Ambuic Acid Inhibits the Biosynthesis of Cyclic Peptide Quormones in Gram-Positive Bacteria

ABSTRACT Quorum sensing is a cell-density-dependent regulatory system in gram-positive bacteria and is often regulated by cyclic peptides called “quormones,” which function as extracellular communication signals. With an aim to discover an antipathogenic agent targeting quorum sensing in gram-positive bacteria, we screened 153 samples of fungal butanol extracts with the guidance of the inhibition of quorum-sensing-mediated gelatinase production in Enterococcus faecalis. Following the screenings, we found that ambuic acid, a known secondary fungal metabolite, inhibited the quorum-sensing-mediated gelatinase production without influencing the growth of E. faecalis. We further demonstrated that ambuic acid targeted the biosynthesis of a cyclic peptide quormone called gelatinase biosynthesis-activating pheromone. Furthermore, ambuic acid also inhibited the biosynthesis of the cyclic peptide quormones of Staphylococcus aureus and Listeria innocua. These results suggest the potential use of ambuic acid as a lead compound of antipathogenic drugs that target the quorum-sensing-mediated virulence expression of gram-positive bacteria.

Genomewide Screening for Genes Associated with Gliotoxin Resistance and Sensitivity in Saccharomyces cerevisiae

ABSTRACT Gliotoxin (GT) is a secondary fungal metabolite with pleiotropic immunosuppressive properties that have been implicated in Aspergillus virulence. However, the mechanisms of GT cytotoxicity and its molecular targets in eukaryotic cells have not been fully characterized. We screened a haploid library of Saccharomyces cerevisiae single-gene deletion mutants (4,787 strains in EUROSCARF) to identify nonessential genes associated with GT increased resistance (GT-IR) and increased sensitivity (GT-IS). The susceptibility of the wild-type parental strain BY4741 to GT was initially assessed by broth microdilution methods using different media. GT-IR and GT-IS were defined as a fourfold increase and decrease, respectively, in MIC, and this was additionally confirmed by susceptibility testing on agar yeast extract-peptone-glucose plates. The specificity of GT-IR and GT-IS mutants exhibiting normal growth compared with the wild-type strain was further tested in studies of their susceptibility to conventional antifungal agents, cycloheximide, and H2O2. GT-IR was associated with the disruption of genes acting in general metabolism (OPI1, SNF1, IFA38), mitochondrial function (RTG2), DNA damage repair (RAD18), and vesicular transport (APL2) and genes of unknown function (YGL235W, YOR345C, YLR456W, YGL072C). The disruption of three genes encoding transsulfuration (CYS3), mitochondrial function (MEF2), and an unknown function (YKL037W) led to GT-IS. Specificity for GT-IR and GT-IS was observed in all mutants. Importantly, the majority (69%) of genes implicated in GT-IR (6/10) and GT-IS (2/3) have human homologs. We identified novel Saccharomyces genes specifically implicated in GT-IR or GT-IS. Because most of these genes are evolutionarily conserved, further characterization of their function could improve our understanding of GT cytotoxicity mechanisms in humans.

Identification and In Vivo Functional Analysis by Gene Disruption of ctnA, an Activator Gene Involved in Citrinin Biosynthesis in Monascus purpureus

ABSTRACT Citrinin, a secondary fungal metabolite of polyketide origin, is moderately nephrotoxic to vertebrates, including humans. From the red-pigment producer Monascus purpureus, a 21-kbp region flanking pksCT, which encodes citrinin polyketide synthase, was cloned. Four open reading frames (ORFs) (orf1, orf2, orf3, and orf4) in the 5′-flanking region and one ORF (orf5) in the 3′-flanking region were identified in the vicinity of pksCT. orf1 to orf5 encode a homolog of a dehydrogenase (similarity, 46%), a regulator (similarity, 38%), an oxygenase (similarity, 41%), an oxidoreductase (similarity, 26%), and a transporter (similarity, 58%), respectively. orf2 (2,006 bp with four introns) encodes a 576-amino-acid protein containing a typical Zn(II)2Cys6 DNA binding motif at the N terminus and was designated ctnA. Although reverse transcriptase PCR analysis revealed that all of these ORFs, except for orf1, were transcribed with pksCT under citrinin production conditions, the disruption of ctnA caused large decreases in the transcription of pksCT and orf5, together with reduction of citrinin production to barely detectable levels, suggesting that these two genes are under control of the ctnA product. Complementation of the ctnA disruptant with intact ctnA on an autonomously replicating plasmid restored both transcription and citrinin production, indicating that CtnA is a major activator of citrinin biosynthesis.

Sex and Age Differences in the Distribution of 14C-Sterigmatocystin in Immature and Mature Rats: A Multiple Dose Study

Sterigmatocystin, a secondary fungal metabolite, produces a toxic reaction upon administration to experimental animals. Examination of rats that have received multiple doses of 14C-sterigmatocystin may indicate the response elicited by repeated exposure to foodstuffs containing the mycotoxin. Daily doses of sterigmatocystin, 8 mg/kg equally divided over 13 days, were administered in the feed. Immature rats (50–150 g) and mature rats (200–300 g) of both sexes were studied. On the 14th day, an 8 mg/kg dose of 14C-sterigmatocystin dissolved in wheat germ oil was orally intubated into the rats. Five rats of each sex and age group were sacrificed at 3, 6, 12, 24, 48, and 96 hours. Biological specimens were collected and analyzed for total radioactivity. Tukey's paired comparison procedure was used to analyze for statistically significant differences in tissue exposure to 14sterigmatocystin due to age and sex. Statistically significant differences in tissue levels were found in 60% or more of the tissues when comparing mature males vs. immature females and mature males vs. mature females. Age and sex differences were also observed in 45% or less of the tissues in other age or sex comparisons. The sterigmatocystin plasma levels vs. time curves contain multiple peaks that may indicate enterohepatic recirculation. The gradual decline in the log-linear phase resulted in long half-lives, ranging from 61.5 to 130 hours.