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1.
mBio ; 15(5): e0057024, 2024 May 08.
Artículo en Inglés | MEDLINE | ID: mdl-38587425

RESUMEN

Fungal resistance to commonly used medicines is a growing public health threat, and there is a dire need to develop new classes of antifungals. We previously described a peptide produced by Enterococcus faecalis, EntV, that restricts Candida albicans to a benign form rather than having direct fungicidal activity. Moreover, we showed that one 12-amino acid (aa) alpha helix of this peptide retained full activity, with partial activity down to the 10aa alpha helix. Using these peptides as a starting point, the current investigation sought to identify the critical features necessary for antifungal activity and to screen for new variants with enhanced activity using both biofilm and C. elegans infection assays. First, the short peptides were screened for residues with critical activity by generating alanine substitutions. Based on this information, we used synthetic molecular evolution (SME) to rationally vary the specific residues of the 10aa variant in combination to generate a library that was screened to identify variants with more potent antifungal activity than the parent template. Five gain-of-function peptides were identified. Additionally, chemical modifications to the peptides to increase stability, including substitutions of D-amino acids and hydrocarbon stapling, were investigated. The most promising peptides were additionally tested in mouse models of oropharyngeal and systemic candidiasis where their efficacy in preventing infection was demonstrated. The expectation is that these discoveries will contribute to the development of new therapeutics in the fight against antimicrobial resistant fungi. IMPORTANCE: Since the early 1980s, the incidence of disseminated life-threatening fungal infections has been on the rise. Worldwide, Candida and Cryptococcus species are among the most common agents causing these infections. Simultaneously, with this rise of clinical incidence, there has also been an increased prevalence of antifungal resistance, making treatment of these infections very difficult. For example, there are now strains of Candida auris that are resistant to all three classes of currently used antifungal drugs. In this study, we report on a strategy that allows for the development of novel antifungal agents by using synthetic molecular evolution. These discoveries demonstrate that the enhancement of antifungal activity from naturally occurring peptides is possible and can result in clinically relevant agents that have efficacy in multiple in vivo models as well as the potential for broad-spectrum activity.


Asunto(s)
Antifúngicos , Biopelículas , Caenorhabditis elegans , Candida albicans , Candidiasis , Enterococcus faecalis , Pruebas de Sensibilidad Microbiana , Antifúngicos/farmacología , Antifúngicos/química , Animales , Ratones , Candida albicans/efectos de los fármacos , Candida albicans/genética , Biopelículas/efectos de los fármacos , Candidiasis/tratamiento farmacológico , Candidiasis/microbiología , Enterococcus faecalis/efectos de los fármacos , Enterococcus faecalis/genética , Caenorhabditis elegans/efectos de los fármacos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/farmacología , Modelos Animales de Enfermedad , Péptidos/farmacología , Péptidos/genética , Péptidos/química
2.
Nat Commun ; 13(1): 6047, 2022 10 13.
Artículo en Inglés | MEDLINE | ID: mdl-36229448

RESUMEN

Fungal pathogens are a continuing challenge due to few effective antifungals and a rise in resistance. In previous work, we described the inhibition of Candida albicans virulence following exposure to the 68 amino acid bacteriocin, EntV, secreted by Enterococcus faecalis. Here, to optimize EntV as a potential therapeutic and better understand its antifungal features, an X-ray structure is obtained. The structure consists of six alpha helices enclosing a seventh 16 amino acid helix (α7). The individual helices are tested for antifungal activity using in vitro and nematode infection assays. Interestingly, α7 retains antifungal, but not antibacterial activity and is also effective against Candida auris and Cryptococcus neoformans. Further reduction of α7 to 12 amino acids retains full antifungal activity, and excellent efficacy is observed in rodent models of C. albicans oropharyngeal, systemic, and venous catheter infections. Together, these results showcase EntV-derived peptides as promising candidates for antifungal therapeutic development.


Asunto(s)
Bacteriocinas , Cryptococcus neoformans , Micosis , Aminoácidos/farmacología , Antifúngicos/farmacología , Antifúngicos/uso terapéutico , Bacteriocinas/metabolismo , Candida albicans , Humanos , Pruebas de Sensibilidad Microbiana , Micosis/tratamiento farmacológico
3.
mBio ; 11(6)2020 11 03.
Artículo en Inglés | MEDLINE | ID: mdl-33144376

RESUMEN

Invasive fungal infections in humans are generally associated with high mortality, making the choice of antifungal drug crucial for the outcome of the patient. The limited spectrum of antifungals available and the development of drug resistance represent the main concerns for the current antifungal treatments, requiring alternative strategies. Antimicrobial peptides (AMPs), expressed in several organisms and used as first-line defenses against microbial infections, have emerged as potential candidates for developing new antifungal therapies, characterized by negligible host toxicity and low resistance rates. Most of the current literature focuses on peptides with antibacterial activity, but there are fewer studies of their antifungal properties. This review focuses on AMPs with antifungal effects, including their in vitro and in vivo activities, with the biological repercussions on the fungal cells, when known. The classification of the peptides is based on their mode of action: although the majority of AMPs exert their activity through the interaction with membranes, other mechanisms have been identified, including cell wall inhibition and nucleic acid binding. In addition, antifungal compounds with unknown modes of action are also described. The elucidation of such mechanisms can be useful to identify novel drug targets and, possibly, to serve as the templates for the synthesis of new antimicrobial compounds with increased activity and reduced host toxicity.


Asunto(s)
Péptidos Catiónicos Antimicrobianos/farmacología , Hongos/efectos de los fármacos , Proteínas Citotóxicas Formadoras de Poros/farmacología , Animales , Antifúngicos/química , Antifúngicos/uso terapéutico , Péptidos Catiónicos Antimicrobianos/biosíntesis , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/uso terapéutico , Vías Biosintéticas , Farmacorresistencia Fúngica , Humanos , Pruebas de Sensibilidad Microbiana , Micosis/tratamiento farmacológico , Micosis/microbiología , Proteínas Citotóxicas Formadoras de Poros/biosíntesis , Proteínas Citotóxicas Formadoras de Poros/química , Proteínas Citotóxicas Formadoras de Poros/uso terapéutico , Relación Estructura-Actividad
4.
PLoS One ; 15(10): e0240351, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-33057423

RESUMEN

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that produces numerous virulence factors and causes serious infections in trauma patients and patients with severe burns. We previously showed that the growth of P. aeruginosa in blood from severely burned or trauma patients altered the expression of numerous genes. However, the specific influence of whole blood from healthy volunteers on P. aeruginosa gene expression is not known. Transcriptome analysis of P. aeruginosa grown for 4 h in blood from healthy volunteers compared to that when grown in laboratory medium revealed that the expression of 1085 genes was significantly altered. Quorum sensing (QS), QS-related, and pyochelin synthesis genes were downregulated, while genes of the type III secretion system and those for pyoverdine synthesis were upregulated. The observed effect on the QS and QS-related genes was shown to reside within serum fraction: growth of PAO1 in the presence of 10% human serum from healthy volunteers significantly reduced the expression of QS and QS-regulated genes at 2 and 4 h of growth but significantly enhanced their expression at 8 h. Additionally, the production of QS-regulated virulence factors, including LasA and pyocyanin, was also influenced by the presence of human serum. Serum fractionation experiments revealed that part of the observed effect resides within the serum fraction containing <10-kDa proteins. Growth in serum reduced the production of many PAO1 outer membrane proteins but enhanced the production of others including OprF, a protein previously shown to play a role in the regulation of QS gene expression. These results suggest that factor(s) within human serum: 1) impact P. aeruginosa pathogenesis by influencing the expression of different genes; 2) differentially regulate the expression of QS and QS-related genes in a growth phase- or time-dependent mechanism; and 3) manipulate the production of P. aeruginosa outer membrane proteins.


Asunto(s)
Pseudomonas aeruginosa/metabolismo , Percepción de Quorum/genética , Virulencia/genética , Bacteriemia/microbiología , Bacteriemia/patología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Medios de Cultivo/química , Regulación hacia Abajo , Humanos , Pseudomonas aeruginosa/aislamiento & purificación , Pseudomonas aeruginosa/patogenicidad , Suero/química , Sideróforos/genética , Transcriptoma , Sistemas de Secreción Tipo III/genética , Sistemas de Secreción Tipo III/metabolismo , Regulación hacia Arriba
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