Chitosan and HPMCAS double-coating as protective systems for alginate microparticles loaded with Ctx(Ile21)-Ha antimicrobial peptide to prevent intestinal infections.

The incorrect use of conventional drugs for both prevention and control of intestinal infections has contributed to a significant spread of bacterial resistance. In this way, studies that promote their replacement are a priority. In the last decade, the use of antimicrobial peptides (AMP), especially Ctx(Ile21)-Ha AMP, has gained strength, demonstrating efficient antimicrobial activity (AA) against pathogens, including multidrug-resistant bacteria. However, gastrointestinal degradation does not allow its direct oral application. In this research, double-coating systems using alginate microparticles loaded with Ctx(Ile21)-Ha peptide were designed, and in vitro release assays simulating the gastrointestinal tract were evaluated. Also, the AA against Salmonella spp. and Escherichia coli was examined. The results showed the physicochemical stability of Ctx(Ile21)-Ha peptide in the system and its potent antimicrobial activity. In addition, the combination of HPMCAS and chitosan as a gastric protection system can be promising for peptide carriers or other low pH-sensitive molecules, adequately released in the intestine. In conclusion, the coated systems employed in this study can improve the formulation of new foods or biopharmaceutical products for specific application against intestinal pathogens in animal production or, possibly, in the near future, in human health.

The Specific Elongation Factor to Selenocysteine Incorporation in Escherichia coli: Unique tRNASec Recognition and its Interactions.

Several molecular mechanisms are involved in the genetic code interpretation during translation, as codon degeneration for the incorporation of rare amino acids. One mechanism that stands out is selenocysteine (Sec), which requires a specific biosynthesis and incorporation pathway. In Bacteria, the Sec biosynthesis pathway has unique features compared with the eukaryote pathway as Ser to Sec conversion mechanism is accomplished by a homodecameric enzyme (selenocysteine synthase, SelA) followed by the action of an elongation factor (SelB) responsible for delivering the mature Sec-tRNASec into the ribosome by the interaction with the Selenocysteine Insertion Sequence (SECIS). Besides this mechanism being already described, the sequential events for Sec-tRNASec and SECIS specific recognition remain unclear. In this study, we determined the order of events of the interactions between the proteins and RNAs involved in Sec incorporation. Dissociation constants between SelB and the native as well as unacylated-tRNASec variants demonstrated that the acceptor stem and variable arm are essential for SelB recognition. Moreover, our data support the sequence of molecular events where GTP-activated SelB strongly interacts with SelA.tRNASec. Subsequently, SelB.GTP.tRNASec recognizes the mRNA SECIS to deliver the tRNASec to the ribosome. SelB in complex with its specific RNAs were examined using Hydrogen/Deuterium exchange mapping that allowed the determination of the molecular envelopes and its secondary structural variations during the complex assembly. Our results demonstrate the ordering of events in Sec incorporation and contribute to the full comprehension of the tRNASec role in the Sec amino acid biosynthesis, as well as extending the knowledge of synthetic biology and the expansion of the genetic code.

Alginate-based microparticles coated with HPMCP/AS cellulose-derivatives enable the Ctx(Ile21)-Ha antimicrobial peptide application as a feed additive.

Microencapsulation is a potential biotechnological tool, which can overcome antimicrobial peptides (AMP) instabilities and reduce toxic side effects. Thus, this study evaluates the antibacterial activities of the Ctx(Ile21)-Ha AMP against multidrug-resistant (MDR) and non-resistant bacteria and develop and characterize peptide-loaded microparticles coated with the enteric polymers hydroxypropylmethylcellulose acetate succinate (HPMCAS) and hydroxypropylmethylcellulose phthalate (HPMCP). Ctx(Ile21)-Ha was obtained by solid phase peptide synthesis (SPPS) method, purified and characterized by HPLC and Mass Spectrometry. The peptide exhibited potent antibiotic activities against Salmonella enteritidis, Salmonella typhimurium, Pseudomonas aeruginosa (MDR), Acinetobacter baumannii (MDR), and Staphylococcus aureus (MDR). Ctx(Ile21)-Ha microencapsulation was performed by ionic gelation with high efficiency, maintaining the physical-chemical stability. Ctx(Ile21)-Ha coated-microparticles were characterized by DSC, TGA, FTIR-Raman, XRD and SEM. Hemolytic activity assay demonstrated that hemolysis was decreased up to 95% compared to single molecule. In addition, in vitro release control profile simulating different portions of gastrointestinal tract was performed and showed the microcapsules' ability to protect the peptide and release it in the intestine, aiming pathogen's location, mainly by Salmonella sp. Therefore, use of microencapsulated Ctx(Ile21)-Ha can be allowed as an antimicrobial controller in monogastric animal production as an oral feed additive (antimicrobial controller), being a valuable option for molecules with low therapeutic indexes or high hemolytic rates.