Biology subjects

Soncini, F. C.

Publications and source records attributed to Soncini, F. C..

4 recordsLinked to original sources

bioRxiv2026

The Cu-induced ScsD reductase is a membrane-bound redox partner of ScsB in the Salmonella envelope

The intracellular environment Salmonella confronts during infection is characterized by multiple redox stressors including reactive oxygen species (ROS) and copper (Cu) ions. Under these conditions, alternative systems of thiol oxidoreductases such as the Cu induced Scs system are required to protect and repair periplasmic proteins. The scsABCD operon encodes three Dsb-like enzymes, ScsB, ScsC, and ScsD, and an accessory protein, ScsA. These proteins are required both for Cu resistance and H2O2 tolerance. ScsB and ScsC function analogously to the canonical DsbD/DsbC redox pair of thiol oxidoreductases. The absence of ScsC was shown to affect the folding/activity of periplasmic proteins involved in amino acid transport and redox homeostasis. Here, we focus in ScsD, the least characterized member of this system. Upon Cu-induced expression, ScsD localizes to the inner membrane, enabling its predicted C-terminal Dsb-like domain to be exposed to the periplasm. Functional analysis indicates that ScsD exists in a reduced state in the Salmonella envelope and serves as a redox partner of ScsB. ScsD exhibits in vivo disulfide reductase activity and restores a deficient disulfide reduction pathway in Salmonella. Similar to ScsC and ScsB, ScsD binds Cu(I) via the Cys residues of its Dsb-like domain; however, this metal interaction appears to lack relevance in Cu detoxification as no impact on intracellular Cu levels was observed. Our results define ScsD as a specialized membrane-bound thiol-disulfide reductase in the Salmonella envelope and highlight the versatility of the Scs system in maintaining periplasmic proteostasis when canonical pathways are compromised by host-imposed Cu stress. ImportanceCopper is a key component of the innate immune system, serving as a primary defense against pathogens like Salmonella. Copper overload targets the bacterial envelope, specifically attacking protein sulfhydryl groups. This causes protein misfolding and inactivation, disrupting essential processes like metabolism, transport and virulence. To survive this stress and restore thiol homeostasis, Salmonella utilizes the scsABCD operon. While the ScsB-ScsC redox pair is well-documented and some protein substrates identified, the role of ScsD remains undefined. This work characterizes ScsD as an inner-membrane-anchored thiol reductase and a new redox partner for ScsB. The ScsD/ScsB pair expands the bacteriums protein quality control capacity, allowing Salmonella to maintain envelope homeostasis within the hostile, copper-rich environment of the host.

microbiology↗

bioRxiv2026

CuiT is a Cu importer required for metal homeostasis in Salmonella enterica

Copper (Cu) is an essential micronutrient that serves as a cofactor for redox enzymes but becomes toxic when unregulated. In bacteria, while Cu efflux systems are well characterized, mechanisms of Cu import remain poorly understood. Here, we characterize the major facilitator superfamily transporter CuiT (STM1486) as a key Cu importer in Salmonella enterica. Comparative genomics revealed that cuiT is evolutionarily conserved across Enterobacteriaceae, and structural modeling predicts a 12-transmembrane-helix architecture with conserved His, Met, and Cys residues suitable for Cu coordination. Functional analyses demonstrated that deletion of cuiT reduces intracellular Cu accumulation, slows Cu uptake kinetics, and diminishes expression of Cu-responsive genes, including copA, cueP, cueO, and golB. Conversely, overexpression of CuiT increases intracellular Cu but sensitizes cells to Cu stress, highlighting the need for tight regulation. Kinetic modeling indicates that CuiT mediates rapid Cu import, supporting larger intracellular Cu pools compared to Pseudomonas influx transporters. These findings position CuiT as a central component of the Salmonella Cu homeostasis network, linking Cu import to transcriptional regulation, redox balance, and stress adaptation. Our work provides mechanistic insights into bacterial Cu acquisition and suggests CuiT and associated pathways as potential targets for antimicrobial strategies. SignificanceCopper (Cu) is essential for bacterial redox enzymes but toxic when dysregulated. While Cu efflux pathways are well studied, mechanisms of Cu import are poorly understood. We identify CuiT, a conserved major facilitator superfamily transporter, as a key Cu importer in Salmonella enterica. CuiT controls intracellular Cu levels, influences Cu-responsive gene expression, and maintains redox balance and stress adaptation. Disruption or overexpression of CuiT perturbs Cu homeostasis, highlighting its regulatory importance. These findings reveal a critical bacterial Cu acquisition pathway and suggest CuiT and its network as potential antimicrobial targets, advancing understanding of metal homeostasis in pathogens.

microbiology↗

bioRxiv2021

Balance between extracellular matrix production and macrophage survival by a Salmonella-specific SPI-2 encoded transcription factor

Cellulose is a major component of the Salmonella biofilm extracellular matrix and it is considered an antivirulence factor because it interferes with Salmonella survival inside macrophages and virulence in mice. Its synthesis is stimulated by CsgD, the master regulator of biofilm extracellular matrix formation in enterobacteria, which in turn is under the control of MlrA, a MerR-like transcription factor. In this work we identified a SPI-2 encoded Salmonella-specific transcription factor homolog to MlrA, MlrB, that represses transcription of its downstream gene, STM1389, also known as orf319, and of csgD inside host cells. MlrB is induced in laboratory media mimicking intracellular conditions and inside macrophages, and it is required for intramacrophage survival. An increased expression of csgD is observed in the absence of MlrB inside host cells. Interestingly, inactivation of the CsgD-controlled cellulose synthase coding-gene, bcsA, restored intramacrophage survival to rates comparable to wild type bacteria in the absence of MlrB. These data indicate that MlrB represses CsgD expression inside host cells and in consequence activation of the cellulose synthase. Our findings provide a novel link between biofilm formation and Salmonella virulence.

microbiology↗

bioRxiv2020

The two-component system CopRS maintains femtomolar levels of free copper in the periplasm of Pseudomonas aeruginosa using a phosphatase-based mechanism

Two component systems control periplasmic Cu+ homeostasis in Gram-negative bacteria. In characterized systems such as Escherichia coli CusRS, upon Cu+ binding to the periplasmic sensing domain of CusS, a cytoplasmic phosphotransfer domain phosphorylates the response regulator CusR. This drives the expression of efflux transporters, chaperones, and redox enzymes to ameliorate metal toxic effects. Here, we show that the Pseudomonas aeruginosa two component sensor histidine kinase CopS exhibits a Cu-dependent phosphatase activity that maintains a non-phosphorylated CopR when the periplasmic Cu levels are below its activation threshold. Upon Cu+ binding to the sensor, the phosphatase activity is blocked and the phosphorylated CopR activates transcription of the CopRS regulon. Supporting the model, mutagenesis experiments revealed that the {Delta}copS strain showed constitutive high expression of the CopRS regulon, lower intracellular Cu+ levels, and larger Cu tolerance when compared to wild type cells. The invariant phospho-acceptor residue His235 of CopS was not required for the phosphatase activity itself, but necessary for its Cu-dependency. To sense the metal, the periplasmic domain of CopS binds two Cu+ ions at its dimeric interface. Homology modeling of CopS based on CusS structure (four Ag+ binding sites) clearly explains the different binding stoichiometries in both systems. Interestingly, CopS binds Cu+/2+ with 30 x 10-15 M affinities, pointing to the absence of free (hydrated) Cu+/2+ in the periplasm. IMPORTANCECopper is a micronutrient required as cofactor in redox enzymes. When free, copper is toxic, mismetallating proteins, and generating damaging free radicals. Consequently, copper overload is a strategy that eukaryotic cells use to combat pathogens. Bacteria have developed copper sensing transcription factors to control copper homeostasis. The cell envelope is the first compartment that has to cope with copper stress. Dedicated two component systems control the periplasmic response to metal overload. This manuscript shows that the copper sensing two component system present in Pseudomonadales exhibits a signal-dependent phosphatase activity controlling the activation of the response regulator, distinct from previously described periplasmic Cu sensors. Importantly, the data show that the sensor is activated by copper levels compatible with the absence of free copper in the cell periplasm. This emphasizes the diversity of molecular mechanisms that have evolved in various bacteria to manage the copper cellular distribution.

microbiology↗