Author: Gorvel, Jean-Pierre ; Salvador-Bescós, Miriam ; Bengoechea, Jose A ; Zúñiga-Ripa, Amaia ; Iriarte, Maite ; Renau-Mínguez, Chantal ; Pellegrini, Joaquin Miguel ; Elizalde-Bielsa, Aitor ; Loperena-Barber, Maite ; Ruiz-Rodríguez, Paula ; Conde-Álvarez, Raquel ; Lancaster, Rebecca ; Coscollá, Mireia ; Moriyón, Ignacio

The genus Pseudochrobactrum encompasses free-living bacteria phylogenetically close to Ochrobactrum opportunistic pathogens and to Brucella, facultative intracellular parasites causing brucellosis, a worldwide-extended and grave zoonosis. Recently, Pseudochrobactrum strains were isolated from Brucella natural hosts on Brucella selective media, potentially causing diagnostic confusions. Strikingly, P. algeriensis was isolated from cattle lymph nodes, organs that are inimical to bacteria. Here, we analyse P. algeriensis potential virulence factors in comparison with Ochrobactrum and Brucella. Consistent with genomic analyses, Western-Blot analyses confirmed that P. algeriensis lacks the ability to synthesize the N-formylperosamine O-polysaccharide characteristic of the lipopolysaccharide (LPS) of smooth Brucella core species. However, unlike other Pseudochrobactrum but similar to some early diverging brucellae, P. algeriensis carries genes potentially synthetizing a rhamnose-based O-polysaccharide LPS. Lipid A analysis by MALDI-TOF demonstrated that P. algeriensis LPS bears a lipid A with a reduced pathogen-associated molecular pattern, a trait shared with Ochrobactrum and Brucella that is essential to generate a highly stable outer membrane and to delay immune activation. Also, although not able to multiply intracellularly in macrophages, the analysis of P. algeriensis cell lipid envelope revealed the presence of large amounts of cationic aminolipids, which may account for the extremely high resistance of P. algeriensis to bactericidal peptides and could favor colonization of mucosae and transient survival in Brucella hosts. However, two traits critical in Brucella pathogenicity are either significantly different (T4SS [VirB]) or absent (erythritol catabolic pathway) in P. algeriensis. This work shows that, while diverging in other characteristics, lipidic envelope features relevant in Brucella pathogenicity are conserved in Brucellaceae. The constant presence of these features strongly suggests that reinforcement of the envelope integrity as an adaptive advantage in soil was maintained in Brucella because of the similarity of some environmental challenges, such as the action of cationic peptide antibiotics and host defense peptides. This information adds knowledge about the evolution of Brucellaceae, and also underlines the taxonomical differences of the three genera compared.

01 Sep 2022·Biochemistry and Biophysics Reports

Polycationic peptide R7-G-Aβ25-35 selectively induces cell death in leukemia Jurkat T cells through speedy mitochondrial depolarization, and CASPASE-3 -independent mechanism

Article

Author: Mendivil-Perez, Miguel ; Jimenez-Del-Rio, Marlene ; Velez-Pardo, Carlos

BackgroundAcute lymphoblastic leukemia (ALL) is still incurable hematologic neoplasia in an important percentage of patients. Therefore, new therapeutic approaches need to be developed.MethodsTo evaluate the cellular effect of cell-penetrating peptides (C-PP) on leukemia cells, Jurkat cells -a model of ALL were exposed to increasing concentration (50-500 μM) Aβ_25-35, R⁷-G-Aβ_25-35 and Aβ_25-35-G-R⁷ peptide for 24 h. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, flow cytometry (FC), and fluorescent microscopy (FM) analysis were used to assess metabolic viability, cell cycle and proliferation, mitochondria functionality, oxidative stress, and cell death markers.ResultsWe report for the first time that the R⁷-G-Aβ_25-35, but not Aβ_25-35 peptide, induced selective cell death in Jurkat cells more efficiently than the Aβ_25-35-G-R⁷ peptide. Indeed, R⁷-G-Aβ_25-35 (200 μM) altered the metabolic activity (-25%), arrested the cell cycle in the G2/M-phase (15%), and induced a significant reduction of cellular proliferation (i.e., -74% reduction of Ki-67 nuclei reactivity). Moreover, R⁷-G-Aβ_25-35 induced the dissipation of mitochondrial membrane potential (ΔΨ_m, 51%) and produced an important amount of reactive oxygen species (ROS, 75% at 8 h) in Jurkat cells. The exposure of cells to antioxidant/cytoprotectant N-acetylcysteine (NAC) did not prevent R⁷-G-Aβ_25-35 from a loss of ΔΨ_m in Jurkat cells. The peptide was also unable to activate the executer CASPASE-3, thereby preserving the integrity of the cellular DNA corroborated by the fact that the caspase-3 inhibitor NSCI was unable to protect cells from R⁷-G-Aβ_25-35 -induced cell damage. Further analysis showed that the R⁷-G-Aβ_25-35 peptide is specifically localized at the outer mitochondria membrane (OMM) according to colocalization with the protein translocase TOMM20. Additionally, the cytotoxic effect of the poly-R⁷ peptide resembles the toxic action of the uncoupler FCCP, mitocan oligomycin, and rotenone in Jurkat cells. Importantly, the R⁷-G-Aβ_25-35 peptide was innocuous to menstrual mesenchymal stromal cells (MenSC) -normal non-leukemia proliferative cells.ConclusionOur findings demonstrated that the cationic Aβ peptide possesses specific anti-leukemia activity against Jurkat cells through oxidative stress (OS)- and CASPASE-3-independent mechanism but fast mitochondria depolarization.

01 Jul 2022·Biochemical and Biophysical Research CommunicationsQ3 · BIOLOGY

Enhanced proteolytic resistance of cationic antimicrobial peptides through lysine side chain analogs and cyclization

Q3 · BIOLOGY

Article

Author: Deber, Charles M ; He, Shelley ; Glibowicka, Mira

While cationic antimicrobial peptides (CAPs) are compelling candidates for antimicrobial therapy, their clinical development is largely hampered by their rapid and non-specific enzymatic degradation in physiological fluids. We have earlier de novo designed and synthesized a novel category of CAPs typified by the sequence KKKKKK-AAFAAWAAFAA-NH₂ (termed "6K-F17") that have remarkable membrane-penetrating power, are highly selective for bacterial rather than host membranes, and are non-cytotoxic. Here we pursue the design and validation of the Lys chain-shortened 6K-F17 analogs 6Dap-F17 (Dap = diaminopropionic acid), 6Dab-F17 (Dab = diaminobutyric acid), and 6Orn-F17 (Orn = ornithine). Intriguingly, although initially designed to specifically resist trypsin vs. their original Lys sites, all three derivatives of 6K-F17 showed markedly improved stability not only against trypsin, but also against the major proteolytic enzymes elastase and proteinase K at a 1:100 enzyme-to-peptide (E:P) ratio. When the least stable analog, 6Dap-F17, was then cyclized ('stapled') - with reduced main chain hydrophobicity to avoid erythrocyte hemolysis - the peptide became robust towards all three enzymes up to 60 min at a 1:100 E:P ratio, and retained strong presence even at an enhanced 1:1 E:P ratio, as determined by HPLC and mass spectrometry. These results suggest that the application of Lys chain-shortening, either alone or in combination with macrocyclization, may enhance metabolic stability of CAPs, and thus their clinical potential.

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Indication	Highest Phase	Country/Location	Organization	Date
Cystic Fibrosis	Preclinical	CA	The Hospital for Sick Children	-
Cystic Fibrosis	Preclinical	CA	University of Toronto	-
Pseudomonas Infections	Preclinical	CA	University of Toronto	-

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