Biology subjects

Alzari, P. M.

Publications and source records attributed to Alzari, P. M..

15 recordsLinked to original sources

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

Structural basis of MfpD, a versatile pathogeny protein from the mfp conservon of Mycobacterium tuberculosis

The mfp conservon of Mycobacterium tuberculosis has been associated with fluoroquinolone resistance and encodes five conserved proteins, including the small GTPase MfpB and its regulatory partner MfpD. In this study, we combined phylogenetic, structural, and biophysical approaches to define the molecular basis of MfpD function. MfpD adopts a Roadblock/LC7-like /{beta} fold and forms a stable dimer in solution, with hydrophobic 2-helix interactions stabilizing the interface. Additional biophysical analyses and AlphaFold3 modeling suggest that MfpD may promote GTP hydrolysis by MfpB through a noncanonical Switch I-dependent mechanism. These findings establish the first structural framework for MfpD-MfpB interactions, building on previously identified in vitro catalytic properties and proposing new insights into MfpDs non-catalytic pathogenesis activity of MfpD in macrophages. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=69 SRC="FIGDIR/small/709265v2_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@77b2d2org.highwire.dtl.DTLVardef@7d1908org.highwire.dtl.DTLVardef@f66eeforg.highwire.dtl.DTLVardef@ed159c_HPS_FORMAT_FIGEXP M_FIG GRAPHICAL ABSTRACT C_FIG

biochemistry↗

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↗

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

High resolution cryo-EM and crystallographic snapshots of the large actinobacterial 2-oxoglutarate dehydrogenase: an all-in-one fusion with unique properties

Actinobacteria possess unique ways to regulate the oxoglutarate node located in the central position of the tricarboxylic acid cycle, a crossroad between energy conservation and nitrogen metabolism. Here, we studied the decarboxylative oxidation route that leads, through the 2-oxoglutarate dehydrogenase (ODH) complex, to the generation of succinyl-CoA and reduced equivalents to feed the respiratory chain. Compared to most organisms in which the oxidative decarboxylation and reductive acylation steps are carried out by different enzymes within the ODH complex, actinobacteria rely on an all-in-one protein (OdhA) in which both activities are carried out by the same polypeptide. We describe high-resolution cryo-EM and X-ray crystallography snapshots of representative enzymes from Mycobacterium smegmatis and Corynebacterium glutamicum, showing that OdhA is an 800-kDa homohexamer that folds into a three-blade propeller shape. The obligate trimeric and dimeric states of the acyltransferase and dehydrogenase domains, respectively, are critical for maintaining the overall assembly, where both domains interact via subtle readjustments of their interfaces. Complexes obtained with substrate analogues, reaction products and allosteric regulators illustrate how these domains operate. Furthermore, we provide additional insights into the phosphorylation-dependent regulation of this enzymatic machinery by the FHA (Fork-Head Associated) signalling protein OdhI, delivering new molecular details on how this actinobacterial-specific switching mechanism operates. Overall, the quaternary organization of OdhA represents a new piece of the fascinating puzzle of the synergistic, mixed pyruvate dehydrogenase/2-oxoglutarate dehydrogenase actinobacterial supercomplex.

biochemistry↗

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↗

bioRxiv2021

A TPR scaffold couples signal detection to OdhI phosphorylation in metabolic control by the protein kinase PknG

Signal transduction is essential for bacteria to adapt to changing environmental conditions. Among many forms of post-translational modifications, reversible protein phosphorylation has evolved as a ubiquitous molecular mechanism of protein regulation in response to specific stimuli. The Ser/Thr protein kinase PknG modulates the fate of intracellular glutamate by controlling the phosphorylation status of the 2-oxoglutarate dehydrogenase regulator OdhI, a function that is conserved among diverse actinobacteria. PknG has a modular organization characterized by the presence of regulatory domains surrounding the catalytic domain. Here we present an investigation through in vivo experiments as well as biochemical and structural methods of the molecular bases of the regulation of PknG from C. glutamicum (CgPknG), in the light of previous knowledge available for the kinase from M. tuberculosis (MtbPknG). We found that OdhI phosphorylation by CgPknG is regulated by a conserved mechanism that depends on a C-terminal domain composed of tetratricopeptide repeats (TPR) essential for metabolic homeostasis. Furthermore, we identified a conserved structural motif that physically connects the TPR domain and a flexible N-terminal extension of the kinase that is involved in docking interactions with OdhI. Based on our results and previous reports, we propose a model in which the TPR domain of PknG couples signal detection to the specific phosphorylation of OdhI. Overall, the available data indicate that conserved PknG domains in distant actinobacteria retain their roles in kinase regulation in response to nutrient availability. IMPORTANCEBacteria control the metabolic processes by which they obtain nutrients and energy in order to adapt to the environment. In this way, the metabolic characteristics of a microorganism determine its ecological role and its usefulness in industrial processes. Here, we use genetic, biochemical, and structural approaches to study a key component in a system that regulates glutamate production in C. glutamicum, a species that is used for the industrial production of amino acids. We elucidated molecular mechanisms involved in metabolic control in C. glutamicum, which are conserved in related pathogenic bacteria. The findings have broader significance for diverse actinobacteria, including microorganisms that cause disease as well as environmental species used to produce billions of dollars of amino acids and antibiotics every year.

biochemistry↗

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

3D architecture and structural flexibility revealed in the subfamily of large glutamate dehydrogenases by a mycobacterial enzyme

Glutamate dehydrogenases (GDHs) are widespread metabolic enzymes that play key roles in nitrogen homeostasis. Large glutamate dehydrogenases composed of 180 kDa subunits (L-GDHs180) contain long N- and C-terminal segments flanking the catalytic core. Despite the relevance of L-GDHs180 in bacterial physiology, the lack of structural data for these enzymes has limited the progress of functional studies. Here we show that the mycobacterial L-GDH180 (mL-GDH180) adopts a quaternary structure that is radically different from that of related low molecular weight enzymes. Intersubunit contacts in mL-GDH180 involve a C-terminal domain that we propose as a new fold and a flexible N-terminal segment comprising ACT-like and PAS-type domains that could act as metabolic sensors for allosteric regulation. These findings uncover unique aspects of the structure-function relationship in the subfamily of L-GDHs.

microbiology↗

bioRxiv2020

Actinobacteria challenge the paradigm: a unique protein architecture for a well-known central metabolic complex

-ketoacid dehydrogenase complexes are large, tripartite enzymatic machineries carrying out key reactions in central metabolism. Extremely conserved across the tree of life, they have so far all considered to be structured around a high molecular weight hollow core, consisting of up to 60 subunits of the acyltransferase component. We provide here evidence that Actinobacteria break the rule by possessing an acetyltranferase component reduced to its minimally active, trimeric unit, characterized by a unique C-terminal helix that affects the oligomerization and the full 3D architecture of the complex. We show that this unique feature is characterized by an insertion, which together with OdhA is found spread over Actinobacteria. This phylum includes organisms or great interest for agriculture, industrial bio-production and many human pathogens as Mycobacterium tuberculosis. Moreover, components of this complex are key for M. tuberculosis survival in the human host, and its unique core and protein-protein interactions represent potentially "druggable" targets.

microbiology↗

bioRxiv2020

Central role and structure of the membrane pseudokinase YukC in the antibacterial Bacillus subtilis Type VIIb Secretion System.

Type VIIb Secretion System (T7SSb) has been recently identified in Firmicutes resembling the mycobacterial T7SSa. Despite limited sequence homology, T7SSa and T7SSb have substrates with striking structural similarities, the WXG100 proteins. Recent advances in Staphylococcus spp. proposed that T7SSb is involved in intra-species competition. However, the architecture and mechanism of action of this secretion complex remain largely obscure. Here, we investigate the T7SSb of Bacillus subtilis as a model system. We report the first evidence of B. subtilis ability to mediate intra- and inter-species antibacterial activity in a T7SSb-dependent manner. Then, we present the first systematic investigation of the T7SSb protein-protein network, revealing novel interactions and highlighting the central role of the pseudokinase subunit YukC in the assembly of the system. Its direct interaction with a T7SSb-secreted toxin supports its role in recruiting substrates to the secretion machinery. Finally, we solved the crystal structure of full-length transmembrane YukC defining novel structural motifs and suggesting that intrinsic flexibility modulates the orientation of the pseudokinase domains and YukC function. Overall, our results provide a better understanding on the role and molecular organisation of the T7SSb, opening new perspectives for the comprehension of this poorly characterized molecular machine.

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↗