The rapid rise of antibiotic resistance and slow discovery of new antibiotics have threatened global health. While novel phage lysins have emerged as potential antibacterial agents, experimental screening methods for novel lysins pose significant challenges due to the enormous workload. Here, the first unified software package, namely DeepLysin, is developed to employ artificial intelligence for mining the vast genome reservoirs (\"dark matter\") for novel antibacterial phage lysins. Putative lysins are computationally screened from uncharacterized Staphylococcus aureus phages and 17 novel lysins are randomly selected for experimental validation. Seven candidates exhibit excellent in vitro antibacterial activity, with LLysSA9 exceeding that of the best-in-class alternative. The efficacy of LLysSA9 is further demonstrated in mouse bloodstream and wound infection models. Therefore, this study demonstrates the potential of integrating computational and experimental approaches to expedite the discovery of new antibacterial proteins for combating increasing antimicrobial resistance.

Brevibacillus laterosporus (Bl) is a Gram-positive and spore-forming bacterium. Insect pathogenic strains have been characterised in New Zealand, and two isolates, Bl 1821L and Bl 1951, are under development for use in biopesticides. However, growth in culture is sometimes disrupted, affecting mass production. Based on previous work, it was hypothesised that Tectiviridae phages might be implicated. While investigating the cause of the disrupted growth, electron micrographs of crude lysates showed structural components of putative phages including capsid and tail-like structures. Sucrose density gradient purification yielded a putative self-killing protein of ~30 kDa. N-terminal sequencing of the ~30 kDa protein identified matches to a predicted 25 kDa hypothetical and a 31.4 kDa putative encapsulating protein homologs, with the genes encoding each protein adjacent in the genomes. BLASTp analysis of the homologs of 31.4 kDa amino acid sequences shared 98.6% amino acid identity to the Linocin M18 bacteriocin family protein of Brevibacterium sp. JNUCC-42. Bioinformatic tools including AMPA and CellPPD defined that the bactericidal potential originated from a putative encapsulating protein. Antagonistic activity of the ~30 kDa encapsulating protein of Bl 1821L and Bl 1951during growth in broth exhibited bacterial autolytic activity. LIVE/DEAD staining of Bl 1821L cells after treatment with the ~30 kDa encapsulating protein of Bl 1821L substantiated the findings by showing 58.8% cells with the compromised cell membranes as compared to 37.5% cells in the control. Furthermore, antibacterial activity of the identified proteins of Bl 1821L was validated through gene expression in a Gram-positive bacterium Bacillus subtilis WB800N. KEY POINTS: • Gene encoding the 31.4 kDa antibacterial Linocin M18 protein was identified • It defined the autocidal activity of Linocin M18 (encapsulating) protein • Identified the possible killing mechanism of the encapsulins.

The Gram-positive and spore-forming bacterium Brevibacillus laterosporus (Bl) belongs to the Brevibacillus brevis phylogenetic cluster. Isolates of the species have demonstrated pesticidal potency against a wide range of invertebrate pests and plant diseases. Two New Zealand isolates, Bl 1821L and Bl 1951, are under development as biopesticides for control of diamondback moth and other pests. However, due to the often-restricted growth of these endemic isolates, production can be an issue. Based on the previous work, it was hypothesised that the putative phages might be involved. During investigations of the cause of the disrupted growth, electron micrographs of crude lysate of Bl 1821L showed the presence of phages’ tail-like structures. A soft agar overlay method with PEG 8000 precipitation was used to differentiate between the antagonistic activity of the putative phage and phage tail-like structures (bacteriocins). Assay tests authenticated the absence of putative phage activity. Using the same method, broad-spectrum antibacterial activity of Bl 1821L lysate against several Gram-positive bacteria was found. SDS-PAGE of sucrose density gradient purified and 10 kD MWCO concentrated lysate showed a prominent protein band of ~48 kD, and transmission electron microscopy revealed the presence of polysheath-like structures. N-terminal sequencing of the ~48 kD protein mapped to a gene with weak predicted amino acid homology to a Bacillus PBSX phage-like element xkdK, the translated product of which shared >90% amino acid similarity to the phage tail-sheath protein of another Bl published genome, LMG15441. Bioinformatic analysis also identified an xkdK homolog in the Bl 1951 genome. However, genome comparison of the region around the xkdK gene between Bl 1821L and Bl 1951 found differences including two glycine rich protein encoding genes which contain imperfect repeats (1700 bp) in Bl 1951, while a putative phage region resides in the analogous Bl 1821L region. Although comparative analysis of the genomic organisation of Bl 1821L and Bl 1951 PBSX-like region with the defective phages PBSX, PBSZ, and PBP 180 of Bacillus subtilis isolates 168 and W23, and Bacillus phage PBP180 revealed low amino acids similarity, the genes encode similar functional proteins in similar arrangements, including phage tail-sheath (XkdK), tail (XkdO), holin (XhlB), and N-acetylmuramoyl-l-alanine (XlyA). AMPA analysis identified a bactericidal stretch of 13 amino acids in the ~48 kD sequenced protein of Bl 1821L. Antagonistic activity of the purified ~48 kD phage tail-like protein in the assays differed remarkably from the crude lysate by causing a decrease of 34.2% in the number of viable cells of Bl 1951, 18 h after treatment as compared to the control. Overall, the identified inducible phage tail-like particle is likely to have implications for the in vitro growth of the insect pathogenic isolate Bl 1821L.

BACKGROUND: Nitraria tangutorum is an important desert shrub that shows resistance to drought, salt and wind erosion stresses. It is a central ecological species in its area. Here, we have studied how N. tangutorum has adapted to achieve a successful reproduction strategy.
RESULTS: We found that N. tangutorum is mainly pollinated by insects of the Hymenoptera, Diptera and Coleoptera orders. Nitraria tangutorum has very small flowers, with the nectary composed of secretive epidermal cells from which nectar is secreted, located within the inner petals. In addition, analyzing the transcriptome of four successive flower developmental stages revealed that mainly differentially expressed genes associated with flower and nectary development, nectar biosynthesis and secretion, flavonoid biosynthesis, plant hormone signal transduction and plant-pathogen interaction show dynamic expression. From the nectar, we could identify seven important proteins, of which the L-ascorbate oxidase protein was first found in plant nectar. Based on the physiological functions of these proteins, we predict that floral nectar proteins of N. tangutorum play an important role in defending against microbial infestation and scavenging active oxygen.
CONCLUSIONS: This study revealed that N. tangutorum is an insect-pollinated plant and its nectary is composed of secretive epidermal cells that specialized into secretive trichomes. We identified a large number of differentially expressed genes controlling flower and nectary development, nectar biosynthesis and secretion, flavonoid biosynthesis, plant hormone signal transduction and plant-pathogen interaction. We suggest that proteins present in N. tangutorum nectar may have both an antibacterial and oxygen scavenging effect. These results provide a scientific basis for exploring how the reproductive system of N. tangutorum and other arid-desert plants functions.

Seroin 1 and seroin 2 are abundant in silkworm cocoon silk and show strong antibacterial activities, and thus are thought to protect cocoon silk from damage by bacteria. In this study, we characterized the expression pattern of silkworm seroin 3, and found that seroin 3 is synthesized in the female ovary and secreted into egg to play its roles. After being infected, seroin 1, 2, and 3 were significantly up-regulated in the silkworm. We synthesized the full-length protein of seroin 1, 2, and 3 and their N/C-terminal domain (seroin-N/C), and compared the antimicrobial activities in vitro. All three seroins showed higher antibacterial activity against Gram-positive bacteria than against Gram-negative bacteria. Seroin 2 showed better antibacterial effect than seroin 1 and 3, whereas seroin 1/2/3-N was better than seroin 1/2/3-C. We found that seroin 2-C has stronger peptidoglycan binding ability than seroin 2-N per the ELISA test. The binding sites of seroin 2 with bacteria were blocked by peptidoglycan, which resulted in the loss of the antibacterial activity of seroin 2. Collectively, these findings suggest that seroin 1 and 2 play antibacterial roles in cocoon silk, whereas seroin 3 functions in the eggs. The three silkworm seroins have the same antibacterial mechanism, that is, binding to bacterial peptidoglycan by the C-terminal domain and inhibiting bacterial growth by the N-terminal domain.

L-amino acid oxidases (LAAOs) have antibacterial activity and play important roles in innate immunity. We have previously identified a LAAO of ~52 kDa in size from the mucus layer of the flounder Platichthys stellate (psLAAO1) and have successfully produced psLAAO1 as a secreted bioactive recombinant protein by using Pichia pastoris (P. pastoris). The recombinant psLAAO1 inhibited the growth of bacteria to the same levels as native psLAAO1 present in the mucus layer. In this study, homology modeling of psLAAO1 predicted metal coordination by residues Y241, H348, and D406. We show that the Michaelis constant (Km) of psLAAO1 decreased and the catalytic constant (Kcat/Km) value increased following pre-treatment of the protein with a chelating agent. In contrast to the non-chelated protein sample, enzymatic activity of EDTA-treated psLAAO1 gradually decreased or was absent after one or two freeze-thaw cycles. The H348A psLAAO1 mutant generated by site-directed mutagenesis and recombinantly produced by P. pastoris did not display antibacterial activity. The results of the metal detection assay revealed that for the non-metal coordinating histidine mutant (H209A, control), the levels of iron, zinc, and magnesium were similar to those of wild-type psLAAO1, whereas magnesium was not detected in the H348A mutant sample. A wild-type psLAAO1 sample treated with chelating agent did not contain zinc and magnesium ions. In conclusion, metal coordination by psLAAO1 affects enzymatic activity, and H348 is involved in the coordination of magnesium, and metal coordination by psLAAO1 provides essential structural stability. KEY POINTS: • Homology modeling of psLAAO1 predicted metal coordination by residue H348 • The H348A psLAAO1 mutant showed no antibacterial activity or magnesium coordination • Metal coordination by H348 affects enzyme activity and structural stability.

In fish, the innate immune system is more important than the adaptive immune system because it responds quickly and nonspecifically to protect against pathogens. Thus, a variety of innate immune molecules have been found in fish. Recently, l-amino acid oxidases (LAOs) were discovered as a new member of the antibacterial protein from fish skin mucus and serum. In this study, we newly found an antibacterial LAO in red-spotted grouper (Epinephelus akaara) serum. It showed a broad range of substrate specificity with aromatic and hydrophobic amino acids. The grouper LAO gene had a low expression level in the kidney under normal conditions; however, it was significantly upregulated by blood loss 1 day after bleeding. In addition, the LAO activity in the serum recovered within 3 days in the same experiment. This quick recovery may indicate that the LAO is an essential innate immune molecule in the whole grouper body.

Bacteriocins are ribosomally synthesized peptides/proteins produced by bacteria. These compounds have antibacterial activity against other bacteria that are usually closely related to the producer strain. Here we describe bacteriocin geobacillin 26 from a thermophilic Gram-positive bacterium Geobacillus stearothermophilus 15. We have purified native bacteriocin, determined its amino acid sequence and heterologously expressed in Gram-negative Escherichia coli. Geobacillin 26 is a heat-labile, high molecular weight antibacterial protein belonging to class III bacteriocins. It has a narrow antibacterial spectrum against other thermophilic bacteria. Our study suggests that this bacteriocin is not a cell wall hydrolyzing enzyme as most of high molecular weight bacteriocins. In addition, geobacillin 26 has no amino acid sequence similarities to other known function proteins. No other class III bacteriocin from a thermophilic bacterium has been reported and well characterized before. Geobacillin 26 as a natural antibacterial agent has a great potential in industry where contamination with other thermophilic bacteria is unwanted. Moreover, this study may prompt to disclose more novel geobacillin 26-like antibacterial proteins, which could find their applications in food industry or medicine.

The mammalian intestine is colonized by trillions of bacteria that perform essential metabolic functions for their hosts. The mutualistic nature of this relationship depends on maintaining spatial segregation between these bacteria and the intestinal epithelial surface. This segregation is achieved in part by the presence of a dense mucus layer at the epithelial surface and by the production of antimicrobial proteins that are secreted by epithelial cells into the mucus layer. Here, we show that resistin-like molecule β (RELMβ) is a bactericidal protein that limits contact between Gram-negative bacteria and the colonic epithelial surface. Mouse and human RELMβ selectively killed Gram-negative bacteria by forming size-selective pores that permeabilized bacterial membranes. In mice lacking RELMβ, Proteobacteria were present in the inner mucus layer and invaded mucosal tissues. Another RELM family member, human resistin, was also bactericidal, suggesting that bactericidal activity is a conserved function of the RELM family. Our findings thus identify the RELM family as a unique family of bactericidal proteins and show that RELMβ promotes host-bacterial mutualism by regulating the spatial segregation between the microbiota and the intestinal epithelium.

Therapeutic LysK-CHAP is a potent anti-staphylococcal protein that could be utilized as an antibiotic substitute. Intein-mediated protein purification is a reasonable and cost-effective method that is most recently used for recombinant therapeutic protein production. Intein (INT) is the internal parts of the protein that can be separated from the immature protein during protein splicing process. This sequence requires no specific enzyme or cofactor for separation. INT sequence and their characteristic of self-cleavage by thiol induction, temperature, and pH changes are used for protein purification. The current study presents the expression of CHAPK262 domain of LysK gene that is fused with INT/chitin-binding sequence while evaluating its purification procedure and antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA). The coding gene sequence of LysK-CHAP (CHAPK262) in pET22-b was amplified with polymerase chain reaction (PCR); the digested product was then cloned into the pTXB1 vector. Electrophoresis confirmed the cloning accuracy of the gene. The pTXB1-CHAPK262 plasmid was transformed to the Escherichia coli ER2566 (E. coli ER2566) expression strain and analyzed for expression of the recombinant protein by SDS-PAGE and Western blotting methods. Finally, CHAPK262 was purified by chitin affinity column using INT tag technology and confirmed by SDS-PAGE. Lytic activity of the purified protein was investigated by disk diffusion method. Cloning of CHAPK262 into the pTXB1 vector, which comprised INT/chitin-binding sequence, was successfully achieved. The SDS-PAGE data also revealed successful expression of the CHAPK262-INT fusion protein and Western blotting method validated the accuracy of the protein. Moreover, purification of CHAPK262 protein was induced by dithiothreitol (DTT) and confirmed by SDS-PAGE. Finally, inhibition zone in MRAS culture medium confirmed antibacterial activity of the protein. Application of intein-mediated antibacterial protein is an appropriate and streamlined method for one-step purification of CHAPK262 as a therapeutic and antibacterial protein. Self-cleaving tags like intein are cost-effective and could be used as a proper purification method for industrial purposes.