Biology subjects

Wehenkel, A. M.

Publications and source records attributed to Wehenkel, A. M..

15 recordsLinked to original sources

bioRxiv2026

Uncoupling mycomembrane biogenesis from mycolic acid synthesis reveals a distinct role for mycoloyltransferases in mycobacterial cell division.

Bacteria of the order Mycobacteriales, including the genera Mycobacterium and Corynebacterium, possess a unique outer membrane, termed the mycomembrane, which is structurally and chemically distinct from the lipopolysaccharide-containing outer membrane of Gram-negative bacteria. A defining feature of the mycomembrane is its enrichment in mycolic acids, long-chain -branched, {beta}-hydroxylated fatty acids that occur as trehalose monomycolate (TMM), trehalose dimycolate (TDM), or are esterified to arabinogalactan, an unusual polymer that is itself covalently linked to peptidoglycan (PG). Mycoloyltransferases are Mycobacteriales-specific enzymes described to catalyze the transfer of mycolic acids from trehalose monomycolate (TMM) to various cell envelope acceptors, including trehalose and arabinogalactan. In several species, including Mycobacterium tuberculosis, these proteins are essential for viability; however, their occurrence as multiple paralogs with partially redundant functions has hindered the precise assignment of their cellular roles. Previously, we showed that Corynebacterium glutamicum remains viable in the absence of mycolic acids, and thus without a mycomembrane, following deletion of pks, the gene required for mycolic acid biosynthesis. Building on this finding, we systematically deleted all genes encoding mycoloyltransferases in C. glutamicum to further disclose their collective function in the cell. The resulting {Delta}myts mutant lacked arabinogalactan-bound mycolates and TDM, yet continued to synthesize TMM. Despite the high abundance of this major glycolipid, the {Delta}myts strain failed to assemble a mycomembrane and displayed pronounced cell aggregation. Unexpectedly, deletion of mycoloyltransferases also caused very severe defects in cell division and morphogenesis that are not observed in a {Delta}pks strain unable to synthesize mycolic acids. Together, these results demonstrate that mycoloyltransferases are essential for mycomembrane assembly but dispensable for TMM biosynthesis, and reveal an unexpected role for these enzymes in cell division that is independent of their canonical mycolic acid transfer activity. SIGNIFICANCEHow the mycomembrane is assembled and anchored to the cell wall remains a central question in Mycobacteriales, where this outer membrane is necessary for envelope integrity and intrinsic antibiotic resistance. By genetically separating mycolic acid synthesis from their incorporation into the outer membrane, we identify arabinogalactan-linked mycolates as the critical determinant for initiating membrane assembly. Unexpectedly, we also uncover a role for mycoloyltransferases beyond their canonical function in lipid metabolism, revealing a functional link with cell division. These findings point to a critical role of Mycoloyltransferases in the coordination between outer membrane biogenesis and bacterial cytokinesis.

microbiology↗

bioRxiv2026

Assessing Corynebacterium glutamicum as a surrogate of Mycobacterium tuberculosis for DNA gyrase inhibitor design.

DNA gyrase is an essential bacterial enzyme and a clinically validated target for the treatment of tuberculosis. However, the discovery of new inhibitors remains limited by the many challenges regarding the manipulation on pathogenic mycobacteria. This study validates Corynebacterium glutamicum (Cglu) as a safe, non-pathogenic surrogate for Mycobacterium tuberculosis (Mtb) to investigate DNA gyrase and facilitate the identification of new inhibitors. Using Cglu as a target allows for fast whole-cell screening under safe conditions while ensuring efficient drug uptake. Cglu shares key physiological features with Mtb, including genome size, complex cell wall structure, and a single type I and type II topoisomerase. Structural and functional comparisons emphasize the similarity of Cglu and Mtb gyrases, which share 70% sequence identity and show comparable catalytic properties and responsiveness to known inhibitors. Thus, the cryo-EM structure of the Cglu gyrase-DNA complex at 3.2 [A] resolution reveals highly conserved drug-binding pockets for known anti-gyrase inhibitors and the genetic depletion of gyrA or gyrB in Cglu causes severe growth and morphological defects, mirroring the effects of chemical inhibition and allowing to link gyrase function to cellular phenotypes. Comparative imaging of different inhibitor classes (fluoroquinolones, aminocoumarins, NBTIs) uncovers distinct morphological signatures that reflect each compounds mode of action. Finally, cross-species complementation confirms functional conservation but also highlights subtle structural differences affecting efficiency. Together, these findings establish Cglu as a robust and biosafe model for dissecting gyrase function, visualizing DNA topology dynamics, and accelerating the discovery of gyrase-targeting antimicrobials. More generally, our studies demonstrate the feasibility of using Cglu as a cell-based screening platform to discover new anti-tuberculous compounds targeting conserved mechanisms, not only for validated TB drug targets such as DNA gyrase but also for new, yet to be identified, targets.

microbiology↗

bioRxiv2026

Deep learning extracts MoA-specific signatures from high-throughput images of chemically and genetically perturbed Corynebacteria

Tuberculosis (TB) is the worldwide leading infectious killer due to a single pathogen and increasing antimicrobial resistance (AMR) makes it imperative to discover and develop new drugs with novel modes of action (MoAs) to treat TB infections. Phenotypic screening of chemical libraries has proven effective at identifying new compounds against bacterial pathogens. However, a major limitation of standard screens is their inability to uncover the MoA of hits thereby preventing targeted selection of compounds with novel MoAs. Linking drug perturbations to mutants from images could potentially enable to predict the targets of compounds that act through novel MoAs. Here, we develop a deep learning (DL)-based method to screen drug-treated Corynebacterium glutamicum (Cglu), a surrogate model for Mycobacterium tuberculosis (Mtb). Our DL model is based on a convolutional neural network architecture that takes high throughput images as input and is trained to distinguish between different MoAs. We show that our approach can robustly differentiate between the MoAs of established antibiotics and correctly recognise the MoA of antibiotics that were not previously seen by the DL model. We also show that inhibitors with the same and previously unseen MoA cluster together and apart from all other reference drugs, allowing for new MoA discovery. Importantly, we show that our model links images of chemical (drugs) and genetic (mutants) perturbations targeting similar pathways, thus paving the way towards mutant-based target prediction of compounds that act through novel MoAs, directly from high-content images. Finally, we explore the phenotypes induced by genetic disruption of pathways and demonstrate that features extracted with our DL model recover known biological relationships from high-throughput images alone using the cell cycle of Cglu as a case study, a finding with promising potential for fundamental mechanistic studies.

microbiology↗

bioRxiv2025

Functional dissection of Wag31 domains for septal recruitment and polar distribution during the cell cycle

Bacterial cell morphogenesis is controlled by the synthesis and organization of peptidoglycan and driven by multi-protein complexes such as the divisome and elongasome. Here we investigate the role of the Corynebacterium glutamicum DivIVA homologue, Wag31, the elongasome scaffold essential for polar growth in Corynebacteriales. Conditional depletion of Wag31 results in viable but coccoid-shaped cells, showing that Wag31 is essential for rod shape maintenance. Our structural phylogenetic analyses of DivIVA homologues revealed that in Actinobacteria, unlike Firmicutes, an intrinsically disordered region spatially separates the N-terminal lipid-binding domain (LBD) from the C-terminal coiled-coil domain (CCD). We show that the LBD is necessary and sufficient for septum localization, independent of its membrane-binding properties, while the CCD domain mediates self-interaction and polar accumulation. Our findings suggest that Wag31 is recruited specifically to the septum through protein-protein interactions, priming the future pole and allowing for a timely divisome-elongasome transition at cytokinesis. Once the pole is formed the self-aggregative properties of the C-terminal CCD dominate and form a stable structure that likely organizes the pole for cell wall biosynthesis.

microbiology↗

bioRxiv2025

Mechanistic insights into the allosteric regulation of cell wall hydrolase RipA in Mycobacterium tuberculosis.

D,L-endopeptidase RipA is the major PG hydrolase required for daughter cell separation in Mycobacterium tuberculosis (Mtb), as RipA defects severely hinder cell division and increase antibiotic vulnerability. Despite extensive studies, the mechanisms governing Mtb RipA regulation remain controversial and poorly understood. Here, we report an integrative structural and functional analysis of the SteAB system, a regulatory complex that has been shown to modulate cell separation in the model organism Corynebacterium glutamicum (Cglu) and is conserved across Mycobacteriales. Although Mtb SteB was previously proposed to act as a mycobacterial outer membrane copper transporter, the crystal structures of the homodimeric protein, alone and in complex with the RipA coiled-coil (CC) domain, rule out this hypothesis. Instead, the high-affinity SteB-RipA interaction, together with computational and biophysical data, strongly supports the role of SteB as a direct RipA activator that releases enzyme autoinhibition upon complex formation. In addition, crystallographic characterization of the cytoplasmic core of SteA revealed a homodimeric organization harboring a conserved functional pocket similar to the phosphonucleotide-binding site of thiamine pyrophosphokinase. These data, coupled with the in vivo phenotypic analysis of a steAB knockout mutant of Cglu, support a model in which the transmembrane SteAB heterotetramer, driven by cytoplasmic ligand binding, orchestrates the productive periplasmic positioning of RipA, leading to PG hydrolysis activation. These findings shed new light on the regulation of mycobacterial cell wall remodeling, with implications for understanding Mtb pathogenesis and identifying novel antimicrobial targets.

microbiology↗

bioRxiv2025

Deep learning recognises antibiotic modes of action from brightfield images

Antimicrobial resistance is a growing public health threat predicted to cause up to 10 million deaths a year by 2050. To circumvent existing bacterial resistance mechanisms, discovering antibiotics with novel modes of action (MoAs) is crucial. While growth inhibition assays can robustly identify antibiotic molecules, they miss promising compounds with subinhibitory phenotypes and do not inform on drug MoA. Microscopy-based cytological profiling of drug-treated bacteria with hand-crafted image descriptors or more recently deep learning (DL) provides complementary information on the MoA. However, current approaches are limited by the need for fluorescent labelling and drug exposure at inhibitory concentrations. It also remains unclear if cytological profiling enables the detection of drugs with novel MoAs. Here, we demonstrate an approach based on supervised DL to identify antibiotic drug MoA from microscopy images without fluorescent labelling. We train a convolutional neural network to predict treatment conditions from brightfield images of Escherichia coli exposed to reference drugs covering multiple MoAs. Our method can detect drug exposure at subinhibitory concentrations and distinguishes individual drug treatments with high accuracy (86.2%). The learned representations implicitly capture MoA-specific phenotypes, enabling perfect MoA recognition (100%) without retraining using a model trained on only 644 images. Our approach can identify the MoA of previously unseen drugs with good accuracy (77.8{+/-}3.3%), as long as the MoA is represented by at least one of the training compounds. Finally, we show that our approach can detect if a drug has a novel MoA with an area under the curve above 0.75 for five out of six MoAs, facilitating microscopy-based identification of novel classes of antibiotics. Our methods and results pave the way towards an automated pipeline for antibiotic drug discovery based on imaging and DL.

microbiology↗

bioRxiv2024

Cryo-EM structure and polar assembly of the PS2 S-layer of Corynebacterium glutamicum

The polar-growing Corynebacteriales have a complex cell envelope architecture characterized by the presence of a specialized outer membrane composed of mycolic acids. In some Corynebacteriales, this mycomembrane is further supported by a proteinaceous surface layer or S-layer, whose function, structure and mode of assembly remain largely enigmatic. Here, we isolated ex vivo PS2 S-layers from the industrially important Corynebacterium glutamicum and determined its atomic structure by 3D cryoEM reconstruction. PS2 monomers consist of a six-helix bundle core, a three-helix bundle arm, and a C-terminal transmembrane (TM) helix. The PS2 core oligomerizes into hexameric units anchored in the mycomembrane by a channel-like coiled-coil of the TM helices. The PS2 arms mediate trimeric lattice contacts, crystallizing the hexameric units into an intricate semipermeable lattice. Using pulse-chase live cell imaging, we show that the PS2 lattice is incorporated at the poles, coincident with the actinobacterial elongasome. Finally, phylogenetic analysis shows a paraphyletic distribution and dispersed chromosomal location of PS2 in Corynebacteriales as a result of multiple recombination events and losses. These findings expand our understanding of S-layer biology and enable applications of membrane-supported self-assembling bioengineered materials.

microbiology↗

bioRxiv2024

FhaA plays a key role in mycobacterial polar elongation and 1 asymmetric growth

Mycobacteria, including pathogens like Mycobacterium tuberculosis, exhibit unique growth patterns and cell envelope structures that challenge our understanding of bacterial physiology. This study sheds light on FhaA, a conserved protein in Mycobacteriales, revealing its pivotal role in coordinating cell envelope biogenesis and asymmetric growth. The elucidation of the FhaA interactome in living mycobacterial cells reveals its participation in the protein network orchestrating cell envelope biogenesis and cell elongation/division. By manipulating FhaA levels, we uncovered its influence on cell morphology, cell envelope organization, and the localization of peptidoglycan biosynthesis machinery. Notably, fhaA deletion disrupted the characteristic asymmetric growth of mycobacteria, highlighting its importance in maintaining this distinctive feature. Our findings position FhaA as a key regulator in a complex protein network, orchestrating the asymmetric distribution and activity of cell envelope biosynthetic machinery. This work not only advances our understanding of mycobacterial growth mechanisms but also identifies FhaA as a potential target for future studies on cell envelope biogenesis and bacterial growth regulation. These insights into the fundamental biology of mycobacteria may pave the way for novel approaches to combat mycobacterial infections addressing the ongoing challenge of diseases like tuberculosis in global health.

microbiology↗

bioRxiv2024

Evolutionary plasticity and functional repurposing of the essential metabolic enzyme MoeA

MoeA, or gephyrin in higher eukaryotes, is crucial for molybdenum cofactor biosynthesis required in redox reactions. Gephyrin is a moonlighting protein also involved in postsynaptic receptor clustering, a feature thought to be a recent evolutionary trait. We showed previously that a repurposed copy of MoeA (Glp) is involved in bacterial cell division. To investigate how MoeA acquired multifunctionality, we used phylogenetic inference and protein structure analyses to understand the diversity and evolutionary history of MoeA. Glp-expressing Bacteria have at least two copies of the gene, and our analysis suggests that Glp has lost its enzymatic role. In Archaea we identified an ancestral duplication where one of the paralogs might bind tungsten instead of molybdenum. In eukaryotes, the acquisition of the moonlighting activity of gephyrin comprised three major events: first, MoeA was obtained from Bacteria by early eukaryotes, second, MogA was fused to the N-terminus of MoeA in the ancestor of opisthokonts, and finally, it acquired the function of anchoring GlyR receptors in neurons. Our results support the functional versatility and adaptive nature of the MoeA scaffold, which has been repurposed independently both in eukaryotes and bacteria to carry out analogous functions in network organization at the cell membrane.

evolutionary biology↗

bioRxiv2023

Eukaryotic-like gephyrin and cognate membrane receptor coordinate corynebacterial cell division and polar elongation.

The order Corynebacteriales includes major industrial and pathogenic actinobacteria such as Corynebacterium glutamicum or Mycobacterium tuberculosis. Their elaborate multi-layered cell wall, composed primarily of the mycolyl-arabinogalactan-peptidoglycan complex, and their polar growth mode impose a stringent coordination between the septal divisome, organized around the tubulin-like protein FtsZ, and the polar elongasome, assembled around the tropomyosin-like protein Wag31. Here, we report the identification of two new divisome members, a gephyrin-like repurposed molybdotransferase (GLP) and its membrane receptor (GLPR). We show that the interplay between the GLPR/GLP module, FtsZ and Wag31 is crucial for orchestrating cell cycle progression. Our results provide a detailed molecular understanding of the crosstalk between two essential machineries, the divisome and elongasome, and reveal that Corynebacteriales have evolved a protein scaffold to control cell division and morphogenesis similar to the gephyrin/GlyR system that in higher eukaryotes mediates synaptic signaling through network organization of membrane receptors and the microtubule cytoskeleton.

microbiology↗

bioRxiv2022

Legionella para-effectors target chromatin and promote bacterial replication

Legionella pneumophila replicates intracellularly by secreting effectors via a type IV secretion system. One of these effectors is a eukaryotic methyltransferase (RomA) that methylates K14 of histone H3 (H3K14me3) to counteract host immune responses. However, it is not known how L. pneumophila infection catalyses H3K14 methylation as this residue is usually acetylated. Here we show that L. pneumophila secretes a eukaryotic-like histone deacetylase (LphD) that specifically targets H3K14ac and works in synergy with RomA. Both effectors target host chromatin and bind the HBO1 histone acetyltransferase complex that acetylates H3K14. Full activity of RomA is dependent on the presence of LphD as H3K14 methylation levels are significantly decreased in a {Delta}lphD mutant. The dependency of these two chromatin-modifying effectors on each other is further substantiated by mutational and virulence assays revealing that the presence of only one of these two effectors impairs intracellular replication, while a double knockout ({Delta}lphD{Delta}romA) can restore intracellular replication. Uniquely, we present evidence for "para-effectors", an effector pair, that actively and coordinately modify host histones to hijack the host response. The identification of epigenetic marks modulated by pathogens opens new vistas for the development of innovative therapeutic strategies to counteract bacterial infection and strengthening host defences.

microbiology↗

bioRxiv2022

A novel nuclease is the executing part of a bacterial plasmid defense system

Cells are continuously facing the risk of taking up foreign DNA that can compromise genomic and cellular integrity. Therefore, bacteria are in a constant arms race with mobile genetic elements such as phages, transposons and plasmids. They have developed several active strategies against invading DNA molecules that can be seen as a bacterial innate immune system. Anti-phage systems are usually organized in defense islands and can consist of restriction-modification (R-M) systems, CRISPR-Cas, and abortive infection (Abi) systems. Despite recent advances in the field, much less is known about plasmid defense systems. We have recently identified the MksBEFG system in Corynebacterium glutamicum as a novel plasmid defense system which comprises homologues of the condensin system MukFEB. Here, we investigated the molecular arrangement of the MksBEFG complex. Importantly, we identified MksG as a novel nuclease that degrades plasmid DNA and is, thus, the executing part of the system. The crystal structure of MksG revealed a dimeric assembly through its DUF2220 C-terminal domains. This domain is homologous to the TOPRIM domain of the topoisomerase II family of enzymes and contains the corresponding divalent ion binding site that is essential for DNA cleavage in topoisomerases, explaining the in vitro nuclease activity of MksG. We further show that the MksBEF subunits exhibit an ATPase cycle similar to MukBEF in vitro and we reason that this reaction cycle, in combination with the nuclease activity provided by MksG, allows for processive degradation of invading plasmids. Super-resolution localization microscopy revealed that the Mks system is spatially regulated via to the polar scaffold protein DivIVA. Introduction of plasmids increases the diffusion rate and alters the localization of MksG, indicating an activation of the system in vivo.

microbiology↗

bioRxiv2021

Proteome remodeling in the Mycobacterium tuberculosis PknG knockout: molecular evidence for the role of this kinase in cell envelope biogenesis and hypoxia response.

Mycobacterium tuberculosis, the etiological agent of tuberculosis, is among the deadliest human pathogens. One of M. tuberculosiss pathogenic hallmarks is its ability to persist in a dormant state in the host for long periods, reinitiating the infectious cycle when favorable environmental conditions are found. Thus, it is not surprising that this pathogen has developed different mechanisms to withstand the stressful conditions found in the host. In particular, the Ser/Thr protein kinase PknG has gained special relevance since it regulates nitrogen metabolism and facilitates bacterial survival inside macrophages. Nevertheless, the molecular mechanisms underlying these effects are far from being elucidated. To further investigate these issues, we performed quantitative proteomics analyses of protein extracts from M. tuberculosis H37Rv and a mutant derivative lacking pknG. Our results showed that in the absence of PknG the mycobacterial proteome was remodeled since 5.7% of the proteins encoded by M. tuberculosis presented significant changes in its relative abundance when compared to the wild-type strain. The main biological processes affected by pknG deletion were the biosynthesis of cell envelope components and the response to hypoxic conditions. As many as 13 DosR-regulated proteins were underrepresented in the pknG deletion mutant, including the distinctive Hrp-1, which was found to be 12-fold decreased according to Parallel Reaction Monitoring experiments. Altogether, the results presented here allow us to postulate that PknG regulation of bacterial adaptation to stress conditions might be an important mechanism underlying its reported effect on intracellular bacterial survival.

biochemistry↗

bioRxiv2020

SepF is the FtsZ-anchor in Archaea: implications for cell division in the Last Universal Common Ancestor

The Archaea present profound differences compared to Bacteria in fundamental molecular and cellular processes. While most Archaea divide by binary fission using an FtsZ-based system similar to Bacteria, they lack the majority of the components forming the complex bacterial divisome. Moreover, how FtsZ precisely functions and interacts with other proteins to assemble the archaeal division machinery remains largely unknown. Notably, among the multiple bacterial factors that tether FtsZ to the membrane during cell constriction, Archaea only possess SepF-like homologues, but their function has not been demonstrated. Here, we combine structural, cellular, and evolutionary approaches to demonstrate that SepF is the FtsZ anchor in the human-associated archaeon Methanobrevibacter smithii. 3D super-resolution microscopy of immunolabeled cells shows that M. smithii SepF co-localizes with FtsZ at the division plane. We also show that M. smithii SepF binds both to membranes and FtsZ, inducing filament bundling. High-resolution crystal structures of archaeal SepF alone and in complex with FtsZCTD reveal that SepF forms a dimer with a specific homodimerization interface. This drives a strikingly different binding mode from what is observed in Bacteria. Finally, analysis of the distribution and phylogeny of SepF and FtsZ indicates that these proteins date back to the Last Universal Common Ancestor (LUCA) and that Archaea may have retained features of an ancestral minimal cell division system, while Bacteria likely diverged to accommodate the emergence of the complex machinery required to coordinate cytokinesis with the rigid peptidoglycan cell wall and the appearance of additional FtsZ tethers. Our results contribute key insights into the largely understudied mechanisms of archaeal cell division, and pave the way for a better understanding of the processes underlying the divide between the two prokaryotic domains.

microbiology↗

bioRxiv2019

Essential dynamic interdependence of FtsZ and SepF for Z-ring and septum formation in Corynebacterium glutamicum

The mechanisms of Z-ring assembly and regulation in bacteria are poorly understood, particularly in non-model organisms. Actinobacteria, one of the largest bacterial phyla that includes the deadly human pathogen Mycobacterium tuberculosis, lack the canonical FtsZ-membrane anchors as well as all positive and negative Z-ring regulators described for E. coli. Here we investigate the physiological function of Corynebacterium glutamicum SepF, the only cell division-associated protein from Actinobacteria known to directly interact with the conserved C-terminal tail of FtsZ but whose actual mode of action in cytokinesis is yet to be elucidated. We used a mechanistic cell biology approach to unveil the essential interdependence of FtsZ and SepF required for the formation of a functional Z-ring in the actinobacterial model organism C. glutamicum. The crystal structure of the SepF-FtsZ complex reveals a hydrophobic FtsZ-binding pocket, which defines the SepF homodimer as the functional unit, and a reversible oligomerization interface regulated via an alpha helical switch. FtsZ filaments and lipid membranes have opposing effects on SepF polymerization, leading to a complex dynamic role of the protein at the division site, involving FtsZ bundling, Z-ring tethering and membrane reshaping activities that are needed for proper Z-ring assembly and function.

microbiology↗