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1. Lung SPLUNC1 Peptide Derivatives in the Lipid Membrane Headgroup Kill Gram-Negative Planktonic and Biofilm Bacteria

   Jakkampudi, Tanvi; Lin, Qiao; Mitra, Saheli; Qin, Weiheng; Kang, Ann; Chen, Jespar; Ryan, Emma; Wang, Runwuan; Gong, Yuqi; Heinrich, Frank; et al (May 2023, Biomacromolecules)

   Lu, Hua (Ed.)

   SPLUNC1 (short palate lung and nasal epithelial clone 1) is a multifunctional host defense protein found in human respiratory tract with antimicrobial properties. In this work we compare the biological activities of four SPLUNC1 antimicrobial peptide (AMP) derivatives using paired clinical isolates of the Gram-negative (G(-)) bacteria Klebsiella pneumoniae, obtained from eleven patients with/without colistin resistance. Secondary structural studies were carried out to study interactions between the AMPs and lipid model membranes (LMMs) utilizing circular dichroism (CD). Two peptides were further characterized using x-ray diffuse scattering (XDS) and neutron reflectivity (NR). A4-153 displayed superior antibacterial activity in both G(-) planktonic cultures and biofilms. NR and XDS revealed that A4-153 (highest activity) is located primarily in membrane headgroups, while A4-198 (lowest activity) is located in hydrophobic region. CD revealed that A4-153 is helical while A4-198 has little helical character, demonstrating that helicity and efficacy are correlated in these SPLUNC1 AMPs. 

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2. Rational Framework for the Design of Trp- and Arg-Rich Peptide Antibiotics Against Multidrug-Resistant Bacteria

   https://doi.org/10.3389/fmicb.2022.889791  

   Xiang, Wenyu; Clemenza, Patrice; Klousnitzer, Jessie; Chen, Jespar; Qin, Weiheng; Tristram-Nagle, Stephanie; Doi, Yohei; Di, Y. Peter; Deslouches, Berthony (May 2022, Frontiers in Microbiology)

   The threat of antibiotic resistance warrants the discovery of agents with novel antimicrobial mechanisms. Antimicrobial peptides (AMPs) directly disrupting bacterial membranes may overcome resistance to traditional antibiotics. AMP development for clinical use has been mostly limited to topical application to date. We developed a rational framework for systematically addressing this challenge using libraries composed of 86 novel Trp- and Arg-rich engineered peptides tested against clinical strains of the most common multidrug-resistant bacteria known as ESKAPE pathogens. Structure-function correlations revealed minimum lengths (as low as 16 residues) and Trp positioning for maximum antibacterial potency with mean minimum inhibitory concentration (MIC) of 2–4 μM and corresponding negligible toxicity to mammalian cells. Twelve peptides were selected based on broad-spectrum activity against both gram-negative and -positive bacteria and <25% toxicity to mammalian cells at maximum test concentrations. Most of the selected PAX remained active against the colistin-resistant clinical strains. Of the selected peptides, the shortest (the 16-residue E35) was further investigated for antibacterial mechanism and proof-of-concept in vivo efficacy. E35 killed an extensively-resistant isolate of Pseudomonas aeruginosa (PA239 from the CDC, also resistant to colistin) by irreversibly disrupting the cell membranes as shown by propidium iodide incorporation, using flow cytometry and live cell imaging. As proof of concept, in vivo toxicity studies showed that mice tolerated a systemic dose of up to 30 mg/kg peptide and were protected with a single 5 mg/kg intravenous (IV) dose against an otherwise lethal intraperitoneal injection of PA239. Efficacy was also demonstrated in an immune-compromised Klebsiella pneumoniae infection model using a daily dose of 4mg/kg E35 systemically for 2 days. This framework defines the determinants of efficacy of helical AMPs composed of only cationic and hydrophobic amino acids and provides a path for a potential departure from the restriction to topical use of AMPs toward systemic application. 

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3. Caerin 1 Antimicrobial Peptides that Inhibit HIV and Neisseria May Spare Protective Lactobacilli

   https://doi.org/10.3390/antibiotics9100661  

   Rollins-Smith, Louise A.; Smith, Patricia B.; Ledeczi, Anna M.; Rowe, Julia M.; Reinert, Laura K. (October 2020, Antibiotics)

   null (Ed.)

   Although acquired immunodeficiency syndrome (AIDS) caused by the human immunodeficiency virus (HIV) is a manageable disease for many, it is still a source of significant morbidity and economic hardship for many others. The predominant mode of transmission of HIV/AIDS is sexual intercourse, and measures to reduce transmission are needed. Previously, we showed that caerin 1 antimicrobial peptides (AMPs) originally derived from Australian amphibians inhibited in vitro transmission of HIV at relatively low concentrations and had low toxicity for T cells and an endocervical cell line. The use of AMPs as part of microbicidal formulations would expose the vaginal microbiome to these agents and cause potential harm to protective lactobacilli. Here, we tested the effects of caerin 1 peptides and their analogs on the viability of two species of common vaginal lactobacilli (Lactobacillus rhamnosus and Lactobacillus crispatus). Several candidate peptides had limited toxicity for the lactobacilli at a range of concentrations that would inhibit HIV. Three AMPs were also tested for their ability to inhibit growth of Neisseria lactamica, a close relative of the sexually transmissible Neisseria gonorrhoeae. Neisseria lactamica was significantly more sensitive to the AMPs than the lactobacilli. Thus, several candidate AMPs have the capacity to inhibit HIV and possible N. gonorrhoeae transmission at concentrations that are significantly less harmful to the resident lactobacilli. 

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4. Synthesis of Lysine Mimicking Membrane Active Antimicrobial Polymers

   Ankita Arora; Wan Zheng; Hongjun Liang; Abhijit Mishra (November 2018, Advances in Polymer Sciences and Technology)

   Bhuvanesh Gupta; Anup K.Ghosh; Atsushi Suzuki; Sunita Rattan (Ed.)

   Antibiotic resistance in bacteria is a major health concern. Antimicrobial Peptides (AMPs) are efficient in killing most microbes and yet the development of resistance to AMPs is rare. Although AMPs show promising antimicrobial activities, commercializing them as antibiotics is difficult as in vitro extraction and purification of AMPs is complicated and expensive. AMP mimicking antimicrobial polymers can overcome such problems while maintaining the necessary features of AMPs. Here, we have developed meth-acrylamide based polymers to mimic AMPs which possess high antimicrobial activities with low cytotoxicity. Bactericidal and scanning electron microscopy studies show that the synthesized polymers are effective against Gram-positive and Gram-negative bacteria. We find that these polymers are lethal to bacteria and at the same time, they are also non-cytotoxic to mammalian cells, thereby increasing the potential of these polymers to be used as antibiotics. 

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5. Ubiquitin-Mimicking Peptides Transfer Differentiates by E1 and E2 Enzymes

   https://doi.org/10.1155/2018/6062520  

   Jin, Bo; Wang, Jiayue; Liu, Xiangnan; Fang, Shuai; Jiang, Bo; Hofmann, Kay; Yin, Jun; Zhao, Bo (August 2018, BioMed Research International)

   Ubiquitin and ubiquitin like proteins (UBLs) play key roles in eukaryotes. These proteins are attached to their target proteins through an E1-E2-E3 cascade and modify the functions of these proteins. Since the discovery of ubiquitin, several UBLs have been identified, including Nedd8, SUMO, ISG15, and Atg8. Ubiquitin and UBLs share a similar three-dimensional structure: β -grasp fold and an X-X-[R/A/E/K]-X-X-[G/X]-G motif at the C-terminus. We have previously reported that ubiquitin, Nedd8, and SUMO mimicking peptides which all contain the conserved motif X-X-[R/A/E/K]-X-X-[G/X]-G still retained their reactivity toward their corresponding E1, E2, and E3 enzymes. In our current study, we investigated whether such C-terminal peptides could still be transferred onto related pathway enzymes to probe the function of these enzymes when they are fused with a protein. By bioinformatic search of protein databases, we selected eight proteins carrying the X-X-[R/A/E/K]-X-X-[G/X]-G motif at the C-terminus of the β -grasp fold. We synthesized the C-terminal sequences of these candidates as short peptides and found that three of them showed significant reactivity with the ubiquitin E1 enzyme Ube1. We next fused the three reactive short peptides to three different protein frames, including their respective native protein frames, a ubiquitin frame and a peptidyl carrier protein (PCP) frame, and measured the reactivities of these peptide-fused proteins with Ube1. Peptide-fused proteins on ubiquitin and PCP frames showed obvious reactivity with Ube1. However, when we measured E2 UbcH7 transfer, we found that the PCP-peptide fusions lost their reactivity with UbcH7. Taken together, these results suggested that the recognition of E2 enzymes with peptide-fused proteins depended not only on the C-terminal sequences of the ubiquitin-mimicking peptides, but also on the overall structures of the protein frames. 

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