Rubén Cebrián

Discovering the Bacterial Circular Proteins: Bacteriocins, Cyanobactins, and Pilins

Over recent years, several examples of natural ribosomally synthesized circular proteins and peptides from diverse organisms have been described. They are a group of proteins for which the precursors must be post-translationally modified to join the N and C termini with a peptide bond. This feature appears to confer a range of potential advantages because these proteins show increased resistance to proteases and higher thermodynamic stability, both of which improve their biological activity. They are produced by prokaryotic and eukaryotic organisms and show diverse biological activities, related mostly to a self-defense or competition mechanism of the producer organisms, with the only exception being the circular pilins. This minireview highlights ribosomally synthesized circular proteins produced by members of the domain Bacteria: circular bacteriocins, cyanobactins, and circular pilins. We pay special attention to the genetic organization of the biosynthetic machinery of these molecules, the role of circularization, and the differences in the possible circularization mechanisms.

The bacteriocin AS-48 requires dimer dissociation followed by hydrophobic interactions with the membrane for antibacterial activity

The molecular mechanism underlining the antibacterial activity of the bacteriocin AS-48 is not known, and two different and opposite alternatives have been proposed. Available data suggested that the interaction of positively charged amino acids of AS-48 with the membrane would produce membrane destabilization and disruption. Alternatively, it has been proposed that AS-48 activity could rely on the effective insertion of the bacteriocin into the membrane. The biological and structural properties of the AS-48G13K/L40K double mutant were investigated to shed light on this subject. Compared with the wild type, the mutant protein suffered an important reduction in the antibacterial activity. Biochemical and structural studies of AS-48G13K/L40K mutant suggest the basis of its decreased antimicrobial activity. Lipid cosedimentation assays showed that the membrane affinity of AS-48G13K/L40K is 12-fold lower than that observed for the wild type. L40K mutation is responsible for this reduced membrane affinity and thus, hydrophobic interactions are involved in membrane association. Furthermore, the high-resolution crystal structure of AS-48G13K/L40K, together with the study of its dimeric character in solution showed that G13K stabilizes the inactive water-soluble dimer, which displays a reduced dipole moment. Our data suggest that the cumulative effect of these three affected properties reduces AS-48 activity, and point out that the bactericidal effect is achieved by the electrostatically driven approach of the inactive water-soluble dimer towards the membrane, followed by the dissociation and insertion of the protein into the lipid bilayer.

Insights into the Functionality of the Putative Residues Involved in Enterocin AS-48 Maturation

ABSTRACT AS-48 is a 70-residue, α-helical, cationic bacteriocin produced by Enterococcus faecalis and is very singular in its circular structure and its broad antibacterial spectrum. The AS-48 preprotein 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. For the study of the specificity of the region of AS-48 that is responsible for maturation, three single mutants have been generated by site-directed mutagenesis in the as-48A 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. The importance of these derivatives in AS-48 processing has been evaluated on the basis of the production and structural characterization of the corresponding derivatives. Notably, only two of them (Trp70Ala and Met1Ala derivatives) could be purified in different forms and amounts and are characterized for their bactericidal activity and secondary structure. We could not detect any production of AS-48 in JH2-2(pAM401-81His-1Ile) by using the conventional chromatographic techniques, despite the high efficiency of the culture conditions applied to produce this enterocin. Our results underline the different important roles of the mutated residues in (i) the elimination of the SP, (ii) the production levels and antibacterial activity of the mature proteins, and (iii) protein circularization. Moreover, our findings suggest that His-1 is critically involved in cleavage site recognition, its substitution being responsible for the blockage of processing, thereby hampering the production of the specific protein in the cellular culture supernatant.

Enterocin AS-48 as Evidence for the Use of Bacteriocins as New Leishmanicidal Agents

ABSTRACT We report the feasibility of enterocin AS-48, a circular cationic peptide produced by Enterococcus faecalis, as a new leishmanicidal agent. AS-48 is lethal to Leishmania promastigotes as well as to axenic and intracellular amastigotes at low micromolar concentrations, with scarce cytotoxicity to macrophages. AS-48 induced a fast bioenergetic collapse of L. donovani promastigotes but only a partial permeation of their plasma membrane with limited entrance of vital dyes, even at concentrations beyond its full lethality. Fluoresceinated AS-48 was visualized inside parasites by confocal microscopy and seen to cause mitochondrial depolarization and reactive oxygen species production. Altogether, AS-48 appeared to have a mixed leishmanicidal mechanism that includes both plasma membrane permeabilization and additional intracellular targets, with mitochondrial dysfunctionality being of special relevance. This complex leishmanicidal mechanism of AS-48 persisted even for the killing of intracellular amastigotes, as evidenced by transmission electron microscopy. We demonstrated the potentiality of AS-48 as a new and safe leishmanicidal agent, expanding the growing repertoire of eukaryotic targets for bacteriocins, and our results provide a proof of mechanism for the search of new leishmanicidal bacteriocins, whose diversity constitutes an almost endless source for new structures at moderate production cost and whose safe use on food preservation is well established.

Analysis of the promoters involved in enterocin AS-48 expression

The enterocin AS-48 is the best characterized antibacterial circular protein in prokaryotes. It is a hydrophobic and cationic bacteriocin, which is ribosomally synthesized by enterococcal cells and post-translationally cyclized by a head-to-tail peptide bond. The production of and immunity towards AS-48 depend upon the coordinated expression of ten genes organized in two operons, as-48ABC (where genes encoding enzymes with processing, secretion, and immunity functions are adjacent to the structural as-48A gene) and as-48C1DD1EFGH. The current study describes the identification of the promoters involved in AS-48 expression. Seven putative promoters have been here amplified, and separately inserted into the promoter-probe vector pTLR1, to create transcriptional fusions with the mCherry gene used as a reporter. The activity of these promoter regions was assessed measuring the expression of the fluorescent mCherry protein using the constitutive pneumococcal promoter PX as a reference. Our results revealed that only three promoters PA, P2(2) and PD1 were recognized in Enterococcus faecalis, Lactococcus lactis and Escherichia coli, in the conditions tested. The maximal fluorescence was obtained with PX in all the strains, followed by the P2(2) promoter, which level of fluorescence was 2-fold compared to PA and 4-fold compared to PD1. Analysis of putative factors influencing the promoter activity in single and double transformants in E. faecalis JH2-2 demonstrated that, in general, a better expression was achieved in presence of pAM401-81. In addition, the P2(2) promoter could be regulated in a negative fashion by genes existing in the native pMB-2 plasmid other than those of the as-48 cluster, while the pH seems to affect differently the as-48 promoter expression.

Antimicrobial characterization and safety aspects of the bacteriocinogenic Enterococcus hirae F420 isolated from Moroccan raw goat milk

The F420 strain, isolated from raw goat milk and identified as Enterococcus hirae, was selected because of its strong activity against gram-positive bacteria, including Listeria monocytogenes. Interestingly, the F420 strain lacks the virulence genes and decarboxylase activity of histidine, lysine, and ornithine, and it is susceptible to 11 of 14 tested antibiotics, including vancomycin. The antimicrobial compounds produced by E. hirae F420 strain showed high resistance to heat treatment and to acidic and basic pHs. The MALDI-TOF mass spectrometry analysis coupled with the sequence of peptide and structural gene analysis of one of the purified enterocins showed 100% identity with enterocin P (EntP), previously described in E. faecium strains. The structural gene for EntP is located on a plasmid of 65 kb. Other enterocins with molecular mass higher than 7 kDa were also detected. This is the first report of the production of EntP by E. hirae species naturally occurring in foods. The biotechnological characteristics of the F420 strain and its enterocins indicate their potential for application in the control of L. monocytogenes and other undesirable bacteria in food systems.

Autophagic-related cell death of Trypanosoma brucei induced by bacteriocin AS-48

The parasitic protozoan Trypanosoma brucei is the causative agent of human African trypanosomiasis (sleeping sickness) and nagana. Current drug therapies have limited efficacy, high toxicity and/or are continually hampered by the appearance of resistance. Antimicrobial peptides have recently attracted attention as potential parasiticidal compounds. Here, we explore circular bacteriocin AS-48s ability to kill clinically relevant bloodstream forms of T. brucei gambiense, T. brucei rhodesiense and T. brucei brucei. AS-48 exhibited excellent anti-trypanosomal activity in vitro (EC50 = 1–3 nM) against the three T. brucei subspecies, but it was innocuous to human cells at 104-fold higher concentrations. In contrast to its antibacterial action, AS-48 does not kill the parasite through plasma membrane permeabilization but by targeting intracellular compartments. This was evidenced by the fact that vital dye internalization-prohibiting concentrations of AS-48 could kill the parasite at 37 °C but not at 4 °C. Furthermore, AS-48 interacted with the surface of the parasite, at least in part via VSG, its uptake was temperature-dependent and clathrin-depleted cells were less permissive to the action of AS-48. The bacteriocin also caused the appearance of myelin-like structures and double-membrane autophagic vacuoles. These changes in the parasites ultrastructure were confirmed by fluorescence microscopy as AS-48 induced the production of EGFP-ATG8.2-labeled autophagosomes. Collectively, these results indicate AS-48 kills the parasite through a mechanism involving clathrin-mediated endocytosis of VSG-bound AS-48 and the induction of autophagic-like cell death. As AS-48 has greater in vitro activity than the drugs currently used to treat T. brucei infection and does not present any signs of toxicity in mammalian cells, it could be an attractive lead compound for the treatment of sleeping sickness and nagana.

Optimization of genotypic and biochemical methods to profile P. acnes isolates from a patient population

Propionibacterium acnes is a key factor in the pathogenesis of acne vulgaris, although currently it is also being associated with medical-device infections. The aim of this work was to validate a safe and quick identification and typing of 24 clinical isolates of Propionibacterium acnes, applying a range of biochemical as well as genetic methods, and investigating the pathogenic potential to associate the different types with human health. RAPD-PCRs revealed the existence of two discernible clusters in correspondence with the phylogroups I and II, according to the PAtig gene polymorphism, leading them to be assigned as P. acnes subsp. acnes subsp. nov. Biotyping according to the pattern of sugar fermentation evidenced that all the isolates from acne and the majority from opportunistic infections fit the biotype I-B3. Consistent with the multiplex touchdown analysis, nearly all the isolates included in this biotype belonged to the subgroups IA1 (the exception being four strains classified as IB). The remaining ones were assigned to phylogroup II, considered to be part of the normal cutaneous microbiota. The susceptibility to three antibiotics was also investigated to explore the relations with the virulence, although no clear trend was identified.

LAB Bacteriocins Controlling the Food Isolated (Drug-Resistant) Staphylococci

Staphylococci are a group of microorganisms that can be often found in processed food and they might pose a risk for human health. In this study we have determined the content of staphylococci in 7 different fresh goat-milk cheeses. These bacteria were present in all of them, ranging from 103 to 106 CFU/g based on growth on selective media. Thus, a set of 97 colonies was randomly picked for phenotypic and genotypic identification. They could be clustered by RAPD-PCR in 10 genotypes, which were assigned by 16S rDNA sequencing to four Staphylococcus species: Staphylococcus aureus, Staphylococcus chromogenes, S. simulans, and S. xylosus. Representative strains of these species (n = 25) were tested for antibiotic sensitivity, and 11 of them were resistant to at least one of the antibiotics tested, including erythromycin, amoxicillin-clavulanic acid and oxacillin. We also tested two bacteriocins produced by lactic acid bacteria (LAB), namely the circular bacteriocin AS-48 and the lantibiotic nisin. These peptides have different mechanism of action at the membrane level. Nevertheless, both were able to inhibit staphylococci growth at low concentrations ranging between 0.16–0.73 μM for AS-48 and 0.02–0.23 μM for nisin, including the strains that displayed antibiotic resistance. The combined effect of these bacteriocins were tested and the fractional inhibitory concentration index (FICI) was calculated. Remarkably, upon combination, they were active at the low micromolar range with a significant reduction of the minimal inhibitory concentration. Our data confirms synergistic effect, either total or partial, between AS-48 and nisin for the control of staphylococci and including antibiotic resistant strains. Collectively, these results indicate that the combined use of AS-48 and nisin could help controlling (pathogenic) staphylococci in food processing and preventing antibiotic-resistant strains reaching the consumer in the final products.

Assessing in vitro digestibility of food biopreservative AS-48

AS-48 is a bacteriocin with potential application as food biopreservative. In order to optimize its use for oral consumption, we assess the impact of gastrointestinal digestion, both in bulk and adsorbed at the air-water interface. Analysis of AS-48 digestion fragments in bulk by SDS-PAGE, RP-HPLC, and MALDI-TOF proves that the previous pepsin exposition promotes digestion by trypsin/chymotrypsin by exposing new cleavage sites. Regarding adsorbed AS-48, the in vitro digestion profile shows that the conformational change undergone by AS-48 upon adsorption affects its digestibility. Gastrointestinal enzymes cleave only susceptible residues, which are oriented into the aqueous phase, while hydrophobic susceptible residues remain undigested. Evaluation of the elasticity of the adsorbed layer confirms also the presence of undigested AS-48. These results are important towards the use of AS-48 in food formulations; assuring that some intact AS-48 resists digestion guarantees its antibacterial activity throughout the gastrointestinal tract.