The GAIIG sequence, common to the amyloid beta peptide (residues 29–33) and to the HIV‐1 gp120 (residues 24–28 in a typical V3 loop), self‐assembles into amyloid fibrils, as suggested by theory and the experiments presented here. The longer YATGAIIGNII sequence from the V3 loop also self‐assembles into amyloid fibrils, of which the first three and the last two residues are outside the amyloid GAIIG core. We postulate that this sequence, with suitably selected modifications at the flexible positions, can serve as a designable scaffold for novel amyloid‐based materials. Moreover, we report the single crystal X‐ray structure of the beta‐breaker peptide GAIPIG at 1.05 Å resolution. The structural information provided in this study could serve as the basis for structure‐based design of potential inhibitors of amyloid formation.

Phage lysins are a source of novel antimicrobials to tackle the bacterial antibiotic-resistance crisis. The engineering of phage lysins is being explored as a game-changing technological strategy to introduce a more precise approach in the way in which antimicrobial therapy is applied. Such engineering efforts will benefit from a better understanding of lysin structure and function. In this work, the antimicrobial activity of the endolysin from Pseudomonas aeruginosa phage JG004, termed Pae87, has been characterized. This lysin had previously been identified as an antimicrobial agent candidate that is able to interact with the Gram-negative surface and disrupt it. Further evidence is provided here based on a structural and biochemical study. A high-resolution crystal structure of Pae87 complexed with a peptidoglycan fragment showed a separate substrate-binding region within the catalytic domain, 18 Å away from the catalytic site and located on the opposite side of the lysin molecule. This substrate-binding region was conserved among phylogenetically related lysins lacking an additional cell-wall-binding domain, but not among those containing such a module. Two glutamic acids were identified to be relevant for the peptidoglycan-degradation activity, although the antimicrobial activity of Pae87 was seemingly unrelated. In contrast, an antimicrobial peptide-like region within the Pae87 C-terminus, named P87, was found to be able to actively disturb the outer membrane and display antibacterial activity by itself. Therefore, an antimicrobial mechanism for Pae87 is proposed in which the P87 peptide plays the role of binding to the outer membrane and disrupting the cell-wall function, either with or without the participation of the catalytic activity of Pae87.

Background/Objectives: Mycobacterium abscessus is an opportunistic pathogen causing infections mainly in patients with immunosuppression and chronic pulmonary pathologies. Extended treatment periods are needed to tackle this pathogen, bacterial eradication is rare, and recurrence can take place with time. New alternative treatments are being investigated, such as bacteriophage therapy. This work describes the characterization of the mycobacteriophage P3MA, showing its ability to infect clinical and standard M. abscessus strains. Methods: Phylogenetic analysis, electron microscopy, growth curves, biofilm assays, checkerboard, and granuloma-like medium studies were performed. Results: P3MA inhibited the growth of clinical samples in both planktonic and biofilm states as well as in a granuloma-like model. The study of the interaction with antibiotics revealed that P3MA exhibited an antagonistic effect combined with clarithromycin, indifference with amikacin, and synergy with imipenem. Conclusions: All these results suggest that, after genetic engineering, P3MA could be a promising candidate for phage therapy in combination with imipenem, including lung infections.

Many bacteriophages use tailspikes to degrade host bacterial polysaccharides, facilitating access to the outer membrane. The homotrimeric tail spikes of the Salmonella phage epsilon15 feature a slender phage-binding domain, a kink, and a barrel-shaped section with three petal-like protrusions. Here, we present the crystal structures of the monomeric protruding petal domain alone and of the trimeric barrel-shaped section with three petal domains. The barrel-shaped section includes a trimeric beta-helix, typical of phage tail spikes, alongside a trimeric beta-sandwich domain. The petal domain exhibits a fold characteristic of the serine-glycine-asparagine-histidine (SGNH) esterase family. Co-crystallisation with O-antigen fragments identified four binding sites on the tailspike: two adjacent sites on the surface of the triple beta-helix, one in the beta-sandwich domain and a fourth near the petal esterase site. These binding sites align with the expected orientation of the phage just before DNA transfer. Nuclear magnetic resonance spectroscopy and site-directed mutagenesis revealed an endorhamnosidase activity, showed that the reaction mechanism proceeds by inversion of the configuration and revealed that the active site is located at the junction of the two beta-helix binding sites. Analogous experiments also revealed an esterase site in the petal domain. Together, the structural and functional insights suggest a dual role for the phage epsilon15 tailspike: de-acetylation of the O-antigen, potentially affecting the local structure and lipopolysaccharide flexibility, plus cleavage of the O-antigen, enabling the phage to approach the bacterial membrane.