Biology subjects

MIYOSHI, S.-I.

Publications and source records attributed to MIYOSHI, S.-I..

3 recordsLinked to original sources

bioRxiv2025

Overexpression of Escherichia coli yaiX Confers Multidrug Resistance and Enhances Virulence in the Silkworm Infection Model

The emergence of bacteria with both antimicrobial resistance and high virulence has become a global health concern, underscoring the urgent need to elucidate the molecular basis underlying these traits. Here, we employed the silkworm (Bombyx mori) infection model, which is suitable for high-throughput screening, together with an Escherichia coli library containing plasmid clones of all genes from strain W3110, to identify genes whose overexpression enhances virulence. We found that overexpression of the uncharacterized protein YaiX promoted bacterial proliferation in silkworms and increased host lethality. Compared with the empty-vector control, the YaiX-overexpressing strain exhibited resistance to multiple antimicrobial agents with diverse mechanisms of action, including {beta}-lactams, tetracyclines, fluoroquinolones, aminoglycosides, cationic surfactants, and hydrogen peroxide. Sequence analysis revealed that amino acids 18-52 of YaiX contain a transferase hexapeptide domain predicted to form a left-handed parallel {beta}-helix. Overexpression of YaiX mutants lacking regions outside this domain conferred ampicillin resistance, whereas deletion of the hexapeptide domain abolished this phenotype. RNA sequencing and GO enrichment analyses further indicated that YaiX overexpression altered the expression of genes encoding RNA-binding proteins and porins. These findings suggest that YaiX overexpression, through its hexapeptide domain, modulates gene expression and contributes to both multidrug resistance and enhanced virulence in E. coli.

microbiology↗

bioRxiv2025

Autoclaving and long-term storage deplete glutamine sources in complex media and affect bacterial phenotypes

Autoclave sterilization is the most common method for sterilizing reagents and media in biology. However, the effects of heat-induced loss or modification of complex medium components on bacterial growth and phenotypes remain poorly understood. Here, we investigated the impact of autoclaving on glutamine sources in bacterial complex media using an Escherichia coli {Delta}glnA mutant, which requires exogenous glutamine for growth. {Delta}glnA exhibited impaired growth in LB medium after autoclaving compared with non-autoclaved LB, whereas its residual growth indicated the presence of heat-stable glutamine sources. Growth assays and HPLC quantification revealed that free glutamine in LB decreased from 58 {micro}M to 12 {micro}M upon autoclaving, while heat-stable glutamine sources remained at 117 {micro}M. Similar growth defects were observed for {Delta}glnA in autoclaved BHI, TSB, and M17 media compared with their non-autoclaved counterparts. Long-term storage of LB at room temperature for 24 weeks also reduced {Delta}glnA growth regardless of autoclaving, compared with freshly prepared LB. Furthermore, supplementation of glutamine sources into glutamine-deficient MHB medium enhanced biofilm formation by Pseudomonas aeruginosa. Collectively, these results demonstrate that autoclaving and storage reduce glutamine sources in complex media, thereby influencing bacterial growth and phenotypes.

microbiology↗

bioRxiv2023

Potential use of Sodium Butyrate (SB) as an anti-virulence agent against Vibrio cholerae targeting ToxT virulence protein.

ABSTRACTCholera, a diarrhoeal disease caused by gram-negative bacterium Vibrio cholerae remains a global health threat in developing countries owing to its high transmissibility and increase in antibiotic resistance. The current issue is to overcome the problem of resistance by antimicrobial therapy. There is a need for alternative strategies with an emphasis on anti-virulent approaches to alter the outcome of bacterial infections. Vibrio cholerae causes cholera by secreting virulence factors in the intestinal epithelial cells. Virulence factors help in cholera toxin production and colonisation during infection. Here, we show that sodium butyrate (SB), a small molecule, had no effect on bacterial viability but was effective in suppressing the virulence attributes of V. cholerae. The production of cholera toxin (CT) was downregulated in a standard V. cholerae El Tor strain and two clinical isolates when grown in presence of sodium butyrate. Analysis of mRNA and protein levels further demonstrated that sodium butyrate reduced the expression of the ToxT-dependent virulence genes like tcpA and ctxAB. DNA-protein interaction assays conducted at cellular (ChIP) and in in vitro conditions (EMSA) indicated that sodium butyrate weakens the binding between ToxT and its downstream promoter DNA, likely by blocking DNA binding. Furthermore, the efficacy of sodium butyrate was confirmed by showing its anti-virulence activity and tissue damage recovery in animal models. Collectively, these findings suggest that sodium butyrate (SB) has the potential to be developed as an anti-virulence agent against V. cholerae in place of conventional antibiotics or as an adjunctive therapy to combat cholera. IMPORTANCEThe world has been facing an upsurge in cholera cases since 2021 with a similar trend continuing into 2022 with over 29 countries reporting cholera outbreaks (World Health Organization 16 December 2022 Disease Outbreak News; Cholera - Global situation). Treatment of cholera involves oral rehydration therapy coupled with antibiotics to reduce the duration of the illness. However, over the last few years, there has been indiscriminate use of antibiotics that contributed largely to the reservoir of antibiotic-resistant strains. In this study, we have addressed the problem of antibiotic resistance by targeting virulence factors. The screening of several compounds led to the identification of a small molecule, sodium butyrate that inhibits the virulence cascade in V. cholerae. We demonstrated that (i) sodium butyrate intervened with ToxT protein-DNA binding and subsequently affected the expression of ToxT-regulated virulence genes (ctxAB and tcpA) (ii) Sodium Butyrate is a potential therapeutic candidate for development of novel antimicrobial agents.

microbiology↗