RESUMEN
The intrinsic ruggedness of Enterococcus faecalis is responsible for its widespread distribution in nature and is often viewed as an important virulence determinant. Previously, we showed that the ClpB ATPase is negatively regulated by CtsR and is required for thermotolerance and virulence in a Galleria mellonella invertebrate model. Here, we used in silico, Northern blot and quantitative real-time PCR analyses to identify additional members of the CtsR regulon, namely the clpP peptidase and the clpC and clpE ATPases. When compared to the parent strain, virulence of the ΔctsR strain in G. mellonella was significantly attenuated.
Asunto(s)
Adenosina Trifosfatasas/biosíntesis , Proteínas Bacterianas/biosíntesis , Enterococcus faecalis/genética , Enterococcus faecalis/patogenicidad , Proteínas de Choque Térmico/biosíntesis , Lepidópteros/microbiología , Proteínas Represoras/biosíntesis , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Endopeptidasa Clp/biosíntesis , Endopeptidasa Clp/metabolismo , Enterococcus faecalis/metabolismo , Regulación Bacteriana de la Expresión Génica , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Respuesta al Choque Térmico/genética , Regiones Promotoras Genéticas , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Virulencia , Factores de Virulencia/biosíntesis , Factores de Virulencia/genéticaRESUMEN
Here, we transcriptionally and phenotypically characterized the clpB gene from Enterococcus faecalis. Northern blot analysis identified a monocistronic mRNA strongly induced at 48 and 50 °C. In silico analysis identified that the clpB gene encodes a protein of 868 aa with a predicted molecular mass of approximately 98 kDa, presenting two conserved ATP-binding domains. Sequence analysis also identified a CtsR-binding box upstream of the putative -10 sequence, and inactivation of the ctsR gene resulted in an approximately 2-log increase in clpB mRNA expression, confirming ClpB as a member of the CtsR regulon. While expression of clpB was induced by heat stress, a ΔclpB strain grew relatively well under many different stressful conditions, including elevated temperatures. However, expression of ClpB appears to play a major role in induced thermotolerance and in pathogenesis, as assessed by using the Galleria mellonella virulence model.