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1.
Molecules ; 29(17)2024 Aug 27.
Artículo en Inglés | MEDLINE | ID: mdl-39274911

RESUMEN

The cell wall is an indispensable element of bacterial cells and a long-known target of many antibiotics. Penicillin, the first discovered beta-lactam antibiotic inhibiting the synthesis of cell walls, was successfully used to cure many bacterial infections. Unfortunately, pathogens eventually developed resistance to it. This started an arms race, and while novel beta-lactams, either natural or (semi)synthetic, were discovered, soon upon their application, bacteria were developing resistance. Currently, we are facing the threat of losing the race since more and more multidrug-resistant (MDR) pathogens are emerging. Therefore, there is an urgent need for developing novel approaches to combat MDR bacteria. The cell wall is a reasonable candidate for a target as it differentiates not only bacterial and human cells but also has a specific composition unique to various groups of bacteria. This ensures the safety and specificity of novel antibacterial agents that target this structure. Due to the shortage of low-molecular-weight candidates for novel antibiotics, attention was focused on peptides and proteins that possess antibacterial activity. Here, we describe proteinaceous agents of various origins that target bacterial cell wall, including bacteriocins and phage and bacterial lysins, as alternatives to classic antibiotic candidates for antimicrobial drugs. Moreover, advancements in protein chemistry and engineering currently allow for the production of stable, specific, and effective drugs. Finally, we introduce the concept of selective targeting of dangerous pathogens, exemplified by staphylococci, by agents specifically disrupting their cell walls.


Asunto(s)
Antibacterianos , Pared Celular , Bacterias Grampositivas , Pared Celular/efectos de los fármacos , Antibacterianos/farmacología , Antibacterianos/química , Bacterias Grampositivas/efectos de los fármacos , Humanos , Bacteriocinas/farmacología , Bacteriocinas/química , Infecciones por Bacterias Grampositivas/tratamiento farmacológico , Infecciones por Bacterias Grampositivas/microbiología , Bacteriófagos
2.
Molecules ; 28(11)2023 May 29.
Artículo en Inglés | MEDLINE | ID: mdl-37298883

RESUMEN

Staphylococcus aureus is a common opportunistic pathogen of humans and livestock that causes a wide variety of infections. The success of S. aureus as a pathogen depends on the production of an array of virulence factors including cysteine proteases (staphopains)-major secreted proteases of certain strains of the bacterium. Here, we report the three-dimensional structure of staphopain C (ScpA2) of S. aureus, which shows the typical papain-like fold and uncovers a detailed molecular description of the active site. Because the protein is involved in the pathogenesis of a chicken disease, our work provides the foundation for inhibitor design and potential antimicrobial strategies against this pathogen.


Asunto(s)
Proteasas de Cisteína , Infecciones Estafilocócicas , Humanos , Staphylococcus aureus , Proteasas de Cisteína/metabolismo , Infecciones Estafilocócicas/microbiología , Papaína/metabolismo , Factores de Virulencia/metabolismo , Proteínas Bacterianas/química
3.
Molecules ; 28(9)2023 Apr 27.
Artículo en Inglés | MEDLINE | ID: mdl-37175180

RESUMEN

Anthracycline antibiotics (ANT) are among the most widely used anticancer drugs. Unfortunately, their use is limited due to the development of drug resistance and cardiotoxicity. ANT metabolism, performed mainly by two enzymes-aldo-keto reductase 1C3 (AKR1C3) and carbonyl reductase 1 (CBR1)-is one of the proposed mechanisms generated by the described effects. In this study, we evaluated the CBR1 inhibitory properties of ASP9521, a compound already known as potent AKR1C3 inhibitor. First, we assessed the possibility of ASP9521 binding to the CBR1 catalytic site using molecular docking and molecular dynamics. The research revealed a potential binding mode of ASP9521. Moderate inhibitory activity against CBR1 was observed in studies with recombinant enzymes. Finally, we examined whether ASP9521 can improve the cytotoxic activity of daunorubicin against human lung carcinoma cell line A549 and assessed the cardioprotective properties of ASP9521 in a rat cardiomyocytes model (H9c2) against doxorubicin- and daunorubicin-induced toxicity. The addition of ASP9521 ameliorated the cytotoxic activity of daunorubicin and protected rat cardiomyocytes from the cytotoxic effect of both applied drugs. Considering the favorable bioavailability and safety profile of ASP9521, the obtained results encourage further research. Inhibition of both AKR1C3 and CBR1 may be a promising method of overcoming ANT resistance and cardiotoxicity.


Asunto(s)
Antineoplásicos , Carbonil Reductasa (NADPH) , Humanos , Ratas , Animales , Simulación del Acoplamiento Molecular , Cardiotoxicidad , Antraciclinas/farmacología , Antraciclinas/metabolismo , Antibióticos Antineoplásicos/farmacología , Daunorrubicina/farmacología , Antineoplásicos/farmacología , Antibacterianos
4.
Cell Commun Signal ; 20(1): 10, 2022 01 20.
Artículo en Inglés | MEDLINE | ID: mdl-35057808

RESUMEN

BACKGROUND: A universal adaptor protein, MyD88, orchestrates the innate immune response by propagating signals from toll-like receptors (TLRs) and interleukin-1 receptor (IL-1R). Receptor activation seeds MyD88 dependent formation of a signal amplifying supramolecular organizing center (SMOC)-the myddosome. Alternatively spliced variant MyD88S, lacking the intermediate domain (ID), exhibits a dominant negative effect silencing the immune response, but the mechanistic understanding is limited. METHODS: Luciferase reporter assay was used to evaluate functionality of MyD88 variants and mutants. The dimerization potential of MyD88 variants and myddosome nucleation process were monitored by co-immunoprecipitation and confocal microscopy. The ID secondary structure was characterized in silico employing I-TASSER server and in vitro using nuclear magnetic resonance (NMR) and circular dichroism (CD). RESULTS: We show that MyD88S is recruited to the nucleating SMOC and inhibits its maturation by interfering with incorporation of additional components. Biophysical analysis suggests that important functional role of ID is not supported by a well-defined secondary structure. Mutagenesis identifies Tyr116 as the only essential residue within ID required for myddosome nucleation and signal propagation (NF-κB activation). CONCLUSIONS: Our results argue that the largely unstructured ID of MyD88 is not only a linker separating toll-interleukin-1 receptor (TIR) homology domain and death domain (DD), but contributes intermolecular interactions pivotal in MyD88-dependent signaling. The dominant negative effect of MyD88S relies on quenching the myddosome nucleation and associated signal transduction. Video abstract.


Asunto(s)
Quinasas Asociadas a Receptores de Interleucina-1 , Factor 88 de Diferenciación Mieloide/metabolismo , Línea Celular , Humanos , Quinasas Asociadas a Receptores de Interleucina-1/química , Quinasas Asociadas a Receptores de Interleucina-1/metabolismo , Factor 88 de Diferenciación Mieloide/genética , Estructura Terciaria de Proteína , Receptores de Interleucina-1/química , Receptores de Interleucina-1/metabolismo , Receptores Toll-Like/metabolismo
5.
Int J Mol Sci ; 23(12)2022 Jun 09.
Artículo en Inglés | MEDLINE | ID: mdl-35742885

RESUMEN

Whilst a large number of regulatory mechanisms for gene expression have been characterised to date, transcription regulation in bacteria still remains an open subject. In clinically relevant and opportunistic pathogens, such as Staphylococcus aureus, transcription regulation is of great importance for host-pathogen interactions. In our study we investigated an operon, exclusive to staphylococci, that we name saoABC. We showed that SaoC binds to a conserved sequence motif present upstream of the saoC gene, which likely provides a negative feedback loop. We have also demonstrated that S. aureus ΔsaoB and ΔsaoC mutants display altered growth dynamics in non-optimal media; ΔsaoC exhibits decreased intracellular survival in human dermal fibroblasts, whereas ΔsaoB produces an elevated number of persisters, which is also elicited by inducible production of SaoC in ΔsaoBΔsaoC double mutant. Moreover, we have observed changes in the expression of saoABC operon genes during either depletion of the preferential carbon or the amino acid source as well as during acidification. Comparative RNA-Seq of the wild type and ΔsaoC mutant demonstrated that SaoC influences transcription of genes involved in amino acid transport and metabolism, and notably of those coding for virulence factors. Our results suggest compellingly that saoABC operon codes for a DNA-binding protein SaoC, a novel staphylococcal transcription factor, and its antagonist SaoB. We linked SaoC to the response to nutrient deficiency, a stress that has a great impact on host-pathogen interactions. That impact manifests in SaoC influence on persister formation and survival during internalisation to host cells, as well as on the expression of genes of virulence factors that may potentially result in profound alternations in the pathogenic phenotype. Investigation of such novel regulatory mechanisms is crucial for our understanding of the dynamics of interactions between pathogenic bacteria and host cells, particularly in the case of clinically relevant, opportunistic pathogens such as Staphylococcus aureus.


Asunto(s)
Infecciones Estafilocócicas , Staphylococcus aureus , Aminoácidos/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación Bacteriana de la Expresión Génica , Humanos , Nutrientes , Operón/genética , Infecciones Estafilocócicas/microbiología , Staphylococcus/genética , Staphylococcus aureus/metabolismo , Factores de Virulencia/metabolismo
6.
Int J Mol Sci ; 22(4)2021 Feb 23.
Artículo en Inglés | MEDLINE | ID: mdl-33672341

RESUMEN

Accumulating evidence suggests that six proteases encoded in the spl operon of a dangerous human pathogen, Staphylococcus aureus, may play a role in virulence. Interestingly, SplA, B, D, and E have complementary substrate specificities while SplF remains to be characterized in this regard. Here, we describe the prerequisites of a heterologous expression system for active SplF protease and characterize the enzyme in terms of substrate specificity and its structural determinants. Substrate specificity of SplF is comprehensively profiled using combinatorial libraries of peptide substrates demonstrating strict preference for long aliphatic sidechains at the P1 subsite and significant selectivity for aromatic residues at P3. The crystal structure of SplF was provided at 1.7 Å resolution to define the structural basis of substrate specificity of SplF. The obtained results were compared and contrasted with the characteristics of other Spl proteases determined to date to conclude that the spl operon encodes a unique extracellular proteolytic system.


Asunto(s)
Péptido Hidrolasas/química , Péptido Hidrolasas/metabolismo , Staphylococcus aureus/enzimología , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cristalografía por Rayos X , Escherichia coli/genética , Metionina/metabolismo , Modelos Moleculares , Péptido Hidrolasas/genética , Péptidos/química , Péptidos/metabolismo , Especificidad por Sustrato
7.
Arch Biochem Biophys ; 671: 1-7, 2019 08 15.
Artículo en Inglés | MEDLINE | ID: mdl-31108049

RESUMEN

Maternal Embryonic Leucine Zipper Kinase (MELK) is overexpressed in various tumors which has been convincingly linked to tumor cell survival. As such, MELK became an interesting target for pharmacological intervention. In this study we present the crystal structure of MELK in complex with dorsomorphin, an inhibitor of VEGFR and AMPK. By defining the mechanistic details of ligand recognition we identify a key residue (Cys89) at the hinge region of MELK responsible for positioning of the ligand at the catalytic pocket. This conclusion is supported by kinetic characterization of Cys89 mutants which show decreased affinity towards both ATP and dorsomorphin. The detailed binding mode of dorsomorphin characterized in this study defines a minimal requirement for MELK ligands, a valuable information for future rational design of inhibitors based on entirely new scaffolds.


Asunto(s)
Inhibidores de Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Pirazoles/metabolismo , Pirimidinas/metabolismo , Dominio Catalítico , Cristalografía por Rayos X , Cisteína/química , Humanos , Estructura Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Unión Proteica , Proteínas Serina-Treonina Quinasas/química , Pirazoles/química , Pirimidinas/química
8.
J Virol ; 91(11)2017 06 01.
Artículo en Inglés | MEDLINE | ID: mdl-28331093

RESUMEN

Coronaviruses are responsible for upper and lower respiratory tract infections in humans. It is estimated that 1 to 10% of the population suffers annually from cold-like symptoms related to infection with human coronavirus NL63 (HCoV-NL63), an alphacoronavirus. The nucleocapsid (N) protein, the major structural component of the capsid, facilitates RNA packing, links the capsid to the envelope, and is also involved in multiple other processes, including viral replication and evasion of the immune system. Although the role of N protein in viral replication is relatively well described, no structural data are currently available regarding the N proteins of alphacoronaviruses. Moreover, our understanding of the mechanisms of RNA binding and nucleocapsid formation remains incomplete. In this study, we solved the crystal structures of the N- and C-terminal domains (NTD, residues 10 to 140, and CTD, residues 221 to 340, respectively) of the N protein of HCoV-NL63, both at a 1.5-Å resolution. Based on our structure of NTD solved here, we proposed and experimentally evaluated a model of RNA binding. The structure of the CTD reveals the mode of N protein dimerization. Overall, this study expands our understanding of the initial steps of N protein-nucleic acid interaction and may facilitate future efforts to control the associated infections.IMPORTANCE Coronaviruses are responsible for the common cold and other respiratory tract infections in humans. According to multiple studies, 1 to 10% of the population is infected each year with HCoV-NL63. Viruses are relatively simple organisms composed of a few proteins and the nucleic acids that carry the information determining their composition. The nucleocapsid (N) protein studied in this work protects the nucleic acid from the environmental factors during virus transmission. This study investigated the structural arrangement of N protein, explaining the first steps of its interaction with nucleic acid at the initial stages of virus structure assembly. The results expand our understanding of coronavirus physiology and may facilitate future efforts to control the associated infections.


Asunto(s)
Coronavirus Humano NL63/química , Proteínas de la Nucleocápside/química , Proteínas de la Nucleocápside/metabolismo , Coronavirus Humano NL63/fisiología , Cristalización , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Conformación Proteica , Multimerización de Proteína , ARN Viral/metabolismo , Ensamble de Virus , Replicación Viral
9.
Langmuir ; 32(22): 5653-62, 2016 06 07.
Artículo en Inglés | MEDLINE | ID: mdl-27124645

RESUMEN

The behavior, secondary structure, and orientation of a recently discovered bacteriocin-like peptide BacSp222 in a lipid model system supported at a gold electrode was investigated by chronocoulometry, polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS), and attenuated total reflectance infrared (ATR-IR) spectroscopy. The IR spectra show that the secondary structure of BacSp222 is predominantly α-helical. Analysis of the spectra in the amide I region shows that the α-helical fragment of the peptide is inserted into bilayer at the potential range at which the bilayer is stable and attached to the Au(111) surface, i.e., from -0.5 to 0.3 V vs Ag/AgCl. Insertion of BacSp222 to the membrane significantly changes the conformation of the acyl chains of lipid molecules, from all-trans to partially melted; however, the chains become less tilted. Based on these results, we propose that BacSp222 interacts with the DMPC bilayer through the barrel-stave pore formation. In this model, α-helix of BacSp222 inserts into the membrane with an angle between the α-helix axis and membrane normal equal to ∼18°. The changes in orientation of the α-helical fragment of the peptide indicate that the orientation of BacSp222 with respect to the bilayer surface is potential-dependent. The peptide is inserted into the membrane driven by the electrostatic field generated by negative charge at the metal surface. It is not inserted at negative potentials where the membrane is detached from the metal and no longer exposed to the electrostatic field of the metal.


Asunto(s)
Bacteriocinas/química , Membrana Dobles de Lípidos/química , Fosfolípidos/química , Estructura Secundaria de Proteína
10.
J Biol Chem ; 289(22): 15544-53, 2014 May 30.
Artículo en Inglés | MEDLINE | ID: mdl-24713703

RESUMEN

Staphylococcal SplB protease belongs to the chymotrypsin family. Chymotrypsin zymogen is activated by proteolytic processing at the N terminus, resulting in significant structural rearrangement at the active site. Here, we demonstrate that the molecular mechanism of SplB protease activation differs significantly and we characterize the novel mechanism in detail. Using peptide and protein substrates we show that the native signal peptide, or any N-terminal extension, has an inhibitory effect on SplB. Only precise N-terminal processing releases the full proteolytic activity of the wild type analogously to chymotrypsin. However, comparison of the crystal structures of mature SplB and a zymogen mimic show no rearrangement at the active site whatsoever. Instead, only the formation of a unique hydrogen bond network, distant form the active site, by the new N-terminal glutamic acid of mature SplB is observed. The importance of this network and influence of particular hydrogen bond interactions at the N terminus on the catalytic process is demonstrated by evaluating the kinetics of a series of mutants. The results allow us to propose a consistent model where changes in the overall protein dynamics rather than structural rearrangement of the active site are involved in the activation process.


Asunto(s)
Serina Proteasas/química , Serina Proteasas/metabolismo , Staphylococcus aureus/enzimología , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Dominio Catalítico , Quimotripsina/química , Quimotripsina/genética , Quimotripsina/metabolismo , Cristalografía por Rayos X , Precursores Enzimáticos/metabolismo , Enlace de Hidrógeno , Modelos Moleculares , Señales de Clasificación de Proteína/fisiología , Estructura Terciaria de Proteína , Serina Proteasas/genética , Staphylococcus aureus/genética , Relación Estructura-Actividad
11.
Chembiochem ; 16(14): 2036-45, 2015 Sep 21.
Artículo en Inglés | MEDLINE | ID: mdl-26212347

RESUMEN

Serine-proteinase-catalyzed peptide splicing was demonstrated in analogues of the trypsin inhibitor SFTI-1: both single peptides and two-peptide chains (C- and N-terminal peptide chains linked by a disulfide bridge). In the second series, peptide splicing with catalytic amount of proteinase was observed only when formation of acyl-enzyme intermediate was preceded by hydrolysis of the substrate Lys-Ser peptide bond. Here we demonstrate that with an equimolar amount of the proteinase, splicing occurs in all the two-peptide-chain analogues. This conclusion was supported by high resolution crystal structures of selected analogues in complex with trypsin. We showed that the process followed a direct transpeptidation mechanism. Thus, the acyl-enzyme intermediate was formed and was immediately used for a new peptide bond formation; products associated with the hydrolysis of the acyl-enzyme were not observed. The peptide splicing was sequence- not structure-specific.


Asunto(s)
Péptidos Cíclicos/química , Péptidos Cíclicos/farmacología , Péptidos/metabolismo , Inhibidores de Tripsina/química , Inhibidores de Tripsina/farmacología , Tripsina/metabolismo , Secuencia de Aminoácidos , Animales , Bovinos , Cristalografía por Rayos X , Helianthus/química , Modelos Moleculares , Datos de Secuencia Molecular , Péptidos/química , Péptidos Cíclicos/síntesis química , Serina Proteasas/síntesis química , Serina Proteasas/química , Serina Proteasas/farmacología , Tripsina/química , Inhibidores de Tripsina/síntesis química
12.
Int J Biol Macromol ; 280(Pt 4): 135510, 2024 Sep 18.
Artículo en Inglés | MEDLINE | ID: mdl-39304044

RESUMEN

Peroxisomal protein import has been identified as a valid target in trypanosomiases, an important health threat in Central and South America. The importomer is built of multiple peroxins (Pex) and structural characterization of these proteins facilitates rational inhibitor development. We report crystal structures of the Trypanosoma brucei and T. cruzi tetratricopeptide repeat domain (TPR) of the cytoplasmic peroxisomal targeting signal 1 (PTS1) receptor Pex5. The structure of the TPR domain of TbPex5 represents an apo-form of the receptor which, together with the previously determined structure of the complex of TbPex5 TPR and PTS1 demonstrate significant receptor dynamics associated with signal peptide recognition. The structure of the complex of TPR domain of TcPex5 with PTS1 provided in this study details the molecular interactions that guide signal peptide recognition at the atomic level in the pathogenic species currently perceived as the most relevant among Trypanosoma. Small - angle X - ray scattering (SAXS) data obtained in solution supports the crystallographic findings on the compaction of the TPR domains of TbPex5 and TcPex5 upon interaction with the cargo.

13.
Chem Res Toxicol ; 26(6): 841-52, 2013 Jun 17.
Artículo en Inglés | MEDLINE | ID: mdl-23621464

RESUMEN

Hepatotoxic microcystins that are produced by freshwater cyanobacteria pose a risk to public health. These compounds may be eliminated by enzymatic degradation. Here, we review the enzymatic pathways for the degradation of these hepatotoxins, some of which are newly discovered processes. The efficiencies of microcystin biodegradation pathways are documented in several papers and are compared here. Additionally, a comprehensive description of the microcystin enzymatic degradation scheme has been supplemented with a proposal for a new biodegradation pathway. Critical comments on less documented hypotheses are also included. The genetic aspects of biodegradation activity are discussed in detail. We also describe some methods that are useful for studying the biological decomposition of microcystins, including screening for microcystin degraders and detecting microcystin degradation products, with an emphasis on mass spectrometric methodology.


Asunto(s)
Microcistinas/metabolismo , Biodegradación Ambiental , Microcistinas/química
14.
Appl Microbiol Biotechnol ; 97(16): 7229-39, 2013 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-23196985

RESUMEN

Staphylococcus aureus strain CH-91, isolated from a broiler chicken with atopic dermatitis, has a highly proteolytic phenotype that is correlated with the disease. We describe the isolation and biochemical and molecular characterization of the AI-type lantibiotic BacCH91 from S. aureus CH-91 culture medium. The bacteriocin was purified using a three-stage procedure comprising precipitation with ammonium sulfate, extraction with organic solvents, and reversed-phase HPLC. The BacCH91 peptide is thermostable and highly resistant to cleavage by both prokaryotic and eukaryotic peptidases. The MIC for the Gram-positive bacteria ranged from 2.5 nM for Microococcus luteus through 1.3-6.0 µM for staphylococcal strains up to more than 100 µM for Lactococcus lactis. BacCH91 was ineffective against the Gram-negative strains tested at the maximal concentration (100 µM). The amino acid sequence of BacCH91 is similar to that of epidermin and gallidermin. The encoding gene (bacCH91) occurred in two allelic variants distinguishable in the restriction fragment length polymorphism assay. Variant I, identified in S. aureus CH-91, dominated in S. aureus strains of poultry origin, although strains with variant II were also identified in this group. S. aureus strains of human origin were characterized exclusively by variant II.


Asunto(s)
Bacteriocinas/farmacología , Staphylococcus aureus/metabolismo , Animales , Bacteriocinas/genética , Bacteriocinas/aislamiento & purificación , Bacteriocinas/metabolismo , Fraccionamiento Químico , Pollos , Cromatografía Líquida de Alta Presión , Clonación Molecular , ADN Bacteriano/química , ADN Bacteriano/genética , Lactococcus lactis/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Micrococcus luteus/efectos de los fármacos , Datos de Secuencia Molecular , Aves de Corral , Enfermedades de las Aves de Corral/microbiología , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Infecciones Estafilocócicas/microbiología , Infecciones Estafilocócicas/veterinaria , Staphylococcus aureus/genética , Staphylococcus aureus/aislamiento & purificación
15.
Chem Res Toxicol ; 25(6): 1192-4, 2012 Jun 18.
Artículo en Inglés | MEDLINE | ID: mdl-22591122

RESUMEN

The MlrC protein from Sphingomonas ACM-3962 strain was heterologously expressed in Escherichia coli strain BL21(DE3) and purified to investigate participation of this enzyme in the biodegradation of two microcystin variants. In contrast with previous reports, our results indicated that MlrC cleaves linear microcystins, thus shedding new light on the role of MlrC enzyme in microcystin biodegradation.


Asunto(s)
Proteínas Bacterianas/metabolismo , Microcistinas/metabolismo , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Microcistinas/química , Reproducibilidad de los Resultados , Sphingomonas , Factores de Tiempo
16.
Life Sci ; 305: 120777, 2022 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-35792180

RESUMEN

Doxorubicin (DOX) is classified by World Health Organization (WHO) as an essential medicine for cancer. However, its clinical application is limited due to resistance development and cardiotoxicity. Many attempts have been made to address these issues with some focused on finding a potential adjuvant therapy. Recently, inhibition of carbonyl reduction of anthracyclines (ANTs), catalyzed by enzymes from carbonyl reductase (CBR) and aldo-keto reductase (AKR) families, emerged as a potential way to simultaneously bypass cancer resistance and alleviate cardiotoxicity of ANTs. In this context, we evaluated the potential application of l synthetic cinnamic acid derivatives (CA) - 1a (2E)-3-(4- chlorophenyl)-1-(4-hydroxypiperidin-1-yl)prop-2-en-1 and 1b (2E)-1-(4-hydroxypiperidin-1-yl)-3-(2-methylphenyl)prop-2-en-1-one. The tested compounds were found to chemosensitize A549 human lung cancer cell line towards DOX-induced viability reduction and apoptosis, while having no effect in non-cancerous lung fibroblasts. Co-treatment with DOX + 1a/1b significantly inhibited the migration of A549 in a Transwell assay. The addition of 1a/1b alleviated menadione-induced viability reduction in H9c2 rat cardiomyoblast cell line. Accordingly, 1a/1b reduced DOX-induced reactive oxygen species (ROS) generation and increased glutathione levels. The compounds were also found to moderate autophagy process and limit inflammatory response in RAW 264.7 macrophage cell line. Inhibitory properties of the compounds towards CBR1 were simulated by molecular modeling and confirmed in vitro in enzyme inhibition assay with recombinant CBR1 protein. In contrast to 1b, 1a has strong CBR1 inhibition, which correlates well with more profound effect elicited by 1a uniformly throughout the other experiments. Finally, no mutagenic, genotoxic or hepatotoxic activity of the compounds were found. The possible products of cytochrome P450 mediated metabolism of 1a and 1b were also established to evaluate the potential impact of first pass effect. Our results suggest that 1a and 1b are promising candidates for DOX adjuvant therapy that may simultaneously chemosensitize cancer cells and alleviate cardiotoxicity. The higher activity of 1a may be linked with CBR1 inhibition.


Asunto(s)
Miocitos Cardíacos , Neoplasias , Oxidorreductasas de Alcohol , Animales , Antibióticos Antineoplásicos/metabolismo , Antibióticos Antineoplásicos/toxicidad , Cardiotoxicidad/metabolismo , Cardiotoxicidad/prevención & control , Cinamatos , Doxorrubicina/toxicidad , Humanos , Miocitos Cardíacos/metabolismo , Neoplasias/metabolismo , Ratas
17.
Genes (Basel) ; 12(5)2021 05 18.
Artículo en Inglés | MEDLINE | ID: mdl-34070083

RESUMEN

Type I toxin-antitoxin (TA) systems are widespread genetic modules in bacterial genomes. They express toxic peptides whose overexpression leads to growth arrest or cell death, whereas antitoxins regulate the expression of toxins, acting as labile antisense RNAs. The Staphylococcus aureus (S. aureus) genome contains and expresses several functional type I TA systems, but their biological functions remain unclear. Here, we addressed and challenged experimentally, by proteomics, if the type I TA system, the SprG1/SprF1 pair, influences the overall gene expression in S. aureus. Deleted and complemented S. aureus strains were analyzed for their proteomes, both intracellular and extracellular, during growth. Comparison of intracellular proteomes among the strains points to the SprF1 antitoxin as moderately downregulating protein expression. In the strain naturally expressing the SprG1 toxin, cytoplasmic proteins are excreted into the medium, but this is not due to unspecific cell leakages. Such a toxin-driven release of the cytoplasmic proteins may modulate the host inflammatory response that, in turn, could amplify the S. aureus infection spread.


Asunto(s)
Antitoxinas/genética , Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica/genética , Expresión Génica/genética , Staphylococcus aureus/genética , Sistemas Toxina-Antitoxina/genética , Citoplasma/genética , Genoma Bacteriano/genética , Proteoma/genética , ARN sin Sentido/genética
18.
Poult Sci ; 100(6): 101119, 2021 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-33975035

RESUMEN

Staphylococcus aureus bacteria are components of physiological biocenosis of skin or mucous membranes in some animals' genera but also they are dangerous opportunistic pathogens responsible for infections of various localization, course or manifestations. Proteins produced by these bacteria destroy tissues, leukocytes and cause haemolysis of erythrocytes. Host organisms respond by defence mechanisms. Production of heat stress proteins (HSPs) is one of defence responses of infected host organism. To evaluate infection and host defence mechanisms some animal models of experimental infection are reported. Use of chick embryo model allows demonstrating adequate differences in staphylococcal virulence depending on the strain genotype. The aim of the study was to examine the changes in heat shock protein HSP70 levels in chick embryo tissues after infection caused by S. aureus strains no. tu2, pa3, ch5, ch10, ch24, and ch25 isolated from chickens. The bacteria were injected directly into fluid of amnion cavity and incubated for 10 days. The mortality of particular chick embryos was reported and the tissues for further analysis were taken every day from day 13 to day 19. The levels of heat stress protein HSP70 were determined by dot-blot method. Results showed that the strains no. ch5, ch24, and ch25 were the most virulent. HSP70 levels increased in all groups of injected embryos at the same time the hatching process was started. The presented study showed that the infected chick embryos were characterized by higher HSP level from 12.3% up to 19.7% compared to the control group. The morphological analysis showed numerous erythrocytes with damaged cell membranes and morphological changes of erythrocytes. Changes in the level of HSP70 protein can be a useful indicator of infection caused by S. aureus bacteria. Additionally, chicken embryo is a helpful research model in studies of pathogenesis of diseases caused by bacteria.


Asunto(s)
Pollos , Proteínas HSP70 de Choque Térmico , Infecciones Estafilocócicas , Animales , Proteínas Aviares/genética , Embrión de Pollo , Proteínas HSP70 de Choque Térmico/genética , Infecciones Estafilocócicas/veterinaria , Staphylococcus aureus
19.
Int J Biol Macromol ; 183: 852-860, 2021 Jul 31.
Artículo en Inglés | MEDLINE | ID: mdl-33932416

RESUMEN

Growing antibiotic resistance of bacteria is a burning problem of human and veterinary medicine. Expansion and introduction of novel microbicidal therapeutics is highly desirable. However, antibiotic treatment disturbs the balance of physiological microbiota by changing its qualitative and/or quantitative composition, resulting in a number of adverse effects that include secondary infections. Although such dysbiosis may be reversed by the treatment with probiotics, a more attractive alternative is the use of antibiotics that target only pathogens, while sparing the commensals. Here, we describe lysostaphin LSp222, an enzyme produced naturally by Staphylococcus pseudintermedius 222. LSp222 is highly effective against S. aureus, including its multi-drug resistant strains. Importantly, the inhibitory concentration for S. epidermidis, the predominant commensal in healthy human skin, is at least two orders of magnitude higher compared to S. aureus. Such significant therapeutic window makes LSp222 a microbiota-friendly antibacterial agent with a potential application in the treatment of S. aureus-driven skin infections.


Asunto(s)
Lisostafina/farmacología , Microbiota/efectos de los fármacos , Piel/microbiología , Staphylococcus/enzimología , Farmacorresistencia Bacteriana/efectos de los fármacos , Humanos , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Piel/efectos de los fármacos , Staphylococcus epidermidis/efectos de los fármacos
20.
Pol J Microbiol ; 59(2): 133-5, 2010.
Artículo en Inglés | MEDLINE | ID: mdl-20734760

RESUMEN

We have reported a bacterial infection in a dog with progressive dysplasia of the hips. Orthopedic surgery was performed. Seven weeks prior to the surgery, the patient was bitten by another dog. The postimplantation wound exuded for four days after the surgery. Microbiological analysis performed by standard identification techniques showed the presence of Staphylococcus intermedius, but an additional molecular analysis indicated S. pseudintermedius. This was followed by an evaluation of antibiotic susceptibility of the strain which showed cefoxitin, ciprofloxacin, clindamycin, trimethoprim/sulfamethoxazole, doksycycline, erythromycin, and gentamicin resistance. Minimal inhibitory concentration (MIC) values for selected antibiotics were reported. Resistance for cefoxitin indicates that methicillin resistant S. pseudintermedius (MRSP) strains were present in individual macroorganisms, but they can expand and persist the colonization of other hosts.


Asunto(s)
Artroplastia de Reemplazo/efectos adversos , Enfermedades de los Perros/microbiología , Complicaciones Posoperatorias/veterinaria , Infecciones Estafilocócicas/veterinaria , Staphylococcus/aislamiento & purificación , Animales , Antibacterianos/farmacología , Artroplastia de Reemplazo/veterinaria , Enfermedades de los Perros/cirugía , Perros , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Staphylococcus aureus Resistente a Meticilina/genética , Staphylococcus aureus Resistente a Meticilina/aislamiento & purificación , Complicaciones Posoperatorias/microbiología , Infecciones Estafilocócicas/microbiología , Staphylococcus/efectos de los fármacos , Staphylococcus/genética
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