Biology subjects

Shen, A.

Publications and source records attributed to Shen, A..

At least 19 recordsLinked to original sources

bioRxiv2026

RasGRP1 promotes efficient intestinal tuft cell differentiation

Intestinal stem and progenitor cells balance proliferation and lineage commitment to maintain epithelial homeostasis and adapt to environmental challenges. Tuft cells are a rare chemosensory lineage that rapidly expands in response to the cytokine IL-13 during helminth infection, yet how epithelial progenitor states influence this response remains incompletely understood. Using genetic mouse models, intestinal organoids, and single-cell approaches, we identify the Ras guanine nucleotide exchange factor RasGRP1 as an epithelial-intrinsic promoter of efficient tuft cell differentiation. Rasgrp1 is expressed in rare crypt epithelial cells distinct from Lgr5-positive stem cells. Once cells entered the tuft cell differentiation trajectory, Rasgrp1 deficiency has little impact on tuft cell maturation, including expression of the Pou2f3 tuft cell transcription factor. Instead, RasGRP1 acts at the progenitor stage, where it restrains EGFR-driven proliferation and supports a transcriptionally heterogeneous transit-amplifying state, including a subset of cells expressing Pou2f3, that is largely lost upon Rasgrp1 deficiency. Consistent with this model, EGFR inhibition restores tuft cell numbers in Rasgrp1-deficient organoids and cooperates with IL-13 to enhance tuft cell differentiation. Rasgrp1-deficient mice exhibit delayed tuft cell hyperplasia and inefficient helminth clearance. Conversely, constitutive epithelial RasGRP1 overexpression, uncoupled from its endogenous regulation, drives spontaneous tuft cell hyperplasia and accelerates parasite clearance, but alters the balance of intestinal epithelial lineages. Together, our findings identify RasGRP1 as a cell-intrinsic regulator that restrains progenitor proliferation and promotes responsiveness to IL-13, thereby coupling growth factor signaling, cell cycle control, and immune-driven epithelial lineage specification.

cell biology↗

bioRxiv2026

Adaptations of the pregnant murine heart to pre-existing hypertension

2-5% of pregnancies are complicated by chronic hypertension [1], increasing the risk of peripartum cardiomyopathy and maternal morbidity [2, 3]. Hypertension reduces stroke volume, whereas pregnancy increases stroke volume. The combined impact of these opposing loads on cardiac function and strain remains unclear. We used a mouse model of pregnancy with pre-existing hypertension to characterize cardiac remodeling. We hypothesized that pregnancy may counteract hypertension-related reductions in cardiac function. Nulliparous female C57Bl6/J mice (7-11 weeks old) were assigned into pregnant (PREG, n=11), angiotensin II-treated (ANGII, n=7), and ANGII+PREG (n=8) groups. Hypertension was induced with subcutaneous angII infusion (1000 ng/kg/min). Cardiac function was measured with high-frequency ultrasound. Ejection fraction (EF) was maintained in PREG animals at 65% but decreased significantly by day 36 in ANGII animals (44.85{+/-}3.27%). In ANGII+PREG mice, EF initially decreased but trended towards PREG values on postpartum day 1. Left ventricle strain was lower in ANGII and ANGII+PREG groups compared to PREG. ANGII and ANGII+PREG animals showed decreased early longitudinal diastolic strain rate at mid-pregnancy compared to baseline (0.82{+/-}0.09% ANGII; 0.73{+/-}0.09% ANGII+PREG), while increasing in PREG (1.76{+/-}0.14%). Minimal physiologically relevant fibrosis was noted across groups via immunohistochemistry. Heart mass-to-body mass was lowest in PREG (6.20{+/-}0.22 mg/g), greatest in ANGII (8.95{+/-}0.39 mg/g), and intermediate in ANGII+PREG (6.92{+/-}0.25 mg/g). Cardiomyocyte cross-sectional area was similar between groups. Overall, pregnancy altered the functional and hypertrophic response to elevated blood pressure. Findings from this work set the foundation for future efforts that may eventually improve cardiovascular care for pregnant patients.

bioengineering↗

bioRxiv2026

Highly efficient genotype compression leveraging genealogical relatedness

Large genetic datasets are terabytes in size, presenting a computational challenge that will intensify as sequencing efforts scale. We present a lossless compression algorithm, kodama, which supports matrix multiplication and is suitable for large-scale statistical analyses. Kodama leverages genealogical relatedness among nominally unrelated individuals and infers a novel data structure similar to the ancestral recombination graph (ARG), called the linear ARG. We applied kodama to whole genome sequencing data from UK Biobank and All of Us. Inferred linear ARGs were 17-89 times smaller on disk compared to the input data; the entire UK Biobank N=200k dataset can be loaded into memory (58GB). Compared with the recently proposed genotype representation graph (GRG), the linear ARG is 2.5 times smaller. Genotype matrix multiplications, which are the bottleneck in most statistical applications, are extremely fast with the linear ARG; we performed a GWAS on the UK Biobank 200k cohort across 89 traits with 42 covariates in 100 seconds, representing a 4,700-fold speedup over PLINK 2.0. We expect that the linear ARG will enable genetic analyses to scale to millions of samples.

genetics↗

bioRxiv2026

MTB-KB: A Curated Knowledgebase of Mycobacterium tuberculosis Related Studies

Tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), has regained its position as the worlds leading killer among infectious diseases. Despite extensive research progress across epidemiology, diagnosis, drug development, treatment regimens, vaccines, drug resistance, virulence factors, and immune mechanisms, MTB-related knowledge remains fragmented across thousands of publications, limiting its effective use. To address this gap, we present MTB-KB, a literature-curated knowledgebase that systematically integrates high-impact findings from eight major sections of TB research. The current release contains 75,170 associations from 1,246 publications, covering 18,439 entities standardized using authoritative databases and WHO-endorsed classifications. A central feature is the interactive knowledge graph, which links cross-section associations to reveal and infer MTB-host interactions, treatment strategies, and vaccine development opportunities. MTB-KB also provides a user-friendly interface with browsing, advanced search, and statistical visualization. Overall, by consolidating dispersed MTB knowledge into a structured and accessible platform, MTB-KB provides a valuable resource for researchers, clinicians, and policymakers, supporting both basic and clinical TB research, enabling evidence-based TB prevention, diagnosis, and treatment, and contributing to global elimination efforts. MTB-KB is accessible at https://ngdc.cncb.ac.cn/mtbkb/.

bioinformatics↗

bioRxiv2026

Medea: An omics AI agent for therapeutic discovery

Therapeutic hypotheses can transfer across diseases but their relevance depends on biological context. The same target, perturbation, or treatment can produce different effects across cell types, disease states, genetic backgrounds, and patients. Therapeutic reasoning therefore requires methods that preserve context, test when evidence supports transfer, and identify where context-specific effects limit it. Although AI agents can perform therapeutic analyses, existing systems often fail to preserve biological context over long workflows, verify intermediate computational steps, or reconcile conflicting evidence across datasets and literature. Here, we present Medea, an AI agent for therapeutic reasoning across biological contexts. Medea executes multi-step analyses using biological tools, machine learning models, and literature retrieval while enforcing verification during planning, execution, and evidence synthesis. We evaluate Medea across 5,673 open-ended analyses in three domains: cell type specific therapeutic target nomination in five diseases and 29 cell types, synthetic lethality prediction in 7 cancer cell lines, and immunotherapy response prediction from multimodal patient profiles. Using a previously unpublished epistatic miniarray profiling screen performed under two DNA-damaging treatments, we evaluate Medea on predicting synthetic lethality among 238,046 gene-gene pairs in yeast. Medea predicts these experimentally measured synthetic lethal interactions, indicating that its performance reflects biological relevance rather than information leakage from benchmark datasets. Across these evaluations, Medea improves performance over large language models, reasoning models, biomedical agents, and specialized machine learning models while maintaining low failure rates and calibrated abstention. These results show that verifiable AI agents can perform therapeutic analyses across biological contexts.

bioinformatics↗

bioRxiv2026

Defining the order of assembly of the Clostridioides difficile divisome complex

Cell division is the ancient pathway by which bacteria synthesize a septum of peptidoglycan, dividing the cell into two. Where all walled bacteria were previously thought to use FtsW-FtsI orthologs to synthesize septal peptidoglycan during division, we recently discovered that the major pathogen Clostridioides difficile is missing FtsW-FtsI and instead relies on the activity of the bifunctional Class A PBP called PBP1 to synthesize the septal peptidoglycan. Furthermore, C. difficile either does not encode or require the majority of canonical divisome proteins described in model bacteria aside from the divisome protein orthologs FtsZ, SepF, and ZapA. Indeed, unlike model systems, SepF and ZapA are essential in C. difficile, suggesting that they have evolved to have a critical function in cell division without the redundant mechanisms present in model organisms. Thus, C. difficile uses a fundamentally different division mechanism compared to previously studied bacteria. To understand how this unusual complex is assembled in C. difficile, we combine CRISPR interference (CRISPRi)-based knock-downs with fluorescent fusions to determine that the hierarchical order of assembly occurs in three phases: (i) FtsZ/ZapA, (ii) SepF, and (iii) PBP1. We further investigate the order of assembly of several non-essential mid-cell localizing proteins and discover that MldA, MldC, DivIVA, FtsK, and PBP3 depend on FtsZ, SepF, and PBP1 for localization, whereas MldB localizes independently of SepF and PBP1. Our work provides a model for divisome assembly in C. difficile and validates genetic and cytological tools that can be used to mechanistically dissect this pathway in the future. IMPORTANCEBacterial cell division has been extensively studied in model systems, but little is known about how this essential process occurs in the clinically important pathogen Clostridioides difficile. Studies in model systems have shown that cell division is carried out by a large multi-protein complex called the "divisome." While components of the divisome are widely conserved and can be traced back to the last bacterial common ancestor billions of years ago, C. difficile uses a unique mechanism of division that is independent of the majority of these canonical divisome genes. In the current study, we characterize the core, essential divisome comprised of FtsZ, ZapA, SepF, and PBP1, and build a model for the order of assembly of this unusual divisome complex.

microbiology↗

bioRxiv2026

The heat-ramp method to study regulated cell death in a pathogenic yeastCryptococcus neoformans

Human fungal pathogens cause a significant public health burden. While no reliable surveilence data are available, estimations suggest that 1 billion infections and over 2 million deaths are attributable to fungal infections annually worldwide. This drove the World Health Organization to generate a priority list of fungal pathogens for reearch, which includes the yeast Cryptococcus neoformans in a top critical priority. With the rise of drug-resistance and emerging fungal pathogens, new conceptual strategies for antifungal therapies are needed in addition to existing antibiotic development pipelines to meet clinical needs. Intrinsic cell death pathways encoded by pathogenic fungi are largely unstudied but could be leveraged for antifungal therapy analogous to anti-cancer therapeutics that activate apoptosis or other cell death mechanisms. Thus far, molecularly defined fungal cell death mechanisms are best characterized for only a few, predominantly model filamentous species. To extend these studies to pathogenic yeast, here we describe and demonstrate a tunable heat-ramp stimulus that when applied to small volumes of yeast cell suspensions reveals a protracted cell death process in the pathogenic yeast Cryptococcus neoformans. This low cost protocol induces robust and reproducible phenotypes to study gene-dependent mechanisms in laboratory strains and clinical isolates.

microbiology↗

bioRxiv2025

A single DNA methylation site regulates cell fate during Clostridioides difficile sporulation.

DNA methylation is a widespread phenomenon in bacteria that can regulate gene expression, although the mechanisms underlying this epigenetic regulation are often poorly understood. In Clostridioides difficile, the orphan DNA methyltransferase CamA promotes sporulation, a process critical for the persistence and transmission of this nosocomial pathogen. However, the specific CamA target genes that drive this increased sporulation phenotype were unknown. Here, we show that methylation of a single CamA motif in the promoter region of spoIIE, which encodes a factor critical for activating the early-acting sporulation sigma factor {sigma}F, is sufficient to promote spoIIE transcription, {sigma}F activation, and spore formation. Surprisingly, the CamA-dependent increase in spoIIE expression also increases the frequency of premature {sigma}F activation prior to asymmetric division, resulting in miscompartmentalization of {sigma}F activity. While this premature activation event triggers cell lysis in the well-studied spore-former Bacillus subtilis, we show that C. difficile cells retain developmental plasticity: predivisional cells that have prematurely activated {sigma}F can abort sporulation and resume vegetative growth, whereas cells that activate {sigma}F in the forespore after asymmetric division remain committed to sporulation. Thus, DNA methylation controls a critical cell fate decision in C. difficile without compromising its capacity to adapt to fluctuating environmental conditions. Finally, we show that CamA confers a significant fitness advantage during murine infection through mechanisms largely independent of its ability to promote sporulation. Since CamA is specific to C. difficile and epigenetically regulates multiple pathways critical for pathogen persistence, these analyses imply that CamA could be a promising antimicrobial target. SignificanceWhile DNA methylation of the bacterial genome is ubiquitous and can regulate diverse processes, identifying the methylation sites that drive a specific phenotype remains challenging. The Clostridioides difficile-specific orphan DNA methyltransferase CamA promotes sporulation and persistence in mice, but the mechanisms underlying these phenotypes were unknown. We identified a single methylation site in the promoter of a key sporulation gene that enhances sporulation, a process critical for this clinically relevant pathogen to transmit disease. Our analyses uncovered an unexpected developmental plasticity to C. difficiles sporulation program while also revealing that CamA regulates processes beyond sporulation that are important for maintaining long-term infection. Thus, targeting CamA may offer therapeutic benefit.

microbiology↗

bioRxiv2025

Integrated Bioinformatics analysis and Metabolomic Responses in finding novel therapeutic approach to treat viral myocarditis

BackgroundMyocarditis is one of the most common health problems in young people. Despite imaging techniques and endomyocardial biopsies advancing, there is still inadequate for myocarditis characterization. Some studies have shown that myocarditis is closely associated with mitochondrial dysfunction. Therefore, this study was aimed to identify mitochondrial-related biomarkers and gene regulatory networks in myocarditis and potential therapeutic targets. MethodsWe downloaded GSE95368 dataset from GEO to get myocarditis gene expression profiles, then used EdgeR and AI algorithm for bioinformatics analysis of DEGs functions. Established CVB3-induced mouse myocarditis model via intraperitoneal injection, and assessed energy metabolism differences using targeted metabolomics. CVB3 stimulus induced myocarditis in H9C2 cells. We assessed mitochondrial dysfunction (ATP, MMP) with commercial kits, MAPK8 release via ELISA, and MAPK8, p-PI3K, p-AKT levels by western blot. Furthermore, a total of 8 patients primarily diagnosed with myocarditis were enrolled, and levels of MAPK8 and CK in serum were detected. SP600125, an inhibitor of MAPK8, was administrated to CVB3-infected mice to study its potential protective effect in viral myocarditis. ResultsWe obtained 77 DEGs enriched in myocarditis. Analysis yielded 11 modules, MEpurple module genes linked to myocarditis progression were identified via WGCNA. MAPK8, NAMPT and ALB are associated with mitochondrial function. CVB3-infected mice showed cardiac inflammation and high MAPK8 expression in serum or heart tissues.The target metabolism results indicated altered central carbon metabolism distinguished CVB3 group from control, with higher D-Glucose-1-phosphate and D-Glucose-6-phosphate and lower L-Cystine, dCMP, IMP and Xylulose-5-phosphate levels. CVB3 treatment caused mitochondrial dysfunction in H9C2 cells (decreased ATP, MMP), and increased MAPK expression. Western blot showed MAPK8 consolidated the levels of p-PI3K and p-AKT. CK activity was notably higher in myocarditis patients than healthy individuals. MAPK8 levels in myocarditis patients serum also exceeded those in healthy individuals. Pearson analysis indicated that MAPK8 and CK may contribute to myocarditis progression via distinct mechanisms. In CVB3-infected mouse model with SP600125 treatment, cardiac inflammation and MAPK8, p-PI3k, p-AKT expression were reduced, confirming MAPK8s crucial role in viral myocarditis. ConclusionOur study identified MAPK8 as a key biomarker of myocarditis, and it may be a potential therapeutic target for myocarditis.

pathology↗

bioRxiv2025

The CspC:CspA heterodimer transduces germinant and co-germinant signals during Clostridioides difficile spore germination.

The clinically significant pathogen Clostridioides difficile, which lacks the transmembrane nutrient germinant receptors conserved in almost all spore-forming bacteria, initiates spore germination by a unique mechanism that requires two signals: a bile acid germinant and an amino acid or divalent cation co-germinant signal. While two soluble pseudoproteases, CspC and CspA, were initially identified as the germinant and co-germinant receptors, respectively, in C. difficile, we previously identified residues in an unstructured region of CspC that regulate the sensitivity of C. difficile spores to both signals. However, the mechanism by which CspC transduces these signals remained unclear. Here, we demonstrate that CspC forms a stable complex with CspA and determine the crystal structure of the CspC:CspA heterodimer. The structure reveals extensive interactions along the binding interface, including direct interactions between the unstructured region of CspC and CspA. Using structure-function analyses, we identify CspC:CspA interactions that regulate the sensitivity of C. difficile spores to germinant signals and show for the first time that CspA regulates C. difficile response to not only co-germinant but also germinant signals. While we also show that CspA can form a homodimer and determine its crystal structure, CspA homodimerization appears unimportant for C. difficile spore germination. Collectively, our analyses establish the CspC:CspA heterodimer, rather than its individual constituents, as a critical signaling node for sensing both germinant and co-germinant signals. They also suggest a new mechanistic model for how C. difficile transduces germinant signals, which could guide the development of therapeutics against this important pathogen.

microbiology↗

bioRxiv2025

Attention robustly dissociates objective performance and subjective visibility reports

Visual experience can sometimes depart from visual performance, providing a powerful lens into the mechanisms generating conscious perception. In one proposed dissociation--subjective inflation--unattended locations in the periphery appear stronger than attended ones despite equated performance. Subjective inflation has played a central role in motivating theories of consciousness that reject the sufficiency of sensory signals for conscious perception. Yet the empirical basis for subjective inflation is limited. Here, in a large-scale adversarial collaboration, we conducted four simultaneously-replicated experiments testing the strength, character, and extent of subjective inflation under inattention. We used a new analytic approach to quantify inattentional inflation over full psychometric functions, beyond single matched-performance levels. We found robust inattentional inflation for contrast-dependent and texture-based perception, at and above the visual threshold. However at suprathreshold, we found inattentional inflation for the overall stimulus but not the specific feature relevant for performance. Finally, we establish the unifying principle that inattentional inflation occurs if and only if attention reduces performance thresholds more than visibility thresholds. Thus what we think we see may regularly exceed what we can visually discriminate, placing constraints on theories of conscious perception.

neuroscience↗

bioRxiv2025

Molecular dissection of Class A PBP function uncovers novel features of the non-canonical Clostridioides difficile divisome complex

Cell division in bacteria is mediated by the "divisome," a multiprotein complex that synthesizes the septal peptidoglycan needed to divide one cell into two. We recently showed that the major nosocomial pathogen Clostridioides difficile assembles a divisome that is fundamentally distinct from previously studied bacteria because it lacks functional orthologs of the septal peptidoglycan-synthesizing enzymes, FtsW and FtsI. While these enzymes were previously thought to mediate cell division in all walled bacteria, C. difficile instead uses the bifunctional Class A Penicillin Binding Protein PBP1 to mediate cell division. Here, we optimized a CRISPRi-based conditional expression system to define features within PBP1 that are critical for its essential functions. Our analyses identify a novel accessory domain that is required for PBP1 function and is conserved across Peptostreptococcaceae family PBP1 homologs. We further show that PBP1s glycosyltransferase and transpeptidase activities are both strictly required for bacterial growth. While PBP1 glycosyltransferase activity is required for septum synthesis during cell division, PBP1s transpeptidase activity is surprisingly dispensable for cell division, although TPase-deficient (PBP1TPase*) cells produce multiple aberrant septa. We demonstrate that the uncontrolled septum synthesis observed in PBP1TPase* cells depends on the non-essential Class B PBP, PBP3, but PBP3s catalytic activity is dispensable for this function. Since we also show that PBP3 is recruited to the divisome complex and forms a complex with PBP1, our analyses reveal a cryptic but important regulatory function for PBP3 in promoting C. difficile cell division. Author SummaryBacterial cell division is an ancient and essential process, but our molecular understanding of this process is primarily based on studies in a select few model systems. Recent work found that the major nosocomial pathogen Clostridioides difficile divides by a fundamentally distinct mechanism because homologs of the canonical septum synthesis complex used by virtually all walled bacteria are either missing from the C. difficile genome or have lost their function during vegetative division. This prior work revealed that septum synthesis in C. difficile is instead driven by the catalytic activity of the Class A Penicillin Binding Protein called PBP1, but the features of PBP1 that enable it to carry out this unusual function were unknown. In the current study, we perform detailed structure-function analyses to determine how C. difficile PBP1 has become specialized for this role. We find that PBP1 carries an unusual regulatory domain that is critical for its function. Our analyses also uncover an unexpected function for the non-essential enzyme PBP3 in cell division, identifying a new component of the C. difficile cell division complex. These analyses provide new insight into how bacteria can repurpose cell wall synthesis enzymes to fulfill essential functions in novel ways.

microbiology↗

bioRxiv2025

Hidden in plain sight: Bacterial genomes reveal thousands of lytic phages with therapeutic potential

Phages are typically classified as temperate, integrating into host genomes, or lytic, replicating and killing bacteria. Lytic phages are not expected in bacterial genome sequences, yet our analysis of 3.6 million bacterial assemblies from 1,226 species revealed >100,000 complete lytic phage genomes, which we term Bacterial Assembly-associated Phage Sequences (BAPS). This represents a [~]five-fold increase in the number of phages with known hosts and fundamentally reshapes our understanding of phage biology. Identifying BAPS has enabled the discovery of entirely novel phage clusters, including clusters distantly related to Salmonella Goslarviruses in E. coli, and Shigella, while significantly expanding known genera such as Seoulvirus (from 16 to >300 members). Notably, close relatives of therapeutic lytic phages were also detected, suggesting clinical isolate sequencing unknowingly archives viable phage candidates. The discovery of complete, lytic phage genomes within bacterial assemblies challenges long-standing assumptions and reveals a vast, untapped reservoir of phages.

microbiology↗

bioRxiv2025

Visualizing phenotypic heterogeneity and single-cell morphology in situ during gut infection

Visualizing phenotypic heterogeneity at single-cell resolution within dense microbial communities is technically challenging. Here, we present a method for visualizing this heterogeneity by combining spectrally compatible reporters to track the spatial distribution of gene expression in individual bacterial cells in the mammalian gut. Using toxin gene expression in Clostridioides difficile as a model for visualizing phenotypic heterogeneity, we demonstrate that, while C. difficile primarily occupies the lumen, a subpopulation of C. difficile associates with the colonic epithelium independent of toxin production. The approach further revealed that heterogeneity in C. difficile toxin gene expression is independent of location in the gut and unexpectedly showed that a toxin gene over-expressing mutant forms filamentous cells during the acute phase of infection. Thus, our reporter system provides quantitative, single-cell resolution of bacterial behavior within the intact gut environment and establishes a broadly applicable platform for investigating phenotypic heterogeneity in dense microbial communities.

microbiology↗

bioRxiv2025

Phyla: Towards a Foundation Model for Phylogenetic Inference

Protein language models (PLMs) are often assumed to capture evolutionary information by training on large protein sequence datasets. Yet it remains unclear whether PLMs can reason about evolution--that is, infer evolutionary relationships between sequences. We test this capability by evaluating whether standard PLM usage, frozen or fine-tuned embeddings with distance-based comparison, supports evolutionary reasoning. Existing PLMs consistently fail to recover phylogenetic structure, despite strong performance on sequence-level tasks such as masked-token and contact prediction. We present PO_SCPLOWHYLAC_SCPLOW, a hybrid state-space and transformer model that jointly processes multiple sequences and is trained using a tree-based objective across 3,000 phylogenies spanning diverse protein families. PO_SCPLOWHYLAC_SCPLOW outperforms the next-best PLM by 9% on tree reconstruction and 23% on taxonomic clustering while remaining alignment- and guide-tree-free. Although classical alignment pipelines achieve higher absolute accuracy, PO_SCPLOWHYLAC_SCPLOW narrows the gap and achieves markedly lower end-to-end runtime. Applied to real data, PO_SCPLOWHYLAC_SCPLOW reconstructs biologically accurate clades in the tree of life and resolves genome-scale relationships among Mycobacterium tuberculosis isolates. These findings suggest that, under standard usage, evolutionary reasoning does not reliably emerge from large-scale sequence modeling. Instead, PO_SCPLOWHYLAC_SCPLOW shows that models trained with phylogenetic supervision can reason about evolution more effectively, offering a biologically grounded path toward evolutionary foundation models.

bioinformatics↗

bioRxiv2025

Strand-swapping in the LolA-like protein GerS promotes the allosteric activation of a bacterial amidase in Clostridioides difficile

GerS is a key lipoprotein regulator of Clostridioides difficile spore germination that shares structural, but not sequence, similarity to LolA in Gram-negative bacteria. GerS and LolA have vastly different biological roles: LolA functions in the trafficking of lipoproteins across the periplasmic space to the outer membrane of Gram-negative bacteria, while GerS allosterically activates the germination-specific amidase CwlD in endospore-forming Gram-positive bacteria. Here, we explore the diversity of LolA-like proteins across bacteria and reveal that GerS homodimer formation is a conserved feature of Peptostreptococcaceae family orthologs. Since dimerization of C. difficile GerS occurs via strand-swapping, we sought to investigate the functional importance of its strand swap by identifying mutations that alter the GerS dimer:monomer equilibrium. Our analyses reveal that GerSK71R and GerSN74L substitutions in the strand-swap hinge of GerS stabilize the homodimer, while a GerST72P substitution promotes monomer formation. Conversely, substituting the highly conserved proline at the equivalent site in LolA for threonine converts LolA from a monomer to a homodimer. Since our data indicate that destabilizing the GerS dimer impairs both GerS:CwlD binding in vitro and CwlD function in C. difficile, strand-swapped dimer formation in LolA-like proteins enhances their affinity for their binding partners. These data are consistent with analyses of equivalent LolA mutants in E. coli and thus provide new insight into the evolution and specialization of this intriguing group of proteins across the bacterial domain.

microbiology↗

bioRxiv2024

EFP Analyzer: A fast, accurate, and easy-to-teach program for analyzing Extracellular Field Potentials from iPSC-derived cardiomyocytes

RationaleInduced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) are an emerging model for determining drug effects and modeling disease. Specialized devices can generate Extracellular Field Potential (EFP) measurements from these cells, analogous to the ventricular complex of the electrocardiogram. ObjectiveThe objective of this study was to develop an easy-to-use, easy-to-teach, reproducible software tool to measure EFPs. Methods-ResultsWe present the EFP-Analyzer (EFPA), a semi-automized analyzer for EFP traces, which identifies and averages beats, identifies landmarks, and calculates intervals. We evaluated the tool in an analysis of 358 EFP traces from 22 patient-derived lines. We analyzed spontaneously beating iPSC-CMs, as well as optically paced iPSC-CMs through channelrhodopsin. We developed stringent quality criteria and measured EFP intervals, including Field Potential Duration (FPD). FPD from optically paced iPSC-CMs were shorter than those of spontaneously beating iPSC-CMs (283.7.0{+/-}54.2 vs. 293.0{+/-}47.5, p: 0.32, respectively). We further analyzed the usability and data replicability of EFPA through an inter-intra observer analysis. Correlation coefficient for inter-reader tangent and threshold measurements for these FPD ranged between r: 0.93-1.00. Bland-Altman plots comparing inter observer results for spontaneously beating and paced iPSC-CMs showed 95% limits of agreement (-13.6 to 19.4ms and -13.2 to 15.3ms, respectively). The EFP-analyzer could accurately detect FPD prolongation due to drug (moxifloxacin) or pathogenic loss of function mutations (CACNA1C N639T). This program is available for download at https://github.com/kroncke-lab/EFPA. The instructions will be available at the same listed website under the README section of the Github main page. ConclusionsThe EFP-Analyzer tool is a useful tool that enables the efficient use of iPSC-CMs as a model to study drug effects and disease.

pharmacology and toxicology↗

bioRxiv2024

Penicillin-binding proteins exhibit catalytic redundancy during asymmetric cell division in Clostridioides difficile

Peptidoglycan synthesis is an essential driver of bacterial growth and division. The final steps of this crucial process involve the activity of the SEDS family glycosyltransferases that polymerize glycan strands and the class B penicillin-binding protein (bPBP) transpeptidases that cross-link them. While many bacteria encode multiple bPBPs to perform specialized roles during specific cellular processes, some bPBPs can play redundant roles that are important for resistance against certain cell wall stresses. Our understanding of these compensatory mechanisms, however, remains incomplete. Endospore-forming bacteria typically encode multiple bPBPs that drive morphological changes required for sporulation. The sporulation-specific bPBP, SpoVD, is important for synthesizing the asymmetric division septum and spore cortex peptidoglycan during sporulation in the pathogen Clostridioides difficile. Although SpoVD catalytic activity is essential for cortex synthesis, we show that it is unexpectedly dispensable for SpoVD to mediate asymmetric division. The dispensability of SpoVDs catalytic activity requires the presence of its SEDS partner, SpoVE, and is facilitated by another sporulation-induced bPBP, PBP3. Our data further suggest that PBP3 interacts with components of the asymmetric division machinery, including SpoVD. These findings suggest a possible mechanism by which bPBPs can be functionally redundant in diverse bacteria and facilitate antibiotic resistance. ImportancePeptidoglycan synthesis requires the transpeptidase activity of penicillin-binding proteins (PBPs), which can have specialized functions during cellular growth, division, and differentiation. However, many bacteria produce PBPs with overlapping functions, and this functional redundancy can increase antibiotic resistance. The spore-forming pathogen, Clostridioides difficile, encodes four PBPs: two are essential for growth and division and another, SpoVD, is essential for spore formation. Here, we show that the transpeptidase activity of SpoVD is dispensable during the first morphological step of sporulation, asymmetric division, because a sporulation-induced PBP, PBP3, partially substitutes for SpoVDs function during this early stage. We find that SpoVD and PBP3 interact during sporulation, suggesting a mechanism by which bPBPs can confer functional redundancy and potentially contribute to antibiotic resistance.

microbiology↗