Biology subjects

Malengo, G.

Publications and source records attributed to Malengo, G..

7 recordsLinked to original sources

bioRxiv2026

Antagonistic small proteins enable magnesium-dependent tuning of two-component signaling

Small proteins are emerging regulators of bacterial virulence via signaling pathways. However, how multiple small proteins coordinate to control a single pathway remains unclear. Here we show that two antagonistic small proteins, SafA and MgrB, forming a magnesium-dependent control module, enhance the signaling sensitivity of the PhoQ/PhoP two-component system in E. coli. Leveraging conditions that enable sequential expression of SafA and MgrB, we dissect and characterize their regulatory dynamics. Our results reveal magnesium as the central control parameter modulating the abundance and affinities of the small proteins for the sensor kinase PhoQ. This coupling together with the competitive binding of the small proteins enables self-adjustment of the signaling network and enhances its sensitivity to environmental magnesium changes. Functionally, loss of either small proteins influences the ability of enteropathogenic E. coli to evade macrophage phagocytosis, linking this regulatory scheme to host interaction. Our findings establish a framework for achieving a high level of input sensitivity through antagonistic regulations in a signaling network.

microbiology↗

bioRxiv2025

Engineering site-specific nucleic acid-protein conjugates by utilizing a natural RNAylation reaction

Nucleic acid-protein conjugates are valuable for synthetic biology, therapeutics, and nanotechnology, but current methods often lack site specificity and rely on non-natural linkages. RNAylation, a one-step enzymatic reaction catalyzed by the bacteriophage T4 enzyme ModB where first discovered in vivo during phage infection, enables site-specific conjugation of nucleic acids to proteins via a natural N-glycosidic bond. Here, we establish RNAylation as a novel and robust in vitro platform for generating nucleic acid-protein conjugates, overcoming key limitations of existing strategies. We define design principles for this approach, demonstrate enhanced nucleic acid stability in human cell lysates, and develop an efficient purification workflow. Furthermore, we achieve successful delivery of purified conjugates into human cells, highlighting the potential for functional in vivo applications. Our work expands RNAylation from a phage-specific phenomenon to a versatile, biologically relevant strategy with broad biotechnological potential.

bioengineering↗

bioRxiv2024

A toolbox of FRET-based c-di-GMP biosensors and its FRET-To-Sort application for genome-wide mapping of the second messenger regulatory network

C-di-GMP is a widespread second messenger, coordinating various cellular functions in bacteria. Levels of c-di-GMP can be highly dynamic and vary over a wide range of concentrations. Here we constructed a large set of FRET-based c-di-GMP biosensors, using homologues of c-di-GMP-binding effector YcgR from different bacterial species. This biosensor library was characterized using a newly established protocol to quantify FRET efficiency using flow cytometry. The resulting toolbox of 18 selected biosensors that undergo large FRET signal change upon c-di-GMP binding displays a [~]100-fold range of c-di-GMP binding affinities. We combined this toolbox with a barcoded Tn5 transposon library and cytometry-based cell sorting to develop FRET-To-Sort, a new application for systematic characterization of gene networks regulating levels of FRET-detected small molecules. Applied to planktonic E. coli cells, FRET-To-Sort identified both known and novel regulatory modules controlling c-di-GMP levels, including flagellum and fimbria biogenesis, lipid metabolism and stress response genes.

microbiology↗

bioRxiv2022

The inhibitory mechanism of a small protein reveals its role in antimicrobial peptide sensing

A large number of small membrane proteins have been discovered in bacteria, but their mechanism of action has remained mostly elusive. Here, we investigate the mechanism of a physiologically important small protein, MgrB, which represses the activity of the sensor kinase PhoQ and is widely distributed among enterobacteria. The PhoQ/PhoP two-component system is a master regulator of the bacterial virulence program and interacts with MgrB to modulate bacterial virulence, fitness, and drug resistance. A combination of crosslinking approaches with functional assays and protein dynamic simulations revealed structural rearrangements due to interactions between MgrB and PhoQ near the membrane/periplasm interface and along the transmembrane helices. These interactions induce the movement of the PhoQ catalytic domain and the repression of its activity. Without MgrB, PhoQ appears to be much less sensitive to antimicrobial peptides, including the commonly used C18G. In the presence of MgrB, C18G promotes MgrB to dissociate from PhoQ, thus activating PhoQ via derepression. Our findings reveal the inhibitory mechanism of the small protein MgrB and uncover its importance in antimicrobial peptide sensing. Significance StatementSmall proteins have high prevalence, vast diversity, and primarily regulatory functions in biological processes across all domains of life. However, their mechanisms of action remain largely elusive. In this study, we investigate the mechanism of the small protein, MgrB. It interacts with the sensor kinase PhoQ, rearranges its conformation, represses its kinase activity, and regulates bacterial response to environmental changes. In particular for antimicrobial peptides, MgrB is required for bacteria to have a selective response to this host-exclusive stimulus. Our findings underline the importance of a small protein in bacterial fitness and drug resistance and provide a molecular basis for engineering novel peptide-based regulators.

microbiology↗

bioRxiv2022

Dependence of diffusion in Escherichia coli cytoplasm on protein size, environmental conditions and cell growth

Inside prokaryotic cells, passive translational diffusion typically limits the rates with which cytoplasmic proteins can reach their locations. Diffusion is thus fundamental to most cellular processes, but the understanding of protein mobility in the highly crowded and non-homogeneous environment of a bacterial cell is still limited. Here we investigated the mobility of a large set of proteins in the cytoplasm of Escherichia coli, by employing fluorescence correlation spectroscopy (FCS) combined with simulations and theoretical modeling. We conclude that cytoplasmic protein mobility could be well described by Brownian diffusion in the confined geometry of the bacterial cell and at the high viscosity imposed by macromolecular crowding. We observed similar size dependence of protein diffusion for the majority of tested proteins, whether native or foreign to E. coli, and, for the faster-diffusing proteins, it is well consistent with the Stokes-Einstein relation once taking into account the specific dumbbell shape of protein fusions. Pronounced subdiffusion and hindered mobility are only observed for proteins with extensive interactions within the cytoplasm. Finally, while protein diffusion becomes markedly faster in actively growing cells, at high temperature, or upon treatment with rifampicin, and slower at high osmolarity, all of these perturbations affect proteins of different sizes in the same proportions, which could thus be described as changes of a well-defined cytoplasmic viscosity.

biophysics↗

bioRxiv2020

Force requirements of endocytic vesicle formation

Mechanical forces are integral to many cellular processes, including clathrin-mediated endocytosis, a principal membrane trafficking route into the cell. During endocytosis, forces provided by endocytic proteins and the polymerizing actin cytoskeleton reshape the plasma membrane into a vesicle. Assessing force requirements of endocytic membrane remodelling is essential for understanding endocytosis. Here, we determined forces applied during endocytosis using FRET-based tension sensors integrated into the major force-transmitting protein Sla2 in yeast. We measured force of approx. 10 pN transmitted over Sla2 molecule, hence a total force of 450-1300 pN required for endocytic vesicle formation. Importantly, decreasing cell turgor pressure and plasma membrane tension reduced force requirements of endocytosis. The measurements in hypotonic conditions and mutants lacking BAR-domain membrane scaffolds then showed the limits of the endocytic force-transmitting machinery. Our study provides force values and force profiles critical for understanding the mechanics of endocytosis and potentially other key cellular membrane-remodelling processes.

cell biology↗

bioRxiv2018

The protein architecture of the endocytic coat analyzed by FRET

Endocytosis is a fundamental cellular trafficking pathway, which requires an organized assembly of the multiprotein endocytic coat to pull the plasma membrane into the cell. Although the protein composition of the endocytic coat is known, its functional architecture is not well understood. Here we determine the nanoscale organization of the endocytic coat by FRET microscopy in yeast. We assessed proximities of 18 conserved coat-associated proteins and used clathrin subunits and protein truncations as molecular rulers to obtain a high-resolution protein map of the coat. Furthermore, we followed rearrangements of coat proteins during membrane invagination and their binding dynamics at the endocytic site. We show that the endocytic coat is stratified into several functional layers situated above and below the clathrin lattice with key proteins transversing through the lattice deeply into the cytoplasm. We propose that this conserved design enables an efficient and regulated function of the endocytic coat during endocytosis.

cell biology↗