RNA silencing in the dermatophyte Microsporum canis

Dermatomycoses caused by Microsporum canis are frequent in domestic animals and easily transmissible to humans. Several proteases secreted by this fungus were identified as potential virulence factors, but the construction of deficient strains is required to investigate their role in the pathogenesis of the disease. Using target genes encoding two of these proteases, a first evaluation of the utility of RNA-mediated silencing as a reverse genetic tool in dermatophytes was carried out. SUB3 and DPPIV, respectively coding for a subtilisin and a dipeptidyl peptidase, were both down-regulated, by means of two plasmid constructs designed to express an RNA hairpin that corresponds to part of their respective sequence. The degree of attenuation was evaluated by enzymatic assay of the transformants culture supernatants, and by real-time reverse transcriptase-polymerase chain reaction. Enzymatic activities and expression levels varied from less than 5% to 100% of that of control transformants obtained with plasmid without hairpin inserts. Inhibition was globally more efficient for SUB3 than for DPPIV. These results show that RNA silencing can be used for functional genomics in M. canis, and particularly to circumvent the limits and technical difficulties of conventional disruption method

RNA silencing in the dermatophyte Microsporum canis

Dermatomycoses caused by Microsporum canis are frequent in domestic animals and easily transmissible to humans. Several proteases secreted by this fungus were identified as potential virulence factors, but the construction of deficient strains is required to investigate their role in the pathogenesis of the disease. Using target genes encoding two of these proteases, a first evaluation of the utility of RNA-mediated silencing as a reverse genetic tool in dermatophytes was carried out. SUB3 and DPPIV, respectively coding for a subtilisin and a dipeptidyl peptidase, were both down-regulated, by means of two plasmid constructs designed to express an RNA hairpin that corresponds to part of their respective sequence. The degree of attenuation was evaluated by enzymatic assay of the transformants culture supernatants, and by real-time reverse transcriptase-polymerase chain reaction. Enzymatic activities and expression levels varied from less than 5% to 100% of that of control transformants obtained with plasmid without hairpin inserts. Inhibition was globally more efficient for SUB3 than for DPPIV. These results show that RNA silencing can be used for functional genomics in M. canis, and particularly to circumvent the limits and technical difficulties of conventional disruption method

Effect of pollen extract supplementation on the varroatosis tolerance of honey bee (Apis mellifera) larvae reared in vitro

International audienc

Impact of the Phoretic Phase on Reproduction and Damage Caused by Varroa destructor (Anderson and Trueman) to Its Host, the European Honey Bee (Apis mellifera L.).

Varroa destructor is a parasitic mite of the honeybee that causes thousands of colony losses worldwide. The parasite cycle is composed of a phoretic and a reproductive phase. During the former, mites stay on adult bees, mostly on nurses, to feed on hemolymph. During the latter, the parasites enter brood cells and reproduce. We investigated if the type of bees on which Varroa stays during the phoretic phase and if the duration of this stay influenced the reproductive success of the parasite and the damage caused to bees. For that purpose, we used an in vitro rearing method developed in our laboratory to assess egg laying rate and the presence and number of fully molted daughters. The expression level of two Varroa vitellogenin genes (VdVg1 and VdVg2), known to vary throughout reproduction, was also quantified. Results showed that the status of the bees or time spent during the phoretic phase impacts neither reproduction parameters nor the Varroa vitellogenin genes levels of expression. However, we correlated these parameters to the gene expression and demonstrated that daughters expressed the vitellogenin genes at lower levels than their mother. Regarding the damage to bees, the data indicated that a longer stay on adult bees during the phoretic phase resulted in more frequent physical deformity in newborn bees. We showed that those mites carry more viral loads of the Deformed Wing Virus and hence trigger more frequently overt infections. This study provides new perspectives towards a better understanding of the Varroa-honeybee interactions

Immunization and Dermatophytes

PURPOSE OF REVIEW: Despite the availability of effective vaccines for certain animal species, vaccination against dermatophytosis requires improvement and further development in both animals and humans. This review provides an update on the current situation and focuses on recent advances in host-dermatophyte relationships that could have implications for future vaccination against the most prevalent of the fungal diseases. RECENT FINDINGS: Numerous dermatophytic virulence factors have recently been isolated and characterized at the molecular level, notably secreted proteases involved in the invasion of the keratin network. Their precise roles in the different steps of the infectious process and in immunopathogenesis are being studied, while all aspects of the host immune response against dermatophytes, including the innate response, are becoming increasingly documented. In addition, new molecular tools are now available for studying dermatophytes, which will accelerate research on this topic. SUMMARY: The growth of knowledge concerning all aspects of the host-dermatophyte relationship should contribute towards sound strategies for the development of effective and safe vaccines against dermatophytosis

Secreted dipeptidyl peptidases as potential virulence factors for Microsporum canis.

Dermatophytoses caused by Microsporum canis are frequently encountered in cats and dogs; they are highly contagious and readily transmissible to humans. In this study, two single genes, respectively coding for dipeptidyl peptidases IV and V (DppIV and DppV), were isolated and characterized. Both proteins share homology with serine proteases of the S9 family, some of which display properties compatible with implication in pathogenic processes. Both genes are expressed in vivo in experimentally infected guinea-pigs and in naturally infected cats, and when the fungus is grown on extracellular matrix proteins as the sole nitrogen and carbon source. DppIV and V were produced as active recombinant proteases in the yeast Pichia pastoris; the apparent molecular weight of rDppV is 83 kDa, whereas rDppIV appears as a doublet of 95 and 98 kDa. Like other members of its enzymatic subfamily, rDppIV has an unusual ability to cleave Pro-X bonds. This activity does not enhance the solubilization of keratin by fungal secreted endoproteases, and the protease probably acts solely on small soluble peptides. RDppV showed no ability to induce delayed-type hypersensitivity (DTH) skin reactions in guinea-pigs, despite the known immunogenic properties of homologous proteins

Reproductive parameters of mites in relation to the treatment and emergence success of the bee.

<p>A) Oviposition proportion and percentage of cells with at least one mature daughter per foraging condition. Bars show the overall rate ± IC95 (not significant). B) Mean numbers of fully molted daughters in relation to the emergence success of the bee. Mean±SE Non molted (n = 11), imago stage (n = 113).</p

Schematic representation of the experimental design applied to the three hives.

<p>Mites experience a different phoretic phase and are then transferred onto spinning larvae to pursue their reproductive cycle in laboratory conditions. Numbers correspond to the moments when mites were sampled to conduct molecular analyses: (i) before the beginning of the phoretic phase; (ii) immediately after the phoretic phase; (iii) three days after the transfer on spinning larvae, at which point the bee is in the last stage of the pharate pupal (or prepupal) period (PP3); (iv) ten days after the transfer on spinning larvae corresponding to the medium pigmented body pupal stage (<i>pbm</i>) for the bees; (v) at the emergence of the bee.</p