Functional characterization and transcriptional repression by Lacticaseibacillus paracasei DinJ-YafQ.

DinJ-YafQ is a bacterial type II TA system formed by the toxin RNase YafQ and the antitoxin protein DinJ. The activity of YafQ and DinJ has been rigorously studied in Escherichia coli, but little has been reported about orthologous systems identified in different microorganisms. In this work, we report an in vitro and in vivo functional characterization of YafQ and DinJ identified in two different strains of Lacticaseibacillus paracasei and isolated as recombinant proteins. While DinJ is identical in both strains, the two YafQ orthologs differ only for the D72G substitution in the catalytic site. Both YafQ orthologs digest ribosomal RNA, albeit with different catalytic efficiencies, and their RNase activity is neutralized by DinJ. We further show that DinJ alone or in complex with YafQ can bind cooperatively to a 28-nt inverted repeat overlapping the -35 element of the TA operon promoter. Atomic force microscopy imaging of DinJ-YafQ in complex with DNA harboring the cognate site reveals the formation of different oligomeric states that prevent the binding of RNA polymerase to the promoter. A single amino acid substitution (R13A) within the RHH DNA-binding motif of DinJ is sufficient to abolish DinJ and DinJ-YafQ DNA binding in vitro. In vivo experiments confirm the negative regulation of the TA promoter by DinJ and DinJ-YafQ and unveil an unexpected high expression-related toxicity of the gfp reporter gene. A model for the binding of two YafQ-(DinJ)2-YafQ tetramers to the promoter inverted repeat showing the absence of protein-protein steric clash is also presented. KEY POINTS: • The RNase activity of L. paracasei YafQ toxin is neutralized by DinJ antitoxin. • DinJ and DinJ-YafQ bind to an inverted repeat to repress their own promoter. • The R13A mutation of DinJ abolishes DNA binding of both DinJ and DinJ-YafQ.

Activity of Membrane-Permeabilizing Lpt Peptides.

Herein, we investigated the toxicity and membrane-permeabilizing capabilities of Lpt and Lpt-like peptides, belonging to type I toxin-antitoxin systems carried by plasmid DNA of Lacticaseibacillus strains. These 29 amino acid peptides are predicted to form α-helical structures with a conserved central hydrophobic sequence and differently charged hydrophilic termini. Like Lpt, the expression of Lpt-like in E. coli induced growth arrest, nucleoid condensation, and cell membrane damage, suggesting membrane interaction as the mode of action. The membrane permeabilization activity of both peptides was evaluated by using liposome leakage assays, dynamic light scattering, and CD spectroscopy. Lpt and Lpt-like showed liposome leakage activity, which did not lead to liposome disruption but depended on peptide concentration. Lpt was generally more effective than Lpt-like, probably due to different physical chemical properties. Leakage was significantly reduced in larger liposomes and increased with negatively charged PCPS liposomes, indicating that electrostatic interactions and membrane curvature influence peptide activity. Contrary to most membrane-active peptides, Lpt an Lpt-like progressively lost their α-helical structure upon interaction with liposomes. Our data are inconsistent with the formation of membrane-spanning peptide pores but support a mechanism relying on the transient failure of the membrane permeability barrier possibly through the formation of "lipid pores".

Molecular Characterization of the Allergenic Arginine Kinase from the Edible Insect Hermetia illucens (Black Soldier Fly).

SCOPE: Arginine kinase (AK) is an important enzyme for energy metabolism of invertebrate cells by participating in the maintenance of constant levels of ATP. However, AK is also recognized as a major allergen in insects and crustaceans capable of cross-reactivity with sera of patients sensitized to orthologous proteins. In the perspective of introducing insects or their derivatives in the human diet in Western world, it is of primary importance to evaluate possible risks for allergic consumers. METHODS AND RESULTS: This work reports the identification and characterization of AK from Hermetia illucens commonly known as the black soldier fly, a promising insect for human consumption. To evaluate allergenicity of AK from H. illucens, putative linear and conformational epitopes are identified by bioinformatics analyses, and Dot-Blot assays are carried out by using sera of patients allergic to shrimp or mites to validate the cross-reactivity. Gastrointestinal digestion reduces significantly the linear epitopes resulting in lower allergenicity, while the secondary structure is altered at increasing temperatures supporting the possible loss or reduction of conformational epitopes. CONCLUSION: The results indicate that the possible allergenicity of AK should be taken in consideration when dealing with novel foods containing H. illucens or its derivatives.

Allergenicity of tropomyosin variants identified in the edible insect Hermetia illucens (black soldier fly).

Insect consumption could address the increasing protein demand in compliance with environmental sustainability. Hermetia illucens (black soldier fly, BSF) is a promising insect for human diet and it is essential to assess the related allergenic risk, meant as primary sensitization or cross-reactivity with known allergens. In this work, we investigate the allergenicity of two tropomyosin variants identified in the BSF genome and produced as recombinant proteins. Immunoblot experiments showed that both proteins were recognized by sera of patients allergic to shrimp or mites highlighting the cross-reactivity risk. CD spectroscopy, cross-linking assays and size-exclusion chromatography showed a structure composed of alpha-helices oligomers for both variants. These proteins were quite stable to pH but sensitive to increasing temperatures. In vitro simulated digestion associated to mass-spectrometry allowed the identification of peptides resistant to gastrointestinal conditions which were compared with epitopes of Arthropoda and Mollusca allergens to predict the persistence of allergenicity upon digestion.

Origin and significance of the human DNase repertoire.

The human genome contains four DNase1 and two DNase2 genes. The origin and functional specialization of this repertoire are not fully understood. Here we use genomics and transcriptomics data to infer the evolutionary history of DNases and investigate their biological significance. Both DNase1 and DNase2 families have expanded in vertebrates since ~ 650 million years ago before the divergence of jawless and jawed vertebrates. DNase1, DNase1L1, and DNase1L3 co-existed in jawless fish, whereas DNase1L2 originated in amniotes by tandem duplication of DNase1. Among the non-human DNases, DNase1L4 and newly identified DNase1L5 derived from early duplications that were lost in terrestrial vertebrates. The ancestral gene of the DNase2 family, DNase2b, has been conserved in synteny with the Uox gene across 700 million years of animal evolution,while DNase2 originated in jawless fish. DNase1L1 acquired a GPI-anchor for plasma membrane attachment in bony fishes, and DNase1L3 acquired a C-terminal basic peptide for the degradation of microparticle DNA in jawed vertebrates. The appearance of DNase1L2, with a distinct low pH optimum and skin localization, is among the amniote adaptations to life on land. The expansion of the DNase repertoire in vertebrates meets the diversified demand for DNA debris removal in complex multicellular organisms.

Actin-Resistant DNase1L2 as a Potential Therapeutics for CF Lung Disease.

In cystic fibrosis (CF), the accumulation of viscous lung secretions rich in DNA and actin is a major cause of chronic inflammation and recurrent infections leading to airway obstruction. Mucolytic therapy based on recombinant human DNase1 reduces CF mucus viscosity and promotes airway clearance. However, the marked susceptibility to actin inhibition of this enzyme prompts the research of alternative treatments that could overcome this limitation. Within the human DNase repertoire, DNase1L2 is ideally suited for this purpose because it exhibits metal-dependent endonuclease activity on plasmid DNA in a broad range of pH with acidic optimum and is minimally inhibited by actin. When tested on CF artificial mucus enriched with actin, submicromolar concentrations of DNase1L2 reduces mucus viscosity by 50% in a few seconds. Inspection of superimposed model structures of DNase1 and DNase1L2 highlights differences at the actin-binding interface that justify the increased resistance of DNase1L2 toward actin inhibition. Furthermore, a PEGylated form of the enzyme with preserved enzymatic activity was obtained, showing interesting results in terms of activity. This work represents an effort toward the exploitation of natural DNase variants as promising alternatives to DNase1 for the treatment of CF lung disease.