RESUMEN
Paired Box (Pax) gene family, a group of transcription regulators have been implicated in diverse physiological processes. However, their role during hematopoiesis which generate a plethora of blood cells remains largely unknown. Using a previously reported single cell transcriptomics data, we analyzed the expression of individual Pax family members in hematopoietic cells in zebrafish. We have identified that Pax9, which is an essential regulator for odontogenesis and palatogenesis, is selectively localized within a single cluster of the hematopoietic lineage. To further analyze the function of Pax9 in hematopoiesis, we generated two independent pax9 knock-out mutants using the CRISPR-Cas9 technique. We found that Pax9 appears to be an essential regulator for granulopoiesis but dispensable for erythropoiesis during development, as lack of pax9 selectively decreased the number of neutrophils with a concomitant decrease in the expression level of neutrophil markers. In addition, embryos, where pax9 was functionally disrupted by injecting morpholinos, failed to increase the number of neutrophils in response to pathogenic bacteria, suggesting that Pax9 is not only essential for developmental granulopoiesis but also emergency granulopoiesis. Due to the inability to initiate emergency granulopoiesis, innate immune responses were severely compromised in pax9 morpholino-mediated embryos, increasing their susceptibility and mortality. Taken together, our data indicate that Pax9 is essential for granulopoiesis and promotes innate immunity in zebrafish larvae.
Asunto(s)
Eritropoyesis/inmunología , Mielopoyesis/inmunología , Factor de Transcripción PAX9/inmunología , Proteínas de Pez Cebra/inmunología , Pez Cebra/inmunología , Animales , Animales Modificados Genéticamente , Infecciones Bacterianas/inmunología , Sistemas CRISPR-Cas , Eritropoyesis/genética , Regulación del Desarrollo de la Expresión Génica , Técnicas de Inactivación de Genes , Granulocitos/inmunología , Inmunidad Innata/genética , Inmunidad Innata/fisiología , Mielopoyesis/genética , Factor de Transcripción PAX9/deficiencia , Factor de Transcripción PAX9/genética , Pez Cebra/embriología , Pez Cebra/genética , Proteínas de Pez Cebra/deficiencia , Proteínas de Pez Cebra/genéticaRESUMEN
Cationic antimicrobial peptides (CAMPs) are important antibiotics because they possess a broad spectrum of activity against both Gram-positive and Gram-negative bacteria, including those resistant to traditional antibiotics. The cyclic peptide bactenecin is a 12-amino acid CAMP that contains one intramolecular disulfide bond. To improve the antibacterial activity of bactenecin, we designed and synthesized several bactenecin analogs by applying multiple approaches, including amino acid substitution, use of the d-enantiomeric form, and lipidation. Among the synthetic analogs, d-enantiomeric bactenecin conjugated to capric acid, which we named dBacK-(cap), exhibited a significantly enhanced antibacterial spectrum with MIC values ranging from 1 to 8⯵M against both Gram-positive and Gram-negative bacteria, including some drug-resistant bacteria. Upon exposure to dBacK-(cap), S. aureus cells were killed within 1â¯hâ¯at the MIC value, but full inactivation of E. coli required over 2â¯h. These results indicate that covalent addition of a d-amino acid and a fatty acid to bactenecin is the most effective approach for enhancing its antibacterial activity.
Asunto(s)
Antibacterianos/farmacología , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Péptidos Cíclicos/farmacología , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Antibacterianos/síntesis química , Antibacterianos/química , Permeabilidad de la Membrana Celular , Diseño de Fármacos , Bacterias Gramnegativas/citología , Bacterias Gramnegativas/ultraestructura , Bacterias Grampositivas/citología , Bacterias Grampositivas/ultraestructura , Cinética , Pruebas de Sensibilidad Microbiana , Microscopía Electrónica de Rastreo , Péptidos Cíclicos/síntesis química , Péptidos Cíclicos/químicaRESUMEN
Antimicrobial peptides (AMPs) are cationic antibiotics that can kill multidrug-resistant bacteria via membrane insertion. However, their weak activity limits their clinical use. Ironically, the cationic charge of AMPs is essential for membrane binding, but it obstructs membrane insertion. In this study, we postulate that this problem can be overcome by locating cationic amino acids at the energetically preferred membrane surface. All amino acids have an energetically preferred or less preferred membrane position profile, and this profile is strongly related to membrane insertion. However, most AMPs do not follow this profile. One exception is protegrin-1, a powerful but neglected AMP. In the present study, we found that a potent AMP, WCopW5, strongly resembles protegrin-1 and that the match between its sequence and the preferred position profile closely correlates with its antimicrobial activity. One of its derivatives, WCopW43, has antimicrobial activity comparable to that of the most effective AMPs in clinical use.
Asunto(s)
Aminoácidos , Péptidos Antimicrobianos , Antibacterianos/farmacología , Antibacterianos/química , CationesRESUMEN
The emergence of drug-resistant pathogenic bacteria threatens human health. Resistance to existing antibiotics is increasing, while the emergence of new antibiotics is slowing. Cationic antimicrobial peptides (CAMPs) are fascinating alternative antibiotics because they possess a broad spectrum of activity, being active against both Gram-positive and Gram-negative bacteria including those resistant to traditional antibiotics. However, low bioavailability resulting from enzymatic degradation and attenuation by divalent cations like Mg2+ and Ca2+ limits their use as antibiotic agents. Here, we report the design of new CAMPs showing both high antibacterial activity and serum stability under physiological ion concentrations. The peptides were designed by applying two approaches, the use of d-enantiomer and lipidation. Based on the sequence of the CopW (LLWIALRKK-NH2), a nonapeptide derived from coprisin, a series of novel d-form CopW lipopeptides with different acyl chain lengths (C6, C8, C10, C12, C14, and C16) were synthesized and evaluated with respect to their activity and salt sensitivity. Among the analogs, the d-form lipopeptide dCopW3 exhibited MIC values ranging from 1.25 to 5⯵M against multidrug-resistant bacteria. Significantly, this compound did not induce bacterial resistance and was highly stable in human serum proteases. The results emphasize the potential of cationic d-form lipopeptide as therapeutically valuable antibiotics for treating drug-resistant bacterial infections.