Biology subjects

Schuh, C. M. A. P.

Publications and source records attributed to Schuh, C. M. A. P..

6 recordsLinked to original sources

bioRxiv2026

Functional export of NDM-7 to outer membrane vesicles in Klebsiella pneumoniae compromises imipenem and cefiderocol activity

The carbapenemases KPC and NDM are the most widespread determinants of carbapenem resistance in Klebsiella pneumoniae. Whereas KPC is a soluble periplasmic serine-{beta}-lactamase, NDM is a membrane-anchored metallo-{beta}-lactamase (MBL), a feature that promotes its incorporation into outer membrane vesicles (OMVs). OMVs are naturally released nanoparticles that deliver diverse bioactive cargo, including enzymes, virulence factors, and signaling molecules, and may contribute to antibiotic resistance. Here, we investigated the export and activity of carbapenemases in OMVs produced by carbapenem-resistant Klebsiella pneumoniae clinical isolates expressing NDM-7, an emerging variant, or KPC-2, as well as in isogenic laboratory-derived K. pneumoniae strains producing NDM-1, NDM-7 or KPC-2. NDM enzymes were detected in vesicles released by NDM-producing strains, whereas KPC-2 remained confined to the cellular fraction and was not observed in OMVs. OMVs contained catalytically active NDM enzyme and conferred protection to susceptible K. pneumoniae against imipenem. Importantly, NDM-positive OMVs also partially restored bacterial growth in the presence of cefiderocol, a siderophore cephalosporin used to treat infections caused by MBL producers. This protective effect was more pronounced for NDM-7 than for NDM-1. Together, these findings show that the clinically emerging NDM-7 variant is efficiently packaged into OMVs in K. pneumoniae and remains enzymatically active, allowing extracellular antibiotic degradation and conferring protection to susceptible bacteria exposed to carbapenems and cefiderocol.

microbiology↗

bioRxiv2025

Royal Jelly Derived Extracellular Vesicles Modulate Microglial Nanomechanics and Inflammatory Responses

BACKGROUND: Microglia, the brain's resident immune cells, undergo profound mechanical and functional changes upon activation contributing to neuroinflammation, a pathological signature of many neurological diseases. Thus, new anti-inflammatory treatment options are needed that tackle these mechanobiological alterations in microglia, which remain strongly understudied. In this context, extracellular vesicles (EVs) are crucial mediators of intercellular and interkingdom communication, yet their influence on the mechanobiological properties of recipient cells remains largely unknown. Honeybee-derived Royal Jelly EVs (RJEVs) have demonstrated remarkable anti-inflammatory properties, but their impact on microglial cellular nanomechanics and uptake mechanisms remains unclear. RESULTS: In this study, we used a multi-disciplinary approach to analyze the resulting biological and nanomechanical changes following the activation of human microglia and the potential effect of RJEV treatment on these mechanobiological parameters. We observed that LPS treatment was associated with decreased cellular Young's modulus, increased membrane fluidity, and enhanced motility of microglia, indicating a more migratory and pro-inflammatory phenotype. Additionally, LPS exposure altered cellular EV uptake mechanisms by shifting preference from an equilibrium of four mechanisms to the predominance of macropinocytosis and clathrin-dependent endocytosis. Remarkably, RJEV treatment counteracted these mechanobiological changes by, in turn, increasing microglial stiffness, reducing motility, and decreasing secretion of pro-inflammatory cytokines. CONCLUSION: This is the first study to demonstrate that microglial activation state dictates EV uptake mechanisms and to establish a direct link between inflammation, cellular and membrane mechanics, and EV-mediated modulation. Our findings highlight RJEVs as promising candidates for regulating neuroinflammation by targeting microglial mechanobiology as well as opening new strategies for EV-based therapeutics.

neuroscience↗

bioRxiv2024

Biofilm formation on glycated collagen modulates Streptococcus mutans bacterial extracellular vesicle production and cargo

Streptococcus mutans is the major microbial etiological agent of dental caries and can adhere to surfaces such as type-I collagen, present in dentin and periodontal tissues. Recent studies have characterized planktonic S. mutans bacterial extracellular vesicles (bEVs) and demonstrated environmental-induced changes due to sugar presence or pH alterations. However, to date there are no studies exploring if surface-derived changes - such as tissue glycation - can modulate bEV production in the context of oral biofilm formation in the elderly. Therefore, the aim of this work was to determine the role of biofilm formation and collagen glycation on the morphology and composition of S. mutans bEVs. For this, bEVs from S. mutans biofilms on native and glycated collagen surfaces were isolated, characterized, and compared to bEVs from planktonic cells. Nanoparticle tracking analysis and microscopy confirmed bEV production and showed that bEVs from biofilms are smaller in size and less abundant than those from planktonic cells. Furthermore, proteome analysis revealed that S. mutans biofilm formation on native and glycated collagen led to the enrichment of several key virulence proteins such as Eno, LuxS, Tpx, and ScrB. Also, a shift towards proteins involved in metabolic processes was found in bEVs following biofilm formation on collagen surfaces, whereas glucan metabolism proteins were overexpressed in vesicles from the planktonic state. These results demonstrate that biofilm formation, as well as the glycation of collagen associated with aging and hyperglycemia, can modulate bEV characteristics and cargo and could play a central role in S. mutans virulence and the development of diseases such as dental caries and periodontal disease.

microbiology↗

bioRxiv2023

Microfabrication-based engineering of biomimetic dentin-like constructs to simulate dental aging

1.Human dentin is a highly organized dental tissue displaying a complex microarchitecture consisting of micrometer-sized tubules encased in a mineralized type-I collagen matrix. As such, it serves as an important substrate for the adhesion of microbial colonizers and oral biofilm formation in the context of dental caries disease, including root caries in the elderly. Despite this issue, there remains a current lack of effective biomimetic in-vitro dentin models that facilitate the study of oral microbial adhesion by considering the surface architecture at the micro- and nanoscales. Therefore, the aim of this study was to develop a novel in-vitro microfabricated biomimetic dentin surface that simulates the complex surface microarchitecture of exposed dentin. For this, a combination of soft lithography microfabrication and biomaterial science approaches were employed to construct a micropitted PDMS substrate functionalized with mineralized type-I collagen. These dentin analogues were subsequently glycated with methylglyoxal (MGO) to simulate dentin matrix aging in-vitro and analyzed utilizing an interdisciplinary array of techniques including atomic force microscopy (AFM), elemental analysis, and electron microscopy. AFM force-mapping demonstrated that the nanomechanical properties of the biomimetic constructs were within the expected biological parameters, and that mineralization was mostly predominated by hydroxyapatite deposition. Finally, dual-species biofilms of Streptococcus mutans and Candida albicans were grown and characterized on the biofunctionalized PDMS microchips, demonstrating biofilm specific morphologic characteristics and confirming the suitability of this model for the study of early biofilm formation under controlled conditions. Overall, we expect that this novel biomimetic dentin model could serve as an in-vitro platform to study oral biofilm formation or dentin-biomaterial bonding in the laboratory without the need for animal or human tooth samples in the future.

bioengineering↗

bioRxiv2022

Nanoscale dynamics of streptococcal adhesion to AGE-modified collagen

The adhesion of initial colonizers such as Streptococcus mutans to collagen is critical for dentinal and root caries progression. One of the most described pathological and aging-associated changes in collagen - including dentinal collagen - is the generation of advanced glycation end-products (AGEs) such as methylglyoxal (MGO)-derived AGEs. Despite previous reports suggesting that AGEs alter bacterial adhesion to collagen, the biophysics driving oral streptococcal attachment to MGO-modified collagen remains largely understudied. Thus, the aim of this work was to unravel the dynamics of the initial adhesion of S. mutans to type-I collagen in the presence and absence of MGO-derived AGEs, by employing bacterial cell force-spectroscopy with atomic force microscopy (AFM). Type-I collagen gels were treated with 10mM MGO to induce AGE formation, which was characterized with microscopy and ELISA. Subsequently, AFM cantilevers were functionalized with living S. mutans UA 159 or S. sanguinis SK 36 cells and probed against collagen surfaces to obtain force-curves displaying bacterial attachment in real-time, from which the adhesion force, number of events, Poisson analysis, and contour and rupture lengths for each individual detachment event were computed. Furthermore, in-silico docking studies between the relevant S. mutans UA 159 collagen-binding protein SpaP and collagen were computed, in the presence and absence of MGO. Overall, results showed that MGO modification increased both the number and adhesion force of single-unbinding events between S. mutans and collagen, without altering the contour or rupture lengths. Both experimental and in-silico simulations suggest that this effect is due to increased specific and non-specific forces and interactions between S. mutans UA 159 and MGO-modified collagen substrates. In summary, these results suggest that collagen alterations due to glycation and AGE formation may play a role in early bacterial adherence to oral tissues, associated with conditions such as aging or chronic hyperglycemia, amongst others.

microbiology↗

bioRxiv2022

Extracellular Vesicles derived from Apis mellifera Royal Jelly promote wound healing by modulating inflammation and cellular responses

Apis mellifera Royal Jelly (RJ) is a well-known remedy in traditional medicine around the world and its versatile effects range from antibacterial to anti-inflammatory properties and pro-regenerative properties. Several active compounds have been identified, however, the mechanisms of action still remain widely unknown. As a glandular product, RJ has been shown to contain a substantial number of extracellular vesicles (EVs) and in this study, we aimed to investigate the extent of involvement of RJEVs in wound healing associated effects. Molecular analysis of RJEVs verified the presence of important conserved exosomal markers such as CD63 and syntenin, as well as cargo molecules MRJP1, defensin-1 and jellein-3. RJEV internalization analysis demonstrated the involvement of membrane fusion as well as macropinocytosis or clathrin-dependent endocytosis into mammalian cells. Furthermore, RJEVs have demonstrated to modulate MSCs differentiation and secretome, as well as decrease LPS-induced inflammation in RAW 264.7 macrophages by blocking the MAPK pathway. In vivo studies confirmed anti-bacterial effects of RJEVs, and demonstrated an acceleration of wound healing in a splinted mouse model. Summarizing, this study suggests that RJEVs of potentially exosomal origin play a crucial role in the known effects of RJ by modulating the inflammatory phase and cellular response in wound healing.

bioengineering↗