RESUMEN
Gram-negative pathogens that produce ß-lactamases pose a serious public health threat as they can render ß-lactam antibiotics inactive via hydrolysis. This action contributes to the waning effectiveness of clinical antibiotics and creates an urgent need for new antimicrobials. Antimicrobial peptides (AMPs) exhibiting multimodal functions serve as a potential source in spite of a few limitations. Thus, the conjugation of conventional antibiotics with AMPs may be an effective strategy to leverage the advantages of each component. In this study, we conjugated meropenem to the AMP Tilapia piscidin 4 (TP4) using a typical coupling reaction. The conjugate was characterized by using HPLC-MS, HR-MS, and MS-MS fragmentation analysis. It was then evaluated in terms of antibacterial potency, hemolysis, and cytotoxicity toward RAW264.7 and CCD-966SK cell lines. The conjugation of meropenem with TP4 significantly reduced the cytotoxicity compared to TP4. Conjugation of unprotected TP4 with meropenem resulted in cross-linking at the N-terminal and lysine sites. The structural activity relationship of the two isomers of the TP4-meropenem conjugate was investigated. Both the isomers showed notable antibacterial activities against NDM-1 Escherichia coli and reduced red blood cell hemolysis as compared to TP4. Lysine conjugate (TP4-K-Mero) showed lesser hemolysis than the N-terminal conjugate (TP4-N-Mero). Molecular modeling further revealed that the conjugates can bind to lipopolysaccharides and inhibit NDM-1 ß-lactamase. Together, these data show that conjugation of antibiotics with AMP can be a feasible approach to increase the therapeutic profile and effectively target multidrug-resistant pathogens. Furthermore, antibiotic conjugation at different AMP sites tends to show unique biological properties.
RESUMEN
Antibiotic therapy is the primary treatment for bovine mastitis, but the drawbacks of this strategy include poor cure rate and economic losses from the need to discard milk with antibiotic residues. Unfortunately, few other treatment options are currently available for mastitis. Failure of antibiotic treatments is often attributed to formation of bacterial biofilms and abscesses in the mammary gland tissue, which lead to chronic infections that are difficult to eradicate and drive recurrent disease. A major mastitis-causing pathogen (MCP) associated with biofilms in bovine mastitis is Staphylococcus aureus. In this study, we demonstrate that octanoic acid has broad-spectrum microbicidal activity against MCPs and effectively inhibits S. aureus biofilm formation in milk (>50% inhibition at 3.13 mM). Octanoic acid effectively clears biofilms (95% eradication at 1X minimum bactericidal concentration, MBC) and infrequently induces S. aureus small colony variants (SCVs) that may cause recurrent mastitis. Additionally, octanoic acid rapidly kills persistent biofilm cells and cells with antibiotic tolerance (within 4 h). In contrast, antibiotics treated at >100X MBC cannot eradicate biofilms but do induce SCVs and antibiotic-tolerant cells. These effects may accelerate the transition from biofilm to chronic infection. Thus, octanoic acid exhibits bactericidal action against S. aureus biofilms, and it is less likely than antibiotic therapy to induce persistent cells and pathogen tolerance. Moreover, octanoic acid acts additively with antibiotics against S. aureus, and it attenuates tetracycline-induced virulence factor gene expression in S. aureus cells. According to these data, octanoic acid may prevent the pathological progression of bovine mastitis and offer a new strategy for treating the condition.
RESUMEN
Antimicrobial peptides (AMPs) are short and positively charged peptides with broad-spectrum antimicrobial activities. AMPs have been investigated as potential antibiotic alternatives to improve growth performance and prevent pathogen infection in the poultry industry. The antimicrobial peptide tilapia piscidin 4 (TP4) was derived from Oreochromis niloticus, possesses antimicrobial activities and immunomodulatory properties, promotes intestinal health, and protects against pathogen infection. The codon-optimized sequence of TP4 was introduced into the pPICZαA vector and transformed into Pichia pastoris. Large-scale expression was induced following culture with methanol in a 500-liter fermenter. Freeze drying of fermented rTP4 broth and then rTP4 evaluation as a feed additive for Gallus gallus domesticus were performed. The in vitro antimicrobial activity of recombinant TP4 (rTP4) against gram-positive and gram-negative pathogens was evaluated. Evaluation of the effect of temperature on the antimicrobial activity of rTP4 showed its high stability at high temperatures. rTP4 significantly enhanced the phagocytic activity of macrophage cells, indicating that rTP4 has a remarkable ability to stimulate macrophages. rTP4 was used as a dietary supplement at 0.75, 1.5, 3.0, 6.0 and 12% in G. g. domesticus for five weeks, and growth performance, gut microbiota composition, and histology were assessed. The 3.0% rTP4 supplement group showed a significant increase in weight gain ratio and feed efficiency compared to those of the basal broiler diet group. Crude rTP4 was expressed by yeast to significantly promote growth efficiency and resistance against pathogens in G. g. domesticus, which could indicate its use as a suitable alternative to antibiotics as feed additives in the poultry industry.
Asunto(s)
Alimentación Animal , Péptidos Catiónicos Antimicrobianos/farmacología , Pollos/crecimiento & desarrollo , Suplementos Dietéticos , Proteínas de Peces/farmacología , Tilapia/genética , Animales , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/genética , Femenino , Proteínas de Peces/química , Proteínas de Peces/genética , Masculino , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologíaRESUMEN
Supplementing chicken feed with antibiotics can improve survival and prevent disease outbreaks. However, overuse of antibiotics may promote the development of antibiotic-resistant bacteria. Recently, antimicrobial peptides have been proposed as alternatives to antibiotics in animal husbandry. Here, we evaluate the effects of antimicrobial peptide, Epinephelus lanceolatus piscidin (EP), in Gallus gallus domesticus. The gene encoding EP was isolated, sequenced, codon-optimized and cloned into a Pichia pastoris recombinant protein expression system. The expressed recombinant EP (rEP) was then used as a dietary supplement for G. g. domesticus; overall health, growth performance and immunity were assessed. Supernatant from rEP-expressing yeast showed in vitro antimicrobial activity against Gram-positive and Gram-negative bacteria, according to an inhibition-zone diameter (mm) assay. Moreover, the antimicrobial peptide function of rEP was temperature independent. The fermentation broth yielded a spray-dried powder formulation containing 262.9 µg EP/g powder, and LC-MS/MS (tandem MS) analysis confirmed that rEP had a molecular weight of 4279 Da, as expected for the 34-amino acid peptide; the DNA sequence of the expression vector was also validated. We then evaluated rEP as a feed additive for G. g. domesticus. Treatment groups included control, basal diet and rEP at different doses (0.75, 1.5, 3.0, 6.0 and 12%). Compared to control, rEP supplementation increased G. g. domesticus weight gain, feed efficiency, IL-10 and IFN-γ production. Our results suggest that crude rEP could provide an alternative to traditional antibiotic feed additives for G. g. domesticus, serving to enhance growth and health of the animals.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/metabolismo , Pollos/inmunología , Sistema Inmunológico/metabolismo , Perciformes/metabolismo , Secuencia de Aminoácidos , Animales , Antiinfecciosos/análisis , Antiinfecciosos/metabolismo , Antiinfecciosos/farmacología , Péptidos Catiónicos Antimicrobianos/análisis , Péptidos Catiónicos Antimicrobianos/clasificación , Péptidos Catiónicos Antimicrobianos/genética , Pollos/crecimiento & desarrollo , Cromatografía Líquida de Alta Presión , Clonación Molecular , Suplementos Dietéticos , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Sistema Inmunológico/efectos de los fármacos , Interferón gamma/metabolismo , Interleucina-10/metabolismo , Filogenia , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/farmacología , Alineación de Secuencia , Espectrometría de Masas en Tándem , TemperaturaRESUMEN
Endothelium (EC) is a key component of blood-brain barrier (BBB), and has an important position in the neurovascular unit. Its dysfunction and death after cerebral ischemic/reperfusion (I/R) injury not only promote evolution of neuroinflammation and brain edema, but also increase the risk of intracerebral hemorrhage of thrombolytic therapies. However, the mechanism and specific interventions of EC death after I/R injury are poorly understood. Here we showed that necroptosis was a mechanism underlying EC death, which promoted BBB breakdown after I/R injury. Treatment of rats with receptor interacting protein kinase 1 (RIPK1)-inhibitor, necrostatin-1 reduced endothelial necroptosis and BBB leakage. We furthermore showed that perivascular M1-like microglia-induced endothelial necroptosis leading to BBB disruption requires tumor necrosis factor-α (TNF-α) secreted by M1 type microglia and its receptor, TNF receptor 1 (TNFR1), on endothelium as the primary mediators of these effects. More importantly, anti-TNFα (infliximab, a potent clinically used drug) treatment significantly ameliorate endothelial necroptosis, BBB destruction and improve stroke outcomes. Our data identify a previously unexplored role for endothelial necroptosis in BBB disruption and suggest infliximab might serve as a potential drug for stroke therapy.