Polyenic Antibiotics and Other Antifungal Compounds Produced by Hemolytic Streptomyces Species.

Streptomyces are of great interest in the pharmaceutical industry as they produce a plethora of secondary metabolites that act as antibacterial and antifungal agents. They may thrive on their own in the soil, or associate with other organisms, such as plants or invertebrates. Some soil-derived strains exhibit hemolytic properties when cultivated on blood agar, raising the question of whether hemolysis could be a virulence factor of the bacteria. In this work we examined hemolytic compound production in 23 ß-hemolytic Streptomyces isolates; of these 12 were soil-derived, 10 were arthropod-associated, and 1 was plant-associated. An additional human-associated S. sp. TR1341 served as a control. Mass spectrometry analysis suggested synthesis of polyene molecules responsible for the hemolysis: candicidins, filipins, strevertene A, tetrafungin, and tetrin A, as well as four novel polyene compounds (denoted here as polyene A, B, C, and D) in individual liquid cultures or paired co-cultures. The non-polyene antifungal compounds actiphenol and surugamide A were also identified. The findings indicate that the ability of Streptomyces to produce cytolytic compounds (here manifested by hemolysis on blood agar) is an intrinsic feature of the bacteria in the soil environment and could even serve as a virulence factor when colonizing available host organisms. Additionally, a literature review of polyenes and non-polyene hemolytic metabolites produced by Streptomyces is presented.

Evaluation and comparison of antibiotic susceptibility profiles of Streptomyces spp. from clinical specimens revealed common and region-dependent resistance patterns.

Notwithstanding the fact that streptomycetes are overlooked in clinical laboratories, studies describing their occurrence in disease and potential pathogenicity are emerging. Information on their species diversity in clinical specimens, aetiology and appropriate therapeutic treatment is scarce. We identified and evaluated the antibiotic susceptibility profile of 84 Streptomyces clinical isolates from the Czech Republic. In the absence of appropriate disk diffusion (DD) breakpoints for antibiotic susceptibility testing (AST) of Streptomyces spp., we determined DD breakpoints by correlation with the broth microdilution method and by the distribution of zone diameters among isolates. Correlation accuracy was high for 9 antibiotics, leading to the establishment of the most valid DD breakpoints for Streptomyces antibiotic susceptibility evaluation so far. Clinical strains belonged to 17 different phylotypes dominated by a cluster of strains sharing the same percentage of 16S rRNA gene sequence identity with more than one species (S. albidoflavus group, S. hydrogenans, S. resistomycificus, S. griseochromogenes; 70% of isolates). AST results showed that Streptomyces exhibited intrinsic resistance to penicillin, general susceptibility to amikacin, gentamycin, vancomycin and linezolid, and high percentage of susceptibility to tetracyclines and clarithromycin. For the remaining antibiotics, AST showed inter- and intra-species variations when compared to available literature (erythromycin, trimethoprim-sulfamethoxazole), indicating a region-dependent rather than species-specific patterns.

The Genome Analysis of the Human Lung-Associated Streptomyces sp. TR1341 Revealed the Presence of Beneficial Genes for Opportunistic Colonization of Human Tissues.

Streptomyces sp. TR1341 was isolated from the sputum of a man with a history of lung and kidney tuberculosis, recurrent respiratory infections, and COPD. It produces secondary metabolites associated with cytotoxicity and immune response modulation. In this study, we complement our previous results by identifying the genetic features associated with the production of these secondary metabolites and other characteristics that could benefit the strain during its colonization of human tissues (virulence factors, modification of the host immune response, or the production of siderophores). We performed a comparative phylogenetic analysis to identify the genetic features that are shared by environmental isolates and human respiratory pathogens. The results showed a high genomic similarity of Streptomyces sp. TR1341 to the plant-associated Streptomyces sp. endophyte_N2, inferring a soil origin of the strain. Putative virulence genes, such as mammalian cell entry (mce) genes were not detected in the TR1341's genome. The presence of a type VII secretion system, distinct from the ones found in Mycobacterium species, suggests a different colonization strategy than the one used by other actinomycete lung pathogens. We identified a higher diversity of genes related to iron acquisition and demonstrated that the strain produces ferrioxamine B in vitro. These results indicate that TR1341 may have an advantage in colonizing environments that are low in iron, such as human tissue.

Activation of a Cryptic Manumycin-Type Biosynthetic Gene Cluster of Saccharothrix espanaensis DSM44229 by Series of Genetic Manipulations.

(1) Background: Manumycins are small actinomycete polyketides with prominent cancerostatic and immunosuppressive activities via inhibition of various eukaryotic enzymes. Their overall activity towards human cells depends on the structural variability of both their polyketide chains, mainly the upper one. In our genetic screening project to find novel producers of anti-inflammatory manumycins, the strain Saccharothrix espanaensis DSM44229 was identified as containing a novel manumycin-type biosynthetic gene cluster (BGC). (2) Methods: The biosynthetic genes appeared to be silent under all assayed laboratory conditions. Several techniques were used to activate the BGC, including: (i) heterologous expression in various hosts, (ii) overexpression of putative pathway-specific regulatory genes, and (iii) overexpression of a bottleneck cyclizing aminolevulinate synthase gene in both natural and heterologous producers. (3) Results: Multiple novel manumycin-type compounds were produced at various levels by genetically-modified strains, sharing a tetraene lower chain structure with a colabomycin subgroup of manumycins, but possessing much shorter and saturated upper chains. (4) Conclusions: A cryptic manumycin-type BGC was successfully activated by genetic means to gain production of novel manumycin-type compounds for future comparative activity assays. Heterologously produced compounds were identical to those found after final activation of the BGC in the original strain, proving the intactness of the cloned BGC.

Inhibition of Pro-Inflammatory Cytokines by Metabolites of Streptomycetes-A Potential Alternative to Current Anti-Inflammatory Drugs?

Current treatment of chronic diseases includes, among others, application of cytokines, monoclonal antibodies, cellular therapies, and immunostimulants. As all the underlying mechanisms of a particular diseases are not always fully clarified, treatment can be inefficient and associated with various, sometimes serious, side effects. Small secondary metabolites produced by various microbes represent an attractive alternative as future anti-inflammatory drug leads. Compared to current drugs, they are cheaper, can often be administered orally, but still can keep a high target-specificity. Some compounds produced by actinomycetes or fungi have already been used as immunomodulators-tacrolimus, sirolimus, and cyclosporine. This work documents strong anti-inflammatory features of another secondary metabolite of streptomycetes-manumycin-type polyketides. We compared the effect of four related compounds: manumycin A, manumycin B, asukamycin, and colabomycin E on activation and survival of human monocyte/macrophage cell line THP-1. The anti-cancer effect of manucycine A has been demonstrated; the immunomodulatory capacities of manumycin A are obvious when using micromolar concentrations. The application of all four compounds in 0.25-5 µM concentrations leads to efficient, concentration-dependent inhibition of IL-1ß and TNF expression in THP-1 upon LPS stimulation, while the three latter compounds show a significantly lower pro-apoptotic effect than manumycin A. We have demonstrated the anti-inflammatory capacity of selected manumycin-type polyketides.

A Human Lung-Associated Streptomyces sp. TR1341 Produces Various Secondary Metabolites Responsible for Virulence, Cytotoxicity and Modulation of Immune Response.

Streptomycetes, typical soil dwellers, can be detected as common colonizers of human bodies, especially the skin, the respiratory tract, the guts and the genital tract using molecular techniques. However, their clinical manifestations and isolations are rare. Recently they were discussed as possible "coaches" of the human immune system in connection with certain immune disorders and cancer. This work aimed for the characterization and evaluation of genetic adaptations of a human-associated strain Streptomyces sp. TR1341. The strain was isolated from sputum of a senior male patient with a history of lung and kidney TB, recurrent respiratory infections and COPD. It manifested remarkably broad biological activities (antibacterial, antifungal, beta-hemolytic, etc.). We found that, by producing specific secondary metabolites, it is able to modulate host immune responses and the niche itself, which increase its chances for long-term survival in the human tissue. The work shows possible adaptations or predispositions of formerly soil microorganism to survive in human tissue successfully. The strain produces two structural groups of cytotoxic compounds: 28-carbon cytolytic polyenes of the filipin type and actinomycin X2. Additionally, we summarize and present data about streptomycete-related human infections known so far.

Secondary Metabolites Produced during the Germination of Streptomyces coelicolor.

Spore awakening is a series of actions that starts with purely physical processes and continues via the launching of gene expression and metabolic activities, eventually achieving a vegetative phase of growth. In spore-forming microorganisms, the germination process is controlled by intra- and inter-species communication. However, in the Streptomyces clade, which is capable of developing a plethora of valuable compounds, the chemical signals produced during germination have not been systematically studied before. Our previously published data revealed that several secondary metabolite biosynthetic genes are expressed during germination. Therefore, we focus here on the secondary metabolite production during this developmental stage. Using high-performance liquid chromatography-mass spectrometry, we found that the sesquiterpenoid antibiotic albaflavenone, the polyketide germicidin A, and chalcone are produced during germination of the model streptomycete, S. coelicolor. Interestingly, the last two compounds revealed an inhibitory effect on the germination process. The secondary metabolites originating from the early stage of microbial growth may coordinate the development of the producer (quorum sensing) and/or play a role in competitive microflora repression (quorum quenching) in their nature environments.

Manumycin A downregulates release of proinflammatory cytokines from TNF alpha stimulated human monocytes.

Macrolide antibiotics such as azithromycin or clarithromycin are known to have potent anti-inflammatory and immunomodulatory effects but these properties cannot be widely used due to a risk of bacterial resistance. We studied another polyketide antibiotic, structurally related manumycin A known as a streptomycete derived farnesyltransferase inhibitor with limited antibacterial effects, with respect to its potential regulation of mRNA expression of several genes associated with proinflammatory responses. Downregulation of mRNA for IL-6, TLR-8, IL-1 beta and IL-10 was found in THP-1 cells after 4h stimulation with TNF alpha in the presence of manumycin A and downregulated TLR-8 and EGR-1 genes were observed after 8h. Among the genes upregulated in response to manumycin were HMOX-1, TNFRSF10A, IL-1R1, TICAM2, NLRP12 after 4h and only IL-1R1 after 8h. Furthermore, manumycin A was found to inhibit IL-1beta, IL-6, and IL-8 production in TNF alpha stimulated THP-1 cells and peripheral blood monocytes in a dose dependent manner (0.25-1 µM of manumycin A) without affecting cell viability. Cell viability of blood monocytes decreased by about 30% at manumycin A doses of 2-5 µM. Manumycin A also inhibited IL-18 release from THP-1 cells, while in cultures of blood monocytes, this cytokine was not detectable. That manumycin A mediated downregulation of proinflammatory genes in human monocytes confirmed by a measurement of cytokine levels in culture supernatants, together with a very limited effect on cell viability, might suggest potential anti-inflammatory properties of this polyketide antibiotic.

Evolution of cyclizing 5-aminolevulinate synthases in the biosynthesis of actinomycete secondary metabolites: outcomes for genetic screening techniques.

A combined approach, comprising PCR screening and genome mining, was used to unravel the diversity and phylogeny of genes encoding 5-aminolevulinic acid synthases (ALASs, hemA gene products) in streptomycetes-related strains. In actinomycetes, these genes were believed to be directly connected with the production of secondary metabolites carrying the C5N unit, 2-amino-3-hydroxycyclopent-2-enone, with biological activities making them attractive for future use in medicine and agriculture. Unlike "classical" primary metabolism ALAS, the C5N unit-forming cyclizing ALAS (cALAS) catalyses intramolecular cyclization of nascent 5-aminolevulinate. Specific amino acid sequence changes can be traced by comparison of "classical" ALASs against cALASs. PCR screening revealed 226 hemA gene-carrying strains from 1,500 tested, with 87% putatively encoding cALAS. Phylogenetic analysis of the hemA homologs revealed strain clustering according to putative type of metabolic product, which could be used to select producers of specific C5N compound classes. Supporting information was acquired through analysis of actinomycete genomic sequence data available in GenBank and further genetic or metabolic characterization of selected strains. Comparison of 16S rRNA taxonomic identification and BOX-PCR profiles provided evidence for numerous horizontal gene transfers of biosynthetic genes or gene clusters within actinomycete populations and even from non-actinomycete organisms. Our results underline the importance of environmental and evolutionary data in the design of efficient techniques for identification of novel producers.

Biosynthesis of colabomycin E, a new manumycin-family metabolite, involves an unusual chain-length factor.

Colabomycin E is a new member of the manumycin-type metabolites produced by the strain Streptomyces aureus SOK1/5-04 and identified by genetic screening from a library of streptomycete strains. The structures of colabomycin E and accompanying congeners were resolved. The entire biosynthetic gene cluster was cloned and expressed in Streptomyces lividans. Bioinformatic analysis and mutagenic studies identified components of the biosynthetic pathway that are involved in the formation of both polyketide chains. Recombinant polyketide synthases (PKSs) assembled from the components of colabomycin E and asukamycin biosynthetic routes catalyzing the biosynthesis of "lower" carbon chains were constructed and expressed in S. aureus SOK1/5-04 ΔcolC11-14 deletion mutant. Analysis of the metabolites produced by recombinant strains provided evidence that in both biosynthetic pathways the length of the lower carbon chain is controlled by an unusual chain-length factor supporting biosynthesis either of a triketide in asukamycin or of a tetraketide in colabomycin E. Biological activity assays indicated that colabomycin E significantly inhibited IL-1ß release from THP-1 cells and might thus potentially act as an anti-inflammatory agent.

The pleiotropic effect of WD-40 domain containing proteins on cellular differentiation and production of secondary metabolites in Streptomyces coelicolor.

The genome of Streptomyces coelicolor encodes six potential WD-40 genes. Two of them, the wdpB (SCO5953) and the wdpC (SCO4422) genes, were studied to determine their function. Deletion of the wdpB gene resulted in a considerable decrease of aerial hyphae formation, leading to a conditionally bald phenotype, and reduced undecylprodigiosin production. In addition, the aerial hyphae of the ΔwdpB mutant strain were unusually branched and showed the signs of irregular septation and precocious lysis. Disruption of wdpC resulted in the reduction of undecylprodigiosin and delayed actinorhodin production. The ΔwdpC mutant strain showed precocious lysis of hyphae and delayed sporulation without typical curling of aerial hyphae in the early sporulation stage. The whole-genome transcriptome analysis revealed that deletion of wdpB affects the expression of genes involved in aerial hyphae differentiation, sporulation and secondary metabolites production. Deletion of wdpC caused downregulation of several gene clusters encoding secondary metabolites. Both the wdp genes seem to possess transcriptional autoregulatory function. Overexpression and genetic complementation studies confirmed the observed phenotype of both mutants. The results obtained suggest that both genes studied have a pleiotropic effect on physiological and morphological differentiation.

Biochemical and genetic insights into asukamycin biosynthesis.

Asukamycin, a member of the manumycin family metabolites, is an antimicrobial and potential antitumor agent isolated from Streptomyces nodosus subsp. asukaensis. The entire asukamycin biosynthetic gene cluster was cloned, assembled, and expressed heterologously in Streptomyces lividans. Bioinformatic analysis and mutagenesis studies elucidated the biosynthetic pathway at the genetic and biochemical level. Four gene sets, asuA-D, govern the formation and assembly of the asukamycin building blocks: a 3-amino-4-hydroxybenzoic acid core component, a cyclohexane ring, two triene polyketide chains, and a 2-amino-3-hydroxycyclopent-2-enone moiety to form the intermediate protoasukamycin. AsuE1 and AsuE2 catalyze the conversion of protoasukamycin to 4-hydroxyprotoasukamycin, which is epoxidized at C5-C6 by AsuE3 to the final product, asukamycin. Branched acyl CoA starter units, derived from Val, Leu, and Ile, can be incorporated by the actions of the polyketide synthase III (KSIII) AsuC3/C4 as well as the cellular fatty acid synthase FabH to produce the asukamycin congeners A2-A7. In addition, the type II thioesterase AsuC15 limits the cellular level of omega-cyclohexyl fatty acids and likely maintains homeostasis of the cellular membrane.

CXC and CC chemokines induced in human renal epithelial cells by inflammatory cytokines.

Human renal epithelial cells might play an important role during the allograft rejection by producing chemokines in response to proinflammatory cytokines such as tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta produced by endothelial and epithelial cells early after transplantation. The production of chemokines allows inflammatory cells to be drawn into the kidney graft and therefore plays a critical role in the pathophysiologic processes that lead to the rejection of renal transplant. In this process, two chemokine superfamilies, the CC and the CXC chemokines, are the most important. The CC chemokines target mainly monocytes and T lymphocytes, while most of the CXC chemokines attract neutrophils. We showed in our study that in vitro, in unstimulated cells, basal mRNA expression of CXC chemokines (Groalpha, Grobeta, Grogamma, ENA-78 and GCP-2, IL-8) that attract neutrophils was detectable and expression of these genes and chemokine release were increased in TNF-alpha- and IL-1beta-induced renal epithelial cells. Most of the CC chemokines [monocyte chemotactic protein-1 (MCP-1), macrophage Inflammatory protein 1 beta (MIP-1beta), regulated upon activation, normal T cell expressed and secreted (RANTES) and macrophage inflammatory protein (MIP-3alpha)] showed detectable mRNA expression only after stimulation with proinflammatory cytokines and not in control cells. TNF-alpha seems to induce preferably the expression of RANTES, MCP-1, interferon-inducible protein (IP-10) and Interferon-Inducible T-cell Alpha Chemoattractant (I-TAC), while IL-1beta induces mainly IL-8 and epithelial neutrophil-activating peptide 78 (ENA-78).

Occurrence of two 5-aminolevulinate biosynthetic pathways in Streptomyces nodosus subsp. asukaensis is linked with the production of asukamycin.

We report the results of cloning genes for two key biosynthetic enzymes of different 5-aminolevulinic acid (ALA) biosynthetic routes from Streptomyces. The genes encode the glutamyl-tRNAGlu reductase (GluTR) of the C5 pathway and the ALA synthase (ALAS) of the Shemin pathway. While Streptomyces coelicolor A3(2) synthesizes ALA via the C5 route, both pathways are operational in Streptomyces nodosus subsp. asukaensis, a producer of asukamycin. In this strain, the C5 route produces ALA for tetrapyrrole biosynthesis; the ALA formed by the Shemin pathway serves as a precursor of the 2-amino-3-hydroxycyclopent-2-enone moiety (C5N unit), an antibiotic component. The growth of S. nodosus and S. coelicolor strains deficient in the GluTR genes (gtr) is strictly dependent on ALA or heme supplementation, whereas the defect in the ALAS-encoding gene (hemA-asuA) abolishes the asukamycin production in S. nodosus. The recombinant hemA-asuA gene was expressed in Escherichia coli and in Streptomyces, and the encoded enzyme activity was demonstrated both in vivo and in vitro. The hemA-asuA gene is situated within a putative cluster of asukamycin biosynthetic genes. This is the first report about the cloning of genes for two different ALA biosynthetic routes from a single bacterium.

The WD-40 repeat protein PkwA of Thermomonospora curvata is associated with rapid growth and is localized in the tips of growing hyphae.

The PkwA protein of the thermophilic actinomycete Thermomonospora curvata has already been reported as the first instance of a WD-40 module-containing protein of prokaryotic origin. This protein is composed of an N-terminal eukaryotic-type protein kinase domain and of seven C-terminal WD-40 repeats. PkwA is a peripheral membrane protein that is linked to the early exponential growth phase of the bacterium. Its intracellular concentrations are extremely low. We have shown that the protein forms high molecular weight complexes and is localized mainly in the tips of the young Thermomonospora vegetative hyphae.

Interleukin 18 (IL-18) upregulation in acute rejection of kidney allograft.

Interleukin 18 (IL-18) is a potent proinflammatory cytokine involved in the host defence by upregulating both innate and acquired immune responses and may be of particular importance also in mechanisms of kidney allograft rejection. Immunohistochemical staining of protocol biopsies showed constitutive IL-18 expression in the epithelium of distal tubules with the induction of immunoreactivity in acute rejection patients where also proximal tubules, infiltrating leukocytes, and endothelium were strongly positive. Furthermore, serum levels of IL-18 were significantly elevated in patients with acute rejection of kidney allograft (1247+/-389 pg/l) as compared to patients with uncomplicated outcome of kidney transplantation (444+/-164 pg/l) and subjects with acute tubulointerstitial nephropathy (385+/-155 pg/l, p<0.0001 for both comparisons). Tissue culture model of renal epithelial cells expressed IL-18 mRNA constitutively and released mature IL-18 in response to TNF-alpha and IFN-gamma. We assume that upregulation of epithelial IL-18 plays an important role in immune and immunopathological reactions in renal parenchyma and contributes to rejection mechanisms of kidney allograft.

Eukaryotic-type protein kinases in Streptomyces coelicolor: variations on a common theme.

The increasing number of genes encoding eukaryotic-type Ser/Thr protein kinases (ESTPKs) in prokaryotes, identified mostly due to genome-sequencing projects, suggests that these enzymes play an indispensable role in many bacterial species. Some prokaryotes, such as Streptomyces coelicolor, carry numerous genes of this type. Though the regulatory pathways have been intensively studied in the organism, experimental proof of the physiological function of ESTPKs is scarce. This review presents a family portrait of the genes identified in the sequence of the S. coelicolor A3(2) genome. Based on the available experimental data on ESTPKs in streptomycetes and related bacteria, and on computer-assisted sequence analyses, possible roles of these enzymes in the regulation of cellular processes in streptomycetes are suggested.