Lysobactin also known as katanosin B is a potent antibiotic with

Lysobactin also known as katanosin B is a potent antibiotic with in vivo efficacy against and (MRSA) and multidrug-resistant streptococcal infections but clinical failure due to Tigecycline vancomycin resistance is increasingly common. attention not only because it represents a new structural class but also because it was shown to bind cell wall precursors from multiple biosynthetic pathways.5 In the course of our efforts to identify potent antimicrobial natural products from novel and known producing organisms we found extracts of is composed of thick layers of PG further modified with covalently bound WTA.7 The PG layers are essential for survival because they Tigecycline stabilize the cell membrane against high turgor pressure thereby preventing osmotic lysis. As shown in Figure 2 the PG precursor Lipid II (LipidIIGly5) is synthesized inside the cell on an undecaprenyl phosphate (Und-P) “carrier lipid” and then flipped outside where it is polymerized and cross-linked to make mature PG.8 Polymerization releases undecaprenyl pyrophosphate (Und-PP) which is dephosphorylated and recycled into the cell so that more Lipid II can be produced.9 The WTA biosynthetic pathway also involves intracellular assembly of a precursor on the Und-P carrier.7 After translocation to the surface of the cell this precursor is attached to the C6 hydroxyl of residues in PG through a phosphodiester bond liberating the carrier lipid.7 Vancomycin inhibits PG biosynthesis by binding to a d-Ala-d-Ala found at the terminus of the stem Tigecycline peptide of Lipid II while ramoplanin and teixobactin bind to a region of Lipid II that includes the pyrophosphate and the first sugar but not the stem peptide.2b 4 5 Teixobactin was also reported to bind a lipid-linked WTA precursor; therefore it was proposed that teixobactin kills by inhibiting both the PG and WTA biosynthetic pathways.5 Figure 2 Schematic of pathways for biosynthesis of lipid-linked PG and WTA precursors from the common intermediate Und-P. Compounds targeting PG and WTA biosynthesis are shown in purple and blue respectively. Lysobactin also known as katanosin B is produced by several genera of Gram-negative gliding bacteria found in soil. First reported in 1987 it was shown to inhibit PG biosynthesis and found to have outstanding in vitro activity against MRSA and vancomycin-resistant (VRE) as well as efficacy against systemic staphylococcal and streptococcal infections in mice.10 Although Tigecycline it was speculated to act as a substrate binder experimental evidence to establish this mechanism of action has not been reported.2 In 2007 Tigecycline two groups independently described the total synthesis of lysobactin and in 2011 the gene cluster was identified and characterized.11 To enable assessment of analogues for possible development we further characterized lysobactin’s activity and determined its mechanism of action. We found that lysobactin is rapidly bactericidal against and also has significant activity against mycobacteria (Figures 3 and S2). The colony forming units (CFUs) of a growing culture treated with lysobactin at 1.5 treated with no antibiotic (black circles) vancomycin (blue triangles) or lysobactin (red squares) at 2× … To determine whether lysobactin could be a substrate binder we added exogenous cell wall precursors to treated with lysobactin. Whereas the stem peptide mimic Lys-d-Ala-d-Ala antagonized the effects of vancomycin it had no effect on the MIC of lysobactin as previously reported.13 In contrast synthetic Lipid I14 and an analogue lacking the stem peptide protected from killing by lysobactin. These results suggested that lysobactin does indeed act via a substrate-binding mechanism (Figure 3c and S3). To confirm a substrate-binding mechanism Tigecycline and characterize lysobactin’s recognition preferences we monitored the reaction rate as a Rabbit polyclonal to AMID. function of substrate concentration for three enzymes that use cell wall precursors MurG SgtB and TagB. MurG catalyzes the formation of Lipid II from Lipid I; SgtB catalyzes the polymerization of the PG precursor Lipid II; TagB catalyzes the transfer of phosphoglycerol to a lipid-linked WTA disaccharide intermediate (Figure 2).14–16 Substrate binders produce a characteristic enzyme inhibition curve in which the reaction rate is negligible at low substrate concentrations because there is no free substrate but jumps as soon as substrate becomes.

The role of nanotopographical extracellular matrix (ECM) cues on vascular endothelial

The role of nanotopographical extracellular matrix (ECM) cues on vascular endothelial cell (EC) organization and function is not well-understood despite the composition of nano- to micro-scale fibrillar ECMs within blood vessels. collagen films that induce parallel EC alignment prior to stimulation with disturbed flow resulting from spatial wall shear stress gradients. Using real time live-cell imaging we tracked the alignment migration trajectories proliferation and anti-inflammatory behavior Bryostatin 1 of ECs when they were cultured on parallel-aligned or randomly oriented nanofibrillar films. Intriguingly ECs cultured on aligned nanofibrillar films remained well-aligned and migrated predominantly along the direction of aligned nanofibrils despite exposure to shear stress orthogonal to the direction of the aligned nanofibrils. Furthermore in stark contrast to ECs cultured on randomly oriented films ECs on aligned nanofibrillar films exposed to disturbed flow had significantly reduced inflammation and proliferation while maintaining intact intercellular junctions. This work reveals fundamental insights into the importance of nanoscale ECM interactions in the maintenance of endothelial function. Importantly it provides new insight into Bryostatin 1 how ECs respond to opposing cues derived from nanotopography and mechanical shear force and has strong implications in the design of polymeric conduits and bioengineered tissues. studies randomly oriented or aligned nanofibrillar films were sterilized with 70% ethanol Bryostatin 1 and rehydrated with 1× PBS for 2 hours. 5×105 primary human dermal microvascular ECs (Lonza P7-10) were seeded onto Itgal the collagen film in EGM-2MV growth media (Lonza) at 37°C and 5% CO2 until they reached approximately 80% confluence. Disturbed flow system A disturbed flow system resulting from spatial wall shear stress gradients was previously characterized15 to recapitulate the pathologic flow profile seen at the bifurcation points of blood vessels (Figure 1a). A Nikon TE-2000 inverted microscope with a motorized stage and enclosed in a plexiglass chamber maintained at 37°C housed the cells and flow orifice. A nine-roller dampened peristaltic pump (Idex) was used to deliver cell culture media at a flow rate of 3 mL/min through 1.3 mm (inner diameter) tubing corresponding to a fluid velocity range of 0-75.3 mm/s. Media flowed downward from the flow orifice (0.7 mm inner diameter) at the conserved flow rate of 3mL/min onto EC-cultured collagen films corresponding to a fluid velocity range between 0-259.8 mm/s and producing a shear stress range of 0-25.1 dynes/cm2 on the cell monolayer (Figure 1b-c) which is within physiological range.40 Cells were exposed to disturbed flow for 24 hours. Phase contrast images were collected every 25 min using Fiji Bryostatin 1 software for 24 hours. All images were bandpass filtered in ImageJ to increase contrast Bryostatin 1 of cell boundaries. To assess shear gradients the cell monolayer was assigned 5 regions of interest defined by concentric rings (R1 R2 R3 R4 R5) each with a radius of 185 μm. The stagnation point directly underneath the flow orifice corresponded to the center of R1 where the cells experience zero shear stress. The magnitude of the shear stress increased radially outward from the jetting center with maximum shear stress peaking within R2 (Figure 1c). The shear stress decreases from R3 to R5. The impinging flow was modeled byaxisymmetric flow using the commercial finite-element analysis (FEA) package COMSOL Multiphysics 3.5a following our previous study.15 A flow rate of 3 ml/min is prescribed at the orifice inlet and a pressure Bryostatin 1 boundary condition is used at the outlet. A “no slip” boundary condition was assumed at the wall (where z=0 at the cell monolayer) such that the velocity of the fluid directly at the wall is zero. The wall shear stress τwas calculated as a function of the velocity gradient
$?u?z$

which quantifies how quickly fluid velocity (u) changes along the z-direction and the fluid viscosity (μ):
$τw=μ?u?z∣z=0$

Quantification of cellular alignment.