Abstract
Gramicidin A (
1
) is a peptide antibiotic that disrupts the transmembrane ion concentration gradient by forming an ion channel in a lipid bilayer. Although long used clinically, it is ...limited to topical application because of its strong hemolytic activity and mammalian cytotoxicity, likely arising from the common ion transport mechanism. Here we report an integrated high-throughput strategy for discovering analogues of
1
with altered biological activity profiles. The 4096 analogue structures are designed to maintain the charge-neutral, hydrophobic, and channel forming properties of
1
. Synthesis of the analogues, tandem mass spectrometry sequencing, and 3 microscale screenings enable us to identify 10 representative analogues. Re-synthesis and detailed functional evaluations find that all 10 analogues share a similar ion channel function, but have different cytotoxic, hemolytic, and antibacterial activities. Our large-scale structure-activity relationship studies reveal the feasibility of developing analogues of
1
that selectively induce toxicity toward target organisms.
The demand for novel antibiotics to combat the global spread of multi drug-resistant pathogens continues to grow. Pathogenic bacteria and fungi that cause fatal human infections can also kill ...silkworms and the infected silkworms can be cured by the same antibiotics used to treat infections in the clinic. As an invertebrate model, silkworm model is characterized by its convenience, low cost, no ethical issues. The presence of conserved immune response and similar pharmacokinetics compared to mammals make silkworm infection model suitable to examine the therapeutic effectiveness of antimicrobial agents. Based on this, we utilized silkworm bacterial infection model to screen the therapeutic effectiveness of various microbial culture broths and successfully identified a therapeutically effective novel antibiotic, lysocin E, which has a novel mode of action of binding to menaquinone, thus leading to membrane damage and bactericidal activity. The similar approach to screen potential antibiotics resulted in the identification of other therapeutically effective novel antibiotics, such as nosokomycin and ASP2397 (VL-2397). In this regard, we propose that the silkworm antibiotic screening model is very effective for identifying novel antibiotics. In this review, we summarize the advantages of the silkworm model and propose that the utilization of silkworm infection model will facilitate the discovery of novel therapeutically effective antimicrobial agents.
Abstract
The regulatory network of virulence factors produced by the opportunistic pathogen Staphylococcus aureus is unclear and the functions of many uncharacterized genes in its genome remain to be ...elucidated. In this study, we screened 380 genes whose function was unassigned, utilizing gene-disrupted transposon mutants of the community-acquired methicillin-resistant S. aureus USA300 for pathogenicity in silkworms. We identified 10 strains with reduced silkworm killing ability. Among them, 8 displayed reduced virulence in a mouse model as evidenced by reduced colony-forming units in organs of infected mice. The role of each gene in pathogenicity was further confirmed by complementation and pathogenicity tests in silkworms, where we found that the phenotype was not restored in 1 strain. Additionally, some of the mutants displayed reduced hemolysis, proteolysis, pigment production, and survival in murine RAW 264.7 monocyte-macrophage cells. These newly identified genes involved in virulence will enhance our understanding of the pathogenicity of S. aureus.
We performed a large-scale screening of gene-disrupted mutants of Staphylococcus aureus using a silkworm infection model and identified 8 novel genes involved in the aggravation of virulence in both silkworm and mouse models.
Lysocin E is a lipopeptide with antibiotic activity against methicillin-resistant Staphylococcus aureus. For unclear reasons, the antibacterial activity of lysocin E in a mouse systemic infection ...model is higher than expected from in vitro results, and the in vitro activity is enhanced by addition of bovine serum. Here, we confirm that serum from various species, including humans, increases lysocin E antimicrobial activity, and identify apolipoprotein A-I (ApoA-I) as an enhancing factor. ApoA-I increases the antibacterial activity of lysocin E when added in vitro, and the antibiotic displays reduced activity in ApoA-I gene knockout mice. Binding of ApoA-I to lysocin E is enhanced by lipid II, a cell-wall synthesis precursor found in the bacterial membrane. Thus, the antimicrobial activity of lysocin E is potentiated through interactions with host serum proteins and microbial components.
We previously reported that disruption of the yjbI gene reduced virulence of Staphylococcus aureus. In this study, we found virulence in both silkworms and mice was restored by introducing the yjbH ...gene but not the yjbI gene to both yjbI and yjbH genes-disrupted mutants, suggesting that yjbH, the gene downstream to the yjbI gene in a two-gene operon-yjbIH, is responsible for this phenomenon. We further observed a decrease in various surface-associated proteins and changes in cell envelope glycostructures in the mutants. RNA-seq analysis revealed that disruption of the yjbI and the yjbH genes resulted in differential expression of a broad range of genes, notably, significant downregulation of genes involved in virulence and oxidative stress. Administration of N-acetyl-L-cysteine, a free-radical scavenger, restored the virulence in both the mutants. Our findings suggested that YjbH plays a role in staphylococcal pathogenicity by regulating virulence gene expression, affecting the bacterial surface structure, and conferring resistance to oxidative stress in a host.
Lysocin E, a 37-membered natural depsipeptide, induces rapid bacteriolysis in methicillin-resistant Staphylococcus aureus via a unique menaquinone-dependent mechanism, presenting a promising ...therapeutic lead. Despite the great medical importance, exploring the potential utility of its derivatives as new platform structures for antibiotic development has remained a significant challenge. Here, we report a high-throughput strategy that enabled the preparation of thousands of analogues of lysocin E and large-scale structure-activity relationship analyses. We integrate 26-step total synthesis of 2401 cyclic peptides, tandem mass spectrometry-sequencing, and two microscale activity assays to identify 23 candidate compounds. Re-synthesis of these candidates shows that 11 of them (A1-A11) exhibit antimicrobial activity superior or comparable to that of lysocin E, and that lysocin E and A1-A11 share L-Leu-6 and L-Ile-11. Therefore, the present strategy allows us to efficiently decipher biologically crucial residues and identify potentially useful agents for the treatment of infectious diseases.
genus from Leuconostocaceae family forms a group of Gram-positive lactic acid bacteria (LAB) that mostly reside in fermented foods and some have been isolated from the environment and vertebrates ...including humans. Currently there are 23 recognized species, 16 complete and 37 draft genome assemblies for this genus.
has been found in various sources and is characterized by their probiotic and bacteriocinogenic properties. Despite its widespread importance, little attention has been paid to genomic characterization of this species with the availability of draft assembly of two species in the public database so far. In this manuscript, we identified
0916-4-2 from fermented kimchi and completed its genome sequence. Comparative genomic analysis identified 88 core genes that had interspecies mean amino acid identity of more than 65%. Whole genome phylogenetic analysis showed that three
strains clustered together and the strain 0916-4-2 was close to strain WiKim14.
analysis for the secondary metabolites biosynthetic gene cluster showed that
are far less producers of secondary metabolites compared to other members of Leuconostocaceae. The availability of the complete genome of
0916-4-2 will facilitate further comparative genomic analysis of
species, including studies of its biotechnological potential and improving the nutritional value of various food products.
Bacillus anthracis is an obligate pathogen and a causative agent of anthrax. Its major virulence factors are plasmid-coded; however, recent studies have revealed chromosome-encoded virulence factors, ...indicating that the current understanding of its virulence mechanism is elusive and needs further investigation. In this study, we established a silkworm (Bombyx mori) infection model of B. anthracis. We showed that silkworms were killed by B. anthracis Sterne and cured of the infection when administered with antibiotics. We quantitatively determined the lethal dose of the bacteria that kills 50% larvae and effective doses of antibiotics that cure 50% infected larvae. Furthermore, we demonstrated that B. anthracis mutants with disruption in virulence genes such as pagA, lef, and atxA had attenuated silkworm-killing ability and reduced colonization in silkworm hemolymph. The silkworm infection model established in this study can be utilized in large-scale infection experiments to identify novel virulence determinants and develop novel therapeutic options against B. anthracis infections.
WAP-8294A2 (lotilibcin, 1) is a potent antibiotic with superior in vivo efficacy to vancomycin against methicillin-resistant Staphylococcus aureus (MRSA). Despite the great medical importance, its ...molecular mode of action remains unknown. Here we report the total synthesis of complex macrocyclic peptide 1 comprised of 12 amino acids with a β-hydroxy fatty-acid chain, and its deoxy analogue 2. A full solid-phase synthesis of 1 and 2 enabled their rapid assembly and the first detailed investigation of their functions. Compounds 1 and 2 were equipotent against various strains of Gram-positive bacteria including MRSA. We present evidence that the antimicrobial activities of 1 and 2 are due to lysis of the bacterial membrane, and their membrane-disrupting effects depend on the presence of menaquinone, an essential factor for the bacterial respiratory chain. The established synthetic routes and the menaquinone-targeting mechanisms provide valuable information for designing and developing new antibiotics based on their structures.
This study was performed with the aim of making a very simple recipe of silkworm diet for research purposes, especially screening of drug candidates. We prepared a diet containing mulberry leaves ...powder and soybean flour at different ratios, fed them to fifth instar silkworm larvae, and observed their growth. We selected the diet with 1:1 ratio of mulberry powder and soybean flour, named MS-11, and used for further experiments. MS-11 diet was available for oral administration of drugs in silkworm hyperglycemic model and infection model. The availability of a simple artificial diet for experiments that require feeding silkworms will enhance the use of silkworms for biological, biotechnological, and pharmacological researches.
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