RESUMEN
Random insertional mutagenesis performed on a Lactococcus lactis reporter strain led us to identify L. lactis ybdD as a protein-overproducing mutant. In different expression contexts, the ybdD mutant shows increased levels of exported proteins and therefore constitutes a new and attractive heterologous protein production host. This study also highlights the importance of unknown regulatory processes that play a role during protein secretion.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Regulación Bacteriana de la Expresión Génica , Técnicas de Inactivación de Genes , Mutagénesis InsercionalRESUMEN
Rhodococcus equi causes severe pneumonia in foals and has recently gained attention as a significant opportunistic pathogen in immunocompromised humans. However, no effective vaccine to prevent rhodococcosis is currently available. In this study, we have engineered the food-grade bacterium Lactococcus lactis to secrete the virulence-associated protein A from R. equi (LL-VapA). The immunogenic potential of LL-VapA strain was then evaluated after either intragastric or intranasal immunization in mice either alone or in combination with LL-Lep, a recombinant strain of L. lactis secreting biologically active leptin, a pleiotropic hormone with significant immunomodulatory properties. Intragastric administration of LL-VapA led to the highest VapA-specific mucosal response whereas intranasal administration led to the highest systemic immune responses. Cytokines released from in vitro-stimulated spleen cells show both a strong IFN-γ response and an increase of IL-4 level in all immunized groups, except for the group intranasally co-administered with both LL-VapA and LL-Lep. Strikingly, a significant reduction in R. equi viable counts in liver and spleen was observed four days after intravenous challenge with a virulent strain of R. equi in all immunized groups except for the group vaccinated by intragastric route with LL-VapA. Altogether, our results demonstrate that LL-VapA can evoke a T(H)1-based protective immune response in intranasally immunized mice. This response is enhanced when co-administered with LL-Lep strain, whereas only co-administration of LL-VapA and LL-Lep can induce a protective immune response in intragastric vaccinated mice, associated with a T(H)1/T(H)2 cytokine response.