Biology subjects

Hatfull, G.

Publications and source records attributed to Hatfull, G..

7 recordsLinked to original sources

bioRxiv2026

Phage RyR-domain proteins degrade ADPR-based immune signals and fuel NAD synthesis

Bacterial, plant, and animal cells synthesize nucleotide immune signals as a conserved strategy to defend against viral infection1-4. In bacteria, Thoeris anti-phage defense systems convert nicotinamide adenine dinucleotide (NAD+) into the cyclic ADP-ribose signals 2'cADPR and 3'cADPR to activate downstream effectors and restrict viral replication5-8. Phage proteins can bind and sequester Thoeris signals6,9-13, but no mechanisms are known to degrade the exceptionally stable 2'cADPR and 3'cADPR molecules and terminate immune activation. Here we use a forward biochemical screen to discover the mycobacteriophage protein RyDEP as the founding member of an enzyme family that cleaves 2'cADPR and 3'cADPR to inactivate Thoeris defense. We show that RyDEP is a glycosidase that cleaves the ribose-ribose linkage in 2' and 3' cADPR immune signals to both inactivate host defense and enable direct restoration of NAD+. A crystal structure of the RyDEP-3'cADPR complex in the post-cleavage state explains the molecular basis of immune signal degradation and reveals surprising homology with the Repeat12 domain of animal ryanodine receptors (RyRs) that control calcium flux and muscle contraction14,15. We demonstrate that diverse phage RyDEP proteins tune RyR-domain activity to either degrade or sequester immune signals. Our results define RyR-domain proteins as regulators of nucleotide immune signaling and explain how viruses subvert host antiviral defense.

microbiology↗

bioRxiv2025

Inactivating conditions of therapeutic mycobacteriophages

There is a need for new therapies to treat drug resistant nontuberculous mycobacteria (NTM) disease. Bacteriophages (phages), which are viruses that infect and kill bacteria, are actively being explored as an alternative approach for treating mycobacterial diseases. Several compassionate-use cases of phage therapy for drug resistant NTM infections exhibit favorable outcomes. To further the development of phage therapy it is important to recognize and avoid conditions that negatively impact phage activity during phage production, storage, formulation, or treatment. Conversely, there is a need to inactivate free phages in certain preclinical phage therapy experiments. In this study, we investigated three mycobacteriophages BPs{Delta}33HTH-HRM10, Muddy, and ZoeJ{Delta}45 from compassionate-use NTM treatment cases for their sensitivity to a variety of conditions that included temperature, acid pH, detergents, mucus, and phage inactivating buffers. Several conditions resulted in dramatic and rapid reductions in the level of active phage while others had no effect. We also observed different sensitivities between the phages. The results provide valuable information to support further investigation and development of these phages as therapeutics.

microbiology↗

bioRxiv2025

Genome editing of phylogenetically distinct bacteria using portable retron-mediated recombineering

Advanced genome editing technologies have enabled rapid and flexible rewriting of the Escherichia coli genome, benefiting fundamental biology and biomanufacturing. Unfortunately, some of the most useful technologies to advance genome editing in E. coli have not yet been ported into other bacterial species. For instance, the addition of bacterial retrons to the genome editing toolbox has increased the efficiency of recombineering in E. coli by enabling sustained, abundant production of ssDNA recombineering donors by reverse transcription that install flexible, precise edits in the prokaryotic chromosome. To extend the utility of this technology beyond E. coli, we surveyed the portability and versatility of retron-mediated recombineering across three different bacterial phyla (Proteobacteria, Bacillota and Actinomycetota) and a total of 15 different species. We found that retron recombineering is functional in all species tested, reaching editing efficiencies above 20% in six of them, above 40% in three of them, and above 90% in two of them. We also tested the extension of the recombitron architecture optimizations and strain backgrounds in a subset of hosts to additionally increase editing rates. The broad recombitron survey carried out in this study forms the basis for widespread use of retron-derived technologies through the whole Bacteria domain.

bioengineering↗

bioRxiv2023

A novel stabilization mechanism accommodating genome length variation in evolutionarily related viral capsids

Tailed bacteriophages are one of the most numerous and diverse group of viruses. They store their genome at quasi-crystalline densities in capsids built from multiple copies of proteins adopting the HK97-fold. The high density of the genome exerts an internal pressure, requiring a maturation process that reinforces their capsids. However, it is unclear how capsid stabilization strategies have adapted to accommodate the evolution of larger genomes in this virus group. Here we characterized a novel capsid reinforcement mechanism in two evolutionary-related actinobacteriophages that modifies the length of a stabilization protein to accommodate a larger genome while maintaining the same capsid size. We used cryo-EM to reveal that capsids contained split hexamers of HK97-fold proteins with a stabilization protein in the chasm. The observation of split hexamers in mature capsids was unprecedented, so we rationalized this result mathematically, discovering that icosahedral capsids can be formed by all split or skewed hexamers as long as their T-number is not a multiple of three. Our results suggest that analogous stabilization mechanisms can be present in other icosahedral capsids, and they provide a strategy for engineering capsids accommodating larger DNA cargoes as gene delivery systems. Significance StatementHow capsids are stabilized and change size is an important part of understanding how to design protein containers and understand viral evolution. We describe a novel capsid stability mechanism that allows the capsid to package a larger genome without changing the capsid architecture and have predicted other capsids using this mechanism. Beyond the evolutionary implications, our findings provide a mechanism to increase the amount of DNA packaged in a capsid, offering a solution to engineer gene delivery systems with larger DNA content, a pressing challenge in gene therapy.

molecular biology↗

bioRxiv2022

A structural dendrogram of the actinobacteriophage major capsid proteins provides important structural insights into the evolution of capsid stability

Many double-stranded DNA viruses, including tailed bacteriophages (phages) and herpesviruses, use the HK97-fold in their major capsid protein to make the capsomers of the icosahedral viral capsid. Following the genome packaging at near-crystalline densities, the capsid is subjected to a major expansion and stabilization step that allows it to withstand environmental stresses and internal high pressure. Several different mechanisms for stabilizing the capsid have been structurally characterized, but how these mechanisms have evolved is still not understood. Using cryo-EM structure determination, structural comparisons, phylogenetic analyses, and Alphafold predictions, we have constructed a detailed structural dendrogram describing the evolution of capsid structural stability within the actinobacteriophages. The cryo-EM reconstructions of ten capsids solved to resolutions between 2.2 and 4 [A]ngstroms revealed that eight of them exhibit major capsid proteins that are linked by a covalent cross-linking (isopeptide bond) between subunits that was first described in the HK97 phage. Those covalent interactions ultimately lead to the formation of mutually interlinked capsomers that has been compared to the structure of chain mail. However, three of the closely related phages do not exhibit such an isopeptide bond as demonstrated by both our cryo-EM maps and the lack of the required residue. This work raises questions about the importance of previously described capsid stabilization mechanisms.

biochemistry↗

bioRxiv2020

Genome analysis of Salmonella enterica serovar Typhimurium bacteriophage L, indicator for StySA (StyLT2III) restriction-modification system action

Bacteriophage L, a P22-like phage of Salmonella enterica sv Typhimurium LT2, was important for definition of mosaic organization of the lambdoid phage family and for characterization of restriction-modification systems of Salmonella. We report the complete genome sequences of bacteriophage L cI-40 13-am43 and L cII-101; the deduced sequence of wildtype L is 40,633 bp long with a 47.5% GC content. We compare this sequence with those of P22 and ST64T, and predict 71 Coding Sequences, 2 tRNA genes and 14 intergenic rho-independent transcription terminators. The overall genome organization of L agrees with earlier genetic and physical evidence; for example, no secondary immunity region (ImmI: ant, arc) or genes for superinfection exclusion (sieA and sieB) are present. Proteomic analysis confirmed identification of virion proteins, along with low levels of assembly intermediates and host cell envelope proteins. The genome of L is 99.9% identical at the nucleotide level to that reported for phage ST64T, despite isolation on different continents ~35 years apart. DNA modification by the epigenetic regulator Dam is generally incomplete. Dam modification is also selectively missing in one location, corresponding to the P22 phase-variation-sensitive promoter region of the serotype-converting gtrABC operon. The number of sites for SenLTIII (StySA) action may account for stronger restriction of L (13 sites) than of P22 (3 sites).

genetics↗

bioRxiv2020

Structures of three actinobacteriophage capsids: Roles of symmetry and accessory proteins

Mycobacterium tuberculosis and abscessus are major human pathogens that are part of the Actinobacteria phylum. Increasing multiple drug resistance in these bacteria has led to a renewed interest in using viruses that infect these bacteria for therapy. In order to understand these viruses, a course-based undergraduate research experience (CURE) program run by SEA-PHAGES at the University of Pittsburgh and HHMI has isolated, sequenced, and annotated over 3000 actinobacteriophages (viruses that infect Actinobacteria). Little work has been done to investigate the structural diversity of these phage, all of which are thought to use a common protein fold, the HK97-fold, in their major capsid protein. Here we describe the structure of three actinobacteriophage capsids isolated by students that infect Mycobacterium smegmatis. The capsid structures were resolved to approximately 6 angstroms, which allowed confirmation that each phage uses the HK97-fold to form their capsid. One phage, Rosebush, has a novel variation of the HK97-fold. Four novel accessory proteins, that form the capsid head along with the major capsid protein, were identified that show limited or no homology to known proteins. The genes that encode the proteins were identified using SDS-PAGE and mass spectrometry. Bioinformatic analysis of the accessory proteins suggest they are used in many actinobacteriophage capsids.

microbiology↗