Bioengineering bacterial outer membrane vesicles as delivery system for RNA therapeutics targeted to lung epithelial cytosols

Intact epithelia lining the airways and alveoli in the lung are essential to maintain lung function. Structural or functional damage of epithelial cells leads in severe diseases, including COPD/emphysema, ibrosis or ALI/ARDS. This central role of epithelia in pulmonary diseases identifies these cells as primary candidates for targeted therapy. With the exception of surface-expressed molecules, however, targeting intracellular components is severely restricted due to poor delivery. We aim to overcome this obstacle using topically administered, bioengineered, biocompatible bacterial outer membrane vesicles (OMVs) as recombinant drug delivery systems for novel biopharmaceuticals. Engineering recombinant surface expression of eukaryotic receptor ligands in ClearColi®, a commercial E.coli BL21 (DE3) strain deficient in lipopolysaccharide production, we have used red fluorescent protein reporters to track OMV loading, transgene expression, and eukaryotic cell trafficking. We demonstrate statistically significant differences in the levels of over 700 proteins between differentially engineered and purified OMV preps with additional differences in transcriptome and lipidome consistency. We also characterised visual and particle size differences observed by transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). Here we report early bioadhesion and culture of re-differentiated lung epithelia. This project aims to bridge the biotechnological gap in the intracellular biopharmaceutics drug delivery challenge for respiratory epithelia through highly controlled, and scalable bio-nanotechnology process. If successful, our work will unlock intracellular imaging and therapeutics research for respiratory diseases with a significant epithelial component, paving the way for other targeting ligands and potentially non-respiratory indications. cellular uptake results in A549 culture as well as air-liquid interface

Genomic and molecular characterization of a novel quorum sensing molecule in Bacillus licheniformis

Abstract Quorum sensing molecules (QSMs) are involved in the regulation of complicated processes helping bacterial populations respond to changes in their cell-density. Although the QS gene cluster (comQXPA) has been identified in the genome sequence of some bacilli, the QS system B. licheniformis has not been investigated in detail, and its QSM (ComX pheromone) has not been identified. Given the importance of this antagonistic bacterium as an industrial workhorse, this study was aimed to elucidate B. licheniformis NCIMB-8874 QS. The results obtained from bioinformatics studies on the whole genome sequence of this strain confirmed the presence of essential quorum sensing-related genes. Although polymorphism was verified in three proteins of this cluster, ComQ, precursor-ComX and ComP, the transcription factor ComA was confirmed as the most conserved protein. The cell–cell communication of B. licheniformis NCIMB-8874 was investigated through further elucidation of the ComX pheromone as 13-amino acid peptide. The peptide sequence of the pheromone has been described through biochemical characterisation

Profiling the Mismatch Tolerance of Argonaute 2 through Deep Sequencing of Sliced Polymorphic Viral RNAs

Low allelic and clonal variability among endogenous RNA interference (RNAi) targets has focused mismatch tolerance studies to RNAi-active guide strands. However, the inherent genomic instability of RNA viruses such as hepatitis C virus (HCV) gives rise to quasi-species mutants within discrete clones: this facilitates mismatch tolerance studies from a target perspective. We recently quantified the slicing imprecision of Argonaute 2 using small interfering RNA (siRNA) analogues of the DNA-directed RNAi drug TT-034 and next generation sequencing of 5’ RNA Ligase-Mediated Rapid Amplification of cDNA Ends (RACE-SEQ). Here, we present an open source, customizable, and computationally light RACE-SEQ bioinformatic pipeline, describing adaptations that semi-quantitatively report the impact of RNAi hybridisation site mismatches from the target perspective. The analysis shows Argonaute 2 has a substitution-specific, 3-5 log activity window between fully complementary targets and targets with mismatches across positions 10-11. It further focuses the endonucleotic Slicer imprecision around positions 13-17, demonstrating its dependence on guide strand central region complementarity, and potentiation by even a single mismatch. We further propose pharmacogenomics value in testing endogenous targets using recombinant replicon systems and RACE-SEQ to report the pharmacodynamics of sequence-specific oligonucleotide therapeutics against all possible polymorphisms in a population, in a minimally-biased, patient-free manner

Introducing EbolaCheck: potential for point-of-need infectious disease diagnosis

The 2013–2015 Ebolavirus disease humanitarian crisis has spurred the development of laboratory-free, point-of-care nucleic acid testing solutions. EbolaCheck is an international consortium of public health, academic and biotechnology industry stakeholders aiming to deliver clinical molecular diagnostic standard-of-care testing suitable for the West African milieu within 12 months. In this article, the current status of the EbolaCheck platform is discussed in the context of the current regulatory framework. Presented here are future goals to achieve differential diagnosis of hemorrhagic fever disease from <5-μl of whole blood samples or mucosal biofluids, in a single tube process, under 40 min and with minimal operator training requirements

Comprehensive analysis of drugs to treat SARS‑CoV‑2 infection: Mechanistic insights into current COVID‑19 therapies (Review)

The major impact produced by the severe acute respiratory syndrome coronavirus 2 (SARS‑CoV‑2) focused many researchers attention to find treatments that can suppress transmission or ameliorate the disease. Despite the very fast and large flow of scientific data on possible treatment solutions, none have yet demonstrated unequivocal clinical utility against coronavirus disease 2019 (COVID‑19). This work represents an exhaustive and critical review of all available data on potential treatments for COVID‑19, highlighting their mechanistic characteristics and the strategy development rationale. Drug repurposing, also known as drug repositioning, and target based methods are the most used strategies to advance therapeutic solutions into clinical practice. Current in silico, in vitro and in vivo evidence regarding proposed treatments are summarized providing strong support for future research efforts

A new threat from an old enemy: Re‑emergence of coronavirus (Review)

The new outbreak of coronavirus from December 2019 has brought attention to an old viral enemy and has raised concerns as to the ability of current protection measures and the healthcare system to handle such a threat. It has been known since the 1960s that coronaviruses can cause respiratory infections in humans; however, their epidemic potential was understood only during the past two decades. In the present review, we address current knowledge on coronaviruses from a short history to epidemiology, pathogenesis, clinical manifestation of the disease, as well as treatment and prevention strategies. Although a great amount of research and efforts have been made worldwide to prevent further outbreaks of coronavirus‑associated disease, the spread and lethality of the 2019 outbreak (COVID‑19) is proving to be higher than previous epidemics on account of international travel density and immune naivety of the population. Only strong, joint and coordinated efforts of worldwide healthcare systems, researchers, and pharmaceutical companies and receptive national leaders will succeed in suppressing an outbreak of this scale

The emerging field of venom-microbiomics for exploring venom as a microenvironment, and the corresponding Initiative for Venom Associated Microbes and Parasites (iVAMP)

Venom is a known source of novel antimicrobial natural products. The substantial, increasing number of these discoveries have unintentionally culminated in the misconception that venom and venom-producing glands are largely sterile environments. Culture-dependent and -independent studies on the microbial communities in venom microenvironments reveal the presence of archaea, algae, bacteria, endoparasites, fungi, protozoa, and viruses. Venom-centric microbiome studies are relatively sparse to date and the adaptive advantages that venom-associated microbes might offer to their hosts, or that hosts might provide to venom-associated microbes, remain unknown. We highlight the potential for the discovery of venom-microbiomes within the adaptive landscape of venom systems. The considerable number of known, convergently evolved venomous animals juxtaposed with the comparatively few studies to identify microbial communities in venom provides new possibilities for both biodiversity and therapeutic discoveries. We present an evidence-based argument for integrating microbiology as part of venomics to which we refer to as venom-microbiomics. We also introduce iVAMP, the Initiative for Venom Associated Microbes and Parasites (https://ivamp-consortium.github.io/), as a growing consortium for interested parties to contribute and collaborate within this subdiscipline. Our consortium seeks to support diversity, inclusion and scientific collaboration among all researchers interested in this subdiscipline

Field-deployable, quantitative, rapid identification of active Ebola virus infection in unprocessed blood

The West African Ebola virus outbreak underlined the importance of delivering mass diagnostic capability outside the clinical or primary care setting in effectively containing public health emergencies caused by infectious disease. Yet, to date, there is no solution for reliably deploying at the point of need the gold standard diagnostic method, real time quantitative reverse transcription polymerase chain reaction (RT- qPCR), in a laboratory infrastructure-free manner. In this proof of principle work, we demonstrate direct performance of RT-qPCR on fresh blood using far-red fluorophores to resolve fluorogenic signal inhibition and controlled, rapid freeze/thawing to achieve viral genome extraction in a single reaction chamber assay. The resulting process is entirely free of manual or automated sample pre-processing, requires no microfluidics or magnetic/mechanical sample handling and thus utilizes low cost consumables. This enables a fast, laboratory infrastructure-free, minimal risk and simple standard operating procedure suited to frontline, field use. Developing this novel approach on recombinant bacteriophage and recombinant human immunodeficiency virus (HIV; Lentivirus), we demonstrate clinical utility in symptomatic EBOV patient screening using live, infectious Filoviruses and surrogate patient samples. Moreover, we evidence assay co-linearity independent of viral particle structure that may enable viral load quantification through pre-calibration, with no loss of specificity across an 8 log- linear maximum dynamic range. The resulting quantitative rapid identification (QuRapID) molecular diagnostic platform, openly accessible for assay development, meets the requirements of resource- limited countries and provides a fast response solution for mass public health screening against emerging biosecurity threats

Bacterial Adaptation to Venom in Snakes and Arachnida

Animal venoms are considered sterile sources of antimicrobial compoundswith strong membrane-disrupting activity against multidrug-resistant bacteria. However,venomous bite wound infections are common in developing nations. Investigating theenvenomation organ and venom microbiota offive snake and two spider species, weobserved venom community structures that depend on the host venomous animal spe-cies and evidenced recovery of viable microorganisms from black-necked spitting cobra(Naja nigricollis) and Indian ornamental tarantula (Poecilotheria regalis) venoms. Amongthe bacterial isolates recovered fromN. nigricollis,weidentified two venom-resistant,novel sequence types ofEnterococcus faecaliswhose genomes feature 16 virulencegenes, indicating infectious potential, and 45 additional genes, nearly half of whichimprove bacterial membrane integrity. Ourfindings challenge the dogma of venom ste-rility and indicate an increased primary infection risk in the clinical management of ven-omous animal bite wounds

Bacterial adaptation to venom in snakes and arachnida

Animal venoms are considered sterile sources of antimicrobial compounds with strong membrane-disrupting activity against multidrug-resistant bacteria. However, venomous bite wound infections are common in developing nations. Investigating the envenomation organ and venom microbiota of five snake and two spider species, we observed venom community structures that depend on the host venomous animal species and evidenced recovery of viable microorganisms from black-necked spitting cobra (Naja nigricollis) and Indian ornamental tarantula (Poecilotheria regalis) venoms. Among the bacterial isolates recovered from N. nigricollis, we identified two venom-resistant, novel sequence types of Enterococcus faecalis whose genomes feature 16 virulence genes, indicating infectious potential, and 45 additional genes, nearly half of which improve bacterial membrane integrity. Our findings challenge the dogma of venom sterility and indicate an increased primary infection risk in the clinical management of venomous animal bite wounds. IMPORTANCE Notwithstanding their 3 to 5% mortality, the 2.7 million envenomation-related injuries occurring annually—predominantly across Africa, Asia, and Latin America—are also major causes of morbidity. Venom toxin-damaged tissue will develop infections in some 75% of envenomation victims, with E. faecalis being a common culprit of disease; however, such infections are generally considered to be independent of envenomation. Here, we provide evidence on venom microbiota across snakes and arachnida and report on the convergent evolution mechanisms that can facilitate adaptation to black-necked cobra venom in two independent E. faecalis strains, easily misidentified by biochemical diagnostics. Therefore, since inoculation with viable and virulence gene-harboring bacteria can occur during envenomation, acute infection risk management following envenomation is warranted, particularly for immunocompromised and malnourished victims in resource-limited settings. These results shed light on how bacteria evolve for survival in one of the most extreme environments on Earth and how venomous bites must be also treated for infections