Biology subjects

Leppla, S. H.

Publications and source records attributed to Leppla, S. H..

6 recordsLinked to original sources

bioRxiv2025

Environmental Regulation of Toxin Production in Bacillus anthracis

Pathogenic Bacillus anthracis strains carry two plasmids - pX01, which encodes a tripartite protein exotoxin complex (PA, LF, and EF); and pX02, which encodes a poly-D-gamma-glutamic acid capsule. A multidomain transcription factor, AtxA, regulates the expression of these virulence genes. AtxA has two DNA-binding Helix-Turn-Helix (HTH) domains, two phosphoenolpyruvate:carbohydrate phosphotransferase system regulatory domains (PRD1 and PRD2), and a putative EIIB domain (a component of PTS sugar transport EII-complexes). Previous studies showed that glucose and CO2 increase AtxA-dependent toxin gene transcription, along with histidine phosphorylation of PRD1 and PRD2. Our transcriptional profiling of virulence factors, PA secretion, and fluorescent reporter strain analyses confirms a synergistic effect of glucose and CO2 on AtxA-dependent toxin production. Deletion of AtxA ({Delta}atxA) significantly reduced glucose uptake in bacteria, suggesting that AtxA may act within the glucose-PTS system. Mutation analysis of the EIIB domain of AtxA identified the cysteine at position 402 as essential for the transcriptional activity of AtxA. Deletion of glucose PTS permease PtsG ({Delta}ptsG) significantly reduced the expression of PA, LF, and EF. Loss of PtsG also caused attenuation in a mouse model of infection. Intracellular imaging using FLIM confirms a physical interaction of PtsG and AtxA through EIIB domain of AtxA. Using phosphomimetic and phosphoablative mutants of AtxA, we confirmed that the physical interaction of PtsG and AtxA is essential for AtxA activity. Finally, the synergy between glucose and CO2 was targeted by deleting pyruvate carboxylase Pyc ({Delta}pyc), which regulates anaplerosis. This deletion confirms that Pyc stimulates the level of phosphoenolpyruvate (PEP) and increases the phosphorelay in glucose-PTS to enhance AtxA activity. Therefore, we propose that a histidine-phosphorelay from PEP regulates AtxA via PTS enzymatic activity, impacting AtxA activity through physical interaction of AtxA and PtsG. Finally, we propose AtxA as an integral component of the glucose-PTS, where transcriptional activity of AtxA is regulated by environmental signals including glucose and CO2.

molecular biology↗

bioRxiv2025

A novel AAA+ ATPase required for sporulation and stress response in Bacillus anthracis

AAA+ proteins function as molecular machines that utilize ATP to perform diverse cellular functions, including protein homeostasis, stress regulation, and cell cycle/developmental processes. In this study, we identified a novel AAA+ ATPase BAS PrkA in B. anthracis Sterne 34F2 which has 88 % protein homology to Bacillus subtilis PrkA. Conserved domain analysis confirms BAS PrkA has an N-terminal AAA+ ATPase domain with characteristic Walker A and Walker B motifs and a conserved secondary region of homology (SRH) domain, along with a C-terminal cAMP-dependent protein kinase domain. Based on Alpha Fold3 predicted structure, we classified BAS PrkA as part of Clade III of the AAA+ superfamily. Contrary to the reported enzymatic activity in B. subtilis PrkA, we observed that BAS PrkA has negligible protease and kinase activity under in-vitro conditions. Nonetheless, BAS PrkA plays a significant role in regulating sporulation. It is temporally expressed during Stages II to VI during sporulation. A null mutant of BAS PrkA exhibits severe sporulation defects, with reduced spore viability, and down regulation of genes related to spore-coat formation. These phenotypes were restored in a complementation strain expressing BAS PrkA ectopically. Additionally, the null mutant strain showed compromised growth under ionic-osmotic stress conditions. Analysis of the BAS PrkA interactome revealed enrichment of two proteins, ProA and EzrA, that are implicated in osmotic stress response and the sporulation process, respectively. These findings show that BAS PrkA plays a critical role in sporulation and osmotic stress response in B. anthracis.

microbiology↗

bioRxiv2025

Specificity and mechanism of the double-stranded RNA-specific J2 monoclonal antibody

Double-stranded (ds) RNAs are major structural components of the transcriptome, hallmarks of viral infection, and primary triggers of innate immune responses. The J2 monoclonal antibody is the gold-standard method to discover and map endogenous dsRNAs across subcellular locations and cell surfaces, detect exogenous RNAs in viral infection, and surveil mRNA prophylactics and therapeutics for inflammatory dsRNAs. To define its epitope, specificity, and mechanism, we determine a 2.85 [A] co-crystal structure of J2 antigen-binding fragment (Fab) bound to dsRNA. J2 uses its heavy and light chains in tandem to track the dsRNA minor groove, recognizing a staggered 8-bp duplex. J2 is exquisitely selective for dsRNAs, requires 14 bp for robust binding, and exhibits greatly diminished binding for GC-rich dsRNAs. J2 and R-loop-specific S9.6 antibody share a common recognition strategy distinct from intracellular dsRNA-binding proteins. This study provides mechanistic insights into dsRNA recognition and establishes a framework for reliable application and data interpretation of the J2 antibody in RNA discovery.

molecular biology↗

bioRxiv2021

Direct imaging of anthrax intoxication in animals reveals shared and individual functions of CMG-2 and TEM-8 in cellular toxin entry

The virulence of Bacillus anthracis is linked to the secretion of anthrax lethal toxin and anthrax edema toxin. These binary toxins consist of a common cell-binding moiety, protective antigen (PA), and the enzymatic moieties, lethal factor (LF) and edema factor (EF). PA binds either of two specific cell surface receptors, capillary morphogenesis protein-2 (CMG-2) or tumor endothelial marker-8 (TEM-8), which triggers the binding, endocytosis, and cytoplasmic translocation of LF and EF. The cellular distribution of functional TEM-8 and CMG-2 receptors during anthrax toxin intoxication in animals is not fully elucidated. Herein, we describe a novel assay to image anthrax toxin intoxication in live animals, and we use the assay to visualize TEM-8- and CMG-2-dependent intoxication. Specifically, we generated a chimeric protein consisting of the N-terminal domain of LF fused to a nuclear localization signal-tagged Cre recombinase (LFn-NLS-Cre). When PA and LFn-NLS-Cre were co-administered to transgenic mice that ubiquitously express a red fluorescent protein in the absence of Cre activity and a green fluorescent protein in the presence of Cre activity, anthrax toxin intoxication could be visualized at single-cell resolution by confocal microscopy. By using this assay, we show that CMG-2 is critical for intoxication in the liver and heart, whereas TEM-8 is required for full intoxication in the kidney and spleen. Other tissues examined were largely unaffected by single deficiences in either receptor, suggesting extensive overlap in TEM-8 and CMG-2 expression. The novel assay will be useful for basic and clinical/translational studies of Bacillus anthracis infection and for identifying on- and off-targets for reengineered toxin variants in the clinical development of cancer treatments. BackgroundAssays for imaging of anthrax toxin intoxication in animals are not available. ResultsAnthrax toxin-Cre fusions combined with fluorescent Cre reporter mice enabled imaging of anthrax toxin intoxication in animals. ConclusionShared and distinct functions of toxin receptors in cellular entry were uncovered. Significance. A simple and versatile assay for anthrax toxin intoxication is described.

pathology↗

bioRxiv2020

Anthrax Toxin as a Molecular Platform to Target Nociceptive Neurons and Modulate Pain

Bacterial toxins are able to act on neurons to modulate signaling and function. Here, we find that nociceptive sensory neurons that mediate pain are enriched in the receptor for anthrax toxins, ANTXR2. Anthrax Edema Toxin (ET) induced cAMP and PKA signaling in Nav1.8+ nociceptive neurons and modulated pain in vivo. Peripherally administered ET mediated mechanical allodynia in naive mice and during B. anthracis infection. Intrathecally administered ET produced analgesic effects, potently blocking pain-like behaviors in multiple mouse models of inflammatory and chronic neuropathic pain. Nociceptor-specific ablation of ANTXR2 attenuated ET-induced signaling and analgesia. Modified anthrax toxin successfully delivered exogenous protein cargo into nociceptive neurons, illustrating utility of the anthrax toxin system as a molecular platform to target pain. ET further induced signaling in human iPSC-derived sensory neurons. Our findings highlight novel interactions between a bacterial toxin and nociceptors that may be utilized for developing new pain therapeutics. SUMMARYANTXR2 expression on nociceptive neurons allows selective targeting and modulation of pain by native and engineered anthrax toxins.

neuroscience↗

bioRxiv2020

Bacillus anthracis chain length, a virulence determinant, is regulated by a transmembrane Ser/Thr protein kinase PrkC

Anthrax is a zoonotic disease caused by Bacillus anthracis, a spore-forming pathogen that displays a chaining phenotype. It has been reported that in a mouse infection model, systemic inoculation with longer bacterial chains caused blockade in lung capillaries. The blockade resulted in increased pathophysiological consequences viz, hypoxia and lung tissue injury. Hence, chaining acts as a virulence factor and molecules that regulate the chaining phenotype can be the potential drug targets. In this study, we have identified the serine/threonine protein kinase of B. anthracis, PrkC, localized at the bacteria-host interface, as a determinant of bacterial chain length. In vitro, prkC disruption strain (BAS {Delta}prkC) grew as shorter chains throughout the bacterial growth cycle as observed through phase-contrast and scanning electron microscopy. Since molecules such as BslO, a septal murein hydrolase, that catalyzes daughter cell separation and Sap, an S-layer structural protein required for the septal localization of BslO, are known to influence chain length, a comparative analysis to determine their levels was done through western-blot analysis. Both BslO and Sap were found to be upregulated in BAS {Delta}prkC at the majority of the time points. Additionally, PrkC disruption was observed to have a significant effect on bacterial growth and cell wall thickness. In BAS {Delta}prkC strain, a decrease in the cell wall thickness and an increase in the multi-septa formation was observed through transmission electron and confocal microscopy respectively. Altogether, we show that PrkC disruption affected chaining phenotype, cell growth and cell wall thickness and also report that the associated molecules were de-regulated. Through this work, we show for the first time that the chaining phenotype is regulated by PrkC, a transmembrane kinase with a sensor domain. During infection, PrkC may regulate the chaining phenotype through the identified signaling mechanism. Authors summaryB. anthracis, a spore-forming pathogen is the causative agent of anthrax, a zoonotic disease that primarily affects livestock and wildlife. Humans are at risk of contracting this disease through exposure to spores generated by infected animals. In the past, B. anthracis spores have been used as a bioterror agent. Hence, there has been a continuous effort to understand the biology of this pathogen to develop both therapeutic and prophylactic treatment. Various virulence factors that are essential for B. anthracis pathogenesis have been identified. The ability of B. anthracis to grow in chains acts as a virulence factor. Longer bacterial chains are reported to cause blockade of lung capillaries in the mouse infection model. In this study, we have shown that the disruption of the lone serine/threonine protein kinase, PrkC, localized at the bacteria-host interface leads to the shortening of the bacterial chains. We have seen that the depletion of PrkC results in an increase in the levels of the proteins responsible for de-chaining. Also, we have analyzed the effect of the disruption on cell growth, bacterial cell wall and septa formation. Since PrkC is a surface localized kinase with an extracellular domain that lacks homology to human proteins, it can be a target for new drugs. Disruption of PrkC activity and hence the longer chains in vivo may prevent pathophysiological consequences associated with the capillary blockade.

microbiology↗