The Repellent DEET Potentiates Carbamate Effects via Insect Muscarinic Receptor Interactions: An Alternative Strategy to Control Insect Vector-Borne Diseases.

Insect vector-borne diseases remain one of the principal causes of human mortality. In addition to conventional measures of insect control, repellents continue to be the mainstay for personal protection. Because of the increasing pyrethroid-resistant mosquito populations, alternative strategies to reconstitute pyrethroid repellency and knock-down effects have been proposed by mixing the repellent DEET (N,N-Diethyl-3-methylbenzamide) with non-pyrethroid insecticide to better control resistant insect vector-borne diseases. By using electrophysiological, biochemichal, in vivo toxicological techniques together with calcium imaging, binding studies and in silico docking, we have shown that DEET, at low concentrations, interacts with high affinity with insect M1/M3 mAChR allosteric site potentiating agonist effects on mAChRs coupled to phospholipase C second messenger pathway. This increases the anticholinesterase activity of the carbamate propoxur through calcium-dependent regulation of acetylcholinesterase. At high concentrations, DEET interacts with low affinity on distinct M1/M3 mAChR site, counteracting the potentiation. Similar dose-dependent dual effects of DEET have also been observed at synaptic mAChR level. Additionally, binding and in silico docking studies performed on human M1 and M3 mAChR subtypes indicate that DEET only displays a low affinity antagonist profile on these M1/M3 mAChRs. These results reveal a selective high affinity positive allosteric site for DEET in insect mAChRs. Finally, bioassays conducted on Aedes aegypti confirm the synergistic interaction between DEET and propoxur observed in vitro, resulting in a higher mortality of mosquitoes. Our findings reveal an unusual allosterically potentiating action of the repellent DEET, which involves a selective site in insect. These results open exciting research areas in public health particularly in the control of the pyrethroid-resistant insect-vector borne diseases. Mixing low doses of DEET and a non-pyrethroid insecticide will lead to improvement in the efficiency treatments thus reducing both the concentration of active ingredients and side effects for non-target organisms. The discovery of this insect specific site may pave the way for the development of new strategies essential in the management of chemical use against resistant mosquitoes.

A novel method using Autographa californica multiple nucleopolyhedrovirus for increasing the sensitivity of insecticide through calcium influx in insect cell line.

Due to an intensive use of chemical insecticides, resistance mechanisms to insecticides together with adverse effects on non-target organisms have been largely reported. Improvement in pest control strategy represents an urgent need to optimize efficiency in the control of pest insects. In this context, a novel method based on the use of insect specific virus applied in combination with chemical insecticide, which could lead to sensitization of the insect target to insecticides is described. Insect virus, the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV), applied onto Sf9 cells induces an increase of intracellular calcium concentration via extracellular calcium influx. Co-application of AcMNPV with chlorpyrifos-ethyl onto Sf9 cells expressing the key enzyme acetylcholinesterase (AChE), known to be targeted by organophosphate insecticides, increases 1.5-fold the sensitivity of AChE to the insecticide. This effect is correlated with intracellular calcium concentration rise since AcMNPV-induced potentiating insecticide effect is counteracted by pretreatment with the calcium channel blocker, cadmium chloride. Increasing insecticide target sensitivity through intracellular calcium modulation by using insect virus co-applied with a chemical insecticide is a very promising strategy allowing optimization of insecticide treatment while reducing the concentration of insecticides used.

In Vitro Antibacterial Activity of Essential Oils against Streptococcus pyogenes.

Streptococcus pyogenes plays an important role in the pathogenesis of tonsillitis. The present study was conducted to evaluate the in vitro antibacterial activities of 18 essential oils chemotypes from aromatic medicinal plants against S. pyogenes. Antibacterial activity of essential oils was investigated using disc diffusion method. Minimum Inhibitory Concentration of essential oils showing an important antibacterial activity was measured using broth dilution method. Out of 18 essential oils tested, 14 showed antibacterial activity against S. pyogenes. Among them Cinnamomum verum, Cymbopogon citratus, Thymus vulgaris CT thymol, Origanum compactum, and Satureja montana essential oils exhibited significant antibacterial activity. The in vitro results reported here suggest that, for patients suffering from bacterial throat infections, if aromatherapy is used, these essential oils, considered as potential antimicrobial agents, should be preferred.

Anopheles gambiae mosquito isolated neurons: a new biological model for optimizing insecticide/repellent efficacy.

To understand better the mode of action of insecticides and repellents used in vector-borne diseases control, we developed a new biological model based on mosquito neurons isolated from adults Anopheles gambiae heads. This cellular model is well adapted to multidisciplinary approaches: electrophysiology, pharmacology, molecular biology and biochemical assays. Using RT-PCR, we demonstrated that isolated neurons express the nicotinic acetylcholine receptor subunit α1 (Agα1 nAchR), two acetylcholinesterases (AChE-1 and AChE-2) and three voltage-gated ion channels required for membrane excitability (AgCav1, AgNav1 and AgKv1). In order to correlate the expression of the different transcripts, encoding functional AgNav channel, nAChR receptor and AChE enzymes detected by RT-PCR, with electrophysiological activity we used patch-clamp technique. We revealed that AgNav and AChE which are targeted by insecticide and/or repellent were sensitive to the pyrethroid permethrin and to the repellent DEET, respectively. In addition, using colorimetric method, we also showed that AChE was sensitive to the carbamate propoxur. These results indicated that this novel neuronal mosquito model will lead to molecular and functional characterization of insecticide/repellent targets and appears as a powerful tool to investigate the development of highly specific and effective strategies for disease vector control.

Disruption of the GPI protein-encoding gene IFF4 of Candida albicans results in decreased adherence and virulence.

Candida albicans is the most important cause of systemic fungal infection in immunocompromised humans. Candidiasis is often initiated by the adherence and the colonization of inert surfaces such as peripheral venous catheters, central catheters, prosthetic cardiac valves, and other prostheses. We have studied the early stage of adherence and have shown that the disruption of C. albicans IFF4 gene encoding a GPI-anchor protein, led to a decrease of adherence of the germ tubes to plastic. Here, we demonstrated the role of the IFF4 gene in adherence to silicone catheter, as well as in virulence using a murine model of disseminated candidiasis. The iff4 Delta null mutant showed both a decrease of adherence to silicone catheter and a reduction of virulence. This work presents evidence for the importance of the IFF4 gene in host-fungal interaction.

Innovative applications for insect viruses: towards insecticide sensitization.

The effective management of emerging insect-borne disease is dependent on the use of safe and efficacious chemical insecticides. Given the inherent ability of insects to develop resistance, it is essential to propose innovative strategies because insecticides remain the most important element of integrated approaches to vector control. Recently, intracellular phosphorylation and dephosphorylation of membrane receptors and ion channels targeted by insecticides have been described as new processes for increasing the sensitivity of insecticides. An efficient method might be to infect host insects with recombinant viruses overexpressing specific protein phosphatases/kinases known to regulate specific insecticide-sensitive targets. This attractive strategy could lead to sensitization of the insects, thus reducing the doses of insecticides and increasing the efficacy of treatments.

Evaluation of an immunomagnetic separation method to capture Candida yeasts cells in blood.

BACKGROUND: Candida species have become the fourth most-frequent cause of nosocomial bloodstream infections in immunocompromised patients. Therefore, rapid identification of pathogenic fungi to species level has been considered critical for treatment. Conventional diagnostic procedures such as blood culture or biochemical tests are lacking both sensitivity and species specificity, so development of rapid diagnostic is essential. RESULTS: An immunomagnetic method involving anti-Candida monoclonal antibodies was developed to capture and concentrate in human blood four different species of Candida cells responsible for invasive yeast infections. In comparison with an automated blood culture, processing time of immunomagnetic separation is shorter, saving at least 24 hours to obtain colonies before identification. CONCLUSION: Thus, this easy to use method provides a promising basis for concentrating all Candida species in blood to improve sensitivity before identification.

Apical loop-internal loop RNA pseudoknots: a new type of stimulator of -1 translational frameshifting in bacteria.

Nearly all members of a widespread family of bacterial transposable elements related to insertion sequence 3 (IS3), therefore called the IS3 family, very likely use programmed -1 ribosomal frameshifting to produce their transposase, a protein required for mobility. Comparative analysis of the potential frameshift signals in this family suggested that most of the insertion sequences from the IS51 group contain in their mRNA an elaborate pseudoknot that could act as a recoding stimulator. It results from a specific intramolecular interaction between an apical loop and an internal loop from two stem-loop structures. Directed mutagenesis, chemical probing, and gel mobility assays of the frameshift region of one element from the IS51 group, IS3411, provided clear evidences of the existence of the predicted structure. Modeling was used to generate a three-dimensional molecular representation of the apical loop-internal loop complex. We could demonstrate that mutations affecting the stability of the structure reduce both frameshifting and transposition, thus establishing the biological importance of this new type of RNA structure for the control of transposition level.

Disruption of Candida albicans IFF4 gene involves modifications of the cell electrical surface properties.

During the past two decades, the prevalence of candidiasis has increased markedly and Candida albicans has now become one of the most important causes of nosocomial infections, especially after colonization of inert surfaces such as catheters or prostheses. In a previous report, we demonstrated the overexpression of 35 unidentified genes in response to adherence of C. albicans germ tubes to plastic. Therefore, a bioinformatic analysis was performed searching for genes encoding surface proteins potentially involved in adherence. Nineteen genes were thus selected, and one of them, CaIFF4, was further investigated. The deduced protein of this CaIFF4 gene revealed a glycosylphosphatidylinositol (GPI)-anchored site as well as the presence of a N-terminal signal peptide. Disruption of both alleles of CaIFF4 gene from C. albicans parent strain BWP17 was performed by PCR method. Then investigations of properties of null mutant for CaIFF4 gene showed a decrease of adherence of germ tubes to plastic in comparison to the parent strain BWP17. Besides, electrophoretic mobilities of germ tubes of CaIFF4 null mutant and of parental strain BWP17 were measured. Data were then analysed with soft particles analysis theory. Results point out a less important electrophoretic mobility of germ tubes of CaIFF4 null mutant in comparison to germ tubes of BWP17 parental strain.

DNA array analysis of Candida albicans gene expression in response to adherence to polystyrene.

Candidiasis is often initiated by the colonization of inert surfaces. In order to elucidate the mechanisms involved in this adherence process, DNA macroarrays were used to analyze the transcriptome of Candida albicans, the main causative agent of this mycoses, in a simple adherence model using germ tubes produced in polystyrene Petri dishes. Non-adherent germ tubes produced on glass surface were used as a control. Analysis of gene expression displayed 77 genes identified as statistically overexpressed in adherent germ tubes. Among these genes, some encoded enzymes participating in metabolism of lipids (such as LIP6), of proteins (such as SAP1) or of carbohydrates (like PGI1, PMI40 and PSA1. Some of these genes have already been reported as playing a role in pathogenesis of C. albicans. However, functions were unknown for a large part (45.5%) of the overexpressed genes which will be analyzed further in order to define their relationship with adherence.

-1 frameshifting at a CGA AAG hexanucleotide site is required for transposition of insertion sequence IS1222.

The discovery of programmed -1 frameshifting at the hexanucleotide shift site CGA_AAG, in addition to the classical X_XXY_YYZ heptanucleotide shift sequences, prompted a search for instances among eubacterial insertion sequence elements. IS1222 has a CGA_AAG shift site. A genetic analysis revealed that frameshifting at this site is required for transposition.

Programmed translational -1 frameshifting on hexanucleotide motifs and the wobble properties of tRNAs.

Programmed -1 ribosomal frameshifting, involving tRNA re-pairing from an AAG codon to an AAA codon, has been reported to occur at the sequences CGA AAG and CAA AAG. In this study, using the recoding region of insertion sequence IS3, we have investigated the influence on frameshifting in Escherichia coli of the first codon of this type of motif by changing it to all other NNA codons. Two classes of NNA codons were distinguished, depending on whether they favor or limit frameshifting. Their degree of shiftiness is correlated with wobble propensity, and base 34 modification, of their decoding tRNAs. A more flexible anticodon loop very likely makes the tRNAs with extended wobble more prone to liberate the third codon base, A, for re-pairing of tRNALys in the -1 frame.

Genetic variability of the frameshift region in IS911 transposable elements from Escherichia coli clinical isolates.

The IS911 bacterial transposable element has been analyzed for its mechanism of transposition and for the way it controls the expression of its genes by programmed -1 translational frameshifting. In the present study the prevalence of IS911 has been determined in the Enterobacteriaceae family and in other Gram-negative bacilli. Three variants, found in Escherichia coli clinical isolates and having mutations in the region implicated in frameshifting, were functionally characterized. All three were altered in their frameshifting and transposition abilities, suggesting that the frameshift region of IS911 may constitute a target for mutations reducing the transposition frequency of this mobile element in natural populations of E. coli.