Targetable nano-delivery vehicles to deliver anti-bacterial small acid-soluble spore protein (SASP) genes.

Interest in phage-based therapeutics is increasing, at least in part due to the need for new treatment options for infections caused by antibiotic-resistant bacteria. It is possible to use wild-type (WT) phages to treat bacterial infections, but it is also possible to modify WT phages to generate therapeutics with improved features. Here, we will discuss features of Phico Therapeutics' SASPject technology, which modifies phages for use as targetable nano-delivery vehicles (NDV), to introduce antibacterial Small Acid Soluble Spore Protein (SASP) genes into specific target bacteria.

Engineered Bacteriophage as a Delivery Vehicle for Antibacterial Protein, SASP.

The difficulties in developing novel classes of antibacterials is leading to a resurgence of interest in bacteriophages as therapeutic agents, and in particular engineered phages that can be optimally designed. Here, pre-clinical microbiology assessment is presented of a phage engineered to deliver a gene encoding an antibacterial small acid soluble spore protein (SASP) and further, rendered non-lytic to give product SASPject PT1.2.

A commentary on the development of engineered phage as therapeutics.

The use of engineered phages offers a unique opportunity to improve on wild-type (WT) phages to generate ever more successful therapeutics to combat bacterial infections. Here, we discuss how phage engineering could be used to overcome some of the technical challenges of phage therapy, and suggest some areas in which more research will be crucial to the development of further novel phage therapeutics.

Mutations in rpsL that confer streptomycin resistance show pleiotropic effects on virulence and the production of a carbapenem antibiotic in Erwinia carotovora.

Spontaneous streptomycin-resistant derivatives of Erwinia carotovora subsp. carotovora strain ATTn10 were isolated. Sequencing of the rpsL locus (encoding the ribosomal protein S12) showed that each mutant was missense, with a single base change, resulting in the substitution of the wild-type lysine by arginine, threonine or asparagine at codon 43.

Management of fistulae in the abdominal region.

Objective: We evaluated a new fistula and wound management system; ostomy and wound care nurses were queried about willingness to use the product in future patients, product wear time and pouch leakage, perifistular skin condition, access for wound care, pouching time, patient mobility and comfort, odor management, pouch flexibility, adhesiveness, and erosion. A health economic assessment was also done.

Method: Twenty-two patients (5 males and 17 females) with an abdominal fistula participated in the study.

Quorum sensing, virulence and secondary metabolite production in plant soft-rotting bacteria.

Quorum sensing describes the ability of bacteria to sense their population density and respond by modulating gene expression. In the plant soft-rotting bacteria, such as Erwinia, an arsenal of plant cell wall-degrading enzymes is produced in a cell density-dependent manner, which causes maceration of plant tissue. However, quorum sensing is central not only to controlling the production of such destructive enzymes, but also to the control of a number of other virulence determinants and secondary metabolites.

Identification of the central quorum sensing regulator of virulence in the enteric phytopathogen, Erwinia carotovora: the VirR repressor.

In the Gram-negative phytopathogen, Erwinia carotovora ssp. atroseptica (Eca) virulence depends on the production of a N-(3-oxohexanoyl)-L-homoserine lactone (OHHL) quorum sensing (QS) signal. This work identifies the elusive 'missing link' between QS and virulence in Erwinia.

Structure-activity relationships of Erwinia carotovora quorum sensing signaling molecules.

Production of virulence factors and secondary metabolites is regulated in the phytopathogen Erwinia carotovora by quorum sensing involving N-acylated homoserine lactone (AHL) signaling molecules. Non-hydrolyzable AHL analogues were synthesized and screened in vivo. The biological activity of each compound was correlated with its ability to bind Erwinia AHL receptor proteins (LuxR homologues) in vitro.

Regulation and biosynthesis of carbapenem antibiotics in bacteria.

Carbapenem antibiotics are members of the beta-lactam family of antibiotics, the most important class of antibiotics currently in clinical use. They are active against many important Gram-positive and Gram-negative pathogens. One important feature of carbapenem antibiotics is their resistance to several beta-lactamases.

Carbapenem antibiotic biosynthesis in Erwinia carotovora is regulated by physiological and genetic factors modulating the quorum sensing-dependent control pathway.

Erwinia carotovora produces the beta-lactam antibiotic, carbapenem, in response to a quorum sensing signalling molecule, N-(3-oxohexanoyl)-L-homoserine lactone (OHHL). We have mapped the OHHL-dependent promoter upstream of the first of the biosynthetic genes, carA. We have also analysed the effect on this promoter of the known genetic regulators of carbapenem expression, carR, carI (encoding homologues of LuxR and LuxI respectively) and hor (encoding a SlyA/MarR-like transcriptional regulator).

Regulation at complex bacterial promoters: how bacteria use different promoter organizations to produce different regulatory outcomes.

Most bacterial promoters are regulated by several signals. This is reflected in the complexity of their organization, with multiple binding sites for different transcription factors. Studies of a small number of complex promoters have revealed different distinct mechanisms that integrate the effects of multiple transcription factors.

Location of the Escherichia coli RNA polymerase alpha subunit C-terminal domain at an FNR-dependent promoter: analysis using an artificial nuclease.

The Escherichia coli FNR protein is a global transcription regulator that activates gene expression via interactions with the RNA polymerase alpha subunit C-terminal domain. Using preparations of E. coli RNA polymerase holoenzyme, specifically labelled with a DNA cleavage reagent, we have determined the location and orientation of the C-terminal domain of the RNA polymerase alpha subunit in transcriptionally competent complexes at a class II FNR-dependent promoter.

Transcription regulation by tandem-bound FNR at Escherichia coli promoters.

FNR is an Escherichia coli transcription factor that regulates the transcription of many genes in response to anaerobiosis. We have constructed a series of artificial FNR-dependent promoters, based on the melR promoter, in which a consensus FNR binding site was centered at position -41.5 relative to the transcription start site.

Transcription activation by FNR: evidence for a functional activating region 2.

The FNR protein of Escherichia coli controls the transcription of target genes in response to anoxia via the assembly-disassembly of oxygen-labile iron-sulfur clusters. Previous work identified patches of surface-exposed amino acids (designated activating regions 1 and 3 [AR1 and AR3, respectively]) of FNR which allow it to communicate with RNA polymerase (RNAP) and thereby activate transcription. Previously it was thought that FNR lacks a functional activating region 2 (AR2), although selecting for mutations that compensate for defective AR1 or a miscoordinated iron-sulfur cluster can reactivate AR2.