NanI sialidase contributes to toxin expression and host cell binding of Clostridium perfringens type G strain CP56 in vitro.

Necrotic enteritis, caused by NetB producing Clostridium perfringens type G strains, is a globally important poultry disease. An initial step in the pathogenesis of necrotic enteritis is the colonization and degradation of the intestinal mucus layer, a process in which C. perfringens sialidases - such as NanI sialidase - may play an important role. Sialidases cleave terminal sialic acid from complex carbohydrates on glycoconjugates, such as mucins. This study shows that NE-associated C. perfringens strain CP56 is able to use sialic acid (Neu5Ac) as a carbon source for bacterial growth. It is shown that supplementation of Neu5Ac in the growth medium does not only induce the production of extracellular sialidases of strain CP56, but also increases the production of both alpha toxin and NetB toxin. Moreover, it was found that pre-treating avian hepatocellular carcinoma cells (LMH cells) with the recombinant NanI sialidase increases the adherence of C. perfringens type G strain CP56 to these cells. As such, the data suggest an important role for sialidases in the pathogenesis of the disease.

Selection of Gut-Resistant Bacteria and Construction of Microbial Consortia for Improving Gluten Digestion under Simulated Gastrointestinal Conditions.

This work aimed to define the microbial consortia that are able to digest gluten into non-toxic and non-immunogenic peptides in the human gastrointestinal tract. METHODS: 131 out of 504 tested Bacillus and lactic acid bacteria, specifically Bacillus (64), lactobacilli (63), Pediococcus (1), and Weissella (3), showed strong gastrointestinal resistance and were selected for their PepN, PepI, PepX, PepO, and PepP activities toward synthetic substrates. Based on multivariate analysis, 24 strains were clearly distinct from the other tested strains based on having the highest enzymatic activities. As estimated by RP-HPLC and nano-ESI-MS/MS, 6 cytoplasmic extracts out of 24 selected strains showed the ability to hydrolyze immunogenic epitopes, specifically 57-68 of α9-gliadin, 62-75 of A-gliadin, 134-153 of γ-gliadin, and 57-89 (33-mer) of α2-gliadin. Live and lysed cells of selected strains were combined into different microbial consortia for hydrolyzing gluten under gastrointestinal conditions. Commercial proteolytic enzymes (Aspergillusoryzae E1, Aspergillusniger E2, Bacillussubtilis Veron HPP, and Veron PS proteases) were also added to each microbial consortium. Consortium activity was evaluated by ELISA tests, RP-HPLC-nano-ESI-MS/MS, and duodenal explants from celiac disease patients. RESULTS: two microbial consortia (Consortium 4: Lactiplantibacillus (Lp.) plantarum DSM33363 and DSM33364, Lacticaseibacillus (Lc.) paracasei DSM33373, Bacillussubtilis DSM33298, and Bacilluspumilus DSM33301; and Consortium 16: Lp. plantarum DSM33363 and DSM33364, Lc. paracasei DSM33373, Limosilactobacillusreuteri DSM33374, Bacillusmegaterium DSM33300, B.pumilus DSM33297 and DSM33355), containing commercial enzymes, were able to hydrolyze gluten to non-toxic and non-immunogenic peptides under gastrointestinal conditions. CONCLUSIONS: the results of this study provide evidence that selected microbial consortia could potentially improve the digestion of gluten in gluten-sensitive patients by hydrolyzing the immunogenic peptides during gastrointestinal digestion.

Production of the Bengamide Class of Marine Natural Products in Myxobacteria: Biosynthesis and Structure-Activity Relationships.

The bengamides, sponge-derived natural products that have been characterized as inhibitors of methionine aminopeptidases (MetAPs), have been intensively investigated as anticancer compounds. We embarked on a multidisciplinary project to supply bengamides by fermentation of the terrestrial myxobacterium M. virescens, decipher their biosynthesis, and optimize their properties as drug leads. The characterization of the biosynthetic pathway revealed that bacterial resistance to bengamides is conferred by Leu 154 of the myxobacterial MetAP protein, and enabled transfer of the entire gene cluster into the more suitable production host M. xanthus DK1622. A combination of semisynthesis of microbially derived bengamides and total synthesis resulted in an optimized derivative that combined high cellular potency in the nanomolar range with high metabolic stability, which translated to an improved half-life in mice and antitumor efficacy in a melanoma mouse model.

A novel N,N-8-amino-8-demethyl-D-riboflavin Dimethyltransferase (RosA) catalyzing the two terminal steps of roseoflavin biosynthesis in Streptomyces davawensis.

Streptomyces davawensis synthesizes the antibiotic roseoflavin (RoF) (8-dimethylamino-8-demethyl-D-riboflavin). It was postulated that RoF is synthesized from riboflavin via 8-amino- (AF) and 8-methylamino-8-demethyl-D-riboflavin (MAF). In a cell-free extract of S. davawensis, an S-adenosyl methionine-dependent conversion of AF into MAF and RoF was observed. The corresponding N,N-8-amino-8-demethyl-d-riboflavin dimethyltransferase activity was enriched by column chromatography. The final most active fraction still contained at least five different proteins that were analyzed by enzymatic digestion and concomitant de novo sequencing by MS/MS. One of the sequences matched a hypothetical peptide fragment derived from an as yet uncharacterized open reading frame (sda77220) located in the middle of a (putative) gene cluster within the S. davawensis genome. Expression of ORF sda77220 in Escherichia coli revealed that the corresponding gene product had N,N-8-amino-8-demethyl-d-riboflavin dimethyltransferase activity. Inactivation of ORF sda77220 led to a S. davawensis strain that synthesized AF but not MAF or RoF. Accordingly, as the first identified gene of RoF biosynthesis, ORF sda77220 was named rosA. RosA (347 amino acids; 38 kDa) was purified from a recombinant E. coli strain (as a His(6)-tagged protein) and was biochemically characterized (apparent K(m) for AF = 57.7 ± 9.2 µm; apparent K(D) for AF = 10.0 µm; k(cat) = 0.37 ± 0.02 s(-1)). RosA is a unique enzyme and may be useful for a variety of applications.

Daptomycin versus Friulimicin B: in-depth profiling of Bacillus subtilis cell envelope stress responses.

The related lipo(depsi)peptide antibiotics daptomycin and friulimicin B show great potential in the treatment of multiply resistant gram-positive pathogens. Applying genome-wide in-depth expression profiling, we compared the respective stress responses of Bacillus subtilis. Both antibiotics target envelope integrity, based on the strong induction of extracytoplasmic function sigma factor-dependent gene expression. The cell envelope stress-sensing two-component system LiaRS is exclusively and strongly induced by daptomycin, indicative of different mechanisms of action in the two compounds.

Aranciamycin analogs generated by combinatorial biosynthesis show improved antitumor activity.

Expression of the aranciamycin biosynthetic gene cluster in Streptomyces diastatochromogenes Tü6028 resulted in production of four novel compounds, aranciamycins E, F, G, and H with different decorations in the tetracyclic backbone. Two derivatives contain a D-amicetose moiety at C7 (aranciamycins F and G), two are hydroxylated at position C1 (aranciamycins E and G), and one is hydroxylated at C13 (aranciamycin F). Analysis of the biological activities of the aranciamycins against two human tumor cell lines--MCF-7 and MATU--shows surprising impact of the hydroxyl group at position C1 on activity. As aranciamycins E and G were the most active derivatives, hydroxylation of the C1 appears to coincide with increased antitumor activity of aranciamycins.

A genomic screening approach to the structure-guided identification of drug candidates from natural sources.

The potential of actinomycetes to produce natural products has been exploited for decades. Recent genomic sequence analyses have revealed a previously unrecognized biosynthetic potential and diversity. In order to rationally exploit this potential, we have developed a sequence-guided genetic screening strategy. In this "genome mining" approach, genes that encode tailoring enzymes from natural product biosyntheses pathways serve as indicator genes for the identification of strains that have the genetic potential to produce natural products of interest. We chose halogenases, which are known to be involved in the synthesis of halometabolites as representative examples. From PCR screening of 550 randomly selected actinomycetes strains, we identified 103 novel putative halogenase genes. A phylogenetic analysis of the corresponding putative halogenases, and the determination of their sequential context with mass spectrometric analysis of cultures filtrates revealed a distinct correlation between the sequence and secondary metabolite class of the halometabolite. The described screening strategy allows rapid access to novel natural products with predetermined structural properties.

Crystal structure of OxyB, a cytochrome P450 implicated in an oxidative phenol coupling reaction during vancomycin biosynthesis.

Gene-inactivation studies point to the involvement of OxyB in catalyzing the first oxidative phenol coupling reaction during glycopeptide antibiotic biosynthesis. The oxyB gene has been cloned and sequenced from the vancomycin producer Amycolatopsis orientalis, and the hemoprotein has been produced in Escherichia coli, crystallized, and its structure determined to 1.7-A resolution. OxyB gave UV-visible spectra characteristic of a P450-like hemoprotein in the low spin ferric state. After reduction to the ferrous state by dithionite or by spinach ferredoxin and ferredoxin reductase, the CO-ligated form gave a 450-nm peak in a UV-difference spectrum. Addition of putative heptapeptide substrates to resting OxyB produced type I changes to the UV spectrum, but no turnover was observed in the presence of ferredoxin and ferredoxin reductase, showing that either the peptides or the reduction system, or both, are insufficient to support a full catalytic cycle. OxyB exhibits the typical P450-fold, with helix L containing the signature sequence FGHGXHXCLG and Cys(347) being the proximal axial thiolate ligand of the heme iron. The structural similarity of OxyB is highest to P450nor, P450terp, CYP119, and P450eryF. In OxyB, the F and G helices are rotated out of the active site compared with P450nor, resulting in a much more open active site, consistent with the larger size of the presumed heptapeptide substrate.