The rapid emergence of antibiotic-resistant infections is prompting increased interest in phage-based antimicrobials. However, acquisition of resistance by bacteria is a major issue in the successful development of phage therapies. Through natural evolution and structural modeling, we identified host-range-determining regions (HRDRs) in the T3 phage tail fiber protein and developed a high-throughput strategy to genetically engineer these regions through site-directed mutagenesis. Inspired by antibody specificity engineering, this approach generates deep functional diversity while minimizing disruptions to the overall tail fiber structure, resulting in synthetic ''phagebodies.'' We showed that mutating HRDRs yields phagebodies with altered host-ranges, and select phagebodies enable long-term suppression of bacterial growth in vitro, by preventing resistance appearance, and are functional in vivo using a murine model. We anticipate that this approach may facilitate the creation of next-generation antimicrobials that slow resistance development and could be extended to other viral scaffolds for a broad range of applications.

26The rapid emergence of antibiotic-resistant infections is prompting increased interest in 27 phage-based antimicrobials. However, acquisition of resistance by bacteria is a major issue in the 28 successful development of phage therapies. Through natural evolution and structural modeling, 29 we identified host-range determining regions (HRDR) in the T3 phage tail fiber protein and 30 developed a high-throughput strategy to genetically engineer these regions through site-directed 31 mutagenesis. Inspired by antibody specificity engineering, this approach generates deep functional 32 diversity (>10 7 different members), while minimizing disruptions to the overall protein structure, 33 resulting in synthetic "phagebodies". We showed that mutating HRDRs yields phagebodies with 34 altered host-ranges. Select phagebodies enable long-term suppression of bacterial growth by 35 preventing the appearance of resistance in vitro and are functional in vivo using a mouse skin 36 infection model. We anticipate this approach may facilitate the creation of next-generation 37 antimicrobials that slow resistance development and could be extended to other viral scaffolds for 38 a broad range of applications. 39 evolution, tail fiber 41 3 Highlights: 42  Vastly diverse phagebody libraries containing 10 7 different members were created. 43  Structure-informed engineering of viral tail fibers efficiently generated host-range 44 alterations. 45  Phagebodies prevented the development of bacterial resistance across long timescales in 46 vitro and are functional in vivo. 47 48 49 59 Schooley et al., 2017). In addition, phages are selective for particular bacterial strains, as opposed 60 conventional chemical antibiotics that exhibit broad-spectrum activity, which contributes to 61 antibiotic-resistance development. Phage selectivity is dependent on binding to cell surface 62 receptors in order to recognize their host and initiate infection (Silva et al., 2016). However, 63 4reliance on receptor recognition for infectivity implies that resistance against a bacteriophage can 64 occur through receptor mutations. As a result, phage-based products are usually composed of 65 multiple unrelated phages that collectively target a range of receptors, thus distributing the 66 selective pressure away from any individual phage receptor. However, these cocktails are often 67 composed of uncharacterized phages whose biology are poorly defined. Furthermore, 68 manufacturing cocktails composed of diverse phages, as well as tracking their pharmacodynamics 69 and immunogenic properties, is complex, which can limit their development as antimicrobial drugs 70 (Cooper et al., 2016). 71 Various approaches have been undertaken to rationally expand the host-range of phages to 72 combat resistance (74 2005; Yosef et al., 2017). However, these approaches depend on hybridization between already 75 characterized bacteriophages with known and desired host-ranges. Natural evolution of phages has 76 also been harnessed to alter phage host-range, but...

In the version of this article originally published, the source of the human skin commensals and the associated acknowledgements, funding and published work were inadvertently omitted. This information has now been added to Methods (in the section 'Bacterial strains and media'), Acknowledgements and References (ref. 25 ), and is also reproduced below. Laurice Flowers, the collaborator who isolated the skin commensals described in ref. 25 , has been added as a coauthor.Moreover, we clarified that many (rather than all) of the encrypted peptides are encoded in proteins unrelated to the immune system (abstract; and page 68, second column). We also corrected the number of encrypted peptides synthesized and characterized (56, rather than 55; pages 68 and 69), the number of encrypted peptides that target both pathogens and commensals (at least 30, rather than 55; page 73, first column), the percentages of peptides that displayed antimicrobial activity against pathogens (62.5%, rather than 63.6%; page 68, second column) and that targeted either pathogens, gut commensals or skin commensals (at least 83.9%, rather than at least 80%; page 69, second column), and the number of human gut microbiota members tested (11, rather than 13; page 68, second column). Incorrect callouts to panels in Fig. 3 were also amended (in particular, the first instances of "Fig. 3a" and "Fig. 3d" on page 70 should have been "Fig. 3d" and "Fig. 3c," respectively; and the two instances of "Fig. 3e" on page 70 should have been "Fig. 3b").

The increasing resistance of bacteria to existing antibiotics constitutes a major public health threat globally. Most current antibiotic treatments are hindered by poor delivery to the infection site, leading to undesired off-target effects and drug resistance development and spread. Here, we describe micro- and nanomachines that effectively and autonomously deliver antibiotic payloads to the target area. The active motion and antimicrobial activity of the silica-based robots are driven by catalysis of the enzyme urease and antimicrobial peptides, respectively. These antimicrobial machines show micromolar bactericidal activity in vitro against different Gram-positive and Gram-negative pathogenic bacterial strains and act by rapidly depolarizing their membrane. Finally, they demonstrated autonomous anti-infective efficacy in vivo in a clinically relevant abscess infection mouse model. In summary, our machines combine navigation, catalytic conversion, and bactericidal capacity to deliver antimicrobial payloads to specific infection sites. This technology represents a much-needed tool to direct therapeutics to their target to help combat drug-resistant infections.

No abstract

No abstract

No abstract

IntroductionMalaria is an infectious disease responsible for approximately one million deaths annually. Peptides such as angiotensin II (AII) and its analogs are known to have antimalarial effects against Plasmodium gallinaceum [1] and Plasmodium falciparum [2]. However, their mechanism of action is still not fully understood at the molecular level. In this work, we investigated this issue by comparing the antimalarial activity of angiotensin II with that of: i) its enantiomer formed by only D-amino acids; ii) its isomer with reversed sequence; and iii) its analogs restricted by lactam bridges -the so-called VC5 peptides. Results and DiscussionThe peptides were synthesized manually with the t-Boc strategy on Merrifield resin (Table 1). AII could inactivate 88% of the Plasmodium gallinaceum sporozoites, which is consistent with its known antimalarial properties. Similar anti-plasmodial effects were measured for ent-AII and other peptides (Figure 1). In contrast to AII, ent-AII and retro-AII could not interact with the membrane AII-receptors ( Figure 2). This indicates that the anti-plasmodial effects of AII analogs depend on direct peptide-phospholipid interactions. This hypothesis was also supported by the anti-plasmodial activities recorded for the lactam bridge-restricted analogs VC5 and ent-VC5. These were comparable to that of AII. A significant change in the anti-plasmodial activity was observed only for retro-AII, which was ~6-fold less effective than AII. We used CD experiments to show that the β-turn was the most frequent conformation adopted by peptides in aqueous and organic solvents. Moreover, the β-turn conformation was correlated with a larger antiplasmodial activity.In the presence of SDS micelles, AII had a β-turn conformation while retro-AII presented a random coiled conformation (Figure 3). Consistently, molecular dynamic simulations revealed that the AII chains were slightly more bent than retro-AII at the surface of a model phospholipid bilayer (Figure 4). We did not observe spontaneous pore formation in either case. This may be an indication that this process involves larger time scales and possibly the organization of the peptide chains into larger assemblies. However, qualitative differences were identified between the behavior of AII and retro-AII by pulling both peptides across the phospholipid bilayer. At the hydrophobic membrane interior, the retro-AII chain was severely coiled and rigid. AII was much more flexible and could experience both straight and coiled conformations. Interactions between AII and phospholipid head groups were kept for a longer time even in the membrane interior. It is conceivable that stronger peptide-head group interactions might be more effective at stabilizing a pore in longer time scales. This contributes to the larger anti-plasmodial activity of AII versus retro-AII. We hope that our results can be used for the systematic design of novel compounds with antimalarial activity.