus ArlSR). Resistance to the glycopeptide antibiotic teicoplanin occurs via efflux regulated by the TcaR regulator in S. aureus. Resistance to vancomycin occurs through modification of the D-Ala-D-Ala target in the cell wall peptidoglycan and removal of high affinity precursors, or by target protection via cell wall thickening. Of the six Van resistance types (VanA-E, VanG), the VanA resistance type is considered in this review, including its regulation by the VanSR TCS. We describe the recent application of biophysical approaches such as the hydrodynamic technique of analytical ultracentrifugation and circular dichroism spectroscopy to identify the possible molecular effector of the VanS receptor that activates expression of the Van resistance genes; both approaches demonstrated that vancomycin interacts with VanS, suggesting that vancomycin itself (or vancomycin with an accessory factor) may be an effector of vancomycin resistance. With 16 and 19 proteins or protein complexes involved in fluoroquinolone and glycopeptide resistances, respectively, and the complexities of bacterial sensing mechanisms that trigger and regulate a wide variety of possible resistance mechanisms, we propose that these antimicrobial resistance mechanisms might be considered complex ‘nanomachines’ that drive survival of bacterial cells in antibiotic environments

The additional value of TGFβ1 and IL-7 to predict the course of prostate cancer progression

Background: Given the fact that prostate cancer incidence will increase in the coming years, new prognostic biomarkers are needed with regard to the biological aggressiveness of the prostate cancer diagnosed. Since cytokines have been associated with the biology of cancer and its prognosis, we determined whether transforming growth factor beta 1 (TGFβ1), interleukin-7 (IL-7) receptor and IL-7 levels add additional prognostic information with regard to prostate cancer

Brevenal Inhibits Pacific Ciguatoxin-1B-Induced Neurosecretion from Bovine Chromaffin Cells

Ciguatoxins and brevetoxins are neurotoxic cyclic polyether compounds produced by dinoflagellates, which are responsible for ciguatera and neurotoxic shellfish poisoning (NSP) respectively. Recently, brevenal, a natural compound was found to specifically inhibit brevetoxin action and to have a beneficial effect in NSP. Considering that brevetoxin and ciguatoxin specifically activate voltage-sensitive Na+ channels through the same binding site, brevenal has therefore a good potential for the treatment of ciguatera. Pacific ciguatoxin-1B (P-CTX-1B) activates voltage-sensitive Na+ channels and promotes an increase in neurotransmitter release believed to underpin the symptoms associated with ciguatera. However, the mechanism through which slow Na+ influx promotes neurosecretion is not fully understood. In the present study, we used chromaffin cells as a model to reconstitute the sequence of events culminating in ciguatoxin-evoked neurosecretion. We show that P-CTX-1B induces a tetrodotoxin-sensitive rise in intracellular Na+, closely followed by an increase in cytosolic Ca2+ responsible for promoting SNARE-dependent catecholamine secretion. Our results reveal that brevenal and β-naphtoyl-brevetoxin prevent P-CTX-1B secretagogue activity without affecting nicotine or barium-induced catecholamine secretion. Brevenal is therefore a potent inhibitor of ciguatoxin-induced neurotoxic effect and a potential treatment for ciguatera

A Factor Graph Nested Effects Model To Identify Networks from Genetic Perturbations

Complex phenotypes such as the transformation of a normal population of cells into cancerous tissue result from a series of molecular triggers gone awry. We describe a method that searches for a genetic network consistent with expression changes observed under the knock-down of a set of genes that share a common role in the cell, such as a disease phenotype. The method extends the Nested Effects Model of Markowetz et al. (2005) by using a probabilistic factor graph to search for a network representing interactions among these silenced genes. The method also expands the network by attaching new genes at specific downstream points, providing candidates for subsequent perturbations to further characterize the pathway. We investigated an extension provided by the factor graph approach in which the model distinguishes between inhibitory and stimulatory interactions. We found that the extension yielded significant improvements in recovering the structure of simulated and Saccharomyces cerevisae networks. We applied the approach to discover a signaling network among genes involved in a human colon cancer cell invasiveness pathway. The method predicts several genes with new roles in the invasiveness process. We knocked down two genes identified by our approach and found that both knock-downs produce loss of invasive potential in a colon cancer cell line. Nested effects models may be a powerful tool for inferring regulatory connections and genes that operate in normal and disease-related processes

Antimicrobial and Efflux Pump Inhibitory Activity of Caffeoylquinic Acids from Artemisia absinthium against Gram-Positive Pathogenic Bacteria

Background: Traditional antibiotics are increasingly suffering from the emergence of multidrug resistance amongst pathogenic bacteria leading to a range of novel approaches to control microbial infections being investigated as potential alternative treatments. One plausible antimicrobial alternative could be the combination of conventional antimicrobial agents/antibiotics with small molecules which block multidrug efflux systems known as efflux pump inhibitors. Bioassay-driven purification and structural determination of compounds from plant sources have yielded a number of pump inhibitors which acted against gram positive bacteria. Methodology/Principal Findings: In this study we report the identification and characterization of 4′,5′-O-dicaffeoylquinic acid (4′,5′-ODCQA) from Artemisia absinthium as a pump inhibitor with a potential of targeting efflux systems in a wide panel of Gram-positive human pathogenic bacteria. Separation and identification of phenolic compounds (chlorogenic acid, 3′,5′-ODCQA, 4′,5′-ODCQA) was based on hyphenated chromatographic techniques such as liquid chromatography with post column solid-phase extraction coupled with nuclear magnetic resonance spectroscopy and mass spectroscopy. Microbial susceptibility testing and potentiation of well know pump substrates revealed at least two active compounds; chlorogenic acid with weak antimicrobial activity and 4′,5′-ODCQA with pump inhibitory activity whereas 3′,5′-ODCQA was ineffective. These intitial findings were further validated with checkerboard, berberine accumulation efflux assays using efflux-related phenotypes and clinical isolates as well as molecular modeling methodology. Conclusions/Significance: These techniques facilitated the direct analysis of the active components from plant extracts, as well as dramatically reduced the time needed to analyze the compounds, without the need for prior isolation. The calculated energetics of the docking poses supported the biological information for the inhibitory capabilities of 4′,5′-ODCQA and furthermore contributed evidence that CQAs show a preferential binding to Major Facilitator Super family efflux systems, a key multidrug resistance determinant in gram-positive bacteria.National Institutes of Health (U.S.) (grant R01GM59903)National Institutes of Health (U.S.) (grant R01AI050875)Netherlands Organization for Scientific Research (VICI grant 700.56.442)Massachusetts Technology Transfer Center (MTTC)National Institutes of Health (U.S.) (grant 5U54MH084690-02

Real-time visualization of heterotrimeric G protein Gq activation in living cells

Contains fulltext : 97296.pdf (publisher's version ) (Open Access)BACKGROUND: Gq is a heterotrimeric G protein that plays an important role in numerous physiological processes. To delineate the molecular mechanisms and kinetics of signalling through this protein, its activation should be measurable in single living cells. Recently, fluorescence resonance energy transfer (FRET) sensors have been developed for this purpose. RESULTS: In this paper, we describe the development of an improved FRET-based Gq activity sensor that consists of a yellow fluorescent protein (YFP)-tagged Ggamma2 subunit and a Galphaq subunit with an inserted monomeric Turquoise (mTurquoise), the best cyan fluorescent protein variant currently available. This sensor enabled us to determine, for the first time, the kon (2/s) of Gq activation. In addition, we found that the guanine nucleotide exchange factor p63RhoGEF has a profound effect on the number of Gq proteins that become active upon stimulation of endogenous histamine H1 receptors. The sensor was also used to measure ligand-independent activation of the histamine H1 receptor (H1R) upon addition of a hypotonic stimulus. CONCLUSIONS: Our observations reveal that the application of a truncated mTurquoise as donor and a YFP-tagged Ggamma2 as acceptor in FRET-based Gq activity sensors substantially improves their dynamic range. This optimization enables the real-time single cell quantification of Gq signalling dynamics, the influence of accessory proteins and allows future drug screening applications by virtue of its sensitivity

A Genetically Hard-Wired Metabolic Transcriptome in Plasmodium falciparum Fails to Mount Protective Responses to Lethal Antifolates

Genome sequences of Plasmodium falciparum allow for global analysis of drug responses to antimalarial agents. It was of interest to learn how DNA microarrays may be used to study drug action in malaria parasites. In one large, tightly controlled study involving 123 microarray hybridizations between cDNA from isogenic drug-sensitive and drug-resistant parasites, a lethal antifolate (WR99210) failed to over-produce RNA for the genetically proven principal target, dihydrofolate reductase-thymidylate synthase (DHFR-TS). This transcriptional rigidity carried over to metabolically related RNA encoding folate and pyrimidine biosynthesis, as well as to the rest of the parasite genome. No genes were reproducibly up-regulated by more than 2-fold until 24 h after initial drug exposure, even though clonal viability decreased by 50% within 6 h. We predicted and showed that while the parasites do not mount protective transcriptional responses to antifolates in real time, P. falciparum cells transfected with human DHFR gene, and adapted to long-term WR99210 exposure, adjusted the hard-wired transcriptome itself to thrive in the presence of the drug. A system-wide incapacity for changing RNA levels in response to specific metabolic perturbations may contribute to selective vulnerabilities of Plasmodium falciparum to lethal antimetabolites. In addition, such regulation affects how DNA microarrays are used to understand the mode of action of antimetabolites