Biology subjects

Brondsted, L.

Publications and source records attributed to Brondsted, L..

9 recordsLinked to original sources

bioRxiv2026

A growth-inhibitory dUTPase from Mosigvirus AV110 disrupts cell division and host physiology in Escherichia coli

dUTPases are essential enzymes for nucleotide metabolism as they catalyze the hydrolysis of dUTP to dUMP, thus preventing the toxic incorporation of uracil into DNA. dUTPases are found in all domains of life, including bacteriophages, where they serve multiple functions to support phage propagation. Phage-encoded dUTPases reduce cellular dUTP levels and provide precursors for de novo dTTP synthesis and the synthesis of modified nucleotides, which are important to avoid DNA-based phage defense systems. In this study, we focused on the dimeric dUTPase Gp135, encoded by the Mosigvirus phage AV110, and examined its effect on growth, cell morphology, DNA replication and cell division of AV110s host, extended-spectrum beta-lactamase-producing E. coli. We observed that Gp135 significantly inhibited growth by disrupting DNA replication and cell division. This resulted in cytoplasmic voids, cell elongation, and ultimately cell death. We further demonstrated the diversity of phage-encoded dUTP/dCTPases through phylogenetic analysis, identifying four distinct clusters: one comprising the most common dUTPases, which form trimers; two more divergent clusters comprising dimeric dUTP/dCTPases; and one new cluster of more diverse, uncharacterized dUTPases and dNTPases. Overall, our results highlight the evolutionary and functional diversity of phage-encoded dUTP/dCTPases, with the Mosigvirus AV110 dUTP/dCTPase illustrating their role in supporting phage propagation.

microbiology↗

bioRxiv2026

A phage-encoded host transcription modulator, Htm, reprograms the transcriptome of ESBL Escherichia coli

During phage infection, phages reprogram their host to secure energy, building blocks for their genome, and proteins, as well as the transcription and translation machinery required to replicate their genome and produce phage particles. This also includes reprogramming the host transcriptome to modify host gene expression and align it with the needs of the phage during infection. In this study, we present a putative early-expressed protein, Gp204 of Mosigvirus AV110, renamed Host transcription modulator (Htm), that has a broad impact on the extended-spectrum beta-lactamase (ESBL)- associated E. coli transcriptome. Overexpression of Htm decreased the growth rate of ESBL-E. coli and caused DNA condensation. Htm is structurally similar to bacterial enhancer-binding proteins, binds ssDNA, and alters the expression levels of over 700 E. coli genes when expressed. The majority of these genes were upregulated and are involved in multiple pathways, such as galactose metabolism and pentose and glucuronate interconversions, which may promote the synthesis of purines, pyrimidines, and histidine, all of which are required for the replication, transcription, and translation of the phage genome. Furthermore, energy production is likely to be upregulated by increased availability of acetyl-CoA, resulting from, among other factors, fatty acid degradation. Htm also downregulated the expression of the structural genes involved in flagellar assembly and multiple two-component systems. In conclusion, Htm could promote phage progeny production by increasing the availability of nucleotides, amino acids, and energy, while inhibiting non-essential processes, such as flagella formation.

microbiology↗

bioRxiv2026

The Mosigvirus AV110 Elongasome-Modulating Peptide (Emp) inhibits Escherichia coli growth by targeting Mre-dependent peptidoglycan synthesis

Extended-spectrum {beta}-lactamase and AmpC-producing Escherichia coli (ESBL/AmpC E. coli) are increasingly recognized as public health threats due to their resistance to commonly used antibiotics. With limited treatment options available, new antibacterials and antibacterial targets can be explored by studying early-expressed phage proteins involved in host reprogramming, as these proteins interact with and disrupt essential bacterial processes. A systematic screening of 45 early-expressed proteins with unknown function of Mosigvirus AV110 identified six proteins that inhibit the growth of ESBL/AmpC E. coli, suggesting a role in host reprogramming. In-depth analysis of Gp94, termed Elongasome-Modulating Peptide (Emp), demonstrated that it alters cell morphology and the localization of peptidoglycan synthesis. This effect could be partially complemented by MreB and MreD. Since Emp interacts directly with MreB, MreC, and MreD, we propose that Emp perturbs elongasome-associated peptidoglycan synthesis, thus providing a scaffold for developing new antibacterials against ESBL/AmpC-producing E. coli.

microbiology↗

bioRxiv2024

The branched receptor binding complex of Ackermannviridae phages promotes adaptative host recognition

Bacteriophages may express multiple receptor binding proteins, enabling the recognition of distinct and diverse bacterial receptors for infection of a broad range of strains. Ackermannviridae phages recognize diverse O-antigens or K-antigens as receptors by expressing multiple tail spike proteins (TSPs). These TSPs interact and form a branched protein complex protruding from the baseplate attached to the distal tail. Here, we aimed to mimic the evolution of the TSP complex by studying the acquisition of new TSPs without disrupting the functionality of the complex. Using kuttervirus phage S117 as a backbone, we demonstrated the acquisition of entire tsp genes from Kuttervirus and Agtrevirus phages within the Ackermannviridae family. A fifth TSP was designed to interact with the complex and provide new host recognition to expand the branched TSP complex. Interestingly, the acquisition of tsp5 resulted in new variants of the branched TSP complex due to the exchange or deletion of tsp genes. Overall, our study provides novel insight into the development of the branched TSP complex, enabling Ackermannviridae phages to adapt to new hosts.

microbiology↗

bioRxiv2023

Campycins are novel broad spectrum antibacterials killing Campylobacter jejuni by targeting the essential major outer membrane protein (MOMP)

Pyocins are high molecular weight bacteriocins produced by Pseudomonas aeruginosa that can be retargeted to new bacterial species by exchanging the pyocin tail fibers with bacteriophage receptor binding proteins (RBPs). Here, we develop retargeted pyocins called campycins as new antibacterials to specifically and effectively kill the major foodborne pathogen Campylobacter jejuni. We used two diverse RBPs (H-fibers) encoded by CJIE1 prophages found in the genomes of C. jejuni strains CAMSA2147 and RM1221 to construct Campycin 1 and Campycin 2, respectively. Together Campycin 1 and 2 could target all C. jejuni strains tested due to complementary antibacterial spectrums. In addition, both campycins led to more than 3 log reductions in C. jejuni counts under microaerobic conditions at 42{degrees}C, whereas the killing efficiency was less efficient under anaerobic conditions at 5{degrees}C. We furthermore discovered that both H-fibers used to construct the campycins bind to the essential major outer membrane protein (MOMP) present in all C. jejuni, in a strain specific manner. Protein sequence alignment and structural modelling suggest that the highly variable extracellular loops of MOMP form the binding sites of the diverse H-fibers. Further in silico analyses of 5000 MOMP sequences suggest that the protein fall into three major clades predicted to be targeted by either Campycin 1 or Campycin 2. Thus, campycins are promising antibacterials against C. jejuni expected to broadly target numerous strains of this human pathogen found in nature and agriculture. IMPORTANCECampylobacter jejuni is the leading cause of bacterial foodborne gastroenteritis and responsible for more than 800 million cases globally each year posing a continuous risk to human health and a huge economic and societal burden. Here, we developed re-targeted R2 pyocins (campycins) as novel antibacterials against C. jejuni by using the receptor binding proteins of CJIE1 prophages observed in many C. jejuni genomes. Notably, campycins broadly target the highly variable yet essential major outer membrane protein (MOMP), and result in more than 3-log reductions in C. jejuni counts under conditions promoting bacterial growth. We therefore propose that campycins have the potential to lower C. jejuni colonization levels in the chicken gut, the main reservoir and cause of human disease, representing a novel efficient antibacterial solution specifically developed to target this widespread foodborne pathogen.

microbiology↗

bioRxiv2023

Engineering of Salmonella phages into novel antimicrobial Tailocins

Due to the extensive use of antibiotics, the increase of infections caused by antibiotic resistant bacteria are now a global health concern. Phages have proven useful for treating bacterial infections and represent a promising alternative or complement to antibiotic treatment. Yet, other alternative exists, such as bacteria-produced non-replicative protein complexes that can kill their targeted bacteria by puncturing their membrane (Tailocins). To expand the repertoire of Tailocins available, we suggest a new approach transforming phages into Tailocins. Here we genetically engineered the virulent Ackermannviridae phage S117, as well as temperate phages Fels-1, -2 and Gifsy-1 and -2 targeting the food pathogen Salmonella, by deleting the portal vertex or major capsid gene using CRISPR-Cas9. We report the production of Tailocin particles from engineered virulent and temperate phages able to kill their native host. Our work represents a steppingstone to tape into the huge diversity of phages and transform them into versatile puncturing new antimicrobials.

synthetic biology↗

bioRxiv2023

Agtrevirus phage AV101 infect diverse extended spectrum β-lactamase E. coli by recognizing four different O-antigens

Bacteriophages in the Agtrevirus genus are known for expressing multiple tail spike proteins (TSPs), but little is known about their genetic diversity and host recognition apart from their ability to infect diverse Enterobacteriaceae species. Here we aim to determine the genetic differences that may account for the diverse host ranges of Agrevirus phages. We performed comparative genomics of 14 Agtrevirus and identified only a few genetic differences including genes involved in nucleotide metabolism. Most notably was the diversity of the tsp gene cluster, specifically in the receptor binding domains that were unique among most of the phages. We further characterized agtrevirus AV101 infecting nine diverse Extended Spectrum {beta}-lactamase (ESBL) E. coli and demonstrated that this phage encoded four unique TSPs among Agtrevirus. Purified TSPs formed translucent zones and inhibited AV101 infection of specific hosts, demonstrating that TSP1, TSP2, TSP3, and TSP4 recognize O8, O82, O153, and O159 O-antigens of ESBL E. coli, respectively. BLASTp analysis showed that the receptor binding domain of TSP1, TSP2, TSP3 and TSP4 are similar to TSPs encoded by E. coli prophages and distant related virulent phages. Thus, Agtrevirus may have gained their receptor binding domains by recombining with prophages or virulent phages. Overall, combining bioinformatic and biological data expands the understanding of TSP host recognition of Agtrevirus and give new insight into the origin and acquisition of receptor binding domains of Ackermannviridae phages. One sentence summaryAgtrevirus phage AV101 express four unique tail spike proteins that recognize different O-antigens of Extended Spectrum {beta}-Lactamase producing E. coli.

microbiology↗

bioRxiv2023

A collection of diverse bacteriophages for biocontrol of ESBL- and AmpC-β-lactamase-producing E. coli

Novel solutions are needed to reduce the risk of transmission of extended spectrum {beta}-lactamase and AmpC {beta}-lactamase producing Escherichia coli (ESBL/AmpC E. coli) in livestock to humans. Since phages are promising biocontrol agents, we established a collection of 28 phages against ESBL/AmpC E. coli and showed by whole genome sequencing that all phages were unique and could be assigned to 15 different genera. Host range analysis showed that 82% of 198 strains, representing the genetic diversity of ESBL/AmpC E. coli, were sensitive to at least one phage. Identifying receptors used for initial binding experimentally as well as in silico predictions, allowed us to combine phages into two different cocktails with broad host range targeting diverse receptors. These phage cocktails inhibit growth and kill ESBL/AmpC E. coli in vitro, thus suggesting the potential of phages as promising biocontrol agents. HIGHLIGHTSO_LI28 unique phages infecting ESBL/AmpC E. coli were isolated and characterized C_LIO_LIBroad host range phages targeting different receptors were used to compose phage cocktails C_LIO_LIPhage cocktails efficiently inhibit growth of ESBL/AmpC E. coli in vitro C_LI

microbiology↗

bioRxiv2018

Innolysins: A novel approach to engineer endolysins to kill Gram-negative bacteria

Bacteriophage-encoded endolysins degrading the essential peptidoglycan of bacteria are promising alternative antimicrobials to handle the global threat of antibiotic resistant bacteria. However, endolysins have limited use against Gram-negative bacteria, since their outer membrane prevents access to the peptidoglycan. Here we present Innolysins, a novel concept for engineering endolysins that allows the enzymes to pass through the outer membrane, hydrolyse the peptidoglycan and kill the target bacterium. Innolysins combine the enzymatic activity of endolysins with the binding capacity of phage receptor binding proteins (RBPs). As our proof of concept, we used phage T5 endolysin and receptor binding protein Pb5, which binds irreversibly to the phage receptor FhuA involved in ferrichrome transport in Escherichia coli. In total, we constructed twelve Innolysins fusing endolysin with Pb5 or the binding domain of Pb5 with or without flexible linkers in between. While the majority of the Innolysins maintained their muralytic activity, Innolysin#6 also showed bactericidal activity against E. coli reducing the number of bacteria by 1 log, thus overcoming the outer membrane barrier. Using an E. coli fhuA deletion mutant, we demonstrated that FhuA is required for bactericidal activity, supporting that the specific binding of Pb5 to its receptor on E. coli is needed for the endolysin to access the peptidoglycan. Accordingly, Innolysin#6 was able to kill other bacterial species that carry conserved FhuA homologs such as Shigella sonnei and Pseudomonas aeruginosa. In summary, the Innolysin approach expands recent protein engineering strategies allowing customization of endolysins by exploiting phage RBPs to specifically target Gram-negative bacteria.\n\nIMPORTANCEThe extensive use of antibiotics has led to the emergence of antimicrobial resistant bacteria responsible for infections causing more than 50,000 deaths per year across Europe and the US. In response, the World Health Organization has stressed an urgent need to discover new antimicrobials to control in particular Gram-negative bacterial pathogens, due to their extensive multi-drug resistance. However, the outer membrane of Gram-negative bacteria limits the access of many antibacterial agents to their targets. Here, we developed a new approach, Innolysins that enable endolysins to overcome the outer membrane by exploiting the binding specificity of phage receptor binding proteins. As proof of concept, we constructed Innolysins against E. coli using the endolysin and the receptor binding protein of phage T5. Given the rich diversity of phage receptor binding proteins and their different binding specificities, our proof of concept paves the route for creating an arsenal of pathogen specific alternative antimicrobials.

microbiology↗