Biology subjects

Filipe, S. R.

Publications and source records attributed to Filipe, S. R..

6 recordsLinked to original sources

bioRxiv2026

The NimB2 opsonin promotes S. aureus recognition by macrophages in Drosophila melanogaster

Phagocytosis is a conserved effector process of innate immunity that enables specialized immune cells to detect, engulf, and degrade invading microbes as well as dying cells and cellular debris. Soluble opsonins enhance this process by coating target surfaces and promoting their recognition and uptake by phagocytic cells. In Drosophila, many phagocytic receptors of the Nimrod family have been characterized, but opsonins remain comparatively poorly understood. Here, we identified the secreted Nimrod protein NimB2 as an insect opsonin that promotes the clearance of Staphylococcus aureus. NimB2 is produced by the fat body, secreted into the hemolymph, and required for resistance to S. aureus infection. NimB2 null mutants showed reduced phagocytosis of S. aureus while maintaining Toll dependent antimicrobial peptide expression. NimB2 promotes S. aureus binding to plasmatocytes (Drosophila macrophages). Using binding assays and bacterial cell wall mutants, we found that NimB2 recognizes a lipoteichoic acid (LTA) dependent determinant on the S. aureus surface and coats the bacterium. We further show that this coating enables efficient recognition by hemocytes via the phagocytic receptor Eater, which is necessary for NimB2 dependent binding. Moreover, Eater overexpression enhances NimB2 mediated association of S. aureus with hemocytes. Together, our results establish NimB2 as an insect opsonin that links a specific bacterial ligand to a phagocytic receptor.

immunology↗

bioRxiv2025

mAIcrobe: an open-source framework for high-throughput bacterial image analysis

Quantitative analysis in bacterial microscopy is often hindered by diverse cell morphologies, population heterogeneity, and the requirement for specialised computational expertise. To address these challenges, mAIcrobe is introduced as an open-source framework that broadens access to advanced bacterial image analysis by integrating a suite of deep learning models. mAIcrobe incorporates multiple segmentation algorithms, including StarDist, CellPose, and U-Net, alongside comprehensive morphological profiling and an adaptable neural network classifier, all within the napari ecosystem. This unified platform enables the analysis of a wide range of bacterial species, from spherical Staphylococcus aureus to rod-shaped Escherichia coli, across various microscopy modalities within a single environment. The biological utility of mAIcrobe is demonstrated through its application to antibiotic phenotyping in E. coli and the identification of cell cycle defects in S. aureus DnaA mutants. The modular design, supported by Jupyter notebooks, facilitates custom model development and extends AI-driven image analysis capabilities to the broader microbiology community. Building upon the foundation established by eHooke, mAIcrobe represents a substantial advancement in automated and reproducible bacterial microscopy.

microbiology↗

bioRxiv2023

Altered PBP4 and GdpP functions synergistically mediate MRSA-like high-level, broad-spectrum β-lactam resistance in Staphylococcus aureus

Infections caused by Staphylococcus aureus are a leading cause of mortality worldwide. S. aureus infections caused by Methicillin-Resistant Staphylococcus aureus (MRSA) are particularly difficult to treat due to their resistance to Next Generation {beta}-lactams (NGB) such as Methicillin, Nafcillin, Oxacillin etc. Resistance to NGBs, which is alternatively known as broad-spectrum {beta}- lactam resistance is classically mediated by PBP2a, a Penicillin-Binding Protein encoded by mecA (or mecC) in MRSA. Thus, presence of mec genes among S. aureus serves as the predictor of resistance to NGBs and facilitates determination of the proper therapeutic strategy for a staphylococcal infection. Although far less appreciated, mecA deficient S. aureus strains can also exhibit NGB resistance. These strains, which are collectively termed as Methicillin-Resistant Lacking mec (MRLM) are currently being identified in increasing numbers among natural resistant isolates of S. aureus. The mechanism/s through which MRLMs produce resistance to NGBs remains unknown. In this study, we demonstrate that mutations that alter PBP4 and GdpP functions, which are often present among MRLMs can synergistically mediate resistance to NGBs. Furthermore, our results unravel that this novel mechanism potentially enables MRLMs to produce resistance towards NGBs at levels comparable to that of MRSAs. Our study, provides a fresh new perspective about alternative mechanisms of NGBs resistance, challenging our current overall understanding of high-level, broad-spectrum {beta}-lactam resistance in S. aureus. It thus suggests reconsideration of the current approach towards diagnosis and treatment of {beta}-lactam resistant S. aureus infections.

microbiology↗

bioRxiv2023

Analysis of the influence of peptidoglycan turnover and recycling on host-pathogen interaction in the Gram-positive pathogen Staphylococcus aureus

During peptidoglycan recycling (PR) bacteria can recover extracellular fragments of peptidoglycan (PGN) liberated by peptidoglycan turnover (PT) during cell growth and division, and reuse them in cell wall biosynthesis or central carbon metabolism. In Gram-negative bacteria, PR has been well studied, and functions in the induction of resistance to certain classes of antibiotics, and in host-pathogen interaction. However, while Gram-negative cell envelope architecture allows for highly efficient PR, Gram-positive bacteria, which lack an outer cell membrane and are instead enclosed by a glycopolymer layer, can shed large quantities of PGN-derived material to the external environment during growth. Nonetheless, the occurrence of PR was recently demonstrated in several Gram-positive bacteria, including the Gram-positive bacterial pathogen Staphylococcus aureus, and its potential adaptive functions are largely unexplored. Given the known roles of PR in Gram-negative bacteria, and that Gram-positive bacteria include several important human pathogens, we asked what role PR may play during Gram-positive pathogen-host interaction. Usingthe model insect host Drosophila melanogaster, we demonstrate that S. aureus mutants impaired in extracellular PGN hydrolysis ({Delta}atl) and PGN fragment uptake ({Delta}murP) show differential virulence compared to their wild-type counterpart. This was linked to increased activation of the D. melanogaster Toll-cascade by spent supernatant from the {Delta}atl mutant. Thus, we propose that S. aureus, and potentially other Gram-positive bacteria, may use extracellular PGN degradation during PT to simultaneously process PGN fragments for recycling and for immune evasion, while recovery and/or metabolism of peptidoglycan fragments during PR may play more subtle roles in determining virulence. Author summaryPGN is a key component of the bacterial cell wall, forming a stress-bearing sacculus surrounding the cell and providing cell shape. During growth and division, the sacculus is dynamically degraded and remodelled to ensure daughter cell separation, resulting in PT. PGN fragments released during PT can be recovered and reutilised by the cell during PR. In Gram-negative pathogens, PR is linked to antibiotic resistance, virulence and modulation of host immune recognition. In Gram-positive bacteria, PR was only recently observed. Here, we explore the roles of PT and PR in host-pathogen interaction in S. aureus, a Gram-positive pathogen of significant clinical relevance. Disruption of PT in S. aureus affected host-pathogen interaction through altering host recognition of shed PGN fragments and PR through modulation of PGN fragment recovery. This improves our understanding of the biology of this important pathogen and may aid development of novel therapeutic approaches to treat S. aureus infections.

microbiology↗

bioRxiv2019

A Molecular Link between Cell Wall Modification and Stringent Response in a Gram-positive Bacteria

To ensure survival during colonization of the human host, bacteria must successfully respond to unfavorable and fluctuating conditions. This study explores the fundamental phenomenon of stress response in a gram-positive bacterium, where we investigate the ability of a cell wall modification enzyme to modulate intracellular stress and prevent the triggering of the stringent response pathway. The Streptococcus pneumoniae cell wall modification proteins MurM and MurN are tRNA-dependent amino acid ligases, which lead to the production of branched muropeptides by generating peptide crossbridges. In addition, MurM has been proposed to contribute to translation quality control by preferentially deacylating mischarged tRNAs mischarged with amino acids that make up the peptidoglycan. Here, we demonstrate that the murMN operon promotes optimal growth under stressed conditions. Specifically, when grown in mildly acidic conditions, a murMN deletion mutant displays early entry into stationary phase and dramatically increased lysis. Surprisingly, these defects are rescued by inhibition of the stringent response pathway or by enhancement of the cells ability to deacylate mischarged tRNA molecules. The increase in lysis results from the activity of LytA, and experiments in macrophages reveal that murMN regulates phagocytosis in a LytA-dependent manner. These results suggest that under certain stresses, these bacterial cells lacking MurMN likely accumulate mischarged tRNA molecules, activate the stringent response pathway, and enter prematurely into stationary phase. Moreover, by virtue of its ability to deacylate mischarged tRNAs while building peptidoglycan crossbridges, MurM can calibrate the stress response with consequences to host-pathogen interactions. Thus, MurM is positioned at the interface of cell wall modification, translation quality control and stringent response. These findings expand our understanding of the functions of the bacterial cell wall: cell wall modifications that impart structural rigidity to the cell are interlinked to the cells ability to signal intracellularly and mount a response to environmental stresses.\n\nSIGNIFICANCEDuring infection, microbes must survive the hostile environmental conditions of the human host. When exposed to stresses, bacteria activate an intracellular response, known as stringent response pathway, to ensure their survival. This study connects two fundamental pathways important for cellular growth in a gram-positive bacterium; it demonstrates that enzymes responsible for cell wall modification are connected to the stringent response pathway via their ability to ameliorate errors in protein translation. Our study was performed on Streptococcus pneumoniae where the cell wall modification enzyme, MurM, is a known determinant of penicillin resistance. We now demonstrate the importance of MurM in translation quality control and establish that it serves as a gatekeeper of the stringent response pathway.

microbiology↗

bioRxiv2018

The pentaglycine bridges of Staphylococcus aureus peptidoglycan are essential for cell integrity

Bacterial cells are surrounded by cell wall, whose main component is peptidoglycan (PG), a macromolecule that withstands the internal turgor of the cell. PG composition can vary considerably between species. The Gram-positive pathogen Staphylococcus aureus possesses highly crosslinked PG due to the presence of cross bridges containing five glycines, which are synthesised by the FemXAB protein family. FemX adds the first glycine of the cross bridge, while FemA and FemB add the second and the third, and the fourth and the fifth glycines, respectively. Of these, FemX was reported to be essential. To investigate the essentiality of FemAB, we constructed a conditional S. aureus mutant of the femAB operon. Depletion of femAB was lethal, with cells appearing as pseudomulticellular forms that eventually lyse due to extensive membrane rupture. This deleterious effect was mitigated by drastically increasing the osmolarity of the medium, indicating that pentaglycine crosslinks are required for S. aureus cells to withstand internal turgor. Despite the absence of canonical membrane targeting domains, FemA has been shown to localise at the membrane. To study its mechanism of localisation, we constructed mutants in key residues present in the putative transferase pocket and the 6 helix of FemA, possibly involved in tRNA binding. Mutations in the 6 helix led to a sharp decrease in protein activity in vivo and in vitro but did not impair correct membrane localisation, indicating that FemA activity is not required for localisation. Our data indicates that, contrarily to what was previously thought, S. aureus cells do not survive in the absence of a pentaglycine cross bridge.

microbiology↗