Cell Lysis Research Articles

Insight into antibacterial effect of titanium nanotubular surfaces with focus on Staphylococcus aureus and Pseudomonas aeruginosa

Materials used for orthopedic implants should not only have physical properties close to those of bones, durability and biocompatibility, but should also exhibit a sufficient degree of antibacterial functionality. Due to its excellent properties, titanium is still a widely used material for production of orthopedic implants, but the unmodified material exhibits poor antibacterial activity. In this work, the physicochemical characteristics, such as chemical composition, crystallinity, wettability, roughness, and release of Ti ions of the titanium surface modified with nanotubular layers were analyzed and its antibacterial activity against two biofilm-forming bacterial strains responsible for prosthetic joint infection (Staphylococcus aureus and Pseudomonas aeruginosa) was investigated. Electrochemical anodization (anodic oxidation) was used to prepare two types of nanotubular arrays with nanotubes differing in dimensions (with diameters of 73 and 118 nm and lengths of 572 and 343 nm, respectively). These two surface types showed similar chemistry, crystallinity, and surface energy. The surface with smaller nanotube diameter (TNT-73) but larger values of roughness parameters was more effective against S. aureus. For P. aeruginosa the sample with a larger nanotube diameter (TNT-118) had better antibacterial effect with proven cell lysis. Antibacterial properties of titanium nanotubular surfaces with potential in implantology, which in our previous work demonstrated a positive effect on the behavior of human gingival fibroblasts, were investigated in terms of surface parameters. The interplay between nanotube diameter and roughness appeared critical for the bacterial fate on nanotubular surfaces. The relationship of nanotube diameter, values of roughness parameters, and other surface properties to bacterial behavior is discussed in detail. The study is believed to shed more light on how nanotubular surface parameters and their interplay affect antibacterial activity.

Preclinical activity of allogeneic SLAMF7-specific CAR T-cells (UCARTCS1) in multiple myeloma

BackgroundAutologous BCMA-specific CAR T-cell therapies have substantial activity in multiple myeloma (MM). However, due to logistical limitations and BCMAlow relapses, there is a need for alternatives. UCARTCS1 cells are ‘off-the-shelf’...

A Zwitterionic Detergent and Catalyst-Based Single-Cell Proteomics Using a Loss-Free Microhole-Collection Disc.

Recent advances in single-cell proteomics have solved many bottlenecks, such as throughput, sample recovery, and scalability via nanoscale sample handling. In this study, we aimed for a sensitive mass spectrometry (MS) analysis capable of handling single cells with a conventional mass spectrometry workflow without additional equipment. We achieved seamless cell lysis and TMT labeling in a micro-HOLe Disc (microHOLD) by developing a mass-compatible single solution based on a zwitterionic detergent and a catalyst for single-cell lysis and tandem mass tag labeling without a heat incubation step. This method was developed to avoid peptide loss by surface adsorption and buffer or tube changes by collecting tandem mass tag-labeled peptide through microholes placed in the liquid chromatography injection vials in a single solution. We successfully applied the microHOLD single-cell proteomics method for the analysis of proteome reprogramming in hormone-sensitive prostate cells to develop castration-resistant prostate cancer cells. This novel single-cell proteomics method is not limited by cutting-edge nanovolume handling equipment and achieves high throughput and ultrasensitive proteomics analysis of limited samples, such as single cells.

Ultrasonic Disintegration of Municipal Sludge: Fundamental Mechanisms, Process Intensification and Industrial Sono-Reactors.

Sludge disintegration is an environmental and industrial challenge that requires intensive research and technological development. Sludge has a complex structure with a high yield of various chemical and biological compounds. Anaerobic digestion is the most commonly used process for sludge disintegration to produce biogas, detoxify sludge, and generate biosolids that can be used in agriculture . Biological cell lysis is the rate-limiting cell lysis. This review discusses the application of sonolysis as a sludge pretreatment for enhanced anaerobic digestion via three combined processes: thermal destruction, hydrochemical shear forces, and radical oxidation. The mechanistic pathways of sono-pretreatment to enhance biogas, sludge-enhanced dewatering, activation of filamentous bacteria, oxidation of organic pollutants, release of heavy metals, reduction of bulking and foaming sludge, and boosting ammonia-oxidizing bacterial activity are discussed in this review. This article also discusses the use of ultrasound in sludge disintegration, highlighting its potential in conjunction with Fenton and cation-binding agents, and reviews common large-scale sonoreactors available on the market..

Enhancement of complement-dependent cytotoxicity by linking factor-H derived short consensus repeats 19-20 to CD20 antibodies.

Antibody-mediated complement-dependent cytotoxicity (CDC) on malignant cells is regulated by several complement control proteins, including the inhibitory complement factor H (fH). fH consists of 20 short consensus repeat elements (SCRs) with specific functional domains. Previous research revealed that the fH-derived SCRs 19-20 (SCR1920) can displace full-length fH on the surface of chronic lymphocytic leukemia (CLL) cells, which sensitizes CLL cells for e.g. CD20-targeting therapeutic monoclonal antibody (mAb) induced CDC. Therefore, we constructed lentiviral vectors for the generation of cell lines that stably produce mAb-SCR-fusion variants starting from the clinically approved parental mAbs rituximab, obinutuzumab and ofatumumab, respectively. Flow-cytometry revealed that the modification of the mAbs by the SCRs does not impair the binding to CD20. Increased in vitro lysis potency compared to their parental mAbs was corroborated by showing specific and dose dependent target cell elimination by CDC when compared to their parental mAbs. Lysis of CLL cells was not affected by the depletion of NK cells, suggesting that antibody-dependent cellular cytotoxicity plays a minor role in this context. Overall, this study emphasizes the crucial role of CDC in the elimination of CLL cells by mAbs and introduces a novel approach for enhancing CDC by directly fusing fH SCR1920 with mAbs.

Prophage-DB: A comprehensive database to explore diversity, distribution, and ecology of prophages.

Viruses that infect prokaryotes (phages) constitute the most abundant group of biological agents, playing pivotal roles in microbial systems. They are known to impact microbial community dynamics, microbial ecology, and evolution. Efforts to document the diversity, host range, infection dynamics, and effects of bacteriophage infection on host cell metabolism are extremely underexplored. Phages are classified as virulent or temperate based on their life cycles. Temperate phages adopt the lysogenic mode of infection, where the genome integrates into the host cell genome forming a prophage. Prophages enable viral genome replication without host cell lysis, and often contribute novel and beneficial traits to the host genome. Current phage research predominantly focuses on lytic phages, leaving a significant gap in knowledge regarding prophages, including their biology, diversity, and ecological roles. Here we develop and describe Prophage-DB, a database of prophages, their proteins, and associated metadata that will serve as a resource for viral genomics and microbial ecology. To create the database, we identified and characterized prophages from genomes in three of the largest publicly available databases. We applied several state-of-the-art tools in our pipeline to annotate these viruses, cluster and taxonomically classify them, and detect their respective auxiliary metabolic genes. In total, we identify and characterize over 350,000 prophages and 35,000 auxiliary metabolic genes. Our prophage database is highly representative based on statistical results and contains prophages from a diverse set of archaeal and bacterial hosts which show a wide environmental distribution. Prophages are particularly overlooked in viral ecology and merit increased attention due to their vital implications for microbiomes and their hosts. Here, we created Prophage-DB to advance our comprehension of prophages in microbiomes through a comprehensive characterization of prophages in publicly available genomes. We propose that Prophage-DB will serve as a valuable resource for advancing phage research, offering insights into viral taxonomy, host relationships, auxiliary metabolic genes, and environmental distribution.

Duffy Antigen Receptor for Chemokines (DARC) Nanodisc-Based Biosensor for Detection of Staphylococcal Bicomponent Pore-Forming Leukocidins.

Staphylococcus aureus (S. aureus) is an opportunistic infectious pathogen, which causes a high mortality rate during bloodstream infections. The early detection of virulent strains in patients' blood samples is of medical interest for rapid diagnosis. The main virulent factors identified in patient isolates include leukocidins that bind to specific membrane receptors and lyse immune cells and erythrocytes. Duffy antigen receptor for chemokines (DARC) on the surface of specific cells is a main target of leukocidins such as gamma-hemolysin AB (HlgAB) and leukocidin ED (LukED). Among them, HlgAB is a conserved and critical leukocidin that binds to DARC and forms pores on the cell membranes, leading to cell lysis. Current methods are based on ELISA or bacterial culture, which takes hours to days. For detecting HlgAB with faster response and higher sensitivity, we developed a biosensor that combines single-walled carbon nanotube field effect transistors (swCNT-FETs) with immobilized DARC receptors as biosensing elements. DARC was purified from a bacterial expression system and successfully reconstituted into nanodiscs that preserve binding capability for HlgAB. Dynamic light scattering (DLS) and scanning electron microscopy (SEM) showed an increase of the DARC-containing nanodisc size in the presence of HlgAB, indicating the formation of HlgAB prepore or pore complexes. We demonstrate that this sensor can specifically detect the leukocidins HlgA and HlgAB in a quantitative manner within the dynamic range of 1 fM to 100 pM with an LOD of 0.122 fM and an LOQ of 0.441 fM. The sensor was challenged with human serum spiked with HlgAB as simulated clinical samples. After dilution for decreasing nonspecific binding, it selectively detected the toxin with a similar detection range and apparent dissociation constant as in the buffer. This biosensor was demonstrated with remarkable sensitivity to detect HlgAB rapidly and has the potential as a tool for fundamental research and clinical applications, although this sensor cannot differentiate between HlgAB and LukED as both have the same receptor.

FlhE functions as a chaperone to prevent formation of periplasmic flagella in Gram-negative bacteria

The bacterial flagellum, which facilitates motility, is composed of ~20 structural proteins organized into a long extracellular filament connected to a cytoplasmic rotor-stator complex via a periplasmic rod. Flagellum assembly is regulated by multiple checkpoints that ensure an ordered gene expression pattern coupled to the assembly of the various building blocks. Here, we use epifluorescence, super-resolution, and transmission electron microscopy to show that the absence of a periplasmic protein (FlhE) prevents proper flagellar morphogenesis and results in the formation of periplasmic flagella in Salmonella enterica. The periplasmic flagella disrupt cell wall synthesis, leading to a loss of normal cell morphology resulting in cell lysis. We propose that FlhE functions as a periplasmic chaperone to control assembly of the periplasmic rod, thus preventing formation of periplasmic flagella.

The Conserved YPX3L Motif in the BK Polyomavirus VP1 Protein Is Important for Viral Particle Assembly but Not for Its Secretion into Extracellular Vesicles.

The BK polyomavirus (BKPyV) is a small DNA non-enveloped virus whose infection is asymptomatic in most of the world's adult population. However, in cases of immunosuppression, the reactivation of the virus can cause various complications, and in particular, nephropathies in kidney transplant recipients or hemorrhagic cystitis in bone marrow transplant recipients. Recently, it was demonstrated that BKPyV virions can use extracellular vesicles to collectively traffic in and out of cells, thus exiting producing cells without cell lysis and entering target cells by diversified entry routes. By a comparison to other naked viruses, we investigated the possibility that BKPyV virions recruit the Endosomal-Sorting Complexes Required for Transport (ESCRT) machinery through late domains in order to hijack extracellular vesicles. We identified a single potential late domain in the BKPyV structural proteins, a YPX3L motif in the VP1 protein, and used pseudovirions to study the effect of point mutations found in a BKPyV clinical isolate or known to ablate the interaction of such a domain with the ESCRT machinery. Our results suggest that this domain is not involved in BKPyV association with extracellular vesicles but is crucial for capsomere interaction and thus viral particle assembly.

Scalable intracellular delivery via microfluidic vortex shedding enhances the function of chimeric antigen receptor T-cells.

Adoptive chimeric antigen receptor T-cell (CAR-T) therapy is transformative and approved for hematologic malignancies. It is also being developed for the treatment of solid tumors, autoimmune disorders, heart disease, and aging. Despite unprecedented clinical outcomes, CAR-T and other engineered cell therapies face a variety of manufacturing and safety challenges. Traditional methods, such as lentivirus transduction and electroporation, result in random integration or cause significant cellular damage, which can limit the safety and efficacy of engineered cell therapies. We present hydroporation as a gentle and effective alternative for intracellular delivery. Hydroporation resulted in 1.7- to 2-fold higher CAR-T yields compared to electroporation with superior cell viability and recovery. Hydroporated cells exhibited rapid proliferation, robust target cell lysis, and increased pro-inflammatory and regulatory cytokine secretion in addition to improved CAR-T yield by day 5 post-transfection. We demonstrate that scaled-up hydroporation can process 5 x 108 cells in less than 10 s, showcasing the platform as a viable solution for high-yield CAR-T manufacturing with the potential for improved therapeutic outcomes.

Biodegradation of the cyanobacterial toxin anatoxin-a by a Bacillus subtilis strain isolated from a eutrophic lake in Saudi Arabia.

Anatoxin-a (ATX-a) is a neurotoxin produced by some species of cyanobacteria. Due to its water solubility and stability in natural water, it could pose health risks to human, animals, and plants. Conventional water treatment techniques are not only insufficient for the removal of ATX-a, but they also result in cell lysis and toxin release. The elimination of this toxin through biodegradation may be a promising strategy. This study examines for the first time the biodegradation of ATX-a to a non-toxic metabolite (Epoxy-ATX-a) by a strain of Bacillus that has a history of dealing with toxic cyanobacteria in a eutrophic lake. The Bacillus strain AMRI-03 thrived without lag phase in a lake water containing ATX-a. The strain displayed fast degradation of ATX-a, depending on initial toxin concentration. At the highest initial concentrations (50 & 100µg L- 1), total ATX-a degradation took place in 4 days, but it took 6 & 7 days at lower concentrations (20, 10, and 1µg L- 1, respectively). The ATX-a biodegradation rate was also influenced by the initial toxin concentration, reaching its maximum value (12.5µg L- 1 day- 1) at the highest initial toxin concentrations (50 & 100µg L- 1). Temperature and pH also had an impact on the rate of ATX-a biodegradation, with the highest rates occurring at 25 and 30 ºC and pH 7 and 8. This nontoxic bacterial strain could be immobilized within a biofilm on sand filters and/or sludge for the degradation and removal of ATX-a and other cyanotoxins during water treatment processes, following the establishment of mesocosm experiments to assess the potential effects of this bacterium on water quality.

Elucidating Physicochemical Features of Holin Proteins Responsible for Bacterial Cell Lysis.

Bacterial resistance to conventional antibiotics stimulated the development of so-called "phage therapies" that rely on cell lysis, which is a process of destroying bacterial cells due to their infections by bacterial viruses. For λ bacteriophages, it is known that the critical role in this process is played by holin proteins that aggregate in cellular membranes before breaking them apart. While multiple experimental studies probed various aspects of cell lysis, the underlying molecular mechanisms remain not well understood. Here we investigate what physicochemical properties of holin proteins are the most relevant for these processes by employing statistical correlation analysis of cell lysis dynamics for different experimentally observed mutant species. Our findings reveal significant correlations between various physicochemical features and cell lysis dynamics. Notably, we uncover a strong inverse correlation between local hydrophobicity and cell lysis times, underscoring the crucial role of hydrophobic interactions in membrane disruption. Stimulated by these observations, a predictive model capable of explicitly estimating cell lysis times for any holin protein mutants based on their mean hydrophobicity values is developed. Our study not only provides important microscopic insights into cell lysis phenomena but also proposes specific routes to optimize medical and biotechnological applications of bacteriophages.

Oncolytic Peptide-Nanoplatform Drives Oncoimmune Response and Reverses Adenosine-Induced Immunosuppressive Tumor Microenvironment.

The application of oncolytic peptides has become a powerful approach to induce complete and long-lasting remission in multiple types of carcinomas, as affirmed by the appearance of tumor-associated antigens and adenosine triphosphate (ATP) in large quantities, which jumpstarts the cancer-immunity cycle. However, the ATP breakdown product adenosine is a significant contributor to forming the immunosuppressive tumor microenvironment, which substantially weakens peptide-driven oncolytic immunotherapy. In this study, a lipid-coated micelle (CA@TLM) loaded with a stapled oncolytic peptide (PalAno) and an adenosine 2A receptor (A2AR) inhibitor (CPI-444) is devised to enact tumor-targeted oncolytic immunotherapy and to overcome adenosine-mediated immune suppression simultaneously. The CA@TLM micelle accumulates in tumors with high efficiency, and the acidic tumor microenvironment prompts the rapid release of PalAno and CPI-444. Subsequently, PalAno induces swift membrane lysis of tumor cells and the release of antigenic materials. Meanwhile, CPI-444 blocks the activation of the immunosuppressive adenosine-A2AR signaling pathway. This combined approach exhibits pronounced synergy at stalling tumor growth and metastasis in animal models for triple-negative breast cancer and melanoma, providing a novel strategy for enhanced oncolytic immunotherapy.

Exploring the destructive synergy between IL-33 and Suilysin hemolysis on blood-brain barrier stability.

Streptococcus suis type 2 (SS2) is a zoonotic pathogen capable of eliciting meningitis, presenting significant challenges to both the swine industry and public health. Suilysin (Sly), one of SS2 most potent virulence determinants, releases a surfeit of inflammatory agents following red blood cell lysis. Notably, while current research on Sly role in SS2-induced meningitis predominantly centers on its interaction with the blood-brain barrier (BBB), the repercussions of Sly hemolytic products on BBB function have largely been sidestepped. In this vein, our study delves into the ramifications of Sly-induced hemolysis on BBB integrity. We discern that Sly hemolytic derivatives exacerbate the permeability of Sly-induced in vitro BBB models. Within these Sly hemolytic products, Interleukin-33 (IL-33) disrupts the expression and distribution of Claudin-5 in brain microvascular endothelial cells, facilitating the release of Interleukin-6 (IL-6) and Interleukin-8 (IL-8), thereby amplifying BBB permeability. Preliminary mechanistic insights suggest that IL-33-driven expression of IL-6 and IL-8 is orchestrated by the p38-mitogen-activated protein kinase signaling, whereas matrix metalloproteinase 9 mediates IL-33-induced suppression of Claudin-5. To validate these in vitro findings, an SS2-infected mouse model was established, and upon intravenous administration of growth stimulation expressed gene 2 (ST2) antibodies, in vivo results further underscored the pivotal role of the IL-33/ST2 axis during SS2 cerebral invasion. In summation, this study pioneerly illuminates the involvement of Sly hemolytic products in SS2-mediated BBB compromise and spotlights the instrumental role and primary mechanism of IL-33 therein. These insights enrich our comprehension of SS2 meningitis pathogenesis, laying pivotal groundwork for therapeutic advancements against SS2-induced meningitis.IMPORTANCEThe treatment of meningitis caused by Streptococcus suis type 2 (SS2) has always been a clinical challenge. Elucidating the molecular mechanisms by which SS2 breaches the blood-brain barrier (BBB) is crucial for the development of meningitis therapeutics. Suilysin (Sly) is one of the most important virulence factors of SS2, which can quickly lyse red blood cells and release large amounts of damage-associated molecular patterns, such as hemoglobin, IL-33, cyclophilin A, and so on. However, the impact of these hemolytic products on the function of BBB is unknown and ignored. This study is the first to investigate the effect of Sly hemolytic products on BBB function. The data are crucial for the study of the pathogenesis of SS2 meningitis and can provide an important reference for the development of meningitis therapeutics.

Optimizing UVA and UVC synergy for effective control of harmful cyanobacterial blooms

Harmful cyanobacterial blooms (HCBs) pose a global ecological threat. Ultraviolet C (UVC) irradiation at 254 nm is a promising method for controlling cyanobacterial proliferation, but the growth suppression is temporary. Resuscitation remains a challenge with UVC application, necessitating alternative strategies for lethal effects. Here, we show synergistic inhibition of Microcystis aeruginosa using ultraviolet A (UVA) pre-irradiation before UVC. We find that low-dosage UVA pre-irradiation (1.5 J cm−2) combined with UVC (0.085 J cm−2) reduces 85% more cell densities compared to UVC alone (0.085 J cm−2) and triggers mazEF-mediated regulated cell death (RCD), which led to cell lysis, while high-dosage UVA pre-irradiations (7.5 and 14.7 J cm−2) increase cell densities by 75–155%. Our oxygen evolution tests and transcriptomic analysis indicate that UVA pre-irradiation damages photosystem I (PSI) and, when combined with UVC-induced PSII damage, synergistically inhibits photosynthesis. However, higher UVA dosages activate the SOS response, facilitating the repair of UVC-induced DNA damage. This study highlights the impact of UVA pre-irradiation on UVC suppression of cyanobacteria and proposes a practical strategy for improved HCBs control.

Potential of β-D-glucan polysaccharide from Ophiocordyceps sinensis OS8 cultivated mycelium on anticancer activity via inducing liver cancer cell death apoptosis

The fungus Ophiocordyceps sinensis OS8 has been sourced from the wild variant of O. sinensis. In this study, the enriched β-D-glucan polysaccharide obtained from cultured mycelium of O. sinensis OS8 (OS8P) was examined for its potential to inhibit the growth of HepG2 liver cancer cells, and the IC50 was 511.79 µg/mL. The study of cellular migration was analyzed by scratch assay. It was found that the HepG2 cell migration was significantly inhibited (P < 0.05) in the OS8P-treated group. The cell apoptotic feature’s reliability was determined via DAPI staining to confirm the experimental results' reliability. The AO/PI double staining assay showed that HepG2 cells treated with OS8P (IC50) had dead cells with condensed and shrieking nuclei that are late apoptotic. Morphological alterations were meticulously examined using scanning electron microscopy. The specific morphological characteristics of apoptotic cells, notably cell lysis, were distinctly observable. The damaged cells demonstrated severe structural disruption, loss of original shape, blebs formation, and apoptotic bodies' appearance. These findings provide pharmacological proof that the OS8P extract could be potentially employed in the food and medical industries for anticancer applications.

Molecular mechanisms of precise timing in cell lysis

Many biological systems exhibit precise timing of events, and one of the most known examples is cell lysis, which is a process of breaking bacterial host cells in the virus infection cycle. However, the underlying microscopic picture of precise timing remains not well understood. We present a novel theoretical approach to explain the molecular mechanisms of effectively deterministic dynamics in biological systems. Our hypothesis is based on the idea of stochastic coupling between relevant underlying biophysical and biochemical processes that lead to noise cancellation. To test this hypothesis, we introduced a minimal discrete-state stochastic model to investigate how holin proteins produced by bacteriophages break the inner membranes of gram-negative bacteria. By explicitly solving this model, the dynamic properties of cell lysis are fully evaluated, and theoretical predictions quantitatively agree with available experimental data for both wild-type and holin mutants. It is found that the observed threshold-like behavior is a result of the balance between holin proteins entering the membrane and leaving the membrane during the lysis. Theoretical analysis suggests that the cell lysis achieves precise timing for wild-type species by maximizing the number of holins in the membrane and narrowing their spatial distribution. In contrast, for mutated species, these conditions are not satisfied. Our theoretical approach presents a possible molecular picture of precise dynamic regulation in intrinsically random biological processes.

Heterogeneous Integration of Acoustic Microextraction with an Optoelectronic Sensor on Glass for Nucleic Acid Testing.

Lab-on-a-chip systems (LOCs), characterized by their high sensitivity, low sample consumption, and portability, have significantly advanced the field of on-site testing. Despite the evolution of integrated LOCs from qualitative to quantitative analyses, on-chip full integration of sample preparation, purification, and multiplexed detection remains a challenge. Here, we propose a strategy for the heterogeneous integration of a set of complementary metal oxide semiconductor-compatible devices including acoustic resonator, thin-film resistors, and temperature/photosensors as a new type of LOC for nucleic acid testing (NAT). Programmed acoustic streaming-based particles and fluid manipulations largely simplify the nucleic acid extraction process including cell lysis, nucleic acid capture, and elution. The design of the acoustic microextraction module and extraction process was thoroughly studied. Benefitted by the microelectromechanical system approach, the conventional mechanical actions and complex flow control are avoided, which enables a compact hand-held NAT instrument without complicated peripherals. Validation experiments conducted on plasma-harboring mutations in the epidermal growth factor receptor (EGFR) gene confirmed the robustness of the system, achieving an impressive nucleic acid (NA) extraction efficiency of approximately 90% within 5 min and a limit of detection of the target NA in the plasma of 1 copy/μL.

Chloride Gradient Is Involved in Ammonium Influx in Human Erythrocytes.

The ammonia/ammonium (NH3/NH4+, AM) concentration in human erythrocytes (RBCs) is significantly higher than in plasma. Two main possible mechanisms for AM transport, including simple and facilitated diffusion, are described; however, the driving force for AM transport is not yet fully characterized. Since the erythroid ammonium channel RhAG forms a structural unit with anion exchanger 1 (eAE1) within the ankyrin core complex, we hypothesized the involvement of eAE1 in AM transport. To evaluate the functional interaction between eAE1 and RhAG, we used a unique feature of RBCs to swell and lyse in isotonic NH4+ buffer. The kinetics of cell swelling and lysis were analyzed by flow cytometry and an original laser diffraction method, adapted for accurate volume sensing. The eAE1 role was revealed according to (i) the changes in cell swelling and lysis kinetics, and (ii) changes in intracellular pH, triggered by eAE1 inhibition or the modulation of eAE1 main ligand concentrations (Cl- and HCO3-). Additionally, the AM import kinetics was analyzed enzymatically and colorimetrically. In NH4+ buffer, RBCs concentration-dependently swelled and lysed when [NH4+] exceeded 100 mM. Cell swelling and hemolysis were tightly regulated by chloride concentration. The complete substitution of chloride with glutamate prevented NH4+-induced cell swelling and hemolysis, and the restoration of [Cl-] dose-dependently amplified the rates of RBC swelling and lysis and the percentage of hemolyzed cells. Similarly, eAE1 inhibition impeded cell swelling and completely prevented hemolysis. Accordingly, eAE1 inhibition, or a lack of chloride anions in the buffer, significantly decreased NH4+ import. Our data indicate that the eAE1-mediated chloride gradient is required for AM transport. Taken together, our data reveal a new player in AM transport in RBCs.

A New Casjensviridae Bacteriophage Isolated from Hospital Sewage for Inactivation of Biofilms of Carbapenem Resistant Klebsiella pneumoniae Clinical Isolates.

Klebsiella pneumoniae, a member of the ESKAPE pathogen group, is a prominent cause of hospital-acquired infections. The WHO has recognized carbapenem-resistant K. pneumoniae as a critical-one priority pathogen. These resilient superbugs have the ability to form biofilms and present a significant global threat. In the present study, we isolated and characterized a bacteriophage SAKp02, from hospital sewage, infectious to carbapenem-resistant K. pneumoniae patient isolates. SAKp02 could infect 43 of 72 clinical isolates, indicating a broad host spectrum. Whole genome analysis classified SAKp02 within the family Casjensviridae, with a 59,343 bp genome encoding 82 ORFs. Comparative genomic analysis revealed significant differences between SAKp02 and its closest viruses, indicating a distinct genetic makeup positioning it as a novel phage strain within the lineage. The SAKp02 genome comprises bacteriolytic enzymes, including holin, endolysin, and phage depolymerase, crucial for bacterial lysis and biofilm disruption. It reduced biofilm biomass by over threefold compared to the control and eradicated 99% of viable cells within a 4 h treatment period. Scanning electron microscopy corroborated the ability of the phage to dismantle biofilm matrices and lyse bacterial cells. Safe and effective treatments are warranted, and hence, the fully characterized lytic phages with therapeutic potential against drug-resistant clinical isolates of bacteria are needed. Our study is the first to report the antibacterial and antibiofilm activity of Casjensviridae phages, and our discovery of a novel K. pneumoniae phage broadens the arsenal against the bacteria.