Differential interactions of the antimicrobial peptide, RQ18, with phospholipids and cholesterol modulate its selectivity for microorganism membranes.

BACKGROUND: Antimicrobial peptides (AMPs) are molecules with potential application for the treatment of microorganism infections. We, herein, describe the structure, activity, and mechanism of action of RQ18, an α-helical AMP that displays antimicrobial activity against Gram-positive and Gram-negative bacteria, and yeasts from the Candida genus. METHODS: A physicochemical-guided design assisted by computer tools was used to obtain our lead peptide candidate, named RQ18. This peptide was assayed against Gram-positive and Gram-negative bacteria, yeasts, and mammalian cells to determine its selectivity index. The secondary structure and the mechanism of action of RQ18 were investigated using circular dichroism, large unilamellar vesicles, and molecular dynamic simulations. RESULTS: RQ18 was not cytotoxic to human lung fibroblasts, peripheral blood mononuclear cells, red blood cells, or Vero cells at MIC values, exhibiting a high selectivity index. Circular dichroism analysis and molecular dynamic simulations revealed that RQ18 presents varying structural profiles in aqueous solution, TFE/water mixtures, SDS micelles, and lipid bilayers. The peptide was virtually unable to release carboxyfluorescein from large unilamellar vesicles composed of POPC/cholesterol, model that mimics the eukaryotic membrane, indicating that vesicles' net charges and the presence of cholesterol may be related with RQ18 selectivity for bacterial and fungal cell surfaces. CONCLUSIONS: RQ18 was characterized as a membrane-active peptide with dual antibacterial and antifungal activities, without compromising mammalian cells viability, thus reinforcing its therapeutic application. GENERAL SIGNIFICANCE: These results provide further insight into the complex process of AMPs interaction with biological membranes, in special with systems that mimic prokaryotic and eukaryotic cell surfaces.

Campylobacter jejuni and Campylobacter coli originated from chicken carcasses modulate their transcriptome to translate virulence genes in human cells / Campylobacter jejuni e Campylobacter coli originadas de carcaças de frango modulam seu transcriptoma para traduzir genes de virulência em células humanas

The aim was to determine the spread of genetically similar profiles of Campylobacter in chicken carcasses and evaluate their ability to produce transcripts for ciaB, dnaJ, p19 and sodB genes, before and after cultivation in Caco-2 cells. The strains used were isolated from 420 samples of chicken carcasses chilled and frozen ready for marketing. The species were identified by PCR-multiplex, the phylogeny was determined by RAPD-PCR and the presence of transcripts was performed by RT-PCR. We identified 74 (17.6%) of Campylobacter strains, being 55 (74.3%) C. jejuni and 19 (25.7%) C. coli. The phylogenetic relationship demonstrated heterogeneity between isolates of the same species, with absence of clones, indicating the high level of diversity of circulating genotypes. The gene transcription showed conflicting results before and after the culture in Caco-2 cell, so that before cultivation isolates showed greater capacity to transcribe genes related to survival and after the interaction with human cells, the strains showed higher potential to transcribe genes associated with virulence. The result of this study contributes to the understanding of how these seemingly fragile microorganisms are the most prevalent bacterial agents in human gastroenteritis.(AU)

O objetivo foi determinar a disseminação de perfis geneticamente semelhantes de Campylobacter em carcaças de frango e avaliar sua capacidade de produzir transcritos para os genes ciaB, dnaJ, p19 e sodB, antes e após o cultivo em células Caco-2. As cepas utilizadas foram isoladas de 420 amostras de carcaças de frango resfriadas e congeladas prontas para comercialização. As espécies foram identificadas por PCR-multiplex, a filogenia foi determinada por RAPD-PCR e a presença de transcritos foi realizada por RT-PCR. Identificamos 74 (17,6%) das cepas de Campylobacter, sendo 55 (74,3%) C. jejuni e 19 (25,7%) C. coli. A relação filogenética demonstrou heterogeneidade entre isolados da mesma espécie, com ausência de clones, indicando o alto nível de diversidade dos genótipos circulantes. A transcrição gênica mostrou resultados conflitantes antes e após a cultura em células Caco-2, de modo que, antes do cultivo, os isolados apresentaram maior capacidade de transcrever genes relacionados à sobrevivência e após a interação com células humanas, as linhagens apresentaram maior potencial para transcrever genes associados à virulência. O resultado deste estudo contribui para a compreensão de como esses microrganismos aparentemente frágeis são os agentes bacterianos mais prevalentes na gastroenterite humana.(AU)

Evolution of Campylobacter jejuni of poultry origin in Brazil.

Campylobacter jejuni is the most common pathogen associated with foodborne diseases. Persistent presence of this pathogen contaminating the environment in slaughterhouses and chicken products have been reported worldwide. Although many efforts have been employed for reducing C. jejuni contamination, few studies have been conducted to understand the dynamics of C. jejuni in slaughterhouses over time. In this study, we evaluated the virulence, antibiotic resistance and genetic diversity profiles of 99 C. jejuni isolated from chilled chicken carcasses collected in Brazilian slaughterhouses during two distinct periods (2011-2012 and 2015-2016). The virulence profile was evaluated for the presence of flaA, ciaB, cadF, pldA and cdtABC genes. Antibiotic resistance was evaluated for amoxicillin-clavulanic acid, gentamicin, erythromycin and tetracycline. Genetic diversity was assessed using RAPD-PCR. The prevalence of C. jejuni was significantly reduced in 2015-2016 as well the number of antibiotic (and multidrug) resistant isolates, except for tetracycline. However, isolates from 2015 to 2016 showed higher prevalence of multiple virulence genes and genetic diversity profile compared to isolates from 2011 to 2012. During the studied period, stricter regulations to control pathogens in poultry farms and slaughterhouses were implemented in Brazil, which may have contributed to the profile variation observed due to changes of selective pressures on bacterial populations.

Micelle Bound Structure and Model Membrane Interaction Studies of the Peptide Hylin a1 from the Arboreal South American Frog Hypsiboas albopunctatus.

Antimicrobial peptides (AMPs) appear as a promising therapeutic candidate against multiresistant pathogens, because they are able to kill microorganisms and have low toxicity of resistance cells. Hylin a1 (Hy-a1, IFGAILPLALGALKNLIK-NH2) is a peptide extracted from the skin secretion of the frog Hypsiboas albopunctatus, which displays antimicrobial and hemolytic activities. We report here structural studies of Hy-a1 using different techniques such as fluorescence, CD and NMR. Our data showed that Hy-a1 acquires a well defined amphipathic α-helix when interacting with a membrane-like environment. Furthermore, Hy-a1 presented different affinity when compared to membranes of zwitterionic or anionic lipid composition. Finally, we proposed a molecular interaction model of this peptide with micelles.