Extracellular vimentin as a modulator of the immune response and an important player during infectious diseases.

Vimentin, an intermediate filament protein primarily recognized for its intracellular role in maintaining cellular structure, has recently garnered increased attention and emerged as a pivotal extracellular player in immune regulation and host-pathogen interactions. While the functions of extracellular vimentin were initially overshadowed by its cytoskeletal role, accumulating evidence now highlights its significance in diverse physiological and pathological events. This review explores the multifaceted role of extracellular vimentin in modulating immune responses and orchestrating interactions between host cells and pathogens. It delves into the mechanisms underlying vimentin's release into the extracellular milieu, elucidating its unconventional secretion pathways and identifying critical molecular triggers. In addition, the future perspectives of using extracellular vimentin in diagnostics and as a target protein in the treatment of diseases are discussed.

Physicochemical Nature of SARS-CoV-2 Spike Protein Binding to Human Vimentin.

Vimentin, a protein that builds part of the cytoskeleton and is involved in many aspects of cellular function, was recently identified as a cell surface attachment site for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The present study investigated the physicochemical nature of the binding between the SARS-CoV-2 S1 glycoprotein receptor binding domain (S1 RBD) and human vimentin using atomic force microscopy and a quartz crystal microbalance. The molecular interactions of S1 RBD and vimentin proteins were quantified using vimentin monolayers attached to the cleaved mica or a gold microbalance sensor as well as in its native extracellular form present on the live cell surface. The presence of specific interactions between vimentin and S1 RBD was also confirmed using in silico studies. This work provides new evidence that cell-surface vimentin (CSV) functions as a site for SARS-CoV-2 virus attachment and is involved in the pathogenesis of Covid-19, providing a potential target for therapeutic countermeasures.

Exposure to polystyrene nanoparticles leads to changes in the zeta potential of bacterial cells.

Polymer molecules, the main components of plastics, are an emerging pollutants in various environmental compartments (water, air, soil) that may induce several ecotoxicological effects on live organisms. Therefore, understanding how plastic particles interact with bacterial cell membranes is crucial in analysing their associated risks in ecosystems and human microbiota. However, relatively little is known about the interaction between nanoplastics and bacteria. The present work focuses on Staphylococcus aureus and Klebsiella pneumoniae, representing the Gram-positive and Gram-negative bacteria respectively, exposed to 100 nm diameter polystyrene nanoparticles (PS NPs). The nanoparticles attach to the cells' membranes of both bacteria, changing their electrical charge, but without the effect of killing the cells. PS NPs caused a change in zeta potential values (both species of bacterial strains), dependent on particle concentration, pH, as well as on exposure time of bacteria to them. Through the application of AFM and FTIR techniques, the presence of PS NPs on bacterial surfaces was detected, suggesting the affinity of the particles to bacterial components, but without any changes in the morphology of the tested bacteria. The zeta potential can be more widely used in the study of interactions between nanostructures and cells.

Plasma Gelsolin Enhances Phagocytosis of Candida auris by Human Neutrophils through Scavenger Receptor Class B.

In addition to its role as an actin-depolymerizing factor in the blood, plasma gelsolin (pGSN) binds bacterial molecules and stimulates the phagocytosis of bacteria by macrophages. Here, using an in vitro system, we assessed whether pGSN could also stimulate phagocytosis of the fungal pathogen Candida auris by human neutrophils. The extraordinary ability of C. auris to evade immune responses makes it particularly challenging to eradicate in immunocompromised patients. We demonstrate that pGSN significantly enhances C. auris uptake and intracellular killing. Stimulation of phagocytosis was accompanied by decreased neutrophil extracellular trap (NET) formation and reduced secretion of proinflammatory cytokines. Gene expression studies revealed pGSN-dependent upregulation of scavenger receptor class B (SR-B). Inhibition of SR-B using sulfosuccinimidyl oleate (SSO) and block lipid transport-1 (BLT-1) decreased the ability of pGSN to enhance phagocytosis, indicating that pGSN potentiates the immune response through an SR-B-dependent pathway. These results suggest that the response of the host's immune system during C. auris infection may be enhanced by the administration of recombinant pGSN. IMPORTANCE The incidence of life-threatening multidrug-resistant Candida auris infections is rapidly growing, causing substantial economic costs due to outbreaks in hospital wards. Primary and secondary immunodeficiencies in susceptible individuals, such as those with leukemia, solid organ transplants, diabetes, and ongoing chemotherapy, often correlate with decreased plasma gelsolin concentration (hypogelsolinemia) and impairment of innate immune responses due to severe leukopenia. Immunocompromised patients are predisposed to superficial and invasive fungal infections. Morbidity caused by C. auris among immunocompromised patients can be as great as 60%. In the era of ever-growing fungal resistance in an aging society, it is critical to seek novel immunotherapies that may help combat these infections. The results reported here suggest the possibility of using pGSN as an immunomodulator of the immune response by neutrophils during C. auris infection.

Ceragenin CSA-13 displays high antibacterial efficiency in a mouse model of urinary tract infection.

Ceragenins (CSAs) are synthetic, lipid-based molecules that display activities of natural antimicrobial peptides. Previous studies demonstrated their high in vitro activity against pathogens causing urinary tract infections (UTIs), but their efficiency in vivo was not explored to date. In this study, we aimed to investigate the bactericidal efficiency of ceragenins against E. coli (Xen14 and clinical UPEC strains) isolates both in vitro and in vivo, as well to explore CSA-13 biodistribution and ability to modulate nanomechanical alterations of infected tissues using animal model of UTI. CSA-44, CSA-131 and particularly CSA-13 displayed potent bactericidal effect against tested E. coli strains, and this effect was mediated by induction of oxidative stress. Biodistribution studies indicated that CSA-13 accumulates in kidneys and liver and is eliminated with urine and bile acid. We also observed that ceragenin CSA-13 reverses infection-induced alterations in mechanical properties of mouse bladders tissue, which confirms the preventive role of CSA-13 against bacteria-induced tissue damage and potentially promote the restoration of microenvironment with biophysical features unfavorable for bacterial growth and spreading. These data justify the further work on employment of CSA-13 in the treatment of urinary tract infections.

Degradation of Polylactide and Polycaprolactone as a Result of Biofilm Formation Assessed under Experimental Conditions Simulating the Oral Cavity Environment.

Polylactide (PLA) and polycaprolactone (PCL) are biodegradable and bioabsorbable thermoplastic polymers considered as promising materials for oral applications. However, any abiotic surface used, especially in areas naturally colonized by microorganisms, provides a favorable interface for microbial growth and biofilm development. In this study, we investigated the biofilm formation of C. krusei and S. mutans on the surface of PLA and PCL immersed in the artificial saliva. Using microscopic (AFM, CLSM) observations and spectrometric measurements, we assessed the mass and topography of biofilm that developed on PLA and PCL surfaces. Incubated up to 56 days in specially prepared saliva and microorganisms medium, solid polymer samples were examined for surface properties (wettability, roughness, elastic modulus of the surface layer), structure (molecular weight, crystallinity), and mechanical properties (hardness, tensile strength). It has been shown that biofilm, especially S. mutans, promotes polymer degradation. Our findings indicate the need for additional antimicrobial strategies for the effective oral applications of PLA and PCL.

Modulation of Biofilm Mechanics by DNA Structure and Cell Type.

Deoxyribonucleic acid (DNA) evolved as a tool for storing and transmitting genetic information within cells, but outside the cell, DNA can also serve as "construction material" present in microbial biofilms or various body fluids, such as cystic fibrosis, sputum, and pus. In the present work, we investigate the mechanics of biofilms formed from Pseudomonas aeruginosa Xen 5, Staphylococcus aureus Xen 30, and Candida albicans 1408 using oscillatory shear rheometry at different levels of compression and recreate these mechanics in systems of entangled DNA and cells. The results show that the compression-stiffening and shear-softening effects observed in biofilms can be reproduced in DNA networks with the addition of an appropriate number of microbial cells. Additionally, we observe that these effects are cell-type dependent. We also identify other mechanisms that may significantly impact the viscoelastic behavior of biofilms, such as the compression-stiffening effect of DNA cross-linking by bivalent cations (Mg2+, Ca2+, and Cu2+) and the stiffness-increasing interactions of P. aeruginosa Xen 5 biofilm with Pf1 bacteriophage produced by P. aeruginosa. This work extends the knowledge of biofilm mechanobiology and demonstrates the possibility of modifying biopolymers toward obtaining the desired biophysical properties.

SARS-CoV-2 Spike Protein (RBD) Subunit Adsorption at Abiotic Surfaces and Corona Formation at Polymer Particles.

The adsorption kinetics of the SARS-CoV-2 spike protein subunit with the receptor binding domain at abiotic surfaces was investigated. A combination of sensitive methods was used such as atomic force microscopy yielding a molecular resolution, a quartz microbalance, and optical waveguide lightmode spectroscopy. The two latter methods yielded in situ information about the protein adsorption kinetics under flow conditions. It was established that at pH 3.5-4 the protein adsorbed on mica and silica surfaces in the form of compact quasi-spherical aggregates with an average size of 14 nm. The maximum coverage of the layers was equal to 3 and 1 mg m-2 at pH 4 and 7.4, respectively. The experimental data were successfully interpreted in terms of theoretical results derived from modeling. The experiments performed for flat substrates were complemented by investigations of the protein corona formation at polymer particles carried out using in situ laser Doppler velocimetry technique. In this way, the zeta potential of the protein layers was acquired as a function of the coverage. Applying the electrokinetic model, these primary data were converted to the dependence of the subunit zeta potential on pH. It was shown that a complete acid-base characteristic of the layer can be acquired only using nanomolar quantities of the protein.

Mechanical Properties of the Extracellular Environment of Human Brain Cells Drive the Effectiveness of Drugs in Fighting Central Nervous System Cancers.

The evaluation of nanomechanical properties of tissues in health and disease is of increasing interest to scientists. It has been confirmed that these properties, determined in part by the composition of the extracellular matrix, significantly affect tissue physiology and the biological behavior of cells, mainly in terms of their adhesion, mobility, or ability to mutate. Importantly, pathophysiological changes that determine disease development within the tissue usually result in significant changes in tissue mechanics that might potentially affect the drug efficacy, which is important from the perspective of development of new therapeutics, since most of the currently used in vitro experimental models for drug testing do not account for these properties. Here, we provide a summary of the current understanding of how the mechanical properties of brain tissue change in pathological conditions, and how the activity of the therapeutic agents is linked to this mechanical state.

Human Vimentin Layers on Solid Substrates: Adsorption Kinetics and Corona Formation Investigations.

Adsorption kinetics of human vimentin on negatively charged substrates (mica, silica, and polymer particles) was analyzed using atomic force microscopy (AFM), quartz microbalance (QCM), and the laser doppler velocimetry (LDV) method. AFM studies realized under diffusion conditions proved that the adsorbed protein layer mainly consisted of aggregates in the form of compact tetramers and hexamers of a size equal to 11-12 nm. These results were consistent with vimentin adsorption kinetics under flow conditions investigated by QCM. It was established that vimentin aggregates efficiently adsorbed on the negatively charged silica sensor at pH 3.5 and 7.4, forming compact layers with the coverage reaching 3.5 mg m-2. Additionally, the formation of the vimentin corona at polymer particles was examined using the LDV method and interpreted in terms of the electrokinetic model. This allowed us to determine the zeta potential of the corona as a function of pH and the electrokinetic charge of aggregates, which was equal to -0.7 e nm-2 at pH 7.4 in a 10 mM NaCl solution. The anomalous adsorption of aggregates exhibiting an average negative charge on the negatively charged substrates was interpreted as a result of a heterogeneous charge distribution. These investigations confirmed that it is feasible to deposit stable vimentin layers both at planar substrates and at carrier particles with well-controlled coverage and zeta potential. They can be used for investigations of vimentin interactions with various ligands including receptors of the innate immune system, immunoglobulins, bacterial virulence factors, and spike proteins of viruses.

Unique Role of Vimentin Networks in Compression Stiffening of Cells and Protection of Nuclei from Compressive Stress.

In this work, we investigate whether stiffening in compression is a feature of single cells and whether the intracellular polymer networks that comprise the cytoskeleton (all of which stiffen with increasing shear strain) stiffen or soften when subjected to compressive strains. We find that individual cells, such as fibroblasts, stiffen at physiologically relevant compressive strains, but genetic ablation of vimentin diminishes this effect. Further, we show that unlike networks of purified F-actin or microtubules, which soften in compression, vimentin intermediate filament networks stiffen in both compression and extension, and we present a theoretical model to explain this response based on the flexibility of vimentin filaments and their surface charge, which resists volume changes of the network under compression. These results provide a new framework by which to understand the mechanical responses of cells and point to a central role of intermediate filaments in response to compression.

Ceragenin CSA-44 as a Means to Control the Formation of the Biofilm on the Surface of Tooth and Composite Fillings.

Recurrent oral infections, as manifested by endodontic and periodontal disease, are often caused by Enterococcus faecalis (E. faecalis) and Candida albicans (C. albicans). Here, we assessed the anti-biofilm activity of ceragenin CSA-44 against these microbes growing as a biofilm in the presence of saliva on the surface of human teeth and dental composite (composite filling) subjected to mechanical stresses. Methods: Biofilm mass analysis was performed using crystal violet (CV) staining. The morphology, viscoelastic properties of the biofilm after CSA-44 treatment, and changes in the surface of the composite in response to biofilm presence were determined by AFM microscopy. Results: CSA-44 prevented biofilm formation and reduced the mass of biofilm formed by tested microorganisms on teeth and dental composite. Conclusion: The ability of CSA-44 to prevent the formation and to reduce the presence of established biofilm on tooth and composite filling suggests that it can serve as an agent in the development of new methods of combating oral pathogens and reduce the severity of oral infections.

Cathelicidin LL-37 in Health and Diseases of the Oral Cavity.

The mechanisms for maintaining oral cavity homeostasis are subject to the constant influence of many environmental factors, including various chemicals and microorganisms. Most of them act directly on the oral mucosa, which is the mechanical and immune barrier of the oral cavity, and such interaction might lead to the development of various oral pathologies and systemic diseases. Two important players in maintaining oral health or developing oral pathology are the oral microbiota and various immune molecules that are involved in controlling its quantitative and qualitative composition. The LL-37 peptide is an important molecule that upon release from human cathelicidin (hCAP-18) can directly perform antimicrobial action after insertion into surface structures of microorganisms and immunomodulatory function as an agonist of different cell membrane receptors. Oral LL-37 expression is an important factor in oral homeostasis that maintains the physiological microbiota but is also involved in the development of oral dysbiosis, infectious diseases (including viral, bacterial, and fungal infections), autoimmune diseases, and oral carcinomas. This peptide has also been proposed as a marker of inflammation severity and treatment outcome.

Biocompatible Materials in Otorhinolaryngology and Their Antibacterial Properties.

For decades, biomaterials have been commonly used in medicine for the replacement of human body tissue, precise drug-delivery systems, or as parts of medical devices that are essential for some treatment methods. Due to rapid progress in the field of new materials, updates on the state of knowledge about biomaterials are frequently needed. This article describes the clinical application of different types of biomaterials in the field of otorhinolaryngology, i.e., head and neck surgery, focusing on their antimicrobial properties. The variety of their applications includes cochlear implants, middle ear prostheses, voice prostheses, materials for osteosynthesis, and nasal packing after nasal/paranasal sinuses surgery. Ceramics, such as as hydroxyapatite, zirconia, or metals and metal alloys, still have applications in the head and neck region. Tissue engineering scaffolds and drug-eluting materials, such as polymers and polymer-based composites, are becoming more common. The restoration of life tissue and the ability to prevent microbial colonization should be taken into consideration when designing the materials to be used for implant production. The authors of this paper have reviewed publications available in PubMed from the last five years about the recent progress in this topic but also establish the state of knowledge of the most common application of biomaterials over the last few decades.

Ceragenin-Coated Non-Spherical Gold Nanoparticles as Novel Candidacidal Agents.

BACKGROUND: Infections caused by Candida spp. have become one of the major causes of morbidity and mortality in immunocompromised patients. Therefore, new effective fungicides are urgently needed, especially due to an escalating resistance crisis. METHODS: A set of nanosystems with rod- (AuR), peanut- (AuP), and star-shaped (AuS) metal cores were synthesized. These gold nanoparticles were conjugated with ceragenins CSA-13, CSA-44, and CSA-131, and their activity was evaluated against Candida strains (n = 21) through the assessment of MICs (minimum inhibitory concentrations)/MFCs (minimum fungicidal concentrations). Moreover, in order to determine the potential for resistance development, serial passages of Candida cells with tested nanosystems were performed. The principal mechanism of action of Au NPs was evaluated via ROS (reactive oxygen species) generation assessment, plasma membrane permeabilization, and release of the protein content. Finally, to evaluate the potential toxicity of Au NPs, the measurement of hemoglobin release from red blood cells (RBCs) was carried out. RESULTS: All of the tested nanosystems exerted a potent candidacidal activity, regardless of the species or susceptibility to other antifungal agents. Significantly, no resistance development after 25 passages of Candida cells with AuR@CSA-13, AuR@CSA-44, and AuR@CSA-131 nanosystems was observed. Moreover, the fungicidal mechanism of action of the investigated nanosystems involved the generation of ROS, damage of the fungal cell membrane, and leakage of intracellular contents. Notably, no significant RBCs hemolysis at candidacidal doses of tested nanosystems was detected. CONCLUSIONS: The results provide rationale for the development of gold nanoparticles of rod-, peanut-, and star-shaped conjugated with CSA-13, CSA-44, and CSA-131 as effective candidacidal agents.

Assessment of Ceragenins in Prevention of Damage to Voice Prostheses Caused by Candida Biofilm Formation.

This study aimed to investigate the potential application of ceragenins (CSAs) as new candidacidal agents to prevent biofilm formation on voice prostheses (VPs). The deterioration of the silicone material of VPs is caused by biofilm growth on the device which leads to frequent replacement procedures and sometimes serious complications. A significant proportion of these failures is caused by Candida species. We found that CSAs have significant candidacidal activities in vitro (MIC; MFC; MBIC), and they effectively eradicate species of yeast responsible for VP failure. Additionally, in our in vitro experimental setting, when different Candida species were subjected to CSA-13 and CSA-131 during 25 passages, no tested Candida strain showed the significant development of resistance. Using liquid chromatography-mass spectrometry (LC-MS), we found that VP immersion in an ethanol solution containing CSA-131 results in silicon impregnation with CSA-131 molecules, and in vitro testing revealed that fungal biofilm formation on such VP surfaces was inhibited by embedded ceragenins. Future in vivo studies will validate the use of ceragenin-coated VP for improvement in the life quality and safety of patients after a total laryngectomy.

Tumor stiffening reversion through collagen crosslinking inhibition improves T cell migration and anti-PD-1 treatment.

Only a fraction of cancer patients benefits from immune checkpoint inhibitors. This may be partly due to the dense extracellular matrix (ECM) that forms a barrier for T cells. Comparing five preclinical mouse tumor models with heterogeneous tumor microenvironments, we aimed to relate the rate of tumor stiffening with the remodeling of ECM architecture and to determine how these features affect intratumoral T cell migration. An ECM-targeted strategy, based on the inhibition of lysyl oxidase, was used. In vivo stiffness measurements were found to be strongly correlated with tumor growth and ECM crosslinking but negatively correlated with T cell migration. Interfering with collagen stabilization reduces ECM content and tumor stiffness leading to improved T cell migration and increased efficacy of anti-PD-1 blockade. This study highlights the rationale of mechanical characterizations in solid tumors to understand resistance to immunotherapy and of combining treatment strategies targeting the ECM with anti-PD-1 therapy.

Nanomechanical Hallmarks of Helicobacter pylori Infection in Pediatric Patients.

BACKGROUND: the molecular mechanism of gastric cancer development related to Helicobacter pylori (H. pylori) infection has not been fully understood, and further studies are still needed. Information regarding nanomechanical aspects of pathophysiological events that occur during H. pylori infection can be crucial in the development of new prevention, treatment, and diagnostic measures against clinical consequences associated with H. pylori infection, including gastric ulcer, duodenal ulcer, and gastric cancer. METHODS: in this study, we assessed mechanical properties of children's healthy and H. pylori positive stomach tissues and the mechanical response of human gastric cells exposed to heat-treated H. pylori cells using atomic force microscopy (AFM NanoWizard 4 BioScience JPK Instruments Bruker). Elastic modulus (i.e., the Young's modulus) was derived from the Hertz-Sneddon model applied to force-indentation curves. Human tissue samples were evaluated using rapid urease tests to identify H. pylori positive samples, and the presence of H. pylori cells in those samples was confirmed using immunohistopathological staining. RESULTS AND CONCLUSION: collected data suggest that nanomechanical properties of infected tissue might be considered as markers indicated H. pylori presence since infected tissues are softer than uninfected ones. At the cellular level, this mechanical response is at least partially mediated by cell cytoskeleton remodeling indicating that gastric cells are able to tune their mechanical properties when subjected to the presence of H. pylori products. Persistent fluctuations of tissue mechanical properties in response to H. pylori infection might, in the long-term, promote induction of cancer development.

Inhomogeneity of stiffness and density of the extracellular matrix within the leukoplakia of human oral mucosa as potential physicochemical factors leading to carcinogenesis.

Oral leukoplakia is a clinical term relating to various morphological lesions, including squamous cell hyperplasia, dysplasia and carcinoma. Leukoplakia morphologically manifested as hyperplasia with epithelial dysplasia is clinically treated as precancerous condition. Nevertheless, there is a lack of good markers indicating the transformation of premalignancies towards cancer. A better understanding of the mechanical environment within the tissues where tumors grow might be beneficial for the development of prevention, diagnostic, and treatment methods in cancer management. Atomic force microscopy (AFM) and immunohistology techniques were used to assess changes in the stiffness and morphology of oral mucosa and leukoplakia samples at different stages of their progression towards cancer. The Young's moduli of the tested leukoplakia samples were significantly higher than those of the surrounding mucus. Robust inhomogeneity of stiffness within leukoplakia samples, reflecting an increase in regeneration and collagen accumulation (increasing density) in the extracellular matrix (ECM), was observed. Within the histologically confirmed cancer samples, Young's moduli were significantly lower than those within the precancerous ones. Inhomogeneous stiffness within leukoplakia might act as "a mechanoagonist" that promotes oncogenesis. In contrast, cancer growth might require the reorganization of tissue structure to create a microenvironment with lower and homogenous stiffness. The immunohistology data collected here indicates that changes in tissue stiffness are achieved by increasing cell/ECM density. The recognition of new markers of premalignancy will aid in the development of new therapies and will expand the diagnostic methods.

Bactericidal Properties of Rod-, Peanut-, and Star-Shaped Gold Nanoparticles Coated with Ceragenin CSA-131 against Multidrug-Resistant Bacterial Strains.

BACKGROUND: The ever-growing number of infections caused by multidrug-resistant (MDR) bacterial strains requires an increased effort to develop new antibiotics. Herein, we demonstrate that a new class of gold nanoparticles (Au NPs), defined by shape and conjugated with ceragenin CSA-131 (cationic steroid antimicrobial), display strong bactericidal activity against intractable superbugs. METHODS: For the purpose of research, we developed nanosystems with rod- (AuR NPs@CSA-131), peanut-(AuP NPs@CSA-131) and star-shaped (AuS NPs@CSA-131) metal cores. Those nanosystems were evaluated against bacterial strains representing various groups of MDR (multidrug-resistant) Gram-positive (MRSA, MRSE, and MLSb) and Gram-negative (ESBL, AmpC, and CR) pathogens. Assessment of MICs (minimum inhibitory concentrations)/MBCs (minimum bactericidal concentrations) and killing assays were performed as a measure of their antibacterial activity. In addition to a comprehensive analysis of bacterial responses involving the generation of ROS (reactive oxygen species), plasma membrane permeabilization and depolarization, as well as the release of protein content, were performed to investigate the molecular mechanisms of action of the nanosystems. Finally, their hemocompatibility was assessed by a hemolysis assay. RESULTS: All of the tested nanosystems exerted potent bactericidal activity in a manner resulting in the generation of ROS, followed by damage of the bacterial membranes and the leakage of intracellular content. Notably, the killing action occurred with all of the bacterial strains evaluated, including those known to be drug resistant, and at concentrations that did not impact the growth of host cells. CONCLUSIONS: Conjugation of CSA-131 with Au NPs by covalent bond between the COOH group from MHDA and NH3 from CSA-131 potentiates the antimicrobial activity of this ceragenin if compared to its action alone. Results validate the development of AuR NPs@CSA-131, AuP NPs@CSA-131, and AuS NPs@CSA-131 as potential novel nanoantibiotics that might effectively eradicate MDR bacteria.