Biology subjects

Sheriff, E. K.

Publications and source records attributed to Sheriff, E. K..

3 recordsLinked to original sources

bioRxiv2024

Enterococcal quorum-controlled protease alters phage infection

Increased prevalence of multidrug resistant bacterial infections has sparked interest in alternative antimicrobials, including bacteriophages (phages). Limited understanding of the phage infection process hampers our ability to utilize phages to their full therapeutic potential. To understand phage infection dynamics we performed proteomics on Enterococcus faecalis infected with the phage VPE25. We discovered numerous uncharacterized phage proteins are produced during phage infection of Enterococcus faecalis. Additionally, we identified hundreds of changes in bacterial protein abundances during infection. One such protein, enterococcal gelatinase (GelE), an fsr quorum sensing regulated protease involved in biofilm formation and virulence, was reduced during VPE25 infection. Plaque assays showed that mutation of either the fsrA or gelE resulted in plaques with a "halo" morphology and significantly larger diameters, suggesting decreased protection from phage infection. GelE-associated protection during phage infection is dependent on the murein hydrolase regulator LrgA and antiholin-like protein LrgB, whose expression have been shown to be regulated by GelE. Our work may be leveraged in the development of phage therapies that can modulate the production of GelE thereby altering biofilm formation and decreasing E. faecalis virulence.

microbiology↗

bioRxiv2024

Pseudo-pac site sequences used by phage P22 in generalized transduction of Salmonella

Salmonella enterica Serovar Typhimurium (Salmonella) and its bacteriophage P22 are a model system for the study of horizontal gene transfer by generalized transduction. Typically, the P22 DNA packaging machinery initiates packaging when a short sequence of DNA, known as the pac site, is recognized on the P22 genome. However, sequences similar to the pac site in the host genome, called pseudo-pac sites, lead to erroneous packaging and subsequent generalized transduction of Salmonella DNA. While the general genomic locations of the Salmonella pseudo-pac sites are known, the sequences themselves have not been determined. We used visualization of P22 sequencing reads mapped to host Salmonella genomes to define regions of generalized transduction initiation and the likely locations of pseudo-pac sites. We searched each genome region for the sequence with the highest similarity to the P22 pac site and aligned the resulting sequences. We built a regular expression (sequence match pattern) from the alignment and used it to search the genomes of two P22-susceptible Salmonella strains-LT2 and 14028S- for sequence matches. The final regular expression successfully identified pseudo-pac sites in both LT2 and 14028S that correspond with generalized transduction initiation sites in mapped read coverages. The pseudo-pac site sequences identified in this study can be used to predict locations of generalized transduction in other P22-susceptible hosts or to initiate generalized transduction at specific locations in P22-susceptible hosts with genetic engineering. Furthermore, the bioinformatics approach used to identify the Salmonella pseudo-pac sites in this study could be applied to other phage-host systems. ImportanceBacteriophage P22 has been a genetic tool and a key model for the study of generalized transduction in Salmonella since the 1950s, yet certain components of the generalized transduction molecular mechanism remain unknown. Specifically, the locations and sequences of pseudo-pac sites, hypothesized to facilitate packaging of Salmonella DNA by P22, to date have not been determined. In this study, we identified the specific locations and sequences of the pseudo-pac sites frequently recognized by P22 in Salmonella genomes. The identification of highly efficient pseudo-pac sites in Salmonella provides fundamental insights into the sequence specificity necessary for P22 pac site recognition and opens the door to more targeted use of generalized transduction with P22.

microbiology↗

bioRxiv2020

Lytic bacteriophages facilitate antibiotic sensitization of Enterococcus faecium

Enterococcus faecium, a commensal of the human intestine, has emerged as a hospital-adapted, multi-drug resistant (MDR) pathogen. Bacteriophages (phages), natural predators of bacteria, have regained attention as therapeutics to stem the rise of MDR bacteria. Despite their potential to curtail MDR E. faecium infections, the molecular events governing E. faecium-phage interactions remain largely unknown. Such interactions are important to delineate because phage selective pressure imposed on E. faecium will undoubtedly result in phage resistance phenotypes that could threaten the efficacy of phage therapy. In an effort to understand the emergence of phage resistance in E. faecium, three newly isolated lytic phages were used to demonstrate that E. faecium phage resistance is conferred through an array of cell wall-associated molecules, including secreted antigen A (SagA), enterococcal polysaccharide antigen (Epa), wall teichoic acids, capsule, and an arginine-aspartate-aspartate (RDD) protein of unknown function. We find that capsule and Epa are important for robust phage adsorption and that phage resistance mutations in sagA, epaR, and epaX enhance E. faecium susceptibility to ceftriaxone, an antibiotic normally ineffective due to its low affinity for enterococcal penicillin binding proteins. Consistent with these findings, we provide evidence that phages potently synergize with cell wall (ceftriaxone and ampicillin) and membrane-acting (daptomycin) antimicrobials to slow or completely inhibit the growth of E. faecium. Our work demonstrates that the evolution of phage resistance comes with fitness defects resulting in drug sensitization and that lytic phages could potentially serve as antimicrobial adjuvants in treating E. faecium infections.

microbiology↗