The sirodesmin biosynthetic gene cluster of the plant pathogenic fungus Leptosphaeria maculans

Sirodesmin PL is a phytotoxin produced by the fungus Leptosphaeria maculans, which causes blackleg disease of canola (Brassica napus). This phytotoxin belongs to the epipolythiodioxopiperazine (ETP) class of toxins produced by fungi including mammalian and plant pathogens. We report the cloning of a cluster of genes with predicted roles in the biosynthesis of sirodesmin PL and show via gene disruption that one of these genes (encoding a two-module non-ribosomal peptide synthetase) is essential for sirodesmin PL biosynthesis. Of the nine genes in the cluster tested, all are co-regulated with the production of sirodesmin PL in culture. A similar cluster is present in the genome of the opportunistic human pathogen Aspergillus fumigatus and is most likely responsible for the production of gliotoxin, which is also an ETP. Homologues of the genes in the cluster were also identified in expressed sequence tags of the ETP producing fungus Chaetomium globosum. Two other fungi with publicly available genome sequences, Magnaporthe grisea and Fusarium graminearum, had similar gene clusters. A comparative analysis of all four clusters is presented. This is the first report of the genes responsible for the biosynthesis of an ETP.     

Bioinformatic and expression analysis of the putative gliotoxin biosynthetic gene cluster of Aspergillus fumigatus

Gliotoxin is a secondary metabolite produced by several fungi including the opportunistic animal pathogen Aspergillus fumigatus. It is a member of the epipolythiodioxopiperazine (ETP) class of toxins characterised by a disulphide bridged cyclic dipeptide. A putative cluster of 12 genes involved in gliotoxin biosynthesis has been identified in A. fumigatus by a comparative genomics approach based on homology to genes from the sirodesmin (another ETP) biosynthetic gene cluster of Leptosphaeria maculans. The physical limits of the cluster in A. fumigatus have been defined by bioinformatics and by identifying the genes that are co-regulated and whose timing of expression correlates with the production of gliotoxin in culture.     

Pathways leading from BarA/SirA to motility and virulence gene expression in Salmonella

The barA and sirA genes of Salmonella enterica serovar Typhimurium encode a two-component sensor kinase and a response regulator, respectively. This system increases the expression of virulence genes and decreases the expression of motility genes. In this study, we examined the pathways by which SirA affects these genes. We found that the master regulator of flagellar genes, flhDC, had a positive regulatory effect on the primary regulator of intestinal virulence determinants, hilA, but that hilA had no effect on flhDC. SirA was able to repress flhDC in a hilA mutant and activate hilA in an flhDC mutant. Therefore, although the flhDC and hilA regulatory cascades interact, sirA affects each of them independently. A form of BarA lacking the two N-terminal membrane-spanning domains, BarA198, autophosphorylates in the presence of ATP and transfers the phosphate to purified SirA. Phosphorylated SirA was found to directly bind the hilA and hilC promoters in gel mobility shift assays but not the flhD, fliA, hilD, and invF promoters. Given that the CsrA/csrB system is known to directly affect flagellar gene expression, we tested the hypothesis that SirA affects flagellar gene expression indirectly by regulating csrA or csrB. The sirA gene did not regulate csrA but did activate csrB expression. Consistent with these results, phosphorylated SirA was found to directly bind the csrB promoter but not the csrA promoter. We propose a model in which SirA directly activates virulence expression via hilA and hilC while repressing the flagellar regulon indirectly via csrB.     

Disruption of a nonribosomal peptide synthetase in Aspergillus fumigatus eliminates gliotoxin production

The fungal secondary metabolite gliotoxin produced by Aspergillus fumigatus has been hypothesized to be important in the development of invasive aspergillosis. In this study, we addressed this hypothesis by disrupting a nonribosomal peptide synthetase (NRPS) (encoded by gliP) predicted to be involved in gliotoxin production. Mutants with a disrupted gliP locus failed to produce gliotoxin, which confirmed the role of the NRPS encoded by gliP in gliotoxin biosynthesis. We found no morphological, developmental, or physiological defects in DeltagliP mutant strains. In addition, disruption of gliP resulted in down regulation of gene expression in the gliotoxin biosynthesis gene cluster, which was restored with addition of exogenous gliotoxin. This interesting result suggests a role for gliotoxin in regulating its own production. Culture filtrates from the DeltagliP mutant were unable to inhibit ionomycin-dependent degranulation of mast cells, suggesting a role for gliotoxin in suppressing mast cell degranulation and possibly in disease development. However, the DeltagliP mutant did not have an impact on survival or tissue burden in a murine inhalational model of invasive aspergillosis. This result suggests that gliotoxin is not required for virulence in an immunosuppressed host with an invasive pulmonary infection.     

致病菌烟曲霉新基因Afu4g13170生孢致毒相关性初步研究

【目的】对烟曲霉Afu4g13170基因功能进行初步研究。【方法】利用Double-jointPCR方法和一步基因敲除技术,构建Afu4g13170基因缺失突变株。【结果】序列比对表明烟曲霉Afu4g13170蛋白与构巢曲霉Ani04163蛋白和新型隐球菌Gib2蛋白的氨基酸序列相似性为88.6%;表型分析表明基因破坏使突变株生长迟缓、梗基伸长、孢子分化能力下降,产孢推迟、产孢量减少,色素产生量降低;色谱分析显示基因缺失突变株的产毒能力下降。【结论】烟曲霉Afu4g13170基因可以作为控制曲霉致毒的一个靶位点。     

Gliotoxin effects on fungal growth: mechanisms and exploitation

Although initially investigated for its antifungal properties, little is actually known about the effect of gliotoxin on Aspergillus fumigatus and other fungi. We have observed that exposure of A. fumigatus to exogenous gliotoxin (14 μg/ml), under gliotoxin-limited growth conditions, results in significant alteration of the expression of 27 proteins (up- and down-regulated >1.9-fold; p<0.05) including de novo expression of Cu, Zn superoxide dismutase, up-regulated allergen Asp f3 expression and down-regulated catalase and a peroxiredoxin levels. Significantly elevated glutathione GSH levels (p<0.05), along with concomitant resistance to diamide, were evident in A. fumigatus ΔgliT, lacking gliotoxin oxidoreductase, a gliotoxin self-protection gene. Saccharomyces cerevisiae deletents (Δsod1 and Δyap1) were hypersensitive to exogenous gliotoxin, while Δgsh1 was resistant. Significant gliotoxin-mediated (5 μg/ml) growth inhibition (p<0.001) of Aspergillus nidulans, Aspergillus terreus, Aspergillus niger, Cochliobolus heterostrophus and Neurospora crassa was also observed. Growth of Aspergillus flavus, Fusarium graminearum and Aspergillus oryzae was significantly inhibited (p<0.001) at gliotoxin (10 μg/ml), indicating differential gliotoxin sensitivity amongst fungi. Re-introduction of gliT into A. fumigatus ΔgliT, at a different locus (ctsD; AFUA_4G07040, an aspartic protease), with selection on gliotoxin, facilitated deletion of ctsD without use of additional antibiotic selection markers. Absence of ctsD expression was accompanied by restoration of gliT expression, and resistance to gliotoxin. Thus, we propose gliT/gliotoxin as a useful selection marker system for fungal transformation. Finally, we suggest incorporation of gliotoxin sensitivity assays into all future fungal functional genomic studies.     

Antifungal activity of microbial secondary metabolites

Secondary metabolites are well known for their ability to impede other microorganisms. Reanalysis of a screen of natural products using the Caenorhabditis elegans-Candida albicans infection model identified twelve microbial secondary metabolites capable of conferring an increase in survival to infected nematodes. In this screen, the two compound treatments conferring the highest survival rates were members of the epipolythiodioxopiperazine (ETP) family of fungal secondary metabolites, acetylgliotoxin and a derivative of hyalodendrin. The abundance of fungal secondary metabolites indentified in this screen prompted further studies investigating the interaction between opportunistic pathogenic fungi and Aspergillus fumigatus, because of the ability of the fungus to produce a plethora of secondary metabolites, including the well studied ETP gliotoxin. We found that cell-free supernatant of A. fumigatus was able to inhibit the growth of Candida albicans through the production of a secreted product. Comparative studies between a wild-type and an A. fumigatus ΔgliP strain unable to synthesize gliotoxin demonstrate that this secondary metabolite is the major factor responsible for the inhibition. Although toxic to organisms, gliotoxin conferred an increase in survival to C. albicans-infected C. elegans in a dose dependent manner. As A. fumigatus produces gliotoxin in vivo, we propose that in addition to being a virulence factor, gliotoxin may also provide an advantage to A. fumigatus when infecting a host that harbors other opportunistic fungi.     

Gliotoxin in <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis>: an example that mycotoxins are potential virulence factors

Moulds produce several different mycotoxins that may improve their chance of survival in particular environments. For example, Aspergillus fumigatus, an important human pathogen, produces several mycotoxins including gliotoxin. This secondary metabolite, a small lipid soluble dipeptide, exerts toxic effects on phagocytic cells and T-lymphocytes at low concentrations in vitro. A. fumigatus also produces high levels of gliotoxin in vivo, and this suggests that host defense mechanisms might be impaired by this metabolite during host infection. In the past few years, the genes responsible for the production of gliotoxin in A. fumigatus have been identified and more recently gliotoxin-minus mutants have been used in animal experiments to ascertain the biological role of this product. Mycotoxins have also been shown to act as virulence factors in some fungal infections of insects and plants.     

Biosynthetic gene clusters for epipolythiodioxopiperazines in filamentous fungi

Epipolythiodioxopiperazines (ETPs) are toxic secondary metabolites made only by fungi. Recent research efforts have provided insight into the molecular mechanisms of the toxicity of these compounds. The availability of complete genome sequences for many fungi has also facilitated the identification of putative ETP biosynthetic gene clusters. Expression and mutational analyses have confirmed the role of such gene clusters in the biosynthesis of two ETPs, sirodesmin PL and gliotoxin. The creation of mutants unable to produce these ETPs has facilitated the assessment of these molecules as virulence factors in interactions between pathogenic fungi and their hosts.     

Gliotoxin is a virulence factor of Aspergillus fumigatus: gliP deletion attenuates virulence in mice immunosuppressed with hydrocortisone

Gliotoxin is an immunosuppressive mycotoxin long suspected to be a potential virulence factor of Aspergillus fumigatus. Recent studies using mutants lacking gliotoxin production, however, suggested that the mycotoxin is not important for pathogenesis of A. fumigatus in neutropenic mice resulting from treatment with cyclophosphomide and hydrocortisone. In this study, we report on the pathobiological role of gliotoxin in two different mouse strains, 129/Sv and BALB/c, that were immunosuppressed by hydrocortisone alone to avoid neutropenia. These strains of mice were infected using the isogenic set of a wild type strain (B-5233) and its mutant strain (gliPDelta) and the the glip reconstituted strain (gliP(R)). The gliP gene encodes a nonribosomal peptide synthase that catalyzes the first step in gliotoxin biosynthesis. The gliPDelta strain was significantly less virulent than strain B-5233 or gliP(R) in both mouse models. In vitro assays with culture filtrates (CFs) of B-5233, gliPDelta, and gliP(R) strains showed the following: (i) deletion of gliP abrogated gliotoxin production, as determined by high-performance liquid chromatography analysis; (ii) unlike the CFs from strains B-5233 and gliP(R), gliPDelta CFs failed to induce proapoptotic processes in EL4 thymoma cells, as tested by Bak conformational change, mitochondrial-membrane potential disruption, superoxide production, caspase 3 activation, and phosphatidylserine translocation. Furthermore, superoxide production in human neutrophils was strongly inhibited by CFs from strain B-5233 and the gliP(R) strain, but not the gliPDelta strain. Our study confirms that gliotoxin is an important virulence determinant of A. fumigatus and that the type of immunosuppression regimen used is important to reveal the pathogenic potential of gliotoxin.     

Gene expression profiling of polymorphonuclear leukocytes treated with the culture filtrate of Aspergillus fumigatus and gliotoxin

Pathogens of the Aspergillus species are frequently seen in deep-seated mycoses. We previously demonstrated that the culture filtrate of Aspergillus fumigatus (CF) has immunosuppressive effects on polymorphonuclear leukocytes (PMNs), which act as the main phagocytes to hyphae of Aspergillus fumigatus (A. fumigatus). But little is known about the gene expression profiles involved in it. Therefore we investigated the changes in gene expression in human PMNs treated with CF or gliotoxin at two time points, using microarray analysis. CF and gliotoxin changed the expression of 548 and 381 genes, respectively. Only 51 genes showed the same expression patterns with the two stimulants, and CF-induced changes in gene expression occurred comparatively earlier than those induced by gliotoxin. Among 31 genes encoding apoptosis, which were up- or down-regulated in this assay, only 3 genes were similarly changed by both kinds of stimulation. Apoptosis was detected and quantified using two apoptosis assays. CF and gliotoxin changed the expessions of only 3 out of 19 regulated genes related to inflammatory mediators and receptors similarly. The up-regulation of the gene encoding annexin 1 (ANXA1), which is known to be involved in extravasation and apoptosis of neutrophils, may play a role in the immunosuppressive effect of A. fumigatus. The difference in expression changes between CF and gliotoxin is presumed to be caused by the interaction among the components of CF and therefore the interaction is an area of interest for further investigation.