Biology subjects

Ensminger, A. W.

Publications and source records attributed to Ensminger, A. W..

10 recordsLinked to original sources

bioRxiv2025

Systematic Discovery of Pathogen Effector Functions across Human Pathogens and Pathways

Pathogens deploy effector proteins to exploit host cell biology, and most pathogen open reading frames (ORFs) are rapidly evolving and lack functional annotation. We developed the eORFeome, a scalable functional genomics platform encompassing 3,835 effector ORFs from diverse viruses, bacteria, and parasites. High-throughput barcoded screens across NF{kappa}B, apoptosis, p53, cGAS-STING and MHC-I pathways revealed functions for hundreds of uncharacterized eORFs, unexpected new activities for known effectors, and distinct pathway-specific functions encoded by single ORFs. Illustrating the power of the approach, we identify HHV6A U14 as a p53 antagonist, HHV7 U21 as a dual-function STING antagonist and MHC-I antigen display inhibitor, and adenoviral 13.6K/i-leader protein as a de novo evolved TAP inhibitor that suppresses MHC-I display. These results establish a general framework for systematic effector annotation, uncover new mechanisms of host-pathogen interaction across kingdoms, and highlight pathogen effectors as a versatile toolkit for rewiring and probing human cellular pathways.

systems biology↗

bioRxiv2024

Global atlas of predicted functional domains in Legionella pneumophila Dot/Icm translocated effectors

Legionella pneumophila utilizes the Dot/Icm type IVB secretion system to deliver hundreds of effector proteins inside eukaryotic cells to ensure intracellular replication. Our understanding of the molecular functions of this largest pathogenic arsenal known to the bacterial world remains incomplete. By leveraging advancements in 3D protein structure prediction, we provide a comprehensive structural analysis of 368 L. pneumophila effectors, representing a global atlas of predicted functional domains summarized in a database (https://pathogens3d.org/legionella-pneumophila). Our analysis identified 157 types of diverse functional domains in 287 effectors, including 159 effectors with no prior functional annotations. Furthermore, we identified 35 unique domains in 30 effector models that have no similarity with experimentally structurally characterized proteins, thus, hinting at novel functionalities. Using this analysis, we demonstrate the activity of thirteen domains, including three unique folds, predicted in L. pneumophila effectors to cause growth defects in the Saccharomyces cerevisiae model system. This illustrates an emerging strategy of exploring synergies between predictions and targeted experimental approaches in elucidating novel effector activities involved in infection.

microbiology↗

bioRxiv2024

A comprehensive two-hybrid analysis to explore the L. pneumophila effector-effector interactome

Legionella pneumophila uses over 300 translocated effector proteins to rewire host cells during infection and create a replicative niche for intracellular growth. To date, several studies have identified L. pneumophila effectors that indirectly and directly regulate the activity of other effectors, providing an additional layer of regulatory complexity. Amongst these are "metaeffectors" - a special class of effectors that regulate the activity of other effectors once inside the host. A defining feature of metaeffectors is direct, physical interaction with a target effector. Metaeffector identification to date has depended on phenotypes in heterologous systems and experimental serendipity. Using a multiplexed, recombinant-barcode-based yeast two-hybrid technology we screened for protein-protein interactions amongst all L. pneumophila effectors and several components of the Dot/Icm type IV secretion system (>167,000 protein combinations). Of the 52 protein interactions identified by this approach, 44 are novel protein interactions, including ten novel effector-effector interactions (doubling the number of known effector-effector interactions).

microbiology↗

bioRxiv2024

Genetic evidence for a regulated cysteine protease catalytic triad in LegA7, a Legionella pneumophila protein that impinges on a stress response pathway

Legionella pneumophila grows within membrane-bound vacuoles in phylogenetically diverse hosts. Intracellular growth requires the function of the Icm/Dot type-IVb secretion system, which translocates more than 300 proteins into host cells. A screen was performed to identify L. pneumophila proteins that stimulate MAPK activation, using Icm/Dot translocated proteins ectopically expressed in mammalian cells. In parallel, a second screen was performed to identify L. pneumophila proteins expressed in yeast that cause growth inhibition in MAPK pathway-stimulatory high osmolarity medium. LegA7 was shared in both screens, a protein predicted to be a member of the bacterial cysteine protease family that has five carboxyl-terminal ankyrin repeats. Three conserved residues in the predicted catalytic triad of LegA7 were mutated. These mutations abolished the ability of LegA7 to inhibit yeast growth. To identify other residues important for LegA7 function, a generalizable selection strategy in yeast was devised to isolate mutants that have lost function and no longer cause growth inhibition on high osmolarity medium. Mutations were isolated in the two carboxyl-terminal ankyrin repeats, as well as an inter-domain region located between the cysteine protease domain and the ankyrin repeats. These mutations were predicted by AlphaFold modeling to localize to the face opposite from the catalytic site, arguing that they interfere with the positive regulation of the catalytic activity. Based on our data, we present a model in which LegA7 harbors a cysteine protease domain with an inter-domain and two carboxyl-terminal ankyrin repeat regions that modulate the function of the catalytic domain.

microbiology↗

bioRxiv2024

A random mutagenesis screen enriched for missense mutations in bacterial effector proteins.

To remodel their hosts and escape immune defenses, many pathogens rely on large arsenals of proteins (effectors) that are delivered to the host cell using dedicated translocation machinery. Effectors hold significant insight into the biology of both the pathogens that encode for them and the host pathways that they manipulate. One of the most powerful systems biology tools for studying effectors is the model organism, Saccharomyces cerevisiae. For many pathogens, the heterologous expression of effectors in yeast is growth inhibitory at a frequency much higher than housekeeping genes, an observation ascribed to targeting conserved eukaryotic proteins. Abrogation of yeast growth inhibition has been used to identify bacterial suppressors of effector activity, host targets, and functional residues and domains within effector proteins. We present here a yeast-based method for enriching for informative, in-frame, missense mutations in a pool of random effector mutants. We benchmark this approach against three effectors from Legionella pneumophila, an intracellular bacterial pathogen that injects a staggering >330 effectors into the host cell. For each protein, we show how in silico protein modeling (AlphaFold2) and missense- directed mutagenesis can be combined to reveal important structural features within effectors. We identify known active site residues within the metalloprotease RavK, highly conserved residues in SdbB, and previously unidentified functional motifs within the C-terminal domain of SdbA. We show that this domain has structural similarity with glycosyltransferases and exhibits in vitro activity consistent with this predicted function.

microbiology↗

bioRxiv2022

Functional diversification despite structural congruence in the HipBST toxin-antitoxin system of Legionella pneumophila

Toxin-antitoxin (TA) systems are abundant genetic modules in bacterial chromosomes and on mobile elements. They are often patchily distributed and their physiological functions remain poorly understood. Here, we characterize a TA system in Legionella pneumophila that is highly conserved across Legionella species. This system is distantly related to Escherichia coli HipBST and we demonstrate that it is a functional tripartite TA system (denoted HipBSTLp). We identify HipBSTLp homologs in diverse taxa, yet in the Gammaproteobacteria these are almost exclusively found in Legionella species. Notably, the toxin HipTLp was previously reported to be a pathogenic effector protein that is translocated by L. pneumophila into its eukaryotic hosts. Contrary to this, we find no signal of HipTLp translocation beyond untranslocated control levels and make several observations consistent with a canonical role as a bacterial toxin. We present structural and biochemical insights into the regulation and neutralization of HipBSTLp, and identify key variations between this system and HipBSTEc. Finally, we show that the target of HipTLp is likely not conserved with any characterized HipA or HipT toxin. This work serves as a unique comparison of a TA system across bacterial species and illustrates the molecular diversity that exists within a single TA family.

microbiology↗

bioRxiv2022

The L. pneumophila effector PieF modulates mRNA stability through association with eukaryotic CCR4-NOT

The eukaryotic CCR4-NOT deadenylase complex is a highly conserved regulator of mRNA metabolism that influences the expression of the complete transcriptome, representing a prime target for a generalist bacterial pathogen. We show that a translocated bacterial effector protein, PieF (Lpg1972) of L. pneumophila Str. Philadelphia-1, interacts specifically with the CNOT7/8 nuclease module of CCR4-NOT, with a dissociation constant in the low nanomolar range. PieF inhibits the catalytic deadenylase subunit CNOT7 of the CCR4-NOT complex in a stoichiometric, dose-dependent manner in vitro. In transfected cells, PieF can silence reporter gene expression and reduce mRNA steady-state levels when artificially tethered. PieF demonstrates molecular similarities to another family of CNOT7-associated factors but demonstrates divergence concerning the interaction interface with CNOT7. In addition, we show that PieF overexpression changes the subcellular localization of CNOT7 and displaces the CNOT6/6L nucleases from CCR4-NOT. Finally, PieF expression phenocopies knockout of the CNOT7 ortholog in S. cerevisiae, resulting in 6-azauracil sensitivity. Collectively, this work suggests that L. pneumophila targets host mRNA stability and expression through a highly conserved host pathway not previously associated with Legionella pathogenesis.

microbiology↗

bioRxiv2021

Mobile element integration reveals an atypical chromosome dimer resolution system in Legionella pneumophila.

In bacteria, the mechanisms used to repair DNA lesions during genome replication include homologous recombination between sister chromosomes. This can lead to the formation of chromosome dimers if an odd number of crossover events occurs. The concatenated DNA must be resolved before cell separation to ensure genomic stability and cell viability. The broadly conserved dif/Xer system counteracts the formation of dimers by catalyzing one additional crossover event immediately prior to cell separation. While dif/Xer systems have been characterized or predicted in the vast majority of proteobacteria, no homologs to dif or xer have been identified in the order Legionellales. Here we report the discovery of a distinct single-recombinase dif/Xer system in the intracellular pathogen Legionella pneumophila. The dif site was uncovered by our analysis of Legionella mobile element-1 (LME-1), which harbors a dif site-mimic and integrates into the L. pneumophila genome via site-specific recombination. We demonstrate that lpg1867 (herein named xerL) encodes a tyrosine recombinase that is necessary and sufficient for catalyzing recombination at the dif site, and that deletion of dif or xerL causes filamentation along with extracellular and intracellular growth defects. We show that the dif/XerL system is present throughout Legionellales and that Coxiella burnetii XerL and its cognate dif site can functionally substitute for the native system in L. pneumophila. Lastly, we describe an unexpected link between C. burnetii dif/Xer and the maintenance of its virulence plasmids. Significance StatementThe maintenance of circular chromosomes depends on the ability to resolve aberrant chromosome dimers as they form. In most proteobacteria, broadly conserved Xer recombinases catalyze single crossovers at short, species-specific dif sites located near the replication terminus. Chromosomal dimerization leads to the formation of two copies of dif, leading to rapid site-specific recombination and elimination of the duplicated intervening sequence. The apparent absence of chromosome-dimer resolution mechanisms in Legionellales has been a mystery to date. By studying a phage-like mobile genetic element, LME-1, we have identified a previously unknown single-recombinase dif/Xer system that is not only widespread across Legionellales but whose activity is linked to virulence in two important human pathogens.

microbiology↗

bioRxiv2018

Working together: cross-priming in two Legionella pneumophila type I-F CRISPR-Cas systems.

In bacteria and archaea, several distinct types of CRISPR-Cas systems provide adaptive immunity through broadly similar mechanisms: short nucleic acid sequences derived from foreign DNA, known as spacers, engage in complementary base pairing with invasive genetic elements setting the stage for nucleases to degrade the target DNA. A hallmark of type I CRISPR-Cas systems is their ability to acquire spacers in response to both new and previously encountered invaders (naive and primed acquisition, respectively). Our phylogenetic analyses of 47 L. pneumophila type I-F CRISPR-Cas systems and their resident genomes suggest that many of these systems have been horizontally acquired. These systems are frequently encoded on plasmids and can co-occur with nearly identical chromosomal loci. We show that two such co-occurring systems are highly protective and undergo efficient primed acquisition in the lab. Furthermore, we observe that targeting by one systems array can prime spacer acquisition in the other. Lastly, we provide experimental and genomic evidence for a model in which primed acquisition can efficiently replenish a depleted type I CRISPR array following a mass spacer deletion event.

microbiology↗

bioRxiv2017

Priming In A Permissive Type I-C CRISPR-Cas System Reveals Distinct Dynamics Of Spacer Acquisition And Loss

CRISPR-Cas is a bacterial and archaeal adaptive immune system that uses short, invader-derived sequences termed spacers to target invasive nucleic acids. Upon recognition of previously encountered invaders, the system can stimulate secondary spacer acquisitions, a process known as primed adaptation. Previous studies of primed adaptation have been complicated by intrinsically high interference efficiency of most systems against bona fide targets. As such, most primed adaptation to date has been studied within the context of imperfect sequence complementarity between spacers and targets. Here, we take advantage of a native type I-C CRISPR-Cas system in Legionella pneumophila that displays robust primed adaptation even within the context of a perfectly matched target. Using next-generation sequencing to survey acquired spacers, we observe strand bias and positional preference that are consistent with a 3' to 5' translocation of the adaptation machinery. We show that spacer acquisition happens in a wide range of frequencies across the plasmid, including a remarkable hotspot that predominates irrespective of the priming strand. We systematically characterize protospacer sequence constraints in both adaptation and interference and reveal extensive flexibilities regarding the protospacer adjacent motif in both processes. Lastly, in a strain with a genetically truncated CRISPR array, we observe greatly increased interference efficiency coupled with a dramatic shift away from spacer acquisition towards spacer loss. Based on these observations, we propose that the Legionella type I-C system represents a powerful model to study primed adaptation and the interplay between CRISPR interference and adaptation.

microbiology↗