Biology subjects

Tsuneda, S.

Publications and source records attributed to Tsuneda, S..

5 recordsLinked to original sources

bioRxiv2026

A Novel Metric for Quantifying the Sustainability of Phage-Mediated Bacterial Suppression

The global spread of antimicrobial-resistant (AMR) bacteria represents one of the greatest threats to modern medicine, making the development of novel therapeutic strategies increasingly urgent. Phage therapy, which exploits bacteriophages (phages, viruses that specifically infect and kill bacteria) has regained attention as a therapeutic approach for multidrug-resistant infections. One critical determinant of treatment outcome is the capacity of phages to sustain bacterial growth suppression; however, no metric has previously existed to directly quantify the duration of effective lytic activity. Here, we propose the Sustainability Index (SusI), a novel metric that quantifies both the duration and extent of phage-mediated bacterial growth suppression, which is restricted to the primary lysis period from lysis initiation to resistance emergence. Evaluation of individual phages and two-phage cocktails against both laboratory and clinical isolates of Escherichia coli demonstrated that SusI provides information independent of the Virulence Index, which primarily reflects bactericidal activity during the initial phase of infection, and serves as a complementary metric to the Suppression Index, which may incorporate behavior beyond primary lysis. Cocktails composed of phages targeting different receptors specificities consistently exhibited higher SusI values, consistent with the notion that multifaceted selective pressure delays resistance emergence. Furthermore, in a mouse model of systemic infection established by intraperitoneal administration, cocktails with higher SusI values demonstrated superior therapeutic efficacy. These results confirm SusI as a practical metric for rational phage cocktail design. As phage therapy advances toward clinical implementation, standardized quantitative metrics such as SusI are expected to facilitate evidence-based selection of therapeutic phages across diverse pathogens and infection conditions. ImportanceThe global spread of antimicrobial-resistant bacteria is making bacterial infections increasingly difficult to treat. Phage therapy, which uses bacteriophages (viruses that specifically infect bacteria), has re-emerged as a therapeutic alternative; however, reliable methods to determine in advance which phages will be therapeutically effective remain limited. Current evaluation metrics are well-suited for quantifying how rapidly phages kill bacteria but were not designed to directly measure how long lytic activity is sustained before resistant bacteria emerge. Here, we developed the Sustainability Index (SusI), a novel metric that specifically quantifies the duration of effective bacterial growth suppression. Evaluation of multiple phages and their combinations (cocktails) against both laboratory and clinical bacterial isolates demonstrated that SusI can distinguish phage combinations that existing metrics fail to differentiate. Moreover, in a mouse model of lethal bacterial infection, higher SusI values correlated with improved therapeutic outcomes. SusI has potential as a practical tool for selecting phages with greater likelihood of therapeutic success.

microbiology↗

bioRxiv2026

Environment-responsive individual cell growth behavior shapes stochastic and deterministic population establishment in ammonia-oxidizing bacteria

Many environmental bacteria do not readily grow under laboratory conditions and population establishment often occurs stochastically. Although the scout hypothesis has been proposed to explain stochastic population establishment in environmental bacteria, how stochastic population establishment is shaped by individual cell growth behaviors in environmental isolates remains unclear. In the present study, we focused on the ammonia-oxidizing bacterium Nitrosomonas sp. PY1 and showed that environmentally responsive individual cell growth behavior, incorporating time-dependent stochastic growth initiation, shapes both deterministic and stochastic population establishment dynamics. Using single-cell observation, we revealed that PY1 altered cell growth behavior in response to surrounding biomass production ({Delta}Vt). These {Delta}Vt-dependent changes in growth behavior were suppressed by the addition of its own cell-free supernatant (CFS), indicating the presence of a growth regulation mechanism via cell-cell communication. Replicate cultures under the same conditions showed that the population establishment of PY1 was stochastic, whereas the model strain Nitrosomonas europaea exhibited synchronized population establishment, consistent with previous reports. This stochasticity in PY1 was also eliminated by the addition of CFS. Finally, a simulation model based on {Delta}Vt-dependent cell growth behavior of PY1 successfully reproduced synchronized population establishment in the presence of CFS. By contrast, the stochastic population establishment observed in the absence of CFS was successfully reproduced by a model incorporating {Delta}Vt-independent growth initiation following a Weibull distribution. Such environmentally responsive changes in population establishment dynamics may contribute to the low isolation success of environmental bacteria and sudden blooms of the rare biosphere.

microbiology↗

bioRxiv2025

Novel Escherichia coli Phages Representing a Distinct Genus within Stephanstirmvirinae: Genome and Host Range Characteristics

Bacteriophages play crucial roles in microbial ecosystems and have potential biotechnological applications. However, our understanding of culturable phages remains limited. This study characterized six novel Escherichia coli phages isolated from pig farm wastewater and urban sewage using comprehensive genomic, morphological, and host-range analyses. Using multiple comparative approaches, including gene-sharing network analysis, average nucleotide identity (ANI), and nucleotide intergenomic similarity (NIS), we demonstrated that five of these phages form a distinct group within the subfamily Stephanstirmvirinae, potentially representing a novel genus provisionally named "Wecvirus". These phages were further classified into two distinct species within the proposed genus, each of which exhibits a unique host range pattern. This host specificity is reflected in the species-specific differences in the amino acid sequences of tail fibers, which are crucial for infection. The remaining phage, which was not classified as Wecvirus exhibited characteristics that challenged the current classification criteria, highlighting the need for more flexible taxonomic approaches. Our findings expand the understanding of phage diversity within Stephanstirmvirinae and contribute to the evolving phage taxonomy framework. ImportanceThe rapid emergence of antibiotic-resistant pathogens necessitates the development of alternative antimicrobial strategies, with phage therapy showing promising potential. However, the successful implementation of phage-based treatments requires a comprehensive understanding of phage diversity and host-pathogen interactions. Our study expands the known diversity within Stephanstirmvirinae by characterizing novel E. coli phages that constitute a distinct genus. The identification of species-specific host recognition mechanisms within this genus provides insights into phage-host adaptation. These findings contribute to our understanding of phage taxonomy and evolution, while also offering practical implications for the development of phage-based therapeutics against pathogenic E. coli strains. This research highlights the importance of continued exploration of novel phages for both fundamental understanding and therapeutic applications.

evolutionary biology↗

bioRxiv2024

From phenotype to receptor: validating physiological clustering of Escherichia coli phages through comprehensive receptor analysis

Understanding the relationship between bacteriophage (phage) classification and target receptors is crucial for phage ecology and applied research. In this study, we compared 13 previously isolated Escherichia coli phages based on physiological characteristics, whole-genome sequences, and tail fiber protein phylogenetics. We improved our previously proposed physiological clustering method by optimizing the bacterial panel for host range assessment, implementing appropriate distance metrics for mixed data types, and applying silhouette coefficient analysis for objective determination of optimal cluster numbers. We combined genomic analysis and lipopolysaccharide (LPS) structural analysis of phage-resistant E. coli strains to identify target receptors of the phages. Complementation experiments further confirmed the direct involvement of identified genes in phage reception. The results revealed that phylogenetically distinct E. coli phages target different sites in the LPS R-core region (modified by WaaV, WaaW, WaaT, and WaaY), membrane proteins (NfrB, TolA, YhaH), or flagella. Our analysis revealed that, subtle chemical modifications of LPS (such as Heptose phosphorylation) were shown to be important for E. coli phage recognition. Furthermore, physiological characteristics, tail fiber phylogenetics, and whole genome analysis independently classified the phages with high correlation to target receptor specificity. The addition of three phages with known receptors further validated our approach. Our results suggest that grouping based on physiological characteristics (such as lysis dynamics and host range) and genotypes (tail fiber phylogenetics or whole genome analysis) independently classified phages with high correlation to target receptor specificity. Here, we elucidated the diversity and specificity of E. coli phage target receptors, providing new insights into the classification of E. coli phages and phage-host interactions. ImportancePhage therapy is gaining attention as an alternative treatment for antibiotic-resistant bacteria. Developing effective phage cocktails requires combining phages with different target receptors, but traditional methods for identifying target receptors are labor-intensive. This study demonstrates that E. coli phages targeting the TK001 strain with different target receptors can be grouped based on their physiological characteristics, tail fiber sequences, or whole genomes. Our approach was enhanced through systematic bacterial panel selection for host range assessment, optimized distance metrics for physiological characteristics, and objective cluster determination using silhouette coefficient analysis for all three classifications. This insight can be used to more efficiently create diverse phage cocktails. Additionally, we identified phages targeting diverse sites, including different regions of LPS, membrane proteins, and flagella. These findings deepen our understanding of phage host recognition mechanisms, enabling the rapid preparation of effective phage cocktails and contributing to new advancements in bacterial infection treatment.

microbiology↗

bioRxiv2020

Global identification of RsmA/N binding sites in Pseudomonas aeruginosa by in vivo UV CLIP-seq

Posttranscriptional regulation of gene expression in bacteria is performed by a complex and hierarchical signaling cascade. Pseudomonas aeruginosa harbors two redundant RNA-binding proteins RsmA/RsmN (RsmA/N), which play a critical role in balancing acute and chronic infections. However, in vivo binding sites on target transcripts and the overall impact on the physiology remains unclear. In this study, we applied in vivo UV crosslinking immunoprecipitation followed by RNA-sequencing (UV CLIP-seq) to detect RsmA/N binding sites at single-nucleotide resolution and mapped more than 500 peaks to approximately 400 genes directly bound by RsmA/N in P. aeruginosa. This also demonstrated the ANGGA sequence in apical loops skewed towards 5UTRs as a consensus motif for RsmA/N binding. Genetic analysis combined with CLIP-seq results identified previously unrecognized RsmA/N targets involved in LPS modification. Moreover, the small non-coding RNAs RsmY/RsmZ, which sequester RsmA/N away from target mRNAs, are positively regulated by the RsmA/N-mediated translational repression of hptB, encoding a histidine phosphotransfer protein, and cafA, encoding a cytoplasmic axial filament protein, thus providing a possible mechanistic explanation for homeostasis of the Rsm system. Our findings present the global RsmA/N-RNA interaction network that exerts pleiotropic effects on gene expression in P. aeruginosa. IMPORTANCEThe ubiquitous bacterium Pseudomonas aeruginosa is notorious as an opportunistic pathogen causing life-threatening acute and chronic infections in immunocompromised patients. P. aeruginosa infection processes are governed by two major gene regulatory systems, namely, the GacA/GacS (GacAS) two-component system and the RNA-binding proteins RsmA/RsmN (RsmA/N). RsmA/N basically function as a translational repressor or activator directly by competing with the ribosome. In this study, we identified hundreds of RsmA/N regulatory target RNAs and the consensus motifs for RsmA/N bindings by UV crosslinking in vivo. Moreover, our CLIP-seq revealed that RsmA/N posttranscriptionally regulate cell wall organization and exert feedback control on GacAS-RsmA/N systems. Many genes including small regulatory RNAs identified in this study are attractive targets for further elucidating the regulatory mechanisms of RsmA/N in P. aeruginosa.

microbiology↗