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Medical Microbiology and Immunology Jun 2024Endogenous antimicrobial peptides (AMPs) play a key role in the host defense against pathogens. AMPs attack pathogens preferentially at the site of entry to prevent...
Endogenous antimicrobial peptides (AMPs) play a key role in the host defense against pathogens. AMPs attack pathogens preferentially at the site of entry to prevent invasive infection. Mycobacterium tuberculosis (Mtb) enters its host via the airways. AMPs released into the airways are therefore likely candidates to contribute to the clearance of Mtb immediately after infection. Since lysozyme is detectable in airway secretions, we evaluated its antimicrobial activity against Mtb. We demonstrate that lysozyme inhibits the growth of extracellular Mtb, including isoniazid-resistant strains. Lysozyme also inhibited the growth of non-tuberculous mycobacteria. Even though lysozyme entered Mtb-infected human macrophages and co-localized with the pathogen we did not observe antimicrobial activity. This observation was unlikely related to the large size of lysozyme (14.74 kDa) because a smaller lysozyme-derived peptide also co-localized with Mtb without affecting the viability. To evaluate whether the activity of lysozyme against extracellular Mtb could be relevant in vivo, we incubated Mtb with fractions of human serum and screened for antimicrobial activity. After several rounds of sub-fractionation, we identified a highly active fraction-component as lysozyme by mass spectrometry. In summary, our results identify lysozyme as an antimycobacterial protein that is detectable as an active compound in human serum. Our results demonstrate that the activity of AMPs against extracellular bacilli does not predict efficacy against intracellular pathogens despite co-localization within the macrophage. Ongoing experiments are designed to unravel peptide modifications that occur in the intracellular space and interfere with the deleterious activity of lysozyme in the extracellular environment.
Topics: Muramidase; Humans; Mycobacterium tuberculosis; Macrophages; Antimicrobial Peptides; Microbial Sensitivity Tests; Microbial Viability
PubMed: 38900248
DOI: 10.1007/s00430-024-00793-0 -
MAbs 2024Over the past two decades, therapeutic antibodies have emerged as a rapidly expanding domain within the field of biologics. tools that can streamline the process of...
Over the past two decades, therapeutic antibodies have emerged as a rapidly expanding domain within the field of biologics. tools that can streamline the process of antibody discovery and optimization are critical to support a pipeline that is growing more numerous and complex every year. High-quality structural information remains critical for the antibody optimization process, but antibody-antigen complex structures are often unavailable and antibody docking methods are still unreliable. In this study, DeepAb, a deep learning model for predicting antibody Fv structure directly from sequence, was used in conjunction with single-point experimental deep mutational scanning (DMS) enrichment data to design 200 potentially optimized variants of an anti-hen egg lysozyme (HEL) antibody. We sought to determine whether DeepAb-designed variants containing combinations of beneficial mutations from the DMS exhibit enhanced thermostability and whether this optimization affected their developability profile. The 200 variants were produced through a robust high-throughput method and tested for thermal and colloidal stability (T, T, T), affinity (K) relative to the parental antibody, and for developability parameters (nonspecific binding, aggregation propensity, self-association). Of the designed clones, 91% and 94% exhibited increased thermal and colloidal stability and affinity, respectively. Of these, 10% showed a significantly increased affinity for HEL (5- to 21-fold increase) and thermostability (>2.5C increase in T), with most clones retaining the favorable developability profile of the parental antibody. Additional tests suggest that these methods would enrich for binding affinity even without first collecting experimental DMS measurements. These data open the possibility of antibody optimization without the need to predict the antibody-antigen interface, which is notoriously difficult in the absence of crystal structures.
Topics: Muramidase; Antibody Affinity; Protein Stability; Humans; Antigens; Animals; Computer Simulation
PubMed: 38899735
DOI: 10.1080/19420862.2024.2362775 -
International Journal of Molecular... May 2024This work unfolds functionalized ABSs composed of FILs ([CCIm][CFSO] and [N1112(OH)][CFSO]), mere fluoro-containing ILs ([CCIm][CFSO] and [CCIm][CFSO]), known globular...
This work unfolds functionalized ABSs composed of FILs ([CCIm][CFSO] and [N1112(OH)][CFSO]), mere fluoro-containing ILs ([CCIm][CFSO] and [CCIm][CFSO]), known globular protein stabilizers (sucrose and [N1112(OH)][CFSO]), low-molecular-weight carbohydrate (glucose), and even high-charge density salt (KPO). The ternary phase diagrams were determined, stressing that FILs highly increased the ability for ABS formation. The functionalized ABSs (FILs vs. mere fluoro-containing ILs) were used to extract lysozyme (Lys). The ABSs' biphasic regions were screened in terms of protein biocompatibility, analyzing the impact of ABS phase-forming components in Lys by UV-VIS spectrophotometry, CD spectroscopy, fluorescence spectroscopy, DSC, and enzyme assay. Lys partition behavior was characterized in terms of extraction efficiency (% EE). The structure, stability, and function of Lys were maintained or improved throughout the extraction step, as evaluated by CD spectroscopy, DSC, enzyme assay, and SDS-PAGE. Overall, FIL-based ABSs are more versatile and amenable to being tuned by the adequate choice of the phase-forming components and selecting the enriched phase. Binding studies between Lys and ABS phase-forming components were attained by MST, demonstrating the strong interaction between Lys and FILs aggregates. Two of the FIL-based ABSs (30 %wt [CCIm][CFSO] + 2 %wt KPO and 30 %wt [CCIm][CFSO] + 25 %wt sucrose) allowed the simultaneous purification of Lys and BSA in a single ABS extraction step with high yield (extraction efficiency up to 100%) for both proteins. The purity of both recovered proteins was validated by SDS-PAGE analysis. Even with a high-charge density salt, the FIL-based ABSs developed in this work seem more amenable to be tuned. Lys and BSA were purified through selective partition to opposite phases in a single FIL-based ABS extraction step. FIL-based ABSs are proposed as an improved extraction step for proteins, based on their biocompatibility, customizable properties, and selectivity.
Topics: Ionic Liquids; Muramidase; Halogenation; Water; Proteins; Animals
PubMed: 38891953
DOI: 10.3390/ijms25115766 -
Microbial Cell Factories Jun 2024Human lysozyme (hLYZ) is a natural antibacterial protein with broad applications in food and pharmaceutical industries. Recombinant production of hLYZ in Komagataella...
BACKGROUND
Human lysozyme (hLYZ) is a natural antibacterial protein with broad applications in food and pharmaceutical industries. Recombinant production of hLYZ in Komagataella phaffii (K. phaffii) has attracted considerable attention, but there are very limited strategies for its hyper-production in yeast.
RESULTS
Here through Atmospheric and Room Temperature Plasma (ARTP)-based mutagenesis and transcriptomic analysis, the expression of two genes MYO1 and IQG1 encoding the cytokinesis core proteins was identified downregulated along with higher hLYZ production. Deletion of either gene caused severe cytokinesis defects, but significantly enhanced hLYZ production. The highest hLYZ yield of 1,052,444 ± 23,667 U/mL bioactivity and 4.12 ± 0.11 g/L total protein concentration were obtained after high-density fed-batch fermentation in the Δmyo1 mutant, representing the best production of hLYZ in yeast. Furthermore, O-linked mannose glycans were characterized on this recombinant hLYZ.
CONCLUSIONS
Our work suggests that cytokinesis-based morphology engineering is an effective way to enhance the production of hLYZ in K. phaffii.
Topics: Muramidase; Recombinant Proteins; Saccharomycetales; Humans; Fermentation; Cytokinesis; Metabolic Engineering; Batch Cell Culture Techniques
PubMed: 38890717
DOI: 10.1186/s12934-024-02434-w -
BMC Veterinary Research Jun 2024Antimicrobial resistance (AMR) is nowadays a major emerging challenge for public health worldwide. The over- and misuse of antibiotics, including those for cell culture,...
BACKGROUND
Antimicrobial resistance (AMR) is nowadays a major emerging challenge for public health worldwide. The over- and misuse of antibiotics, including those for cell culture, are promoting AMR while also encouraging the research and employment of alternative drugs. The addition of antibiotics to the cell media is strongly recommended in sperm preservation, being gentamicin the most used for boar semen. Because of its continued use, several bacterial strains present in boar semen have developed resistance to this antibiotic. Antimicrobial peptides and proteins (AMPPs) are promising candidates as alternative antibiotics because their mechanism of action is less likely to promote AMR. In the present study, we tested two AMPPs (lysozyme and nisin; 50 and 500 µg/mL) as possible substitutes of gentamicin for boar semen preservation up to 48 h of storage.
RESULTS
We found that both AMPPs improved sperm plasma membrane and acrosome integrity during semen storage. The highest concentration tested for lysozyme also kept the remaining sperm parameters unaltered, at 48 h of semen storage, and reduced the bacterial load at comparable levels of the samples supplemented with gentamicin (p > 0.05). On the other hand, while nisin (500 µg/mL) reduced the total Enterobacteriaceae counts, it also decreased the rapid and progressive sperm population and the seminal oxidation-reduction potential (p < 0.05).
CONCLUSIONS
The protective effect of lysozyme on sperm function together with its antimicrobial activity and inborn presence in body fluids, including semen and cervical mucus, makes this enzyme a promising antimicrobial agent for boar semen preservation.
Topics: Animals; Semen Preservation; Male; Anti-Bacterial Agents; Swine; Muramidase; Nisin; Semen; Spermatozoa; Antimicrobial Peptides; Cell Membrane; Gentamicins; Acrosome
PubMed: 38867200
DOI: 10.1186/s12917-024-04105-9 -
Bioconjugate Chemistry Jun 2024Bioconjugation of polymers to proteins is a method to impart improved stability and pharmacokinetic properties to biologic systems. However, the precise effects of... (Comparative Study)
Comparative Study
Bioconjugation of polymers to proteins is a method to impart improved stability and pharmacokinetic properties to biologic systems. However, the precise effects of polymer architecture on the resulting bioconjugates are not well understood. Particularly, cyclic polymers are known to possess unique features such as a decreased hydrodynamic radius when compared to their linear counterparts of the same molecular weight, but have not yet been studied. Here, we report the first bioconjugation of a cyclic polymer, poly(ethylene glycol) (PEG), to a model protein, T4 lysozyme, containing a single engineered cysteine residue (V131C). We compare the stability and activity of this conjugate with those of a linear PEG-T4 lysozyme analogue of similar molecular weight. Furthermore, we used molecular dynamics (MD) simulations to determine the behavior of the polymer-protein conjugates in solution. We introduce cyclic polymer-protein conjugates as potential candidates for the improvement of biologic therapeutics.
Topics: Polyethylene Glycols; Muramidase; Molecular Dynamics Simulation; Bacteriophage T4
PubMed: 38809040
DOI: 10.1021/acs.bioconjchem.4c00202 -
Nutrients May 2024Human milk (HM) contains the essential macronutrients and bioactive compounds necessary for the normal growth and development of newborns. The milk collected by human...
Human milk (HM) contains the essential macronutrients and bioactive compounds necessary for the normal growth and development of newborns. The milk collected by human milk banks is stored frozen and pasteurized, reducing its nutritional and biological value. The purpose of this study was to determine the effect of hyperbaric storage at subzero temperatures (HS-ST) on the macronutrients and bioactive proteins in HM. As control samples, HM was stored at the same temperatures under 0.1 MPa. A Miris HM analyzer was used to determine the macronutrients and the energy value. The lactoferrin (LF), lysozyme (LYZ) and lactalbumin (LAC) content was checked using high-performance liquid chromatography, and an ELISA test was used to quantify secretory immunoglobulin A (sIgA). The results showed that the macronutrient content did not change significantly after 90 days of storage at 60 MPa/-5 °C, 78 MPa/-7 °C, 111 MPa/-10 °C or 130 MPa/-12 °C. Retention higher than 90% of LYZ, LAC, LF and sIgA was observed in the HM stored at conditions of up to 111 MPa/-10 °C. However, at 130 MPa/-12 °C, there was a reduction in LYZ and LF, by 39 and 89%, respectively. The storage of HM at subzero temperatures at 0.1 MPa did not affect the content of carbohydrates or crude and true protein. For fat and the energy value, significant decreases were observed at -5 °C after 90 days of storage.
Topics: Humans; Milk, Human; Nutritive Value; Lactoferrin; Food Storage; Muramidase; Lactalbumin; Immunoglobulin A, Secretory; Nutrients; Milk Proteins; Female
PubMed: 38794693
DOI: 10.3390/nu16101455 -
Gut Microbes 2024Chemotherapy remains a major treatment for malignant tumors, yet the application of standard dose intensity chemotherapy is limited due to the side effects of cytotoxic...
Chemotherapy remains a major treatment for malignant tumors, yet the application of standard dose intensity chemotherapy is limited due to the side effects of cytotoxic drugs, especially in old populations. The underlying mechanisms of cytotoxicity and strategies to increase the safety and tolerance of chemotherapy remain to be explored. Using 5-fluorouracil (5-FU), a cornerstone chemotherapeutic drug, we demonstrate that the main cause of death in (AL) fed mice after 5-FU chemotherapy was infection caused by translocation of intestinal opportunistic pathogens. We show that these opportunistic pathogens greatly increase in the intestine after chemotherapy, which was closely related to loss of intestinal lysozyme. Of note, two weeks of dietary restriction (DR) prior to chemotherapy significantly protected the loss of lysozyme and increased the content of the beneficial genera, resulting in a substantial inhibition of intestinal opportunistic pathogens and their translocation. The rescue effect of DR could be mimicked by Lysozyme or gavage. Our study provides the first evidence that DR achieved a comprehensive protection of the intestinal physical, biological and chemical barriers, which significantly improved the overall survival of 5-FU-treated mice. Importantly, the above findings were more prominent in old mice. Furthermore, we show that patients over 65 years old have enriched opportunistic pathogens in their gut microbiota, especially after 5-FU based chemotherapy. Our study reveals important mechanisms for the poor chemotherapy tolerance of the elderly population, which can be significantly improved by short-term DR. This study generates new insights into methods for improving the chemotherapeutic prognosis by increasing the chemotherapy tolerance and safety of patients with malignant tumors.
Topics: Animals; Fluorouracil; Mice; Bacterial Translocation; Gastrointestinal Microbiome; Humans; Intestines; Muramidase; Caloric Restriction; Mice, Inbred C57BL; Male; Lactobacillus; Bacteria; Female; Opportunistic Infections
PubMed: 38780487
DOI: 10.1080/19490976.2024.2355693 -
International Journal of Pharmaceutics Jun 2024Amino acids (AAs) have been used as excipients in protein formulations both in solid and liquid state products due to their stabilizing effect. However, the mechanisms...
Amino acids (AAs) have been used as excipients in protein formulations both in solid and liquid state products due to their stabilizing effect. However, the mechanisms by which they can stabilize a protein have not been fully elucidated yet. The purpose of this study was to investigate the effect of AAs with distinct physicochemical properties on the stability of a model protein (lysozyme, LZM) during the spray-drying process and subsequent storage. Molecular descriptor based multivariate data analysis was used to select distinct AAs from the group of 20 natural AAs. Then, LZM and the five selected AAs (1:1 wt ratio) were spray-dried (SD). The solid form, residual moisture content (RMC), hygroscopicity, morphology, secondary/tertiary structure and enzymatic activity of LZM were evaluated before and after storage under 40 °C/75 % RH for 30 days. Arginine (Arg), leucine (Leu), glycine (Gly), tryptophan (Trp), aspartic acid (Asp) were selected because of their distinct properties by using principal component analysis (PCA). The SD LZM powders containing Arg, Trp, or Asp were amorphous, while SD LZM powders containing Leu or Gly were crystalline. Recrystallization of Arg, Trp, Asp and polymorph transition of Gly were observed after the storage under accelerated conditions. The morphologies of the SD particles vary upon the different AAs formulated with LZM, implying different drying kinetics of the five model systems. A tertiary structural change of LZM was observed in the SD powder containing Arg, while a decrease in the enzymatic activity of LZM was observed in the powders containing Arg or Asp after the storage. This can be attributed to the extremely basic and acidic conditions that Arg and Asp create, respectively. This study suggests that when AAs are used as stabilizers instead of traditional disaccharides, not only do classic vitrification theory and water replacement theory play a role, but the microenvironmental pH conditions created by basic or acidic AAs in the starting solution or during the storage of solid matter are also crucial for the stability of SD protein products.
Topics: Muramidase; Amino Acids; Excipients; Drug Storage; Spray Drying; Powders; Drug Stability; Wettability; Chemistry, Pharmaceutical
PubMed: 38734275
DOI: 10.1016/j.ijpharm.2024.124217 -
The Journal of Physical Chemistry. B May 2024The disaccharide trehalose is generally acknowledged as a superior stabilizer of proteins and other biomolecules in aqueous environments. Despite many theories aiming to... (Comparative Study)
Comparative Study
The disaccharide trehalose is generally acknowledged as a superior stabilizer of proteins and other biomolecules in aqueous environments. Despite many theories aiming to explain this, the stabilization mechanism is still far from being fully understood. This study compares the stabilizing properties of trehalose with those of the structurally similar disaccharide sucrose. The stability has been evaluated for the two proteins, lysozyme and myoglobin, at both low and high temperatures by determining the glass transition temperature, , and the denaturation temperature, . The results show that the sucrose-containing samples exhibit higher than the corresponding trehalose-containing samples, particularly at low water contents. The better stabilizing effect of sucrose at high temperatures may be explained by the fact that sucrose, to a greater extent, binds directly to the protein surface compared to trehalose. Both sugars show elevation with an increasing sugar-to-protein ratio, which allows for a more complete sugar shell around the protein molecules. Finally, no synergistic effects were found by combining trehalose and sucrose. Conclusively, the exact mechanism of protein stabilization may vary with the temperature, as influenced by temperature-dependent interactions between the protein, sugar, and water. This variability can make trehalose to a superior stabilizer under some conditions and sucrose under others.
Topics: Trehalose; Sucrose; Muramidase; Calorimetry, Differential Scanning; Myoglobin; Protein Stability; Animals; Temperature
PubMed: 38733344
DOI: 10.1021/acs.jpcb.4c00022