The dwarf neon rainbowfish Melanotaenia praecox, a small spiny-rayed fish with potential as a new Acanthomorpha model fish: II. Establishment of a microinjection procedure for genetic engineering.

BACKGROUND: Rainbowfish is a clade of colorful freshwater fish. Melanotaenia praecox is a small rainbowfish species with biological characteristics that make it potentially useful as an experimental model species. We anticipate that M. praecox could become a new model used in various fields, such as ecology, evolution, and developmental biology. However, few previous studies have described experimental set-ups needed to understand the molecular and genetic mechanisms within this species. RESULTS: We describe detailed procedures for genetic engineering in the rainbowfish M. praecox. By using these procedures, we successfully demonstrated CRISPR/Cas-mediated knockout and Tol2 transposon-mediated transgenesis in this species. Regarding the CRISPR/Cas system, we disrupted the tyrosinase gene and then showed that injected embryos lacked pigmentation over much of their body. We also demonstrated that a Tol2 construct, including a GFP gene driven by a ubiquitous promoter, was efficiently integrated into the genome of M. praecox embryos. CONCLUSIONS: The establishment of procedures for genetic engineering in M. praecox enables investigation of the genetic mechanisms behind a broad range of biological phenomena in this species. Thus, we suggest that M. praecox can be used as a new model species in various experimental biology fields.

Direct interaction between fd phage pilot protein pIII and the TolQ-TolR proton-dependent motor provides new insights into the import of filamentous phages.

Filamentous phages are one of the simplest examples of viruses with a protein capsid that protects a circular single-stranded DNA genome. The infection is very specific, nonlytic, and can strongly affect the physiology or provide new pathogenic factors to its bacterial host. The infection process is proposed to rely on a pore-forming mechanism similar to that of certain nonenveloped eukaryotic viruses. The Ff coliphages (including M13, fd, and f1) have been intensively studied and were used to establish the sequence of events taking place for efficient crossing of the host envelope structure. However, the mechanism involved in the penetration of the cell inner membrane is not well understood. Here, we identify new host players involved in the phage translocation mechanism. Interaction studies by a combination of in vivo biochemical methods demonstrate that the adhesion protein pIII located at the tip of the phage binds to TolQ and TolR, two proteins that form a conserved proton-dependent molecular motor in the inner membrane of the host cell. Moreover, in vivo cysteine cross-linking studies reveal that the interactions between the pIII and TolQ or TolR occur between their transmembrane helix domains and may be responding to the proton motive force status of the cell. These results allow us to propose a model for the late stage of filamentous phage translocation mediated by multiple interactions with each individual component of the host TolQRA complex.

Modulation of abscisic acid signaling via endosomal TOL proteins.

The phytohormone abscisic acid (ABA) functions in the control of plant stress responses, particularly in drought stress. A significant mechanism in attenuating and terminating ABA signals involves regulated protein turnover, with certain ABA receptors, despite their main presence in the cytosol and nucleus, subjected to vacuolar degradation via the Endosomal Sorting Complex Required for Transport (ESCRT) machinery. Collectively our findings show that discrete TOM1-LIKE (TOL) proteins, which are functional ESCRT-0 complex substitutes in plants, affect the trafficking for degradation of core components of the ABA signaling and transport machinery. TOL2,3,5 and 6 modulate ABA signaling where they function additively in degradation of ubiquitinated ABA receptors and transporters. TOLs colocalize with their cargo in different endocytic compartments in the root epidermis and in guard cells of stomata, where they potentially function in ABA-controlled stomatal aperture. Although the tol2/3/5/6 quadruple mutant plant line is significantly more drought-tolerant and has a higher ABA sensitivity than control plant lines, it has no obvious growth or development phenotype under standard conditions, making the TOL genes ideal candidates for engineering to improved plant performance.

Axonal Growth and Fasciculation of Spinal Neurons Promoted by Aldynoglia in Alkaline Fibrin Hydrogel: Influence of Tol-51 Sulfoglycolipid.

Traumatic spinal cord injury (tSCI) has complex pathophysiological events that begin after the initial trauma. One such event is fibroglial scar formation by fibroblasts and reactive astrocytes. A strong inhibition of axonal growth is caused by the activated astroglial cells as a component of fibroglial scarring through the production of inhibitory molecules, such as chondroitin sulfate proteoglycans or myelin-associated proteins. Here, we used neural precursor cells (aldynoglia) as promoters of axonal growth and a fibrin hydrogel gelled under alkaline conditions to support and guide neuronal cell growth, respectively. We added Tol-51 sulfoglycolipid as a synthetic inhibitor of astrocyte and microglia in order to test its effect on the axonal growth-promoting function of aldynoglia precursor cells. We obtained an increase in GFAP expression corresponding to the expected glial phenotype for aldynoglia cells cultured in alkaline fibrin. In co-cultures of dorsal root ganglia (DRG) and aldynoglia, the axonal growth promotion of DRG neurons by aldynoglia was not affected. We observed that the neural precursor cells first clustered together and then formed niches from which aldynoglia cells grew and connected to groups of adjacent cells. We conclude that the combination of alkaline fibrin with synthetic sulfoglycolipid Tol-51 increased cell adhesion, cell migration, fasciculation, and axonal growth capacity, promoted by aldynoglia cells. There was no negative effect on the behavior of aldynoglia cells after the addition of sulfoglycolipid Tol-51, suggesting that a combination of aldynoglia plus alkaline fibrin and Tol-51 compound could be useful as a therapeutic strategy for tSCI repair.

A Novel Coenzyme A Analogue in the Anaerobic, Sulfate-Reducing, Marine Bacterium Desulfobacula toluolica Tol2T.

Coenzyme A (CoA) thioesters are formed during anabolic and catabolic reactions in every organism. Degradation pathways of growth-supporting substrates in bacteria can be predicted by differential proteogenomic studies. Direct detection of proposed metabolites such as CoA thioesters by high-performance liquid chromatography coupled with high-resolution mass spectrometry can confirm the reaction sequence and demonstrate the activity of these degradation pathways. In the metabolomes of the anaerobic sulfate-reducing bacterium Desulfobacula toluolica Tol2T grown with different substrates various CoA thioesters, derived from amino acid, fatty acid or alcohol metabolism, have been detected. Additionally, the cell extracts of this bacterium revealed a number of CoA analogues with molecular masses increased by 1 dalton. By comparing the chromatographic and mass spectrometric properties of synthetic reference standards with those of compounds detected in cell extracts of D. toluolica Tol2T and by performing co-injection experiments, these analogues were identified as inosino-CoAs. These CoA thioesters contain inosine instead of adenosine as the nucleoside. To the best of our knowledge, this finding represents the first detection of naturally occurring inosino-CoA analogues.

The Tol-Pal system of Acinetobacter baumannii is important for cell morphology, antibiotic resistance and virulence.

Acinetobacter baumannii is an opportunistic human pathogen that has become a global threat to healthcare institutions. This Gram-negative bacterium is one of the most successful human pathogens worldwide and responsible for hospital-acquired infections. This is due to its outstanding potential to adapt to very different environments, to persist in the human host and most important, its ability to develop multidrug resistance. Our combined approach of genomic and phenotypic analyses led to the identification of the envelope spanning Tol-Pal system in A. baumannii. We found that the deletion of the tolQ, tolR, tolA, tolB, and pal genes affects cell morphology and increases antibiotic sensitivity, such as the ∆tol-pal mutant exhibits a significantly increased gentamicin and bacitracin sensitivity. Furthermore, Galleria mellonella caterpillar killing assays revealed that the ∆tol-pal mutant exhibits a decreased killing phenotype. Taken together, our findings suggest that the Tol-Pal system is important for cell morphology, antibiotic resistance, and virulence of A. baumannii.

Recruitment of the TolA Protein to Cell Constriction Sites in Escherichia coli via Three Separate Mechanisms, and a Critical Role for FtsWI Activity in Recruitment of both TolA and TolQ.

The Tol-Pal system of Gram-negative bacteria helps maintain the integrity of the cell envelope and ensures that invagination of the envelope layers during cell fission occurs in a well-coordinated manner. In Escherichia coli, the five Tol-Pal proteins (TolQ, -R, -A, and -B and Pal) accumulate at cell constriction sites in a manner that normally requires the activity of the cell constriction initiation protein FtsN. While septal recruitment of TolR, TolB, and Pal also requires the presence of TolQ and/or TolA, the latter two can recognize constriction sites independently of the other system proteins. What attracts TolQ or TolA to these sites is unclear. We show that FtsN indirectly attracts both proteins and that PBP1A, PBP1B, and CpoB are dispensable for their septal recruitment. However, the ß-lactam aztreonam readily interferes with the septal accumulation of both TolQ and TolA, indicating that FtsN-stimulated production of septal peptidoglycan by the FtsWI synthase is critical to their recruitment. We also discovered that each of TolA's three domains can separately recognize division sites. Notably, the middle domain (TolAII) is responsible for directing TolA to constriction sites in the absence of other Tol-Pal proteins and CpoB, while recruitment of TolAI requires TolQ and that of TolAIII requires a combination of TolB, Pal, and CpoB. Additionally, we describe the construction and use of functional fluorescent sandwich fusions of the ZipA division protein, which should be more broadly valuable in future studies of the E. coli cell division machinery. IMPORTANCE Cell division (cytokinesis) is a fundamental biological process that is incompletely understood for any organism. Division of bacterial cells relies on a ring-like machinery called the septal ring or divisome that assembles along the circumference of the mother cell at the site where constriction will eventually occur. In the well-studied bacterium Escherichia coli, this machinery contains over 30 distinct proteins. We studied how two such proteins, TolA and TolQ, which also play a role in maintaining the integrity of the outer membrane, are recruited to the machinery. We find that TolA can be recruited by three separate mechanisms and that both proteins rely on the activity of a well-studied cell division enzyme for their recruitment.

Reconstructing the Last Common Ancestor: Epistemological and Empirical Challenges.

Reconstructing the genetic traits of the Last Common Ancestor (LCA) and the Tree of Life (TOL) are two examples of the reaches of contemporary molecular phylogenetics. Nevertheless, the whole enterprise has led to paradoxical results. The presence of Lateral Gene Transfer poses epistemic and empirical challenges to meet these goals; the discussion around this subject has been enriched by arguments from philosophers and historians of science. At the same time, a few but influential research groups have aimed to reconstruct the LCA with rich-in-detail hypotheses and high-resolution gene catalogs and metabolic traits. We argue that LGT poses insurmountable challenges for detailed and rich in details reconstructions and propose, instead, a middle-ground position with the reconstruction of a slim LCA based on traits under strong pressures of Negative Natural Selection, and for the need of consilience with evidence from organismal biology and geochemistry. We defend a cautionary perspective that goes beyond the statistical analysis of gene similarities and assumes the broader consequences of evolving empirical data and epistemic pluralism in the reconstruction of early life.

Phase Transitions and Stabilities among Three Phases of Di-p-tolyl Disulfides.

Di-p-tolyl disulfides (p-Tol2S2) are employed as load-carrying additives because of their anti-wear and extreme load-bearing qualities. External pressure triggers conformational up-conversion (leads to phase transition) in the molecules of p-Tol2S2, by compensating for the stress and absorbing its energy. These features make p-Tol2S2 a potential candidate for next-generation energy storage devices. Upon lithiation, MoS2 expands up to 103% which causes stress and affects battery stability and performance. Therefore, it is essential to study these materials under different physical conditions. In this work, we used density functional theory (DFT) at ωB97XD/6-31G* functional level, to calculate lattice parameters, Gibbs free energies, and vibrational spectra of three phases (i.e., α, ß, and γ) of p-Tol2S2 under different pressure and temperature conditions. The phase transition between phases α and ß occurred at a pressure and temperature of 0.65 GPa and 463 K, respectively. Furthermore, phase transition between phases α and γ was found at a pressure and temperature of 0.35 GPa and 400 K, respectively. Moreover, no phase transition was observed between phases ß and γ under the pressure range studied (0 GPa to 5.5 GPa). We also computed and compared the FT-IR spectra of the three phases. These results can guide scientists and chemists in designing more stable battery materials.

Mutations in enterobacterial common antigen biosynthesis restore outer membrane barrier function in Escherichia coli tol-pal mutants.

The outer membrane (OM) is an essential component of the Gram-negative bacterial envelope that protects the cells against external threats. To maintain a functional OM, cells require distinct mechanisms to ensure balance of proteins and lipids in the membrane. Mutations in OM biogenesis and/or homeostasis pathways often result in permeability defects, but how molecular changes in the OM affect barrier function is unclear. Here, we seek potential mechanism(s) that can alleviate permeability defects in Escherichia coli cells lacking the Tol-Pal complex, which accumulate excess PLs in the OM. We identify mutations in enterobacterial common antigen (ECA) biosynthesis that re-establish OM barrier function against large hydrophilic molecules, yet did not restore lipid homeostasis. Furthermore, we demonstrate that build-up of biosynthetic intermediates, but not loss of ECA itself, contributes to the rescue. This suppression of OM phenotypes is unrelated to known effects that accumulation of ECA intermediates have on the cell wall. Finally, we reveal that an unusual diacylglycerol pyrophosphoryl-linked lipid species also accumulates in ECA mutants, and might play a role in the rescue phenotype. Our work provides insights into how OM barrier function can be restored independent of lipid homeostasis, and highlights previously unappreciated effects of ECA-related species in OM biology.

Toll and Toll-like receptor signalling in development.

The membrane receptor Toll and the related Toll-like receptors (TLRs) are best known for their universal function in innate immunity. However, Toll/TLRs were initially discovered in a developmental context, and recent studies have revealed that Toll/TLRs carry out previously unanticipated functions in development, regulating cell fate, cell number, neural circuit connectivity and synaptogenesis. Furthermore, knowledge of their molecular mechanisms of action is expanding and has highlighted that Toll/TLRs function beyond the canonical NF-κB pathway to regulate cell-to-cell communication and signalling at the synapse. Here, we provide an overview of Toll/TLR signalling and discuss how this signalling pathway regulates various aspects of development across species.

Optogenetics on Zebrafish.

Optogenetics brought noninvasive neural activation in living organisms. Transparent zebrafish larva is one of the suitable animal models that receive the full benefit of this technique and provides behavioral studies based on intact individual nervous system. In this chapter, we describe methods to introduce optogenetic genes into zebrafish, and desirable apparatus for photostimulation and motion analysis with an example from our studies.

Calcium Signaling in the Cerebellar Radial Glia and Its Association with Morphological Changes during Zebrafish Development.

Radial glial cells are a distinct non-neuronal cell type that, during development, span the entire width of the brain walls of the ventricular system. They play a central role in the origin and placement of neurons, since their processes form structural scaffolds that guide and facilitate neuronal migration. Furthermore, glutamatergic signaling in the radial glia of the adult cerebellum (i.e., Bergmann glia), is crucial for precise motor coordination. Radial glial cells exhibit spontaneous calcium activity and functional coupling spread calcium waves. However, the origin of calcium activity in relation to the ontogeny of cerebellar radial glia has not been widely explored, and many questions remain unanswered regarding the role of radial glia in brain development in health and disease. In this study we used a combination of whole mount immunofluorescence and calcium imaging in transgenic (gfap-GCaMP6s) zebrafish to determine how development of calcium activity is related to morphological changes of the cerebellum. We found that the morphological changes in cerebellar radial glia are quite dynamic; the cells are remarkably larger and more elaborate in their soma size, process length and numbers after 7 days post fertilization. Spontaneous calcium events were scarce during the first 3 days of development and calcium waves appeared on day 5, which is associated with the onset of more complex morphologies of radial glia. Blockage of gap junction coupling inhibited the propagation of calcium waves, but not basal local calcium activity. This work establishes crucial clues in radial glia organization, morphology and calcium signaling during development and provides insight into its role in complex behavioral paradigms.

Germ cell-specific expression of Venus by Tol2-mediated transgenesis in endangered endemic cyprinid Honmoroko (Gnathopogon caerulescens).

Fishes expressing a fluorescent protein in germ cells are useful to perform germ cell transfer experiments for conservation study. Nonetheless, no such fish has been generated in endangered endemic fishes. In this study, we tried to produce a fish expressing Venus fluorescent protein in germ cells using Honmoroko (Gnathopogon caerulescens), which is one of the threatened small cyprinid endemic to the ancient Lake Biwa in Japan. To achieve germ cell-specific expression of Venus, we used piwil1 (formally known as ziwi) promoter and Tol2 transposon system. Following the co-injection of the piwil1-Venus expression vector and the Tol2 transposase mRNA into fertilized eggs, presumptive transgenic fish were reared. At 7 months of post-fertilization, about 19% (10/52) of the examined larvae showed Venus fluorescence in their gonad specifically. Immunohistological staining and in vitro spermatogenesis using gonads of the juvenile founder fish revealed that Venus expression was detected in spermatogonia and spermatocyte in male, and oogonia and stage I and II oocytes in female. These results indicate that the Tol2 transposon and zebrafish piwil1 promoter enabled gene transfer and germ cell-specific expression of Venus in G. caerulescens. In addition, in vitro culture of juvenile spermatogonia enables the rapid validation of temporal expression of transgene during spermatogenesis.

Diphtheria toxin-mediated transposon-driven poly (A)-trapping efficiently disrupts transcriptionally silent genes in embryonic stem cells.

Random gene trapping is the application of insertional mutagenesis techniques that are conventionally used to inactivate protein-coding genes in mouse embryonic stem (ES) cells. Transcriptionally silent genes are not effectively targeted by conventional random gene trapping techniques, thus we herein developed an unbiased poly (A) trap (UPATrap) method using a Tol2 transposon, which preferentially integrated into active genes rather than silent genes in ES cells. To achieve efficient trapping at transcriptionally silent genes using random insertional mutagenesis in ES cells, we generated a new diphtheria toxin (DT)-mediated trapping vector, DTrap that removed cells, through the expression of DT that was induced by the promoter activity of the trapped genes, and selected trapped clones using the neomycin-resistance gene of the vector. We found that a double-DT, the dDT vector, dominantly induced the disruption of silent genes, but not active genes, and showed more stable integration in ES cells than the UPATrap vector. The dDT vector disrupted differentiated cell lineage genes, which were silent in ES cells, and labeled trapped clone cells by the expression of EGFP upon differentiation. Thus, the dDT vector provides a systematic approach to disrupt silent genes and examine the cellular functions of trapped genes in the differentiation of target cells and development.

Decoupling Filamentous Phage Uptake and Energy of the TolQRA Motor in Escherichia coli.

Filamentous phages are nonlytic viruses that specifically infect bacteria, establishing a persistent association with their host. The phage particle has no machinery for generating energy and parasitizes its host's existing structures in order to cross the bacterial envelope and deliver its genetic material. The import of filamentous phages across the bacterial periplasmic space requires some of the components of a macrocomplex of the envelope known as the Tol system. This complex uses the energy provided by the proton motive force (pmf) of the inner membrane to perform essential and highly energy-consuming functions of the cell, such as envelope integrity maintenance and cell division. It has been suggested that phages take advantage of pmf-driven conformational changes in the Tol system to transit across the periplasm. However, this hypothesis has not been formally tested. In order to decouple the role of the Tol system in cell physiology and during phage parasitism, we used mutations on conserved essential residues known for inactivating pmf-dependent functions of the Tol system. We identified impaired Tol complexes that remain fully efficient for filamentous phage uptake. We further demonstrate that the TolQ-TolR homologous motor ExbB-ExbD, normally operating with the TonB protein, is able to promote phage infection along with full-length TolA.IMPORTANCE Filamentous phages are widely distributed symbionts of Gram-negative bacteria, with some of them being linked to genome evolution and virulence of their host. However, the precise mechanism that permits their uptake across the cell envelope is poorly understood. The canonical phage model Fd requires the TolQRA protein complex in the host envelope, which is suspected to translocate protons across the inner membrane. In this study, we show that phage uptake proceeds in the presence of the assembled but nonfunctional TolQRA complex. Moreover, our results unravel an alternative route for phage import that relies on the ExbB-ExbD proteins. This work provides new insights into the fundamental mechanisms of phage infection and might be generalized to other filamentous phages responsible for pathogen emergence.

Lipid trafficking across the Gram-negative cell envelope.

The outer membrane (OM) of Gram-negative bacteria exhibits unique lipid asymmetry, with lipopolysaccharides (LPS) residing in the outer leaflet and phospholipids (PLs) in the inner leaflet. This asymmetric bilayer protects the bacterium against intrusion of many toxic substances, including antibiotics and detergents, yet allows acquisition of nutrients necessary for growth. To build the OM and ensure its proper function, the cell produces OM constituents in the cytoplasm or inner membrane and transports these components across the aqueous periplasmic space separating the two membranes. Of note, the processes by which the most basic membrane building blocks, i.e. PLs, are shuttled across the cell envelope remain elusive. This review highlights our current understanding (or lack thereof) of bacterial PL trafficking, with a focus on recent developments in the field. We adopt a mechanistic approach and draw parallels and comparisons with well-characterized systems, particularly OM lipoprotein and LPS transport, to illustrate key challenges in intermembrane lipid trafficking. Pathways that transport PLs across the bacterial cell envelope are fundamental to OM biogenesis and homeostasis and are potential molecular targets that could be exploited for antibiotic development.

Outer Membrane Lipid Secretion and the Innate Immune Response to Gram-Negative Bacteria.

The outer membrane (OM) of Gram-negative bacteria is an asymmetric lipid bilayer that consists of inner leaflet phospholipids and outer leaflet lipopolysaccharides (LPS). The asymmetric character and unique biochemistry of LPS molecules contribute to the OM's ability to function as a molecular permeability barrier that protects the bacterium against hazards in the environment. Assembly and regulation of the OM have been extensively studied for understanding mechanisms of antibiotic resistance and bacterial defense against host immunity; however, there is little knowledge on how Gram-negative bacteria release their OMs into their environment to manipulate their hosts. Discoveries in bacterial lipid trafficking, OM lipid homeostasis, and host recognition of microbial patterns have shed new light on how microbes secrete OM vesicles (OMVs) to influence inflammation, cell death, and disease pathogenesis. Pathogens release OMVs that contain phospholipids, like cardiolipins, and components of LPS molecules, like lipid A endotoxins. These multiacylated lipid amphiphiles are molecular patterns that are differentially detected by host receptors like the Toll-like receptor 4/myeloid differentiation factor 2 complex (TLR4/MD-2), mouse caspase-11, and human caspases 4 and 5. We discuss how lipid ligands on OMVs engage these pattern recognition receptors on the membranes and in the cytosol of mammalian cells. We then detail how bacteria regulate OM lipid asymmetry, negative membrane curvature, and the phospholipid-to-LPS ratio to control OMV formation. The goal is to highlight intersections between OM lipid regulation and host immunity and to provide working models for how bacterial lipids influence vesicle formation.

Suppressor Mutations in degS Overcome the Acute Temperature-Sensitive Phenotype of ΔdegP and ΔdegP Δtol-pal Mutants of Escherichia coli.

In Escherichia coli, the periplasmic protease DegP plays a critical role in degrading misfolded outer membrane proteins (OMPs). Consequently, mutants lacking DegP display a temperature-sensitive growth defect, presumably due to the toxic accumulation of misfolded OMPs. The Tol-Pal complex plays a poorly defined but an important role in envelope biogenesis, since mutants defective in this complex display a classical periplasmic leakage phenotype. Double mutants lacking DegP and an intact Tol-Pal complex display exaggerated temperature-sensitive growth defects and the leaky phenotype. Two revertants that overcome the temperature-sensitive growth phenotype carry missense mutations in the degS gene, resulting in D102V and D320A substitutions. D320 and E317 of the PDZ domain of DegS make salt bridges with R178 of DegS's protease domain to keep the protease in the inactive state. However, weakening of the tripartite interactions by D320A increases DegS's basal protease activity. Although the D102V substitution is as effective as D320A in suppressing the temperature-sensitive growth phenotype, the molecular mechanism behind its effect on DegS's protease activity is unclear. Our data suggest that the two DegS variants modestly activate RseA-controlled, σE-mediated envelope stress response pathway and elevate periplasmic protease activity to restore envelope homeostasis. Based on the release of a cytoplasmic enzyme in the culture supernatant, we conclude that the conditional lethal phenotype of ΔtolB ΔdegP mutants stems from a grossly destabilized envelope structure that causes excessive cell lysis. Together, the data point to a critical role for periplasmic proteases when the Tol-Pal complex-mediated envelope structure and/or functions are compromised.IMPORTANCE The Tol-Pal complex plays a poorly defined role in envelope biogenesis. The data presented here show that DegP's periplasmic protease activity becomes crucial in mutants lacking the intact Tol-Pal complex, but this requirement can be circumvented by suppressor mutations that activate the basal protease activity of a regulatory protease, DegS. These observations point to a critical role for periplasmic proteases when Tol-Pal-mediated envelope structure and/or functions are perturbed.

Safety and Efficacy of a Novel Vaginal Anti-infective, TOL-463, in the Treatment of Bacterial Vaginosis and Vulvovaginal Candidiasis: A Randomized, Single-blind, Phase 2, Controlled Trial.

BACKGROUND: Bacterial vaginosis (BV) and vulvovaginal candidiasis (VVC) present serious reproductive health risks and management challenges, with poor control attributed to survival of treatment-resistant biofilm communities. Boric acid is used in various regimens for non-albicans VVC and recurrent BV. We investigated safety and efficacy of a novel boric acid-based vaginal anti-infective with enhanced antibiofilm activity (TOL-463) in treating BV and VVC. METHODS: In this phase 2 randomized, investigator-blinded trial conducted at 2 sexual health clinics, women with BV or VVC were randomly assigned (1:1) to 7 nights of TOL-463 vaginal gel or insert. The primary test of cure (TOC) was clinical cure at day 9-12; safety was assessed at TOC and day 21-30. RESULTS: One hundred six participants (53 with BV, 36 VVC, 17 both) were enrolled; most were African American (69%). Clinical cure rate of BV at TOC was 59% (95% confidence interval [CI], 41%-75%) for TOL-463 insert and 50% (95% CI, 31%-69%) for TOL-463 gel, and for VVC, 92% (95% CI, 67%-99%) for TOL-463 insert and 81% (95% CI, 57%-93%) for TOL-463 gel. Both products were safe and well tolerated with no secondary cases of VVC; vulvovaginal burning was the most common adverse event (9.6%). CONCLUSIONS: TOL-463, especially in vaginal insert form, is effective and safe in treating BV and VVC. Future studies should assess the potential role of TOL-463 as a biofilm disrupter in enhancing likelihood of cure relative to approved therapies, reducing recurrence rates, and combined with traditional antimicrobials. CLINICAL TRIALS REGISTRATION: NCT02866227.