Análisis funcionales de los determinantes genéticos del clúster as-48

Resumen

The experimental work presented in this Doctoral Thesis Report was designed to investigate molecular and functional aspects on AS¿ 48 bacteriocin. This is a 70¿residues, helical, cationic bacteriocin produced by Enterococcus faecalis and is very singular in its circular structure and its broad antibacterial activity at the membrane cytoplasmic level in a broad spectrum of Gram ¿positive bacteria. The first objective of this report was to investigate the molecular mechanism underlining the antibacterial activity of the bacteriocin AS¿48. Our experimental data support that the antimicrobial activity of AS¿48 could be achieved by a mechanism known as molecular electroporation but alternatively, the x¿ray structure indicated that the protein displays two different oligomeric states according to the physicochemical environment: a water¿soluble dimeric form (DF¿I) where hydrophobic interactions mediate dimer formation and an amphipathic, membrane¿bound dimeric form II (DF¿II), and the transition between the two dimeric forms is followed by the insertion of the protein into the lipid bilayer. The results here presented show the consequences of two point mutations in the AS¿48G13K/L40K double mutant, in the antibacterial activity of AS¿48 against susceptible cells. Thus, our data suggest that the additive effect of each mutation along the different steps of the molecular mechanism of action decreases the antimicrobial character of the protein. In fact we have observed that mutations strengthens the inactive and water¿soluble dimeric form of AS¿48.Secondly, both mutations, would decrease the ability of the protein to recognize the bacterial membrane by decreasing its dipole moment and thirdly, the mutation would hinder proper membrane insertion due to repulsion between the lysine and the phospholipids. Thus, our data suggest that the additive effect of each mutation along the different steps of the molecular mechanism of action decreases the antimicrobial character of the protein. Thus, our experimental data support that the antimicrobial function of AS¿48 is achieved by the dissociation of the AS¿48 water¿soluble dimer followed by the insertion of the protein into the lipid bilayer. The 2nd objective was to analyze the functionality of three residues involved in the maturation of the AS¿48 preprotein by expressing the respective single derivatives. AS¿48 is translated as a preprotein, which consists of an N¿terminal signal peptide (SP) (35 residues) followed by a proprotein moiety that undergoes posttranslational modifications to yield the mature and active circular protein. In this study, three single mutants have been generated by site¿directed mutagenesis in the as48A structural gene. The substitutions were made just in the residues that are thought to constitute a recognition site for the SP cleavage enzyme (His¿1, Met1) and in those involved in circularization (Met1, Trp70). Each derivative was expressed in the enterococcal JH2¿2 strain containing the necessary native biosynthetic machinery for enterocin production. Our results convincingly show that the residue His¿1 should not be mutated to Ile, because this change affects the efficiency of a still unidentified enzyme involved in the cleavage of the SP. In fact, only the His¿1Ile mutation categorically restrains the cleavage of the precursor. However, JH2¿2 cells harboring pAM401¿ 81Met1Ala plasmid produced mature and mainly processed AS¿48 derivatives, although the protein yield in culture decreased to values of approximately 2%. The strikingly low expression of this mutant could be caused by an inefficient processing of the preprotein induced by the absence of a Met in such a critical position. Finally, the change Trp 70Ala also showed significant levels of production reduction, but the more interesting was that the existence of linear forms of AS¿48 found in the supernatants of JH2¿2(pAM401¿81Trp70Ala) transformants. This is the most significant finding of this work, since linear species have never before been described in AS¿48 or in other circular proteins. (Cebrian et al., 2010. App Environ. Microbiol. 76:72687276). To further aspects of molecular interactions between determinants as¿ 48 cluster is prior identification of the promoters involved in the expression of enterocin AS¿ 48 , the production and immunity depends on the coordinated expression of ten genes which are organized in two operons ( as¿ as¿ 48ABC and 48C1DD1EFGH). In this report we have investigated the potential promoter regions of the cluster, which were separately amplified and transcriptionally fused to the mCherry reporter gene. The activity of the promoters cloned in JH2 ¿2 plasmid¿free strain, was assessed by measuring the fluorescence emitted by the protein mCherry, using the Px Streptococcus pneumoniae promoter as control. The results confirmed the existence of three promoters (PA , P2(2) and PD1 ) which were functional in E. faecalis, but, interestingly also in Lactococcus lactis and Escherichia coli. The increased activity in the assay conditions was obtained with the PX promoter in all cases, followed by P2(2), which showed twice the activity of PA and 4¿fold more than PD . Analysis of factors that could affect the activity of the promoters showed that, in general, there are a better expression in the presence of pAM401 ¿81 plasmid containing the gene cluster as48. Besides, P2(2) promoter appears to be subject to a negative regulation by some determinant present on pMB2 (the plasmid where the as¿48 cluster was identified in the native S¿48 strain), while the pH differently affected the expression of the three identified promoters (Cebrian et al. 2014. PlosOne . Vol.9 , No.3 , e90603 ) The ultimate objective of this work was to investigate the functionality of the genetic determinants responsible for AS¿48 expression. This objective was addressed from two different ways: i) through the over¿expression of genes involved in their biogenesis (structural gene and those involved in the maturation process) or responsible for secretion and immunity against AS¿48, and ii) modifying the expression levels of the promoters as48 through the use of alternative carbon sources or by replacing the native promoters. In addition, we carried out an analysis in silico on the As¿48B and ¿C proteins (related to AS¿48 biogenesis) to establish their secondary and 3D structure and the structurefunction relationships. For these purposes, a series of constructions, in which the as48A, BC,C1D and D1 genes cloned under its own promoter or under stronger promoters were designed to assess their stability and the effects of the gene overdose in single and double JH2¿2 transformants. This allows us to assess the influence of gene dosage on growth, resistance and the ability to produce AS¿48. As expected, only the presence of the immunity determinant had relevance in the resistance against AS¿48 exogenously added (with MIC 8¿fold highest), while a double dose of the structural as48A gene conferred an over¿producer phenotype. Moreover, the presence of galactose and trehalose in the medium increased the AS¿48 production. Growing on galactose the PA expression was induced, although this effect was not observed on trehalose. Finally the replacement of the native promoters by another strongest ( Px or P32) only was possible for ¿C1D or ¿C1DD1 genes, but such changes did not improve the producer AS¿48 character. It has been also confirmed that as48D1 gene is not translationally coupled to as48C1D, in spite of the three genes are overlapped into the operon.