Biology subjects

Massidda, O.

Publications and source records attributed to Massidda, O..

8 recordsLinked to original sources

bioRxiv2026

GpsB acts as an adapter for MacP-mediated activation of class A penicillin-binding protein aPBP2a in Streptococcus pneumoniae, independently of MacP phosphorylation

Regulation of class A penicillin-binding proteins (aPBPs) in peptidoglycan biosynthesis is incompletely understood in Gram-positive bacteria. One example is activation of aPBP2a by GpsB and phosphorylated MacP in the ovoid-shaped pathogen, Streptococcus pneumoniae. We set out to examine whether phosphorylation of Thr residues other than Thr32 contributed to MacP activation of aPBP2a. We also wanted to determine whether GpsB and MacP activation of aPBP2a were related. Here we report that MacP was phosphorylated about equally at Thr32 and Thr56 in physiological and biochemical assays. However, based on transformation and growth assays, phosphorylation of MacP was not required for aPBP2a activation. A structure-function analysis confirmed that most of the MacP cytoplasmic domain, which was predicted by AlphaFold3 to be disordered, was not required for aPBP2a activation. These analyses further identified amino acids in the MacP transmembrane domain and the aPBP2a juxtamembrane region, as well as a variant of the GpsB-binding motif in the membrane-proximal cytoplasmic region of MacP, required for aPBP2a activation. Together, these results support a tripartite model in which GpsB acts as an adapter for activation of aPBP2a by MacP. Finally, additional interaction, Tn-seq, and growth assays suggested other modes of direct or indirect regulation of aPBP2a activity.

microbiology↗

bioRxiv2026

Environmental context reveals a conditional role of the Tol-Pal system in envelope organization in Acinetobacter baumannii

Gram-negative bacteria must coordinate remodeling of the peptidoglycan cell wall with invagination of the outer membrane to preserve envelope integrity during growth and division. The conserved Tol-Pal system has been implicated in coordinating these processes, yet its physiological contribution to envelope organization remains unclear and may depend on environmental context. Here, we examined the role of Tol-Pal in coordinating envelope remodeling in Acinetobacter baumannii across distinct growth environments. Loss of Tol-Pal did not cause a major population growth defect, and septal peptidoglycan incorporation remained largely preserved under standard laboratory growth conditions. In contrast, under specific environmental conditions--including nutrient-rich media, altered osmotic conditions, and host-like environments--Tol-Pal deficiency disrupted the spatial organization of cell division and cell morphology. Tol-Pal mutants also exhibited modest but reproducible reductions in outer membrane barrier robustness and decreased fitness in environmental and host-associated contexts. Together, these findings demonstrate that Tol-Pal is not an essential component of the core division machinery but instead contributes to the coordinated organization of the Gram-negative envelope under conditions that impose additional physiological demands. More broadly, our results highlight how environmental context can reveal conditional roles for conserved envelope systems that are not apparent during standard laboratory growth. ImportanceThe Gram-negative envelope is a complex, multilayered structure that must remain intact as cells grow and divide across diverse and often challenging environments. Coordination between peptidoglycan remodeling and outer membrane invagination is therefore critical for maintaining envelope organization and cellular fitness. Here, we show that the conserved Tol-Pal system in Acinetobacter baumannii contributes to the spatial organization of cell division and outer membrane robustness under specific environmental conditions. Although Tol-Pal deficiency permits sustained population growth under standard laboratory conditions, its absence disrupts envelope organization and compromises bacterial fitness in environmental and host-associated contexts. These findings demonstrate how environmental conditions can expose conditional roles for conserved envelope systems and highlight the importance of physiological context in shaping bacterial cell envelope organization.

microbiology↗

bioRxiv2026

Outer membrane remodeling via lipid-peptidoglycan crosstalk enables lipooligosaccharide-deficient colistin resistance

Gram-negative bacteria rely on an asymmetric outer membrane (OM) for barrier integrity, with phospholipids confined to the inner leaflet and glycolipids such as lipopolysaccharide (LPS) or lipooligosaccharide (LOS) forming the outer leaflet. Although LPS/LOS was long considered essential, recent findings challenge this view, leaving the mechanistic basis and evolutionary flexibility unclear. Here, we identify lipid asymmetry as a structural checkpoint that governs access to LOS-independent survival. Using Acinetobacter baumannii as a model, we show that disrupting retrograde phospholipid transport and surface phospholipid degradation destabilizes OM lipid balance, creating a permissive state that enables emergence of LOS-deficient, colistin-resistant variants. Integrated lipidomic and transcriptomic analyses reveal a staged remodeling program that reinforces lipoprotein scaffolds, rewires peptidoglycan synthesis, and expands trafficking pathways to stabilize a glycolipid-free envelope. Critically, loss of LOS coincides with sharp repression of PBP1A, and maintaining its activity blocks adaptation, demonstrating interdependence between OM and peptidoglycan homeostasis. We propose a three-state model--basal, permissive, adapted--that explains how envelope architecture gates evolutionary trajectories to antibiotic resistance. SignificanceColistin is a last-resort antibiotic targeting lipopolysaccharide (LPS) or lipooligosaccharide (LOS) in Gram-negative pathogens, and many emerging antimicrobials aim to inhibit LPS/LOS biosynthesis and transport. Resistance usually arises via lipid A modification, which preserves LPS/LOS while reducing colistin binding. Resistance to colistin can also arise via complete loss of LOS, which occurs in some Acinetobacter baumannii strains but is constrained in others, such as strain ATCC 17978. Here, we demonstrate that LOS essentiality is not fixed but dictated by outer membrane architecture. Disrupting phospholipid homeostasis creates a permissive envelope that allows LOS-deficient, colistin-resistant variants to emerge, while reducing peptidoglycan synthesis further promotes this state. These findings identify lipid asymmetry as a structural checkpoint in resistance evolution and suggest that preserving envelope homeostasis could limit bacterial escape from colistin and guide strategies for next-generation antibiotic development.

microbiology↗

bioRxiv2026

PBP1A and LdtJ support cell envelope homeostasis and impact selection of Colistin-resistance in Acinetobacter baumannii

The multilayered cell envelope of Acinetobacter baumannii is an essential structure that maintains cellular integrity and protects the bacterial cell against external stresses and antibiotics. It consists of an inner membrane, a thin peptidoglycan (PG) layer and an asymmetric outer membrane (OM) enriched in lipooligosaccharide (LOS), whose lipid A moiety is the target of colistin, a last-resource antibiotic. Although lipid A is essential in most Gram-negatives, A. baumannii can survive without LOS through envelope remodeling, particularly in strains producing low levels of the bifunctional penicillin-binding protein PBP1A (encoded by mrcA) or in {Delta}mrcA mutants. Here, we identify a functional interplay between the LD-transpeptidase LdtJ, which generates 3-3 cross-links, and PBP1A, which catalyzes 4-3 transpeptidation during PG synthesis. We show that simultaneous inactivation of both enzymes severely affected growth, viability, morphology, and OM homeostasis. PG analyses revealed that the {Delta}ldtJ {Delta}mrcA mutants displays reduced overall cross-linkage and shorter glycan chains, producing a weakened sacculus. Co-immunoprecipitation demonstrated that PBP1A associates with LdtJ, supporting their coordinated activity at sites of PG synthesis. Notably, {Delta}ldtJ {Delta}mrcA mutants exhibited the highest recovery frequency of colistin-resistant, LOS-deficient variants compared with wild type or single mutants. Together, our findings demonstrate that coupling between 4-3 and 3-3 transpeptidation is critical for envelope stability in A. baumannii and highlight how disrupting this coordination favors the emergence of colistin resistance. This work identifies a conserved PG remodeling vulnerability that directly links PG integrity to the evolution of antibiotic resistance, offering a new conceptual framework for destabilizing the A. baumannii envelope. ImportanceThe global rise of multidrug-resistant Acinetobacter baumannii represents an urgent clinical threat, largely driven by its extraordinary capacity to withstand cell envelope damages and escape last-resort antibiotics such as colistin. Although peptidoglycan synthesis and remodeling are known to influence outer membrane stability, how these processes are coordinated in A. baumannii has remained unclear. Here, we identify a crucial interplay between two critical envelope biogenesis and remodeling activities, 4-3 and 3-3 transpeptidations, mediated by the bifunctional PBP1A and the LD-transpeptidase LdtJ, respectively. Disrupting this coordination weakens the peptidoglycan, destabilizes the outer membrane, and increases the emergence frequency of colistin-resistant, LOS-deficient variants. These findings highlight a previously unrecognized vulnerability in the envelope homeostasis of A. baumannii, suggesting that simultaneously targeting DD and LD-transpeptidation could potentiate therapeutic strategies aimed at limiting antibiotic resistance development.

microbiology↗

bioRxiv2025

Peptidoglycan DD-peptidases have distinct activities that impact fitness of Acinetobacter baumannii

The Gram-negative cell envelope is a vital interface between the bacterial cell and its environment. It acts as a selective barrier, blocking harmful agents while permitting nutrient uptake. Additionally, it enables environmental sensing and adaptive responses. Structurally, it is composed of the outer membrane, the cytoplasmic (inner) membrane, and the periplasm, which contains the peptidoglycan layer. Peptidoglycan is a conserved polymer that provides structural integrity, allowing the cell to withstand the internal turgor. It consists of glycan strands connected by short peptides, forming a mesh-like structure. In Gram-negative bacteria, the majority of the peptidoglycan subunits contain tetrapeptides. Tetrapeptides are generated through the action of DD-carboxypeptidases (DD-CPases), which cleave the terminal D-alanine from pentapeptides. Gram-negative bacteria encode multiple DD-CPases, but their precise role in maintaining cell shape and structural integrity remain poorly understood. The nosocomial pathogen Acinetobacter baumannii encodes three putative DD-CPases. To investigate the role of these enzymes, we generated single mutants, as well as double mutants in dacC, dacD, and pbpG, which encode the homologs of Escherichia coli DD-CPases PBP5, PBP6a, PBP6b, and the endopeptidase (DD-EPase) PBP7, respectively. We assessed the mutants for changes in cell morphology, growth dynamics, and stress tolerance. Additionally, we analyzed the composition of their peptidoglycan layers to determine the biochemical consequences of their inactivation. Each mutant exhibited distinct alterations in coccobacillary morphology and growth. Peptidoglycan analysis confirmed the enzymes possess DD-CPase activity, and PBP6b also demonstrated endopeptidase activity. Together, our results demonstrate that each peptidoglycan-modifying enzyme contributes uniquely to cell growth and morphology. These findings underscore their non-redundant functions and suggest their specific activities may serve as valuable targets for developing new antimicrobial therapies. ImportanceDD-peptidases, including carboxypeptidases and endopeptidases are crucial for maintaining cell envelope homeostasis, with distinct roles for each enzyme in cell wall biogenesis and structural integrity. The enzymatic characterization presented in this study not only advance our understanding of fundamental A. baumannii biology but also highlight these enzymatic activities as targets for development of innovative therapeutic strategies to combat infections caused by this multidrug-resistant microbe.

microbiology↗

bioRxiv2023

Chromosomal Duplications of MurZ (MurA2) or MurA (MurA1), Amino Acid Substitutions in MurZ (MurA2), and Absence of KhpAB Obviate the Requirement for Protein Phosphorylation in Streptococcus pneumoniae D39

GpsB links peptidoglycan synthases to other proteins that determine the shape of the respiratory pathogen Streptococcus pneumoniae (pneumococcus; Spn) and other low-GC Gram-positive bacteria. GpsB is also required for phosphorylation of proteins by the essential StkP(Spn) Ser/Thr protein kinase. Here we report three classes of frequently arising chromosomal duplications ({approx}21-176 genes) containing murZ (MurZ-family homolog of MurA) or murA that suppress {Delta}gpsB or {Delta}stkP. These duplications arose from three different repeated sequences and demonstrate the facility of pneumococcus to modulate gene dosage of numerous genes. Overproduction of MurZ or MurA alone or overexpression of MurZ caused by {Delta}khpAB mutations suppressed {Delta}gpsB or {Delta}stkP phenotypes to varying extents. {Delta}gpsB and {Delta}stkP were also suppressed by MurZ amino-acid changes distant from the active site, including one in commonly studied laboratory strains, and by truncation or deletion of the homolog of IreB(ReoM). Unlike in other Gram-positive bacteria, MurZ is predominant to MurA in pneumococcal cells. However, {Delta}gpsB and {Delta}stkP were not suppressed by {Delta}clpCP, which did not alter MurZ or MurA amounts. These results support a model in which regulation of MurZ and MurA activity, likely by IreB(Spn), is the only essential requirement for protein phosphorylation in exponentially growing D39 pneumococcal cells.

microbiology↗

bioRxiv2022

Roles of RodZ and Class A PBP1b in the Assembly and Regulation of the Peripheral Peptidoglycan Elongasome in Ovoid-Shaped Cells of Streptococcus pneumoniae D39

RodZ of rod-shaped bacteria functions to link MreB filaments to the Rod peptidoglycan (PG) synthase complex that moves circumferentially perpendicular to the long cell axis, creating hoop-like sidewall PG. Ovoid-shaped bacteria, such as Streptococcus pneumoniae (pneumococcus; Spn) that lack MreB, use a different modality for peripheral PG elongation that emanates from the midcell of dividing cells. Yet, S. pneumoniae encodes a RodZ homolog similar to RodZ in rod-shaped bacteria. We show here that the helix-turn-helix and transmembrane domains of RodZ(Spn) are essential for growth at 37{degrees}C. {Delta}rodZ mutations are suppressed by {Delta}pbp1a, mpgA(Y488D), and {Delta}khpA mutations that suppress {Delta}mreC, but not {Delta}cozE. Consistent with a role in PG elongation, RodZ(Spn) co-localizes with MreC and aPBP1a throughout the cell cycle and forms complexes and interacts with PG elongasome proteins and regulators. Depletion of RodZ(Spn) results in aberrantly shaped, non-growing cells and mislocalization of elongasome proteins MreC, PBP2b, and RodA. Moreover, Tn-seq reveals that RodZ(Spn), but not MreCD(Spn), displays a specific synthetic-viable genetic relationship with aPBP1b, whose function is unknown. We conclude that RodZ(Spn) acts as a scaffolding protein required for elongasome assembly and function and that aPBP1b, like aPBP1a, plays a role in elongasome regulation and possibly peripheral PG synthesis. Graphical Summary O_FIG O_LINKSMALLFIG WIDTH=192 HEIGHT=200 SRC="FIGDIR/small/494439v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@f1cfb9org.highwire.dtl.DTLVardef@17cac68org.highwire.dtl.DTLVardef@1af20borg.highwire.dtl.DTLVardef@108d83c_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2018

The cell cycle regulator GpsB functions as cytosolic adaptor for multiple cell wall enzymes

Bacterial growth and cell division requires precise spatiotemporal regulation of the synthesis and remodelling of the peptidoglycan layer that surrounds the cytoplasmic membrane. GpsB is a cytosolic protein that affects cell wall synthesis by binding to the cytoplasmic mini-domains of peptidoglycan synthases to ensure their correct subcellular localisation. Here we have discovered critical structural features for the interaction of GpsB with peptidoglycan synthases from three different bacteria and demonstrated their importance for cell wall growth and viability. We have used these structural motifs to predict and confirm novel partners of GpsB in Bacillus subtilis, illuminating the role of this key regulator of peptidoglycan synthesis. GpsB thus functions as an adaptor, to mediate the interaction between membrane proteins, scaffolding proteins, signalling proteins and enzymes to generate larger protein complexes at specific sites in a bacterial cell cycle-dependent manner. Given the importance of GpsB in pathogenic bacteria, this study has not only revealed mechanistic details of how cell wall synthesis is co-ordinated with the bacterial cell cycle but could also represent a starting point for the design of much needed new antibiotics.

microbiology↗