Small molecule inhibitors of Ago2 decrease Venezuelan equine encephalitis virus replication.

Venezuelan equine encephalitis virus (VEEV) is classified as a Category B Select Agent and potential bioterror weapon for its severe disease course in humans and equines and its potential for aerosol transmission. There are no current FDA licensed vaccines or specific therapies against VEEV, making identification of potential therapeutic targets a priority. With this aim, our research focuses on the interactions of VEEV with host microRNA (miRNA) machinery. miRNAs are small non-coding RNAs that act as master regulators of gene expression by downregulating or degrading messenger RNA, thus suppressing production of the resultant proteins. Recent publications implicate miRNA interactions in the pathogenesis of various viral diseases. To test the importance of miRNA processing for VEEV replication, cells deficient in Ago2, an important component of the RNA-induced silencing complex (RISC), and cells treated with known Ago2 inhibitors, notably acriflavine (ACF), were utilized. Both conditions caused decreased viral replication and capsid expression. ACF treatment promoted increased survival of neuronal cells over a non-treated, infected control and reduced viral titers of fully virulent VEEV as well as Eastern and Western Equine Encephalitis Viruses and West Nile Virus, but not Vesicular Stomatitis Virus. ACF treatment of VEEV TC-83 infected mice resulted in increased in vivo survival, but did not affect survival or viral loads when mice were challenged with fully virulent VEEV TrD. These results suggest that inhibition of Ago2 results in decreased replication of encephalitic alphaviruses in vitro and this pathway may be an avenue to explore for future therapeutic development.

Acriflavine treatment promotes dyskinetoplasty in Trypanosoma cruzi as revealed by ultrastructural analysis.

Trypanosomatid mitochondrial DNA is structured as a giant network of thousands of interlocked DNA molecules enclosed within the kinetoplast. The structure and replication mechanism of kinetoplast DNA (kDNA) is unique, thereby making it an excellent chemotherapeutic target. Alteration in the structural organization of kDNA can give rise to dyskinetoplastic (Dk) strains. In Dk cells, the kDNA is dispersed in clumps throughout the mitochondrial matrix and not organized into a network. In this work, Trypanosoma cruzi epimastigotes were treated with acriflavine, a DNA intercalating drug, which promoted a decrease in cell proliferation and induced the appearance of Dk protozoa. In treated cells, the kinetoplast lost its normal disc-shaped structure because the fibrillar arrangement was reduced to a compact, amorphous mass within the mitochondrion. Moreover, basic proteins associated with kDNA were redistributed throughout the Dk protozoal kinetoplast. We sought to understand how the disruption of the kDNA leads to the emergence of the Dk phenotype with atomic force microscopy (AFM) analysis of isolated networks. Our results demonstrate that the detachment of minicircles from the kDNA disk promotes the disassembly of the network, thereby generating Dk cells. Our data strongly suggest that acriflavine inhibits T. cruzi multiplication by interfering with kDNA replication.

SmvA, and not AcrB, is the major efflux pump for acriflavine and related compounds in Salmonella enterica serovar Typhimurium.

OBJECTIVES: The aim was to study the role played by SmvA pump in the efflux of quaternary ammonium compounds (QACs) in Salmonella enterica serovar Typhimurium (Salmonella Typhimurium). METHODS: Mutants in the smvA, acrB and tolC genes were constructed by the red swap method. P22 was used to transduce tolC to acrB and smvA mutant strains. The susceptibility of these strains to acriflavine and a variety of QACs was determined by MIC assays. RESULTS: In comparison with the Salmonella Typhimurium wild-type strain, the smvA mutant was more susceptible to QACs than the acrB mutant strain. A tolC single mutant was more susceptible than an acrB mutant to QACs, acriflavine, ethidium bromide, malachite green and pyronin B. The tolC-acrB double mutant was as susceptible as the single tolC mutant to QACs. Additionally, the smvA mutant strain was more susceptible to acriflavine than the acrB mutant (MICs = 31.3 versus 125 mg/L, i.e. 4-fold). Finally, the tolC-smvA double mutant (3.9 mg/L) was approximately 10 times more susceptible to acriflavine than either smvA (31.3 mg/L) or tolC (31.3 mg/L) single mutants. CONCLUSIONS: It is the SmvA efflux pump, and not AcrB, that plays the major role in the efflux of acriflavine and other QACs from Salmonella Typhimurium. This apparently conflicting report is due to the fact that in Escherichia coli the smvA gene does not exist. Our results suggest that tolC and smvA genes encode components of two different efflux systems with overlapping specificities that work in parallel to export acriflavine and other QACs.

Over-expression of genes coding for proline oxidase, riboflavin kinase, cytochrome c oxidase and an MFS transporter induced by acriflavin in Trichophyton rubrum.

Acriflavin (3,6-acridinediamine) and other acridine derivatives act in both prokaryotic and eukaryotic cells at the level of DNA-coiling enzymes (topoisomerases) causing the stabilization of the enzyme-DNA cleavable complex. In order to better understand the mode of action of acriflavin, Differential Display RT-PCR was used to isolate transcripts specifically over-expressed during exposure of Trichophyton rubrum mycelia to this drug. Five transcripts, whose differential expressions were confirmed by Northern blotting, revealed genes not previously described in this dermatophyte. Functional grouping identified putative enzymes possibly involved in the mitochondrial respiratory electron-transport chain and in iron transport. These results may be relevant to our understanding of the molecular events involved in the stress response of T. rubrum to acriflavin.

The gene that determines resistance to tioconazole and to acridine derivatives in Aspergillus nidulans may have a corresponding gene in Trichophyton rubrum.

Understanding the genetic mechanisms involved in resistance to antifungal agents is important in the fight against pathogenic fungi. In the present investigation we studied a strain of the model fungus Aspergillus nidulans which presents resistance to tioconazole and behaves as the wild strain in the presence of other azole derivatives. Genetic analysis revealed that this resistance is due to a mutation in a single gene located on chromosome II, closely linked to the allele responsible for resistance to acriflavine and other acridine derivatives, i.e., acrA1. This result suggests that a multidrug resistance (MDR)-type mechanisms may be involved. Two tioconazole-resistant strains of the pathogenic fungus Trichophyton rubrum obtained after mutagenic treatment also became simultaneously resistant to acriflavine and ethidium bromide, suggesting the existence of a resistance mechanism similar to that observed with the acrA1 mutation in A. nidulans.

[Effect of different dairy products on the growth of Listeria monocytogenes in selective media]. / Influencia de diferentes derivados lácteos sobre el crecimiento de Listeria monocytogenes en medios selectivos.

We have studied the influence of the incorporation of milk cream and skim milk on the growth kinetic of Listeria monocytogenes in listeria enrichment broth with 15 mg/l or 7.5 mg/l of acriflavine. Acriflavine was responsible, at least partially, for delayed growth of Listeria monocytogenes in the enrichment broths. A longer lag phase of the growth was produced by the addition of milk cream or skim milk to the enrichment broth containing 7.5 mg/l of acriflavine. However, the maximum population obtained at 48 h did not show significant differences. In the presence of 15 mg/l of acriflavine, we observed a decrease of the viable counts during the early phase of the growth cycle, which was enhanced by the addition of milk cream or skim milk. Moreover, the maximum growth rate was reduced by the addition of skim milk and maximum population was not reached at 48 h. These results suggest the need to validate the methodology of recuperation of Listeria monocytogenes from each dairy product, since its efficiency may be affected by product composition, specially when the sample biocharge is low.

A plasmid involved in chloramphenicol production in Streptomyces venezuelae: evidence from genetic mapping.

To test the hypothesis that chloramphenicol production in Streptomyces venezuelae depends on the presence of a plasmid, mapping analysis was carried out by using eight markers in addition to chloramphenicol production and melanoid pigment formation. The sequence of the eight markers was determined on a circular linkage map as follows: -his-ade-str-leu-lys-met-ilv-pro-(his-). This sequence resulted in the frequency of quadruple crossover (q.c.o.) recombinants having the lowest value, 3-2 to 4-9%. However, the character of chloramphenicol non-production, which was obtained by incubating mycelia with acriflavin, was not required to explain the results. From these results and other tests, it is concluded that chloramphenicol production is controlled by a plasmid. This plasmid appeared to be non-transferable in conjugation.