RESUMEN
The first line of the Arthropods defense against infections is the hard-structured exoskeleton, a physical barrier, usually rich in insoluble chitin. For entomopathogenic fungi that actively penetrate the host body, an arsenal of hydrolytic enzymes (as chitinases and N-acetylglucosaminidases), that break down chitin, is essential. Notably, twenty-one putative chitinase genes have been identified in the genome of Metarhizium anisopliae, a generalist entomopathogenic fungus. As a multigenic family, with enzymes that, presumably, perform redundant functions, the main goal is to understand the singularity of each one of such genes and to discover their precise role in the fungal life cycle. Specially chitinases that can act as virulence determinants are of interest since these enzymes can lead to more efficient biocontrol agents. Here we explored a horizontally acquired chitinase from M. anisopliae, named chiMaD1. The deletion of this gene did not lead to phenotypic alterations or diminished supernatant's chitinolytic activity. Surprisingly, chiMaD1 deletion enhanced M. anisopliae virulence to the cattle tick (Rhipicephalus microplus) larvae and engorged females, while did not alter the virulence to the mealworm larvae (Tenebrio molitor). These results add up to recent reports of deleted genes that enhanced entomopathogenic virulence, showing the complexity of host-pathogen interactions.
Asunto(s)
Quitinasas/genética , Proteínas Fúngicas/genética , Metarhizium/patogenicidad , Rhipicephalus/microbiología , Animales , Quitina/metabolismo , Quitinasas/metabolismo , Proteínas Fúngicas/metabolismo , Eliminación de Gen , Transferencia de Gen Horizontal , Interacciones Huésped-Patógeno , Larva/microbiología , Metarhizium/clasificación , Metarhizium/enzimología , Metarhizium/genética , Control Biológico de Vectores , Filogenia , Tenebrio/microbiología , VirulenciaRESUMEN
BACKGROUND: Metarhizium anisopliae is an entomopathogenic fungus used in the biological control of some agricultural insect pests, and efforts are underway to use this fungus in the control of insect-borne human diseases. A large repertoire of proteins must be secreted by M. anisopliae to cope with the various available nutrients as this fungus switches through different lifestyles, i.e., from a saprophytic, to an infectious, to a plant endophytic stage. To further evaluate the predicted secretome of M. anisopliae, we employed genomic and transcriptomic analyses, coupled with phylogenomic analysis, focusing on the identification and characterization of secreted proteins. RESULTS: We determined the M. anisopliae E6 genome sequence and compared this sequence to other entomopathogenic fungi genomes. A robust pipeline was generated to evaluate the predicted secretomes of M. anisopliae and 15 other filamentous fungi, leading to the identification of a core of secreted proteins. Transcriptomic analysis using the tick Rhipicephalus microplus cuticle as an infection model during two periods of infection (48 and 144 h) allowed the identification of several differentially expressed genes. This analysis concluded that a large proportion of the predicted secretome coding genes contained altered transcript levels in the conditions analyzed in this study. In addition, some specific secreted proteins from Metarhizium have an evolutionary history similar to orthologs found in Beauveria/Cordyceps. This similarity suggests that a set of secreted proteins has evolved to participate in entomopathogenicity. CONCLUSIONS: The data presented represents an important step to the characterization of the role of secreted proteins in the virulence and pathogenicity of M. anisopliae.
Asunto(s)
Proteínas Fúngicas/genética , Genoma Fúngico , Metarhizium/genética , Animales , Hibridación Genómica Comparativa , Perfilación de la Expresión Génica , Interacciones Huésped-Patógeno/genética , Metarhizium/clasificación , Filogenia , Rhipicephalus/metabolismo , Rhipicephalus/microbiología , Análisis de Secuencia de ARNRESUMEN
The production of lipase by twenty-nine yeasts isolated from the phylloplane of Hibiscus rosa-sinensis was evaluated. The highest lipase producers were Pseudozyma hubeiensis HB85A, Debaryomyces occidentalis-like HB83 and Cryptococcus sp. HB80. P. hubeiensis HB85A batch fermentations were carried out in a bioreactor and lipase production improved 3.2-fold as compared to flask submerged cultures. The production process was significantly reduced from 48 h (in flasks) to 18 h (in the bioreactor). The better hydrolytic activity was achieved with C16 p-nitrophenyl ester. Maximal activity was observed at pH 7.0, the optimum temperature was 50 degrees C at pH 7.0 and the enzyme was stable at 30 and 40 degrees C. The lipolytic activity was stimulated by Mg(2+), K(+) and Ba(2+) salts and EDTA and slightly inhibited by Ca(2+) salts. Non-ionic detergents such as Triton X-100, Tween 80 and Tween 20 strongly stimulated lipase activity, whereas SDS inhibited it. The lipase was stable in iso-octane and hexane at 80%.