Molecular mechanism of topoisomerase poisoning by the peptide antibiotic albicidin.

The peptide antibiotic albicidin is a DNA topoisomerase inhibitor with low-nanomolar bactericidal activity towards fluoroquinolone-resistant Gram-negative pathogens. However, its mode of action is poorly understood. We determined a 2.6 Å resolution cryoelectron microscopy structure of a ternary complex between Escherichia coli topoisomerase DNA gyrase, a 217 bp double-stranded DNA fragment and albicidin. Albicidin employs a dual binding mechanism where one end of the molecule obstructs the crucial gyrase dimer interface, while the other intercalates between the fragments of cleaved DNA substrate. Thus, albicidin efficiently locks DNA gyrase, preventing it from religating DNA and completing its catalytic cycle. Two additional structures of this trapped state were determined using synthetic albicidin analogues that demonstrate improved solubility, and activity against a range of gyrase variants and E. coli topoisomerase IV. The extraordinary promiscuity of the DNA-intercalating region of albicidins and their excellent performance against fluoroquinolone-resistant bacteria holds great promise for the development of last-resort antibiotics.

Molecular mechanism of SbmA, a promiscuous transporter exploited by antimicrobial peptides.

Antibiotic metabolites and antimicrobial peptides mediate competition between bacterial species. Many of them hijack inner and outer membrane proteins to enter cells. Sensitivity of enteric bacteria to multiple peptide antibiotics is controlled by the single inner membrane protein SbmA. To establish the molecular mechanism of peptide transport by SbmA and related BacA, we determined their cryo­electron microscopy structures at 3.2 and 6 Å local resolution, respectively. The structures show a previously unknown fold, defining a new class of secondary transporters named SbmA-like peptide transporters. The core domain includes conserved glutamates, which provide a pathway for proton translocation, powering transport. The structures show an outward-open conformation with a large cavity that can accommodate diverse substrates. We propose a molecular mechanism for antibacterial peptide uptake paving the way for creation of narrow-targeted therapeutics.

Inhibitory Compounds Targeting Plasmodium falciparum Gyrase B.

Malaria persists as a major health problem due to the spread of drug resistance and the lack of effective vaccines. DNA gyrase is a well-validated and extremely effective therapeutic target in bacteria, and it is also known to be present in the apicoplast of malarial species, including Plasmodium falciparum. This raises the possibility that it could be a useful target for novel antimalarials. To date, characterization and screening of this gyrase have been hampered by difficulties in cloning and purification of the GyrA subunit, which is necessary together with GyrB for reconstitution of the holoenzyme. To overcome this, we employed a library of compounds with specificity for P. falciparum GyrB and assessed them in activity tests utilizing P. falciparum GyrB together with Escherichia coli GyrA to reconstitute a functional hybrid enzyme. Two inhibitory compounds were identified that preferentially inhibited the supercoiling activity of the hybrid enzyme over the E. coli enzyme. Of these, purpurogallin (PPG) was found to disrupt DNA binding to the hybrid gyrase complex and thus reduce the DNA-induced ATP hydrolysis of the enzyme. Binding studies indicated that PPG showed higher-affinity binding to P. falciparum GyrB than to the E. coli protein. We suggest that PPG achieves its inhibitory effect on gyrase through interaction with P. falciparum GyrB leading to disruption of DNA binding and, consequently, reduction of DNA-induced ATPase activity. The compound also showed an inhibitory effect against the malaria parasite in vitro and may be of interest for further development as an antimalarial agent.

Pentapeptide repeat protein QnrB1 requires ATP hydrolysis to rejuvenate poisoned gyrase complexes.

DNA gyrase, a type II topoisomerase found predominantly in bacteria, is the target for a variety of 'poisons', namely natural product toxins (e.g. albicidin, microcin B17) and clinically important synthetic molecules (e.g. fluoroquinolones). Resistance to both groups can be mediated by pentapeptide repeat proteins (PRPs). Despite long-term studies, the mechanism of action of these protective PRPs is not known. We show that a PRP, QnrB1 provides specific protection against fluoroquinolones, which strictly requires ATP hydrolysis by gyrase. QnrB1 binds to the GyrB protein and stimulates ATPase activity of the isolated N-terminal ATPase domain of GyrB (GyrB43). We probed the QnrB1 binding site using site-specific incorporation of a photoreactive amino acid and mapped the crosslinks to the GyrB43 protein. We propose a model in which QnrB1 binding allosterically promotes dissociation of the fluoroquinolone molecule from the cleavage complex.

The Importance of Interdisciplinary Treatment in an Esthetically Challenging Case.

A single missing tooth is a common occurrence among young patients and impacts esthetics and long-term oral health in terms of compromised bone, gum tissue, and, if warranted, an implant and final prosthesis. In this case report, after years of poorly executed orthodontic therapy, the patient's dental growth complicated the development of an esthetically pleasing smile. An interdisciplinary approach was utilized comprising periodontal surgery, a second course of orthodontics, and prosthodontics to provide comprehensive patient care that included evaluation of occlusion and esthetics. Orthodontic treatment was performed to position the teeth in the most esthetic, functionally optimal position. An implant crown in the maxillary left central incisor position and direct bonding on the maxillary right central incisor were indicated to treat a large edentulous area. Final orthodontic treatment achieved a substantial reduction of incisor protrusion and proper mesial-distal distance between the future implant and adjacent teeth. An ideal emergence profile, appealing esthetics, and a provisional restoration were created before the final crown. Optimal alignment of teeth relative to the arch was achieved, and adequate tissue dimensions were created by combining surgical augmentations with provisional restorative therapy.

A Role for Low-Density Lipoprotein Receptor-Related Protein 6 in Blood Vessel Regression in Wound Healing.

Objective: The healing of skin wounds is typified by a pattern of robust angiogenesis followed by vascular regression. Pigment epithelium-derived factor (PEDF), a recognized endogenous antiangiogenic protein, regulates vascular regression in resolving wounds through an unknown receptor. Among the multiple receptors for PEDF that have been identified, low-density lipoprotein receptor-related protein 6 (Lrp6) has been described as a regulator of angiogenesis in multiple systems. The purpose of the current study was to determine if the Lrp6 receptor plays a role in vessel regression in wounds. Approach: Excisional skin wounds were prepared on C57BL/6 mice. RT-PCR and immunoblots were performed to measure Lrp6 expression over a time course of wound healing. Immunohistochemistry was performed to localize Lrp6 in both recombinant PEDF (rPEDF)-treated and control wounds. To examine whether Lrp6 is critical to the regulation of capillary regression in vivo, wounds were treated with Lrp6 siRNA to minimize its presence in wounds. Immunohistochemistry for CD31 was performed to quantify blood vessel density. Results: PCR and immunoblots revealed significant increases in Lrp6 expression during the vascular regression phase of wound healing. Lrp6 was found to colocalize with CD31+ endothelial cells in wounds. The addition of rPEDF to wounds caused an increase in Lrp6-CD31+ endothelial cell colocalization. Inhibition of Lrp6 by siRNA impeded the vascular regression phase of healing. Innovation: This study is the first to demonstrate an association between Lrp6 and vessel regression in wound healing. Conclusion: Lrp6 is expressed in wounds in a temporal and spatial manner that suggests it may be a receptor for PEDF during vascular regression. PEDF increases Lrp6 expression in the wound vasculature, and inhibition of Lrp6 blocked vascular regression in wounds. The results suggest that Lrp6 is important to vascular regression in wounds, possibly through direct interaction with PEDF.

Pigment Epithelium-Derived Factor (PEDF) as a Regulator of Wound Angiogenesis.

Although the inflammatory and proliferative phases of wound healing have been well described, much less is known about how healing resolves. During the resolution phase, pruning of the capillary bed and maturation of capillaries occurs and influences the final strength and fidelity of the wound. PEDF, an endogenous anti-angiogenic factor, is produced in wounds and may contribute to the removal of capillaries during wound resolution. This study utilized PEDF-/- mice to examine how PEDF influences wound angiogenesis, particularly capillary density and permeability. The absence of PEDF led to transient changes in dermal wound closure and collagen content, but caused substantial changes in wound angiogenesis. Compared to wild type (WT) mice, wounds from PEDF-/- mice exhibited a significant increase in capillaries during the proangiogenic phase of repair, and a delay in capillary pruning. Conversely, the addition of rPEDF caused a reduction in capillary density within skin wounds in WT mice. In vitro studies showed that PEDF inhibited migration and tube formation by dermal microvascular endothelial cells, and caused a decrease in the expression of VEGFR2, VCAM-1, and other surface receptors. The results demonstrate that loss of PEDF causes a distinctive wound healing phenotype that is characterized by increased angiogenesis and delayed resolution. The findings suggest that PEDF most likely acts through multiple mechanisms to regulate proper capillary refinement in wounds.

Pigment epithelium-derived factor as a multifunctional regulator of wound healing.

During dermal wound repair, hypoxia-driven proliferation results in dense but highly permeable, disorganized microvascular networks, similar to those in solid tumors. Concurrently, activated dermal fibroblasts generate an angiopermissive, provisional extracellular matrix (ECM). Unlike cancers, wounds naturally resolve via blood vessel regression and ECM maturation, which are essential for reestablishing tissue homeostasis. Mechanisms guiding wound resolution are poorly understood; one candidate regulator is pigment epithelium-derived factor (PEDF), a secreted glycoprotein. PEDF is a potent antiangiogenic in models of pathological angiogenesis and a promising cancer and cardiovascular disease therapeutic, but little is known about its physiological function. To examine the roles of PEDF in physiological wound repair, we used a reproducible model of excisional skin wound healing in BALB/c mice. We show that PEDF is abundant in unwounded and healing skin, is produced primarily by dermal fibroblasts, binds to resident microvascular endothelial cells, and accumulates in dermal ECM and epidermis. PEDF transcript and protein levels were low during the inflammatory and proliferative phases of healing but increased in quantity and colocalization with microvasculature during wound resolution. Local antibody inhibition of endogenous PEDF delayed vessel regression and collagen maturation during the remodeling phase. Treatment of wounds with intradermal injections of exogenous, recombinant PEDF inhibited nascent angiogenesis by repressing endothelial proliferation, promoted vascular integrity and function, and increased collagen maturity. These results demonstrate that PEDF contributes to the resolution of healing wounds by causing regression of immature blood vessels and stimulating maturation of the vascular microenvironment, thus promoting a return to tissue homeostasis after injury.