Biology subjects

Tehrani, A. F.

Publications and source records attributed to Tehrani, A. F..

2 recordsLinked to original sources

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↗

bioRxiv2025

Molecular interplay between peptidoglycan integrity and outer membrane asymmetry in maintaining cell envelope homeostasis

The bacterial cell envelope is a critical interface with the environment, particularly in Gram-negative species where the outer membrane and peptidoglycan layers coordinate to maintain structural integrity and resist turgor. Although this coordination is essential for survival, the molecular mechanisms linking outer membrane and peptidoglycan homeostasis remain poorly understood. LD-transpeptidases (LDTs) are enzymes that crosslink peptides in peptidoglycan and incorporate D-amino acids, but their physiological roles are not fully defined. Here, we characterize the activity of the LDT enzyme LdtJ in Acinetobacter baumannii and investigate the consequences of its deletion. Loss of LdtJ disrupts cell morphology, downregulates peptidoglycan precursor genes (e.g., dadA, alr), and activates the stringent response, including elevated ppGpp levels and dksA upregulation. These defects are fully suppressed in a {Delta}ldtJ {Delta}mla double mutant, implicating the outer membrane lipid transport Mla pathway in compensatory regulation. RNA sequencing revealed that transcriptional changes in the {Delta}ldtJ mutant are reversed in the double mutant, highlighting a functional interplay between peptidoglycan remodeling and outer membrane lipid asymmetry. Our findings suggest that LdtJ contributes to envelope integrity not only through peptidoglycan modification but also by influencing broader regulatory and metabolic networks. IMPORTANCEAcinetobacter baumannii is a leading cause of hospital-acquired infections and is highly resistant to antibiotics. Its survival relies on the integrity of the cell envelope, composed of the peptidoglycan layer and outer membrane. While LD-transpeptidases (LDTs) are traditionally known for reinforcing peptidoglycan structure through non-canonical crosslinking, our findings reveal that the LdtJ enzyme also plays a critical role in regulating cellular metabolism and stress responses. Deletion of ldtJ results in pronounced growth defects and abnormal cell morphology - phenotypes that are fully suppressed by disrupting the outer membrane lipid asymmetry transport system, Mla. This genetic interaction uncovers a previously unrecognized link between peptidoglycan remodeling and outer membrane lipid homeostasis. These insights deepen our understanding of envelope coordination in Gram-negative bacteria and suggest that targeting interconnected stress response pathways could offer novel strategies to undermine bacterial resilience.

microbiology↗