Biology subjects

Andersen, S. E.

Publications and source records attributed to Andersen, S. E..

5 recordsLinked to original sources

bioRxiv2025

A prophage-encoded sRNA limits lytic phage infection of adherent-invasive E. coli

Prophages are prevalent features of bacterial genomes that can reduce susceptibility to lytic phage infection, yet the mechanisms involved are often elusive. Here, we identify a small RNA (svsR) encoded by the lambdoid prophage NC-SV in adherent-invasive Escherichia coli (AIEC) strain NC101 that confers resistance to lytic coliphages. Comparative genomic analyses revealed that NC-SV-like prophages and svsR homologs are conserved across diverse Enterobacteriaceae. Transcriptional analyses reveal that svsR represses maltodextrin transport genes, including lamB, which encodes the outer membrane maltoporin LamB--a known receptor for multiple phages. Nutrient supplementation experiments show that maltodextrin enhances phage adsorption, while glucose suppresses it, consistent with established effects of these sugars on lamB expression. In vivo, we compared wild-type NC101 and a prophage-deletion strain (NC101{Delta}NC-SV) in mice to assess the impact of NC-SV on lytic phage susceptibility. Although intestinal E. coli densities remained stable across groups, animals colonized with NC101 exhibited markedly reduced phage burdens in both the intestinal lumen and mucosa compared to mice colonized with NC101{Delta}NC-SV. This reduced phage pressure was associated with increased dissemination of NC101 to extraintestinal tissues, including the spleen and liver. Together, these findings highlight a nutrient-responsive, prophage-encoded mechanism that protects AIEC from phage predation and may promote bacterial persistence and dissemination in the inflamed gut.

microbiology↗

bioRxiv2024

Comparative analysis of 43 distinct RNA modifications by nanopore tRNA sequencing

Transfer RNAs are the fundamental adapter molecules of protein synthesis and the most abundant and heterogeneous class of noncoding RNA molecules in cells. The study of tRNA repertoires remains challenging, complicated by the presence of dozens of post transcriptional modifications. Nanopore sequencing is an emerging technology with promise for both tRNA sequencing and the detection of RNA modifications; however, such studies have been limited by the throughput and accuracy of direct RNA sequencing methods. Moreover, detection of the complete set of tRNA modifications by nanopore sequencing remains challenging. Here we show that recent updates to nanopore direct RNA sequencing chemistry (RNA004) combined with our own optimizations to tRNA sequencing protocols and analysis workflows enable high throughput coverage of tRNA molecules and characterization of nanopore signals produced by 43 distinct RNA modifications. We share best practices and protocols for nanopore sequencing of tRNA and further report successful detection of low abundance mitochondrial and viral tRNAs, providing proof of concept for use of nanopore sequencing to study tRNA populations in the context of infection and organelle biology. This work provides a roadmap to guide future efforts towards de novo detection of RNA modifications across multiple organisms using nanopore sequencing.

molecular biology↗

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

Global mapping of the Chlamydia trachomatis conventional secreted effector-host interactome reveals CebN interacts with nucleoporins and Rae1 to impede STAT1 nuclear translocation.

To usurp host defenses and establish a replicative niche, obligate intracellular pathogens are tasked with remodeling the host cell using a comparatively small repertoire of effector proteins. For Chlamydia trachomatis (C.t), discovery of secreted proteins and their host targets has been particularly challenging due to the bacteriums historical genetic intractability. Using affinity purification-mass spectrometry, we defined host interaction partners for 21 secreted effector proteins, providing the first comprehensive type III secretion system (T3SS) effector- host interactome generated during infection. Among these, we show that the C-terminus of CebN (CT584) binds multiple nucleoporins and Rae1, host factors previously associated only with viral immune evasion. Remarkably, we shown that CebN localizes to the nuclear envelope not only in infected cells but also in uninfected bystander cells. Functionally, CebN is both necessary and sufficient to perturb STAT1 nuclear import following IFN-{gamma} stimulation and its expression is critical for C.t. survival, as evidenced by reduced bacterial replication and smaller inclusions in cells infected with a CebN mutant. Together, these finds expand our understanding of chlamydia effector biology and highlight novel bacterial strategies for manipulating host defenses at the nuclear pore. SIGNIFICANCEChlamydia trachomatis (C.t.) is a leading cause of sexually transmitted infections and blindness, yet the molecular mechanisms it uses to manipulate host defenses remain poorly defined. Unlike many pathogens, C.t. relies on a limited set of effectors to remodel the host cell and establish its niche. We identified host targets for 21 C.t. effector proteins. Focusing on CebN, we show that it binds nucleoporins and Rae1, host factors previously linked only to viral immune antagonism. CebN localizes to the nuclear envelope of both infected and bystander cells, and is critical for replication, inclusion development, and perturbation of STAT1 nuclear import following IFN-{gamma} stimulation. These findings uncover a novel strategy by which C.t. manipulates nuclear pore function to evade host defenses and establish infection.

microbiology↗

bioRxiv2023

Evolution of a small phage protein confers resistance to antiphage defense in Enterococcus faecalis

The prevalence of multidrug resistant (MDR) bacterial infections continues to rise as the development of antibiotics needed to combat these infections remains stagnant. MDR enterococci are a major contributor to this crisis. A potential therapeutic approach for combating MDR enterococci is bacteriophage (phage) therapy, which uses lytic viruses to infect and kill pathogenic bacteria. While phages that lyse some strains of MDR enterococci have been identified, other strains display high levels of resistance and the mechanisms underlying this resistance are poorly defined. Here, we use a CRISPR interference (CRISPRi) screen to identify a genetic locus found on a mobilizable plasmid from Enterococcus faecalis involved in phage resistance. This locus encodes a putative serine recombinase followed by a Type IV restriction enzyme (TIV-RE) that we show restricts the replication of phage phi47 in E. faecalis. We further find that phi47 evolves to overcome restriction by acquiring a missense mutation in a TIV-RE inhibitor protein. We show that this inhibitor, termed type IV restriction inhibiting factor A (tifA), binds and inactivates diverse TIV-REs. Overall, our findings advance our understanding of phage defense in drug-resistant E. faecalis and provide mechanistic insight into how phages evolve to overcome antiphage defense systems.

microbiology↗