Pedro Maria Alzari

Serine/threonine phosphorylation has emerged as a central regulatory mechanism of the bacterial cell cycle. However, the dynamics and temporal selectivity of protein phosphorylation through the cell cycle remain unamenable to existing analytical tools. Although fluorescent phosphorylation biosensors are widely available in single eukaryotic cells, such tools have yet to be adapted for bacteria. To address this gap, we developed an in vivo fluorescent biosensor specifically designed to monitor serine/threonine phosphorylation in single bacterial cells. Specifically, this sensor reports protein phosphorylation mediated by the serine/threonine kinase StkP, the main regulator of cell division in Streptococcus pneumoniae . We showed that StkP activity is constitutive throughout the cell cycle. However, we observed that StkP-mediated protein phosphorylation is heterogeneous from cell to cell in early stages of the cell cycle. Importantly, our results revealed that the cell division protein DivIVA is found phosphorylated at the cell poles, suggesting an additional regulatory layer controlling its function at this location. Collectively, this work provides original insights into the temporal dynamics of StkP activity and the regulation of the pneumococcal cell cycle by phosphorylation. IMPORTANCE: How bacterial serine/threonine phosphorylation is dynamically regulated in living single cells remains inaccessible, largely because efficient reporters required to monitor phosphorylation in vivo are lacking. In this study, we designed a fluorescent biosensor approach that enables direct visualization of serine/threonine phosphorylation dynamics in single bacterial cells. Using this approach, we revealed that the activity of StkP, the serine/threonine kinase that regulates cell division in Streptococcus pneumoniae , is constitutive throughout the cell cycle, yet StkP-mediated protein phosphorylation is unexpectedly heterogeneous across individual cells in the early stages of division. Importantly, our reporter also showed that the phosphorylation of the key cell division protein DivIVA is highly dynamic. Together, these findings provide the conceptual basis to improve our understanding of the role of phosphorylation in pneumococcal cell division and pave the way for investigating phosphorylation dynamics in other bacterial species.

To address drug-resistant tuberculosis, the leading single-pathogen infectious killer, drugs with novel modes of action (MoAs) are urgently needed. Phenotypic screening of chemical libraries can identify antimicrobial compounds, but standard screens cannot reveal the MoA of hits, limiting targeted selection of compounds with novel MoAs. Here, we develop a deep learning (DL) model to screen drug-treated Corynebacterium glutamicum ( Cglu ), a surrogate for Mycobacterium tuberculosis . We train our DL model to distinguish between MoAs directly from high-throughput images. Our model robustly classifies MoAs of established antibiotics and recognizes the MoA of previously unseen antibiotics. Inhibitors with a previously unseen MoA cluster together and apart from reference drugs, enabling the detection of novel MoAs. Moreover, our model links images of chemical (drugs) and genetic (mutants) perturbations targeting similar pathways, supporting mutant-based target prediction of compounds with novel MoAs directly from images. Last, our DL model recovers known biological relationships from images alone using the Cglu cell cycle as a case study.

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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 homolog , Wag31, the elongasome scaffold that is required for polar growth in Corynebacteriales . Conditional depletion of Wag31 results in coccoid-shaped cells, able to divide over several generations, showing that Wag31 is essential for rod shape maintenance, but not for viability in the conditions tested. Wag31/DivIVA homologs are formed by two distinct coiled-coil domains, a highly conserved N-terminal domain (NtD, also referred to as the DivIVA domain), and a more variable C-terminal domain (CtD). Our structural phylogenetic analyses revealed that in Actinobacteria , unlike Firmicutes , an intrinsically disordered region spatially separates the NtD from the CtD. We show that the NtD is necessary and sufficient for septum localization, while the CtD mediates self-interaction and polar accumulation. Our findings suggest that Wag31 is recruited specifically to the septum through protein–protein interactions involving the NtD, 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 CtD dominate and form a stable structure that likely organizes the pole for cell wall biosynthesis. IMPORTANCE: Wag31, the elongasome scaffold in Corynebacteriales , is a cytoskeletal protein that remains poorly understood. In Firmicutes, the Wag31 homolog, DivIVA, has been largely studied, but its function differs from that of Actinobacteria . Using AlphaFold predictions and evolutionary analysis, we found a clear sequence and structure cutoff between Firmicutes and Actinobacteria , correlating with their different functions. An intrinsically disordered insertion in Wag31 appeared early in the actinobacterial tree, separating the membrane/septum binding function of the N-terminal DivIVA domain from the C-terminal coiled-coil domain’s self-aggregation function. We analyzed each domain’s role and assigned a dominant role in cellular distribution. The N-ter is necessary for septal recruitment, independent of membrane binding capacities. Timely recruitment is essential for correct cellular distribution, as interference with this domain leads to asymmetric Wag31 localization and septum misplacement. We propose that Wag31’s early role at the septum is to prime the future pole, where it organizes polar growth upon cytokinesis.

Nerve growth factor (NGF) is a member of the neurotrophin family, essential for neuronal survival and phenotypic maintenance. However, in vitro, its function can be disrupted by oxidative posttranslational modifications such as tyrosine nitration. Nitrated NGF (NO ₂ NGF) has been shown to have a gain-of-toxic, pro-apoptotic, activity in motoneuron cultures. Herein, we report the generation and characterization of monoclonal antibodies (mAbs) that specifically recognize NO ₂ NGF to unravel its formation in vivo. Using hybridoma technology, we produced mAbs with high affinity and selectivity for NO ₂ NGF, as demonstrated immunochemically and by surface plasmon resonance. The antibodies elicit neutralizing capacity to NO ₂ NGF in neuronal cells. Nitrated Tyr52 within the NGF _48–58 sequence was identified as the primary antigenic determinant by crystallographic analysis of antibody:peptide complexes at atomic resolution, peptide-based epitope mapping and molecular dynamics simulations, whereas local sequence NGF motifs around the nitrated tyrosine are important for protein specificity. The antibodies revealed NO ₂ NGF accumulation in amyotrophic lateral sclerosis (ALS) rodent models and human subjects. Indeed, immunofluorescence showed selective accumulation of NO ₂ NGF in spinal cord regions undergoing motor neuron degeneration, as well as in sciatic nerves and neuromuscular junctions. Our findings establish NGF nitration as an oxidative hallmark in ALS and demonstrate that monoclonal antibodies targeting this chemical modification are powerful tools for both mechanistic studies and biomarkers development. This work proposes a link between neurotrophin nitration and neurodegenerative disease progression and opens avenues for therapeutic exploration along the peroxynitrite-tyrosine nitration pathway.

D,L-endopeptidase RipA is the major peptidoglycan (PG) hydrolase required for 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 Mtb SteAB system, a regulatory transmembrane septal complex encoded by adjacent genes Rv1697 ( steA ) and Rv1698/mctB ( steB ) that are conserved across Mycobacteriales . The separate crystal structures of the cytoplasmic core of Mt SteA and the periplasmic core of Mt SteB in complex with the RipA coiled-coil domain (RipA _CC ), along with biochemical evidence that these proteins form a stable transmembrane physical complex, define the function of the SteAB complex as a regulator of RipA-mediated cell separation in Mtb , arguing against a previously proposed role of MctB (Rv1698) as a putative mycobacterial outer membrane copper transporter. Our structural findings showed that, upon Mt SteB binding, RipA _CC is oriented perpendicular to the membrane, bringing its endopeptidase catalytic domain in physical contact with the PG layer, while the homodimeric structural core of SteA revealed 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 SteAB heterotetramer orchestrates the productive 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. IMPORTANCE: Peptidoglycan (PG) is a major component of the bacterial cell wall. A flexible but strong PG mesh encloses the cell, conferring mechanical resistance and preventing cell lysis. This PG mesh is continually remodeled during the bacterial life by the coordinated action of tightly regulated PG-hydrolases and synthetases. Here, we report the structural characterization of the M. tuberculosis SteAB system, which regulates the action of the major enzyme responsible for disassembling the PG mesh to allow daughter cell separation at the end of cell division. The proposed model for the septal control of PG hydrolysis illustrates how the transmembrane SteAB complex can promote enzyme activation and provides structural information that may help target the activation mechanism for antibiotic development.

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 alpha/beta fold and forms a stable dimer in solution, with hydrophobic alpha2-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 MfpD non-catalytic pathogenesis activity of MfpD in macrophages.

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.

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.

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.