Identification of a New Antimicrobial, Desertomycin H, Utilizing a Modified Crowded Plate Technique.

The antibiotic-resistant bacteria-associated infections are a major global healthcare threat. New classes of antimicrobial compounds are urgently needed as the frequency of infections caused by multidrug-resistant microbes continues to rise. Recent metagenomic data have demonstrated that there is still biosynthetic potential encoded in but transcriptionally silent in cultivatable bacterial genomes. However, the culture conditions required to identify and express silent biosynthetic gene clusters that yield natural products with antimicrobial activity are largely unknown. Here, we describe a new antibiotic discovery scheme, dubbed the modified crowded plate technique (mCPT), that utilizes complex microbial interactions to elicit antimicrobial production from otherwise silent biosynthetic gene clusters. Using the mCPT as part of the antibiotic crowdsourcing educational program Tiny EarthTM, we isolated over 1400 antibiotic-producing microbes, including 62 showing activity against multidrug-resistant pathogens. The natural product extracts generated from six microbial isolates showed potent activity against vancomycin-intermediate resistant Staphylococcus aureus. We utilized a targeted approach that coupled mass spectrometry data with bioactivity, yielding a new macrolactone class of metabolite, desertomycin H. In this study, we successfully demonstrate a concept that significantly increased our ability to quickly and efficiently identify microbes capable of the silent antibiotic production.

Differences in the Relative Abundance of ProBDNF and Mature BDNF in A549 and H1299 Human Lung Cancer Cell Media.

Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, has been linked to several human malignancies and shown to promote tumorigenesis. The purpose of this study was to explore the relative abundance of pro-brain-derived neurotrophic factor (proBDNF) and mature BDNF (mBDNF) in A549 (p53 wild-type) and H1299 (p53-null) lung cancer cell media. Higher levels of proBDNF were detected in the media of A549 cells than in H1299 cell media. Using inhibitors, we found that the levels of proBDNF and mBDNF in the media are likely regulated by PI3K, AKT, and NFκB. However, the largest change in these levels resulted from MMP2/9 inhibition. Blocking p53 function in A549 cells resulted in increased mBDNF and decreased proBDNF, suggesting a role for p53 in regulating these levels. The ratio of proBDNF/mBDNF was not affected by MMP2 knockdown but increased in the media of both cell lines upon knockdown of MMP9. Downregulation of either MMP2 or MMP9 by siRNA showed that MMP9 siRNA treatment of either A549 or H1299 cells resulted in decreased cell viability and increased apoptosis, an effect diminished upon the same treatment with proBDNF immunodepleted media, suggesting that MMP9 regulates the cytotoxic effects induced by proBDNF in lung cancer cells.

Regulation of amyloid-ß levels by matrix metalloproteinase-2/9 (MMP2/9) in the media of lung cancer cells.

In this study, we set out to identify regulators of intact amyloid-ß40/42 (Aß) levels in A549 (p53 wild-type) and H1299 (p53-null) lung cancer cell media. Higher Aß levels were detected in the media of A549 than H1299 cells without or with treatment with 4-methylumbelliferone (4-MU) and/or the anti-CD44 antibody (5F12). Using inhibitors, we found that PI3K, AKT, and NFκB are likely involved in regulating Aß levels in the media. However, increased Aß levels that more closely resembled those found upon 4-MU co-treatment resulted from MMP2/9 inhibition, suggesting that MMP2/9 maybe the main contributors to regulation of Aß levels in the media. Differences in Aß levels might be accounted for, in part, by p53 since blocking p53 function in A549 cells resulted in decreased Aß levels, increased MMP2/9 levels, increased PI3K/AKT activities and the phospho/total NFκB ratio. Using siRNA targeted against MMP2 or MMP9, we found increased Aß levels in the media, however, MMP2 knockdown led to Aß levels closely mimicking those detected by co-treatment with 4-MU. Cell viability or apoptosis upon treatment with either MMP2 or MMP9 siRNA along with Aß immunodepletion, showed that MMP2 is the predominant regulator of the cytotoxic effects induced by Aß in lung cancer cells.

Interaction of amyloid beta with humanin and acetylcholinesterase is modulated by ATP.

Humanin (HN) is known to bind amyloid beta (Aß)-inducing cytoprotective effects, while binding of acetylcholinesterase (AChE) to Aß increases its aggregation and cytotoxicity. Previously, we showed that binding of HN to Aß blocks aggregation induced by AChE and that HN decreases but does not abolish Aß-AChE interactions in A549 cell media. Here, we set out to shed light on factors that modulate the interactions of Aß with HN and AChE. We found that binding of either HN or AChE to Aß is not affected by heparan sulfate, while ATP, thought to reduce misfolding of Aß, weakened interactions between AChE and Aß but strengthened those between Aß and HN. Using media from either A549 or H1299 lung cancer cells, we observed that more HN was bound to Aß upon addition of ATP, while levels of AChE in a complex with Aß were decreased by ATP addition to A549 cell media. Exogenous addition of ATP to either A549 or H1299 cell media increased interactions of endogenous HN with Aß to a comparable extent despite differences in AChE expression in the two cell lines, and this was correlated with decreased binding of exogenously added HN to Aß. Treatment with exogenous ATP had no effect on cell viability under all conditions examined. Exogenously added ATP did not affect viability of cells treated with AChE-immunodepleted media, and there was no apparent protection against the cytotoxicity resulting from immunodepletion of HN. Moreover, exogenously added ATP had no effect on the relative abundance of oligomer versus total Aß in either cell line.

Biochemical determinants of the IGFBP-3-hyaluronan interaction.

IGFBP-3, the most abundant IGFBP and the main carrier of insulin-like growth factor I (IGF-I) in the circulation, can bind IGF-1 with high affinity, which attenuates IGF/IGF-IR interactions, thereby resulting in antiproliferative effects. The C-terminal domain of insulin-like growth factor-binding protein-3 (IGFBP-3) is known to contain an 18-basic amino acid motif capable of interacting with either humanin (HN) or hyaluronan (HA). We previously showed that the 18-amino acid IGFBP-3 peptide is capable of binding either HA or HN with comparable affinities to the full-length IGFBP-3 protein and that IGFBP-3 can compete with the HA receptor, CD44, for binding HA. Blocking the interaction between HA and CD44 reduced viability of A549 human lung cancer cells. In this study, we set out to better characterize IGFBP-3-HA interactions. We show that both stereochemistry and amino acid identity are important determinants of the interaction between the IGFBP-3 peptide and HA and for the peptide's ability to exert its cytotoxic effects. Binding of IGFBP-3 to either HA or HN was unaffected by glycosylation or reduction of IGFBP-3, suggesting that the basic 18-amino acid residue sequence of IGFBP-3 remains accessible for interaction with either HN or HA upon glycosylation or reduction of the full-length protein. Removing N-linked oligosaccharides from CD44 increased its ability to compete with IGFBP-3 for binding HA, while reduction of CD44 rendered the protein relatively ineffective at blocking IGFBP-3-HA interactions. We conclude that both deglycosylation and disulfide bond formation are important for CD44 to compete with IGFBP-3 for binding HA.

Humanin Blocks the Aggregation of Amyloid-ß Induced by Acetylcholinesterase, an Effect Abolished in the Presence of IGFBP-3.

It is known that the humanin (HN) peptide binding to amyloid-ß (Aß) protects against its cytotoxic effects, while acetylcholinesterase (AChE) binding to Aß increases its aggregation and cytotoxicity. HN is also known to bind the insulin-like growth factor binding protein-3 (IGFBP-3). Here, we examined the regulation of Aß conformations by HN, AChE, and IGFBP-3 both in vitro and in the conditioned media from A549 and H1299 lung cancer cells. Our in vitro results showed the following: IGFBP-3 binds HN and blocks it from binding Aß in the absence or presence of AChE; HN and AChE can simultaneously bind Aß but not when in the presence of IGFBP-3; HN is unable to reduce the aggregation of Aß in the presence of IGFBP-3; and HN abolishes the aggregation of Aß induced by the addition of AChE in the absence of IGFBP-3. In the media, AChE and HN can simultaneously bind Aß. While both AChE and HN are detected when using 6E10 Aß antibodies, only AChE is detected when using the Aß 17-24 antibody 4G8, the anti-oligomer A11, and the anti-amyloid fibril LOC antibodies. No signal was observed for IGFBP-3 with any of the anti-amyloid antibodies used. Exogenously added IGFBP-3 reduced the amount of HN found in a complex when using 6E10 antibodies and correlated with a concomitant increase in the amyloid oligomers. Immunodepletion of HN from the media of the A549 and H1299 cells increased the relative abundance of the oligomer vs the total amount of Aß, the A11-positive prefibrillar oligomers, and to a lesser extent the LOC-positive fibrillar oligomers, and was also correlated with diminished cell viability and increased apoptosis.