Biology subjects

Costa, S. F.

Publications and source records attributed to Costa, S. F..

6 recordsLinked to original sources

bioRxiv2025

Spatially Organized DNA-templated Silver Nanoclusters as Potent Antimicrobial Agents for ESKAPE Infections

Antibiotic-resistant bacteria cause more than one million deaths annually worldwide. The rapid evolution and gene exchange among pathogens often render new antibiotic drugs ineffective soon after deployment, underscoring the urgent need for alternative therapeutic strategies. Nanoscale silver is well known for its innate bacteriostatic and bactericidal activity but typically requires high concentrations for efficacy that causes toxicities and limits broader clinical applications. To address this, we introduce programmable self-assembling DNA scaffolds that template, stabilize, and spatially organize multiple copies of monodisperse silver nanoclusters (DNA-AgNCs). These assemblies enhance antimicrobial potency of formulations while also exhibiting intrinsic fluorescence, providing dual functionality for therapeutic and bioimaging applications. Detailed characterization identified DNA-AgNC scaffolds with improved stability and enhanced activity against clinically relevant antibiotic-resistant planktonic ESKAPE pathogens. We also revealed the DNA-AgNC constructs that significantly lowered intracellular infections of human bone cells with Staphylococcus aureus. Collectively, the results highlight spatially organized DNA-AgNCs as a promising modular platform for next-generation antibacterial therapy and real-time bioimaging.

biochemistry↗

bioRxiv2025

FtsW protein-protein interactions visualized in live Staphylococcus aureus cells by FLIM-FRET

The bacterial cell cycle relies on the coordinated and dynamic interactions between division proteins and those involved in peptidoglycan (PG) synthesis. However, visualizing these interactions in vivo remains technically challenging. Here, we established fluorescence-lifetime imaging microscopy combined with Forster resonance energy transfer (FLIM-FRET) as a robust, spatially resolved technique to visualize protein interactions in living Staphylococcus aureus using fluorescent proteins. After systematically optimizing growth conditions and the analysis pipeline, we validated the method with cytosolic and membrane-anchored control proteins, achieving FRET efficiencies of up to 40%. Using FLIM-FRET, we mapped the protein interactions of the critical glycosyltransferase FtsW within the septal PG synthesizing complex. We confirmed its interaction with the cognate transpeptidase PBP1 and the regulatory protein DivIB. Notably, we found that FtsW also self-interacts, an observation corroborated by an alternative FLIM-FRET method employing Halo-Tag labelled with Janelia Fluor dyes. These findings support the hypothesis that septal PG synthesis may be carried out by a complex of multimers, capable of simultaneously synthesizing more than one glycan strand. Inhibition of PG synthesis by directly targeting PBP1 with the beta-lactam antibiotic imipenem weakened the interaction between PBP1 and FtsW, whereas the FtsW self-interaction was enhanced in a dose-dependent manner. In contrast, inhibition of PG synthesis by targeting the lipid II flippase, therefore depleting the FtsW-PBP1 substrate from the outer surface of the cell membrane, had little effect on these interactions. This suggests that alterations in FtsW interactions result primarily from antibiotic-induced conformational changes or from uncoupling the activities of FtsW and PBP1, resulting in the presence of uncrosslinked glycans, rather than merely from a loss of PG synthesis activity.

microbiology↗

bioRxiv2023

The role of GpsB in cell morphogenesis of Staphylococcus aureus

For decades, cells of the gram-positive bacterial pathogen Staphylococcus aureus were thought to lack a dedicated elongation machinery. However, S. aureus cells were recently shown to elongate before division, in a process that requires a SEDS (Shape Elongation Division and Sporulation) / PBP (Penicillin Binding Protein) pair for peptidoglycan synthesis, consisting of the glycosyltransferase RodA and the transpeptidase PBP3. In ovococci and rod-shaped bacteria the elongation machinery, known as elongasome, is composed of various proteins besides a dedicated SEDS/PBP pair. To identify proteins involved in the elongation of S. aureus, we screened the Nebraska Transposon Mutant Library, which contains transposon mutants in virtually all non-essential staphylococcal genes, for mutants with modified cell shape. We confirmed the roles of RodA/PBP3 in S. aureus elongation and identified GpsB, SsaA, and RodZ as additional proteins involved in this process. The gpsB mutant showed the strongest phenotype, mediated by the partial delocalization from the division septum of PBP2, the only bifunctional PBP in S. aureus, with both glycosyltransferase and transpeptidase activity, and of the PBP4 transpeptidase. Increased levels of these PBPs at the cell periphery result in higher levels of peptidoglycan insertion throughout the entire cell, overriding the RodA/PBP3-mediated peptidoglycan synthesis at the outer edge of the septum, which leads to cell elongation. As a consequence, in the absence of GpsB, S. aureus cells become more spherical.

microbiology↗

bioRxiv2022

Conformational changes in the essential E. coli septal cell wall synthesis complex suggest an activation mechanism

The bacterial divisome, a macromolecular machine that is composed of more than thirty proteins in E. coli, orchestrates the essential process of cell wall constriction during cell division. Novel antimicrobial strategies can target protein-protein interactions within the divisome and will benefit from insights into divisome structure and dynamics. In this work, we combined structure prediction, molecular dynamics simulation, single-molecule imaging, and mutagenesis to construct a model of the core complex of the E. coli divisome composed of the essential septal cell wall synthase complex formed by FtsW and FtsI, and its regulators FtsQ, FtsL, FtsB, and FtsN. We observed extensive interactions in four key regions in the periplasmic domains of the complex. FtsQ, FtsL, and FtsB scaffold FtsI in an extended conformation with the FtsI transpeptidase domain lifted away from the membrane through interactions among the C-terminal domains. FtsN binds between FtsI and FtsL in a region rich in residues with superfission (activating) and dominant negative (inhibitory) mutations. Mutagenesis experiments in cellulo and in silico revealed that the essential domain of FtsN functions as a tether to tie FtsI and FtsL together, impacting interactions between the anchor-loop of FtsI and the putative catalytic region of FtsW, suggesting a mechanism of how FtsN activates the cell wall synthesis activities of FtsW and FtsI.

biophysics↗

bioRxiv2020

Blocking IL-10 signaling with soluble IL-10 receptor restores specific lymphoproliferative response in dogs with leishmaniasis caused by Leishmania infantum

rIL-10 plays a major role in restricting exaggerated inflammatory and immune responses, thus preventing tissue damage. However, the restriction of inflammatory and immune responses by IL-10 can also favor the development and/or persistence of chronic infections or neoplasms. Dogs that succumb to leishmaniasis caused by L. infantum (CanL) develop exhaustion of T lymphocytes and are unable to mount appropriate cellular immune responses to control the infection. These animals fail to mount specific lymphoproliferative responses and produce interferon gamma and TNF-alpha that would activate macrophages and promote destruction of intracellular parasites. Blocking IL-10 signaling may contribute to the treatment of CanL. In order to obtain a tool for this blockage, the present work endeavored to identify the canine casIL-10R1 amino acid sequence, generate a recombinant baculovirus chromosome encoding this molecule, which was expressed in insect cells and subsequently purified to obtain rcasIL-10R1. In addition, rcasIL-10R1 was able to bind to homologous IL-10 and block IL-10 signaling pathway, as well as to promote lymphoproliferation in dogs with leishmaniasis caused by L. infantum.

immunology↗

bioRxiv2018

Carbapenem-resistant Pseudomonas aeruginosa bloodstream infection: in vitro synergy, virulence and clinical outcome

In the current study carbapanem-resistant P. aeruginosa bloodstream infection (CRPA-BSI) was associated with high 14-day mortality. Although, highly resistant to meropenem, half of the isolates achieved in vitro synergy with the combination of meropenem with colistin, and the patients that received this combination therapy showed a tendency towards lower mortality. Our strains did not carry exoU gene, on the other hand, patients with CRPA-BSI caused by strains harboring gene lasB evolved more frequent to death.

microbiology↗