Conjugation is a major route of horizontal gene transfer, the driving force in the evolution of bacterial genomes. Antibiotic producing soil bacteria of the genus Streptomyces transfer DNA in a unique process involving a single plasmid-encoded protein TraB and a double-stranded DNA molecule. However, the molecular function of TraB in directing DNA transfer from a donor into a recipient cell is unknown. Here, we show that TraB constitutes a novel conjugation system that is clearly distinguished from DNA transfer by a type IV secretion system. We demonstrate that TraB specifically recognizes and binds to repeated 8 bp motifs on the conjugative plasmid. The specific DNA recognition is mediated by helix a3 of the C-terminal winged-helix-turn-helix domain of TraB. We show that TraB assembles to a hexameric ring structure with a central B3.1 nm channel and forms pores in lipid bilayers. Structure, sequence similarity and DNA binding characteristics of TraB indicate that TraB is derived from an FtsK-like ancestor protein, suggesting that Streptomyces adapted the FtsK/SpoIIIE chromosome segregation system to transfer DNA between two distinct Streptomyces cells.

Bacteria often colonize in-dwelling medical devices and grow as complex biofilm communities of cells embedded in a self-produced extracellular polymeric matrix, which increases their resistance to antibiotics and the host immune system. During biofilm growth, bacterial cells cooperate through specific quorum-sensing (QS) signals. Taking advantage of this mechanism of biofilm formation, we hypothesized that interrupting the communication among bacteria and simultaneously degrading the extracellular matrix would inhibit biofilm growth. To this end, coatings composed of the enzymes acylase and α-amylase, able to degrade bacterial QS molecules and polysaccharides, respectively, were built on silicone urinary catheters using a layer-by-layer deposition technique. Multilayer coatings of either acylase or amylase alone suppressed the biofilm formation of corresponding Gram-negative Pseudomonas aeruginosa and Gram-positive Staphylococcus aureus. Further assembly of both enzymes in hybrid nanocoatings resulted in stronger biofilm inhibition as a function of acylase or amylase position in the layers. Hybrid coatings, with the QS-signal-degrading acylase as outermost layer, demonstrated 30% higher antibiofilm efficiency against medically relevant Gram-negative bacteria compared to that of the other assemblies. These nanocoatings significantly reduced the occurrence of single-species (P. aeruginosa) and mixed-species (P. aeruginosa and Escherichia coli) biofilms on silicone catheters under both static and dynamic conditions. Moreover, in an in vivo animal model, the quorum quenching and matrix degrading enzyme assemblies delayed the biofilm growth up to 7 days.

Pristinamycin production in Streptomyces pristinaespiralis Pr11 is tightly regulated by an interplay between different repressors and activators. A ␥-butyrolactone receptor gene (spbR), two TetR repressor genes (papR3 and papR5), three SARP (Streptomyces antibiotic regulatory protein) genes (papR1, papR2, and papR4), and a response regulator gene (papR6) are carried on the large 210-kb pristinamycin biosynthetic gene region of Streptomyces pristinaespiralis Pr11. A detailed investigation of all pristinamycin regulators revealed insight into a complex signaling cascade, which is responsible for the fine-tuned regulation of pristinamycin production in S. pristinaespiralis. Streptomycetes are filamentous, Gram-positive soil bacteria that are well known for their ability to produce varieties of bioactive secondary metabolites, including more than 70% of the commercially important antibiotics (1). The production of antibiotics is controlled by a vast array of physiological and nutritional conditions, communicated by extracellular and intracellular signaling molecules (2). The beginning of antibiotic biosynthesis is often coordinated with processes of morphological differentiation. The characteristic Streptomyces life cycle involves the formation of a feeding substrate mycelium and subsequent development of aerial hyphae, which finally septate into spores (3). Generally, antibiotic production begins as the culture enters stationary growth in liquid culture and coincidences with the onset of morphological differentiation in agar-grown cultures (reviewed in reference 4). In many Streptomyces strains, antibiotic production is regulated by low-molecular-weight compounds, called ␥-butyrolactone autoregulators (GBLs) (5, 6). GBLs are small diffusible signaling molecules that are synthesized and gradually accumulated in a growth-dependent manner, at or near the middle of the exponential phase of Streptomyces growth, when they trigger the onset of antibiotic biosynthesis and/or morphological differentiation at nanomolar concentrations (7). Often, the GBL signal is transmitted via a hierarchical signaling cascade including pleiotropic and pathway-specific regulators, which all together control the antibiotic production: when the GBL concentration reaches a critical level, the signal is transmitted into the cells by binding to specific cytoplasmic receptor proteins, the GBL receptors (7). GBL receptors belong to the TetR family of transcriptional regulators (8). In the absence of the corresponding ligand, the GBL receptor binds to conserved AT-rich, partially palindromic sequences (9), the so-called "ARE" sequences (autoregulatory element) (10), within the promoter regions of its target genes and thereby represses the transcription of these genes. By binding of the GBLs to their receptors, the latter undergo a conformational change and dissociate from the target DNA, allowing expression of the derepressed genes (11). Predominantly, targets of GBL receptors are transcriptional regulatory genes, such as TetR and SARP (Streptomyce...

Modulation of Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling is a promising method of treating autoimmune diseases, and the profound potency of clinical compounds makes this mode of action particularly attractive. Other questions that remain unanswered also include: What is the ideal selectivity between JAK1 and JAK3? Which cells are most relevant to JAK blockade? And what is the ideal tissue distribution pattern for addressing specific autoimmune conditions? We hypothesized that JAK3 selectivity is most relevant to low-dose clinical effects and interleukin-10 (IL-10) stimulation in particular, that immune cells are the most important compartment, and that distribution to inflamed tissue is the most important pharmacokinetic characteristic for in vivo disease modification. To test these hypotheses, we prepared modified derivatives of JAK3 specific inhibitors that target C909 near the ATP binding site based on FM-381, first reported in 2016; a compound class that was hitherto limited in uptake and exposure in vivo. These limits appear to be due to metabolic instability of side groups binding in the selectivity pocket. We identified derivatives with improved stability and tissue exposure. Conjugation to macrolide scaffolds with medium chain linkers was sufficient to stabilize the compounds and improve transport to organs while maintaining JAK3 affinity. These conjugates are inflammation targeted JAK3 inhibitors with long tissue half-lives and high exposure to activated immune cells.

Short-chain fatty acids (SCFAs) have a range of effects in metabolism and immune regulation. We have observed that delivery of SCFAs to lysosomes has potent immune regulatory effects, possibly as a surrogate signal for the presence of anaerobic organisms. To better understand the pharmacology of lysosomal SCFA donors, we investigated the distribution and metabolism of propionate and butyrate donors. Each analog (1 a and 2 a) can donate three SCFA equivalents via ester hydrolysis through six intermediate metabolites. The compounds are stabilized by low pH, and stability in cells is usually higher than in medium, but is cell-type specific. Butyrate derivatives were found to be more stable than propionates. Triesters were more stable than di-or mono-esters. The donors were surprisingly stable in vivo, and hydrolysis of each position was organ specific. Jejunum and liver caused rapid loss of 4'' esters. The gut metabolite pattern by i. v. differed from that of p.o. application, suggesting luminal and apical enzyme effects in the gut epithelium. Central organs could de-esterify the 11position. Levels in lung relative to other organs were higher by p.o. than via i. v., suggesting that delivery route can influence the observed pharmacology and that gut metabolites distribute differently. The donors were largely eliminated by 24 h, following near linear decline in organs. The observed levels and distribution were found to be consistent with pharmacodynamic effects, particularly in the gut.