Biology subjects

Jacobs-Sera, D.

Publications and source records attributed to Jacobs-Sera, D..

4 recordsLinked to original sources

bioRxiv2026

Genome-wide CRISPRi mapping of mycobacteriophage interaction determinants in Mycobacterium smegmatis mc2155

Bacteriophages are highly specific predators of bacterial hosts, but the molecular mechanisms governing their entry across the structurally complex mycobacterial cell envelope remain poorly defined. Utilizing a genome-wide CRISPR interference library in the model organism Mycobacterium smegmatis against a phylogenetically representative collection of 70 mycobacteriophages, we mapped the functional architecture of host susceptibility determinants. This parallelized genetic screen revealed a spatial continuum of infection requirements spanning from outer leaflet surface structures down to deep cytoplasmic machinery. The four major outer membrane Mycobacterium smegmatis porins (Msp) emerged as the dominant candidate receptor family for 46 diverse phages, representing the first protein receptors identified within this group. Interrogating the asymmetric outer bilayer also implicated cell envelope glycolipids as candidate receptors for 14 phages, resolving into glycopeptidolipid sub-lineages. Beneath the mycomembrane, arabinogalactan functions as a candidate secondary receptor for 5 phages, while the inner membrane lipid transporter MmpL11 and the global nucleoid-associated protein Lsr2 represent determinant factors for 14 and 31 phages respectively. While physical deletions of major porins trigger spontaneous regulatory pathways that upregulate silent backup paralogues, acute transcriptional knockdowns circumvent suppressor accumulation, revealing unmasked single-gene dependencies. Structural analysis demonstrated that host determinant usage decouples entirely from whole-genome viral phylogeny, indicating that standard phylogenetic groupings fail to predict surface recognition profiles. Ultimately, mapping this host interactome reveals an evolutionary strategy where phages target highly redundant or abundant host machinery to exploit fixed physiological liabilities, presenting a robust molecular blueprint for the rational engineering of escape-resistant therapeutic phage cocktails.

microbiology↗

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

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↗