Biology subjects

Feher, T.

Publications and source records attributed to Feher, T..

5 recordsLinked to original sources

bioRxiv2026

Novel Synthetic Polymyxin Variants Inspired by Newly Uncovered Natural Sequences Explored as Potential Antibiotics

The incidence of infections caused by multidrug-resistant bacterial agents has increased at an alarming rate worldwide. With the aim of identifying novel antimicrobial peptides (AMPs), we screened bacteria isolated from various environmental samples. Our hypothesis was that the sequence space of natural AMPs belonging to known AMP classes is far from saturation. We used a classical pipeline of bacterial culturing, overlay assays, extraction and fractionation to purify AMPs for identification. Using LC/MS analysis, the structure of AMPs isolated from a strain of Paenibacillus was narrowed down and a new subclass of polymyxins was uncovered. These harbor three aliphatic residues within the cyclic C-terminal, and in certain cases Ser replaces Thr at position A2. Four discrete polymyxins fitting the new class, but not identical to the natural polymyxins were synthesized and tested against 29 human pathogenic bacteria. Each displayed an antibacterial spectrum different from that of colistin, with the best candidate surpassing it in potency against ten bacterial strains, but underperforming against three others. These results demonstrate that current screening of natural bacterial isolates can still permit the design of novel antimicrobial peptide variants without the substantial threat of diminishing returns.

microbiology↗

bioRxiv2024

Directional Immobilisation of SpyTag Bacteriophage on PDMS surfaces for Phage based Microfluidics

The increasing incidence of bacterial infections caused by antibiotic-resistant pathogens worldwide underlines the need to develop novel diagnostic tools enabling the early initiation of targeted antimicrobial therapy. One promising possibility is to unite the high specificity and sensitivity of phage-based applications with the speed and sensitivity provided by microfluidic devices. As a prerequisite of developing such systems, we aimed at the directional immobilization of phages on the surface of Polydimethylsiloxane (PDMS), a material commonly used for building such devices. Our work utilised the covalent interaction between two proteins: SpyTag, genetically encoded on the capsid of the phage, and BslA-SpyCatcher fusion protein, purified and surface displayed on PDMS. We demonstrate a simple methodology for the directional tail up immobilisation of SpyTagged Phage on to user defined locations on the surface of a PDMS device and subsequent on chip capture and infection of a cognate host. Our technique serves to illustrate a generally applicable solution to develop the next generation of phage based bio-sensors.

bioengineering↗

bioRxiv2024

TXTL-Powered K1F Internal Capsid Protein Engineering for Specific, Orthogonal and Rapid Phage-based Pathogen Detection

The internal capsid proteins that reside within phage of the Podoviridae family hold high potential for being used as sensitive and reliable diagnostic tools. The concealed nature of the capsid interior ensures that any encapsulated signal or signal generating enzyme, e.g., fused to an internal capsid protein, is suppressed whilst the phage is unaccompanied by its host. Furthermore, the only naturally occurring mechanism for releasing the internal capsid proteins, and therefore exposing their amalgamated signal/enzyme, is for them to be passed through the tail and subsequently ejected out of the phage, a post-adsorption phenomenon which occurs when the host is present, thus presenting a precise model for signal/enzyme release only upon pathogen presence. Here, a small N terminal subunit of the NanoLuc luciferase is fused and incorporated into the K1F internal capsid structure using a simple, non-genomic method. This internalised subunit is exposed to the test solution containing its C terminal counterpart (natural complementation immediately forms the full NanoLuc enzyme) and substrate furimazine in an inducible manner which mimics the presence of the K1F host, E. coli K1 thereby presenting a novel method for rapidly detecting this disease causing pathogen. Finally, it is expected that by building upon this internal capsid protein engineering approach, which completely bypasses the time-inducing processes of intracellular nucleic acid transcription and translation, an unprecedentedly rapid detection device can be developed for an array of bacterial pathogens.

synthetic biology↗

bioRxiv2021

inPOSE: a flexible toolbox for chromosomal cloning and amplification of bacterial transgenes

Cloning genes and operons encoding heterologous functions in bacterial hosts is almost exclusively carried out today using plasmid vectors. This has multiple drawbacks, including the need for constant selection and the variation in copy numbers. Chromosomal integration of transgenes has always offered a viable alternative, however, to date it has been of limited use due to its tedious nature and to being limited often to a single copy. We introduce here a strategy that uses bacterial insertion sequences, the simplest autonomous transposable elements to insert and amplify genetic cargo into a bacterial chromosome. Transgene insertion can take place either as transposition or homologous recombination, and copy-number amplification is achieved using controlled copy-paste transposition. We display successful use of IS1 and IS3 for this purpose in Escherichia coli cells, using various selection markers. We demonstrate the insertion of selectable genes, an unselectable gene, and a five-gene operon in up to two copies in a single step. We continue with the amplification of the inserted cassette to double-digit copy numbers within two rounds of transposase induction and selection. Finally, we analyze the stability of the cloned genetic constructs in the lack of selection and find it to be superior to all investigated plasmid-based systems. Due to the ubiquitous nature of transposable elements, we believe that with proper design, this strategy can be adapted to numerous further bacterial species.

molecular biology↗

bioRxiv2018

CRISPR-interference based modulation of mobile genetic elements in bacteria

Spontaneous mutagenesis of synthetic genetic constructs by mobile genetic elements frequently results in the rapid loss of advantageous functions. Previous efforts to minimize such mutations required the exceedingly time-consuming manipulation of bacterial chromosomes and the complete removal of insertional sequences (ISes). To this aim, we developed a single plasmid-based system (pCRIS) that applies CRISPR-interference to inhibit the transposition of bacterial ISes. pCRIS expresses multiple guide RNAs to direct inactivated Cas9 (dCas9) to simultaneously silence IS1, IS3, IS5, and IS150 at up to 38 chromosomal loci in Escherichia coli, in vivo. As a result, the transposition rate of all four targeted ISes dropped to negligible levels at both chromosomal and episomal targets, increasing the half-life of exogenous protein expression. Most notably, pCRIS, while requiring only a single plasmid delivery performed within a single day, provided a reduction of IS-mobility comparable to that seen in genome-scale chromosome engineering projects. Global transcriptomics analysis revealed nevertheless only minute alterations in the expression of untargeted genes. Finally, the transposition-silencing effect of pCRIS was easily transferable across multiple E. coli strains. The plasticity and robustness of our IS-silencing system make it a promising tool to stabilize bacterial genomes for synthetic biology and industrial biotechnology applications.

synthetic biology↗