Atsushi Miyashita

Lysin motif (LysM) domain-containing receptors are evolutionarily conserved pattern recognition receptors (PRRs) that serve as key mediators of glycan sensing and innate immune activation in plants and mammals. In invertebrates, however, their role in activating innate immunity remains poorly understood, although some evidence for immunosuppressive functions exists. In this study, we performed in silico structural analyses and identified a putative Bombyx mori LYSMD3 homolog ( XP_004933441.1 ). This protein exhibits high structural similarity in the LysM domain to human LYSMD3, with a root-mean-square deviation (RMSD) of 0.559 Å, indicating close structural alignment. RNA-seq analysis of hemocytes isolated from silkworm larvae injected with N -acetylchitohexaose (GN6), a chitin-derived oligosaccharide and known ligand of human LYSMD3, revealed transcriptional activation of innate immune effectors, including antimicrobial peptide (AMP) genes such as cecropins . GN6 also induced cecropin transcription in isolated hemocytes in vitro , and Western blotting of hemolymph confirmed elevated cecropin B protein levels. Furthermore, GN6 and chitin significantly improved survival outcomes against P. aeruginosa infection, with median effective doses (ED₅₀) values of 0.62 and 0.48 µg/larva, respectively. In contrast, N -acetylglucosamine (GlcNAc) and shorter oligosaccharides (GN2–GN5) were ineffective. These findings provide the first molecular-level evidence of a putative glycan receptor in silkworms based on the structural similarity to known LysM domains. Moreover, GN6-induced antimicrobial peptide expression and enhanced infection resistance demonstrate immune activation in this model, supporting an evolutionarily conserved glycan-sensing pathway in invertebrates.

Pathogenic fungi pose significant societal challenges due to limited therapeutic targets resulting from the eukaryotic nature of fungi. This limitation emphasizes the importance of enhancing susceptibility to inhibitors of Cyp51, a crucial enzyme in ergosterol biosynthesis targeted by azole antifungals. In Cyp51 isozyme deletion strains (Δ cyp51A and Δ cyp51B ) of Trichophyton rubrum , the predominant dermatophyte species, we found that Cyp51B is essential for basal mycelial growth, while Cyp51A functions as an inducible isozyme associated with azole tolerance. Based on these differential functions, we hypothesized that each isozyme would show distinct susceptibility to azole antifungals. Our study demonstrated that most azoles exhibited increased antifungal activity against Δ cyp51A , while select agents demonstrated increased antifungal activity against Δ cyp51B . Remarkably, fluconazole, sulconazole, and imazalil exhibited relatively increased activity against Δ cyp51A , whereas prochloraz demonstrated increased activity against Δ cyp51B . Combining these isozyme-selective agents exerted synergistic effects against the wild-type strain and the parent ku80 -knockout strain but not against individual Cyp51 knockout mutants. Our data revealed that the two Cyp51 isozymes can be selectively inhibited by different azole antifungals, resulting in a synergistic effect when combined. This synergistic effect was also observed on another fungal species, Aspergillus welwitschiae , which also has two Cyp51 isozymes. These data demonstrate that combining azole antifungals with different Cyp51 isozyme selectivities exerts synergistic effects against fungi possessing multiple Cyp51 isozymes. These findings advance antifungal therapeutic strategies by demonstrating that the combination of antifungals with different Cyp51 isozyme selectivities offers a promising approach for treating fungal infections, opening new avenues for isozyme-specific drug development.

Mucorales fungi cause life-threatening mucormycosis in patients with clinical risk factors, such as immunodeficiency. Moreover, they are resistant to several antifungal drugs, highlighting the urgent need for novel therapies. Traditional mammalian models are expensive and raise ethical concerns, thereby limiting their suitability for large-scale studies. We established a silkworm (Bombyx mori) infection model to investigate the pathogenicity of Mucorales and evaluated its relevance to human infection. Strains of Rhizopus arrhizus, Mucor circinelloides, and Cunninghamella bertholletiae induced fatal infections in the silkworm. Grocott-stained silkworm tissue sections revealed hyphal invasion patterns closely resembling those observed in human mucormycosis. In addition, experimental simulation of clinical risk factors (steroid use and iron overload) significantly reduced the median lethal dose (LD₅₀). Treatment with the antifungal drug isavuconazonium prolonged the survival of R. arrhizus-infected silkworms, suggesting the potential use of this model for novel antifungal screening. LD₅₀ estimation for four R. arrhizus strains revealed up to a 100-fold difference in pathogenicity to the silkworm among the tested strains, corresponding to variations in cell surface characteristics. Interestingly, the presence of high-molecular weight (50–100 kDa) cell surface proteins was associated with high pathogenicity among R. arrhizus strains. In conclusion, the silkworm is a viable, human-relevant alternative model to investigate Mucorales infections, with potential for large-scale antimucormycosis drug screening.

Dominance of Bacillus species on conventional agar media often hampers the isolation of taxonomically diverse soil bacteria (i.e., “ Bacillus dominance”). To overcome this Bacillus dominance, we developed a novel agar medium containing leaf mold extract (LME) and assessed its utility for isolating microorganisms from soil samples. For comparison, conventional yeast malt extract agar (YME agar) was used as a control. 16S rRNA gene sequencing revealed that colonies cultured on YME agar predominantly consisted of Bacillus -related bacteria, whereas LME agar enabled the growth of a wider variety of taxa, particularly actinomycetes such as Streptomyces . Notably, LME agar supported a higher diversity of soil bacterial isolates even from samples with a low abundance of Bacillus -related species. Among the 138 isolates recovered from LME agar, some either exhibited very low 16S rRNA gene sequence identity to known species in public databases, or failed to yield 16S rRNA amplicons with conventional universal primers, suggesting the presence of previously uncharacterized or novel taxa. Furthermore, one unidentified isolate failed to grow on standard nutrient media (YME, BHI, LB10, or TSB), but proliferated in liquid leaf mold extract, indicating a specific nutrient dependency. These findings demonstrate that LME agar can facilitate the isolation of a broader spectrum of soil microorganisms (including rare and previously uncultured species) not readily recoverable on common basal media. Incorporation of ecosystem-derived substrates into culture media may greatly enhance the discovery and study of novel microbial diversity from environmental samples. Importance: Soil contains a vast number of microorganisms, many of which remain undiscovered due to limitations in standard laboratory methods. A major issue is the overgrowth of Bacillus species on conventional media, which suppresses the growth of other bacteria. To address this, we developed a new agar medium using leaf mold extract (LME) and found it successfully supported the growth of a much wider variety of soil bacteria, including rare species. Some of the isolated microbes could not be identified using standard genetic tools, and one unique strain only grew in LME-based media, indicating a special nutrient requirement. These findings show that using natural environmental components like leaf mold can help scientists grow and study bacteria that were previously missed. This approach can expand our understanding of microbial diversity and may lead to the discovery of new microbes with potential applications in agriculture, medicine, or environmental science.

Global concerns about drug-resistant bacteria have underscored the need for new antimicrobial drugs. Emerging strategies in drug discovery include considering the third factors that influence drug activity. These factors include host-derived elements, adjuvants, and drug combinations, which are crucial in regulating antimicrobial efficacy. Traditional in vivo assessments have relied on animal models to study drug absorption, distribution, metabolism, excretion, and toxicity (ADMET). Alternative models, such as silkworms, are being explored to overcome the ethical and financial barriers associated with mammalian models. The silkworm has been proven effective in evaluating ADMET and in highlighting the therapeutic potential enhanced by third factors. Host factors (either mammalian or non-mammalian) enhance the antimicrobial activity of antimicrobial agents such as lysocin E. Additionally, using d-cycloserine to potentiate vancomycin has successfully combated vancomycin-resistant infections in silkworms. Leveraging silkworms in drug discovery could establish a novel screening method incorporating interactions with third factors, whether host related or non-host-related, thus promising new pathways for identifying antimicrobial drugs with unique mechanisms of action.

We have established several models of infectious diseases in silkworms to explore disease-causing mechanisms and identify new antimicrobial substances. These models involve injecting laboratory-cultured pathogens into silkworms and monitoring their survival over a period of days. The use of silkworms is advantageous because they are cost-effective and raise fewer ethical concerns than mammalian subjects, allowing for larger experimental group sizes. To capitalize on these benefits, there is a growing importance in mechanizing and automating the experimental processes that currently require manual labor. This paper discusses the future of laboratory automation, specifically through the mechanization and automation of silkworm-based experimental procedures.

In this study, we developed a new agar medium using leaf mold extract and evaluated its microbial cultivation performance with soil samples. As a control for performance evaluation, the general-purpose nutrient medium YME agar was used. For YME agar, isolated microbes were confirmed to belong mainly to the Bacillus genus through 16S rRNA gene sequencing. In contrast, for the leaf mold agar, bacteria belonging to either Streptomyces or Rhizobium were frequently isolated. Also, of the 51 sequenced isolates on the leaf mold agar medium, an unidentified species (i.e., not listed in databases) with 16S rRNA sequence identity below 98.7% was found. The unidentified species did not grow in standard nutrient media (YME, BHI, LB10, or TSB) but grew in 10% leaf mold extract. The findings of this study suggest that the use of leaf mold agar medium can effectively isolate soil microbes that are difficult to culture in general-purpose nutrient media such as YME. Moreover, this approach indicates a viable method for discovering unknown, previously-uncultured microbial species from soil samples.

The molecular mechanisms that allow pathogenic bacteria to infect animals have been intensively studied. On the other hand, the molecular mechanisms by which bacteria acquire virulence functions are not fully understood. In the present study, we experimentally evaluated the evolution of a non-pathogenic strain of Escherichia coli in a silkworm infection model and obtained pathogenic mutant strains. As one cause of the high virulence properties of E. coli mutants, we identified amino acid substitutions in LptD (G580S) and LptE (T95I) constituting the lipopolysaccharide (LPS) transporter, which translocates LPS from the inner to the outer membrane and is essential for E. coli growth. The growth of the LptD and LptE mutants obtained in this study was indistinguishable from that of the parent strain. The LptD and LptE mutants exhibited increased secretion of outer membrane vesicles containing LPS and resistance against various antibiotics, antimicrobial peptides, and host complement. In vivo cross-linking studies revealed that the conformation of the LptD-LptE complex was altered in the LptD and LptE mutants. Furthermore, several clinical isolates of E. coli carried amino acid substitutions of LptD and LptE that conferred resistance against antimicrobial substances. This study demonstrated an experimental evolution of bacterial virulence properties in an animal infection model and identified functional alterations of the growth-essential LPS transporter that led to high bacterial virulence by conferring resistance against antimicrobial substances. These findings suggest that non-pathogenic bacteria can gain virulence traits by changing the functions of essential genes, and provide new insight to bacterial evolution in a host environment. Pathogenic bacteria developed their virulence properties by changing the functions of various genes after the emergence of the host animals on earth. The types of gene function alterations that confer bacterial virulence properties, however, have remained unclear. We utilized a silkworm infection model to perform an experimental evolution of bacterial virulence activity. From a non-pathogenic strain of Escherichia coli, we obtained a mutant strain that exhibited 500-fold higher virulence than the original strain and identified mutations of the lipopolysaccharide (LPS) transporter, which translocates LPS onto the bacterial surface, as one cause of the high virulence. The mutations changed the structure of the LPS transporter, increased the secretion of outer membrane vesicles, and enabled bacterial survival in the presence of host antimicrobial substances. This mechanism to gain high virulence occurs naturally, as several E. coli clinical isolates carried mutations of the LPS transporter that confer resistance against antimicrobial substances. Our study unveiled a novel mechanism by which bacteria increase their virulence through modifying their gene function.

Should females alter their reproductive strategy when attacked by pathogens? Two hypotheses provide opposite predictions. Terminal reproductive investment theory predicts that reproduction should increase when the risk of death increases. However, physiological trade-offs between reproduction and immune function might be expected to produce a decrease in reproduction during a robust immune response. There is evidence for both hypotheses. We examine whether age determines the effect of an immune challenge on reproductive strategy in long-winged females of the Texas field cricket, Gryllus texensis, when fed an ecologically valid (i.e. limited) diet. The limited diet reduced reproductive output. However, even under resource-limited conditions, immune challenge had no effect on the reproductive output of young or middle-aged females. Both reproductive output and immune function (lysozyme-like activity and phenoloxidase (PO) activity) increased with age, which is contrary to both hypotheses. We hypothesize that PO activity is pleiotropic and represents an investment in both reproduction and immune function. Three proPO genes (identified in a published RNA-seq dataset (transcriptome)) were expressed either in the fat body or the ovaries (supporting the hypothesis that PO is bifunctional). The possible bifunctionality of PO suggests that it may not be an appropriate immune measure for studies on immune/reproductive trade-offs. This study also suggests that the threshold for terminal reproductive investment may not decrease prior to senescence in some species.

1. Should females increase or decrease reproduction when attacked by pathogens? Two hypotheses provide opposite predictions. Terminal reproductive investment theory predicts an increase in reproduction, but hypothesized physiological trade-offs between reproduction and immune function might be expected to produce a decrease. There is evidence for both hypotheses. What determines the choice between the two responses remains unclear. We examine the effect of age on the reproductive response to immune challenge in long-wing females of the Texas field cricket, Gryllus texensis, when fed an ecologically valid (limited) diet. 2. The limited diet reduced reproductive output. However, immune challenge had no effect on their reproductive output either in young or middle-aged crickets, which is contrary to either prediction. 3. Flight muscle maintenance correlated negatively with reproductive output, suggesting a physiological trade-off between flight muscle maintenance and reproduction. Within the long-wing variant there was considerable variability in flight muscle maintenance. This variability may mask physiological trade-offs between immunity and reproduction. 4. Middle-aged crickets had higher total phenoloxidase (PO) activity in their hemolymph, compared to young females, which is contrary to the terminal investment theory. Given that PO is involved in both immunity and reproduction, the increased PO may reflect simultaneous investment in both functions. 5. We identified four proPO transcripts in a published RNA-seq dataset (transcriptome). Three of the proPO genes were expressed either in the fat body or the ovaries (supporting the hypothesis that PO is bifunctional); however, the two organs expressed different subsets. The possible bifunctionality of PO suggests that it may not be an appropriate immune measure for immune/reproductive trade-offs in some species. 6. Increasing age may not cue terminal reproductive investment prior to senescence.