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Antimicrobial peptides (AMPs), which can be modified to kill a broad spectrum of microoganisms or a specific microorganism, are considered as promising alternatives to combat the rapidly widespread, resistant bacterial infections. However, there are still several obstacles to overcome. These include toxicity, stability, and the ability to interfere with the immune response and bacterial resistance. To overcome these challenges, we herein replaced the regular peptide bonds with isopeptide bonds to produce new AMPs based on the well-known synthetic peptides Amp1L and MSI-78 (pexiganan). Two new peptides Amp1EP and MSIEP were generated while retaining properties such as size, sequence, charge, and molecular weight. These new peptides have reduced toxicity toward murine macrophage (RAW 264.7) cells, human monocytic (THP-1) cells, and human red blood cells (hRBCs) and enhanced the stability toward proteolytic degradation. Importantly, the new peptides do not repress the pro-inflammatory cytokine and hence should not modulate the immune response. Structurally, the new peptides, Amp1EP and MSIEP, have a structure of random coils in contrast to the helical structures of the parental peptides as revealed by circular dichroism (CD) analysis. Their mode of action, assessed by flow cytometry, includes permeabilization of the bacterial membrane. Overall, we present here a new approach to modulate AMPs to develop antimicrobial peptides for future therapeutic purposes.

Multidrug resistant bacteria possess various mechanisms that can sense environmental stresses such as antibiotics and antimicrobial peptides and rapidly respond to defend themselves. Two known defense strategies are biofilm formation and lipopolysaccharide (LPS) modification. Though LPS modifications are observed in biofilm-embedded bacteria, their effect on biofilm formation is unknown. Using biochemical and biophysical methods coupled with confocal microscopy, atomic force microscopy, and transmission electron microscopy, we show that biofilm formation is promoted in a Pseudomonas aeruginosa PAO1 strain with a loss of function mutation in the arnB gene. This loss of function prevents the addition of the positively charged sugar 4-amino-4-deoxy-l-arabinose to lipid A of LPS under restrictive magnesium conditions. The data reveal that the arnB mutant, which is susceptible to antimicrobial peptides, forms a biofilm that is more robust than that of the wild type. This is in line with the observations that the arnB mutant exhibits outer surface properties such as hydrophobicity and net negative charge that promote the formation of biofilms. Moreover, when grown under Mg2+ limitation, both the wild type and the arnB mutant exhibited a reduction in the level of membrane-bound polysaccharides. The data suggest that the loss of polysaccharides exposes the membrane and alters its biophysical properties, which in turn leads to more biofilm formation. In summary, we show for the first time that blocking a specific lipid A modification promotes biofilm formation, suggesting a trade-off between LPS remodeling and resistance mechanisms of biofilm formation.

For successful infection and propagation viruses must overcome many obstacles such as the immune system and entry into their host cells. The human immunodeficiency virus (HIV), utilizes its trimeric envelope protein gp160, specifically the gp41 subunit, to enter its host cell. During this process, a gp41-central coiled-coil is formed from three N and three C terminal heptad repeats, termed the six helix bundle, which drives membrane fusion. Recently, T-cell suppression has been reported as an additional function for several regions of gp41 by interfering with the T-cell receptor signaling cascade. One of these regions encompasses the conserved pocket binding domain that is situated in the C terminal heptad repeat and stabilizes six-helix bundle formation. This could indicate that the pocket binding domain plays a role in T-cell suppression in addition to its role in membrane fusion. To investigate this dual function, we used two independent cell cultures coupled with biophysical techniques. The data reveal that the pocket binding domain mediates T-cell suppression by stabilizing a T-cell receptor binding conformation in the membrane. Moreover, we show that the clinically used HIV fusion inhibitor T-20 did not show suppressive abilities, in contrast to the potent fusion inhibitor C34. In addition, by focusing on six helix bundle conformation post its assembly, we shed light on a mechanism by which gp41’s function alternates from membrane fusion facilitation to suppression of T-cell receptor activation.