Biology subjects

Kosaki, T.

Publications and source records attributed to Kosaki, T..

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↗

bioRxiv2024

Overexpression of Ribosomal Proteins Leads to Zn Resistance in Escherichia coli

Knockout of ribosomal protein bL36 (RpmJ) leads to Zn resistance in Escherichia coli, and the expression of ribosomal protein genes other than RpmJ increase in the rpmJ-knockout strain. In this study, we examined whether the overexpression of ribosomal proteins causes Zn resistance using an E. coli overexpression gene library (ASKA clone library). The overexpression of 48 of the 54 ribosomal proteins led to Zn resistance. However, the overexpression of proteins other than ribosomal proteins did not lead to Zn resistance, suggesting that Zn resistance is a phenomenon specific to the overexpression of ribosomal proteins. In addition, the overexpression of ribosomal proteins did not lead to resistance to metal ions other than Zn (Cu2+, Ni2+, Mn2+, and Ag+), suggesting a Zn-specific resistance mechanism. Deletion of ZntA, a Zn efflux pump, resulted in the loss of Zn resistance in a ribosomal protein-overexpressing strain. Deletion of Lon protease, which is responsible for degrading misfolded proteins, in a ribosomal protein-overexpressing strain resulted in the accumulation of overexpressed ribosomal proteins and loss of Zn resistance. These results suggest that the overexpression of ribosomal proteins leads to Zn resistance in E. coli via ZntA and Lon protease. ImportanceThe ribosome is a complex comprising ribosomal RNAs and more than 50 types of ribosomal proteins. Ribosomal proteins play an important role in ribosomal function responsible for protein translation; however, their involvement in other cellular processes is not fully understood. Based on the finding that ribosomal protein expression increases in a Zn-resistant E. coli mutant, we analyzed 54 ribosomal proteins and found that the overexpression of 48 ribosomal proteins led to Zn resistance. This finding suggests a role for ribosomal proteins in resistance to zinc stress.

microbiology↗

bioRxiv2022

Knockout of ykcB, a putative glycosyltransferase, leads to vancomycin resistance in Bacillus subtilis

Vancomycin resistance of gram-positive bacteria poses a serious health concern around the world. In this study, we searched for vancomycin-resistant mutants from a gene deletion library of a model gram-positive bacterium, Bacillus subtilis, to elucidate the mechanism of vancomycin resistance. We found that knockout of ykcB, a glycosyltransferase that is expected to utilize C55-P-glucose to glycosylate cell surface components, caused vancomycin resistance in B. subtilis. Knockout of ykcB altered the susceptibility to multiple antibiotics, including sensitization to {beta}-lactams, and increased the pathogenicity to silkworms. Furthermore, the ykcB-knockout mutant had: i) an increased content of diglucosyl diacylglycerol, a glycolipid that shares a precursor with C55-P-glucose, ii) a decreased amount of lipoteichoic acid, and iii) decreased biofilm formation ability. These phenotypes and vancomycin resistance were abolished by knockout of ykcC, a ykcB-operon partner involved in C55-P-glucose synthesis. Overexpression of ykcC enhanced vancomycin resistance in both wild-type B. subtilis and the ykcB-knockout mutant. These findings suggest that ykcB deficiency induces structural changes of cell surface molecules depending on the ykcC function, leading to resistance to vancomycin, decreased biofilm formation ability, and increased pathogenicity to silkworms. IMPORTANCEAlthough vancomycin is effective against gram-positive bacteria, vancomycin-resistant bacteria is a major public health concern. While the vancomycin resistance mechanisms of clinically important bacteria such as Staphylococcus aureus, Enterococcus faecium, and Streptococcus pneumoniae are well-studied, they remain unclear in other gram-positive bacteria. In the present study, we searched for vancomycin-resistant mutants from a gene deletion library of a model gram-positive bacterium, Bacillus subtilis, and found that knockout of a putative glycosyltransferase, ykcB, caused vancomycin resistance in B. subtilis. Notably, unlike the previously reported vancomycin-resistant bacterial strains, ykcB-deficient B. subtilis exhibited increased virulence while maintaining its growth rate. Our results broaden the fundamental understanding of vancomycin-resistance mechanisms in gram-positive bacteria.

microbiology↗